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Comparison of Functional Proteomic Analyses of Human Breast Cancer Cell Lines T47D and MCF7
T47D and MCF7 are two human hormone-dependent breast cancer cell lines which are widely used as experimental models for in vitro and in vivo (tumor xenografts) breast cancer studies. Several proteins involved in cancer development were identified in these cell lines by proteomic analyses. Although these studies reported the proteomic profiles of each cell line, until now, their differential protein expression profiles have not been established. Here, we used two-dimensional gel and mass spectrometry analyses to compare the proteomic profiles of the two cell lines, T47D and MCF7. Our data revealed that more than 164 proteins are differentially expressed between them. According to their biological functions, the results showed that proteins involved in cell growth stimulation, anti-apoptosis mechanisms and cancerogenesis are more strongly expressed in T47D than in MCF7. These proteins include G1/S-specific cyclin-D3 and prohibitin. Proteins implicated in transcription repression and apoptosis regulation, including transcriptional repressor NF-X1, nitrilase homolog 2 and interleukin-10, are, on the contrary, more strongly expressed in MCF7 as compared to T47D. Five proteins that were previously described as breast cancer biomarkers, namely cathepsin D, cathepsin B, protein S100-A14, heat shock protein beta-1 (HSP27) and proliferating cell nuclear antigen (PCNA), are found to be differentially expressed in the two cell lines. A list of differentially expressed proteins between T47D and MCF7 was generated, providing useful information for further studies of breast cancer mechanisms with these cell lines as models.
Adjo Aka, Juliette; Lin, Sheng-Xiang
Comparison of functional proteomic analyses of human breast cancer cell lines T47D and MCF7.
T47D and MCF7 are two human hormone-dependent breast cancer cell lines which are widely used as experimental models for in vitro and in vivo (tumor xenografts) breast cancer studies. Several proteins involved in cancer development were identified in these cell lines by proteomic analyses. Although these studies reported the proteomic profiles of each cell line, until now, their differential protein expression profiles have not been established. Here, we used two-dimensional gel and mass spectrometry analyses to compare the proteomic profiles of the two cell lines, T47D and MCF7. Our data revealed that more than 164 proteins are differentially expressed between them. According to their biological functions, the results showed that proteins involved in cell growth stimulation, anti-apoptosis mechanisms and cancerogenesis are more strongly expressed in T47D than in MCF7. These proteins include G1/S-specific cyclin-D3 and prohibitin. Proteins implicated in transcription repression and apoptosis regulation, including transcriptional repressor NF-X1, nitrilase homolog 2 and interleukin-10, are, on the contrary, more strongly expressed in MCF7 as compared to T47D. Five proteins that were previously described as breast cancer biomarkers, namely cathepsin D, cathepsin B, protein S100-A14, heat shock protein beta-1 (HSP27) and proliferating cell nuclear antigen (PCNA), are found to be differentially expressed in the two cell lines. A list of differentially expressed proteins between T47D and MCF7 was generated, providing useful information for further studies of breast cancer mechanisms with these cell lines as models. PMID:22384035
Aka, Juliette Adjo; Adjo Aka, Juliette; Lin, Sheng-Xiang
Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.
The human breast tumor cloned cell lines T47D-A8 and All are estrogen dependent for cell proliferation in the nude mouse model. In contrast, these cells multiplied at similar rates when grown in serum-free cultures, regardless of the presence of 17 beta-estradiol (3 X 10(-11) to 3 X 10(-8) M estradiol). Addition of 10% charcoal-dextran stripped human female serum to the culture medium resulted in a marked inhibition of cell proliferation. The addition of 3 X 10(-11) M estradiol overcame the inhibitory effect of serum. Similar results were obtained with the human breast tumor C7MCF7 cell line. Both cell lines contain similar estrophilin levels. The Kd of the estrophilin-estradiol complex was 0.39 X 10(-10) M for C7MCF7 cells and 4.4 X 10(-10) M for T47D-A11 cells. Maximal cell yields were achieved at 5 X 10(-12) M free estradiol levels in 10% charcoal-dextran stripped serum supplemented medium. These data are compatible with the following interpretation: (a) estradiol-sensitive cells are inhibited from proliferating by a serum-borne factor; and (b) estradiol neutralizes this inhibitory effect. This mechanism seems not to be mediated by estradiol binding to the cellular estrophilins because (a) the free estradiol levels needed for maximal response are significantly lower than the estrophilin Kds, and (b) maximal proliferation rates occur at similar estradiol concentrations for these three cell lines, regardless of the binding properties of their estrophilins. PMID:3697972
Soto, A M; Murai, J T; Siiteri, P K; Sonnenschein, C
Modulation of vitamin D receptor and estrogen receptor by 1,25(OH) 2-vitamin D 3 in T-47D human breast cancer cells
1,25(OH)2-Vitamin D3 inhibits breast cancer cell proliferation through interaction with the vitamin D receptor (VDR). Regulation of VDR is under the influence of several factors which include the functional ligand for this receptor (1,25(OH)2-vitamin D3) as well as heterologous steroid hormones. We evaluated the nature of homologous regulation in T-47D human breast cancer cells with a radiolabelled ligand binding assay
Fatemeh Davoodi; Richard V. Brenner; Stephen R. T. Evans; Lisa M. Schumaker; Mohsen Shabahang; Russell J. Nauta; Robert R. Buras
Resveratrol-Induced Apoptosis Is Associated with Activation of p53 and Inhibition of Protein Translation in T47D Human Breast Cancer Cells
Background and Purpose:Trans-resveratrol (RSVL; 3,4?,5-trihydroxystilbene), a natural compound found in grapes, berries, peanuts and red wine exerts certain anticancer roles in different human cancer types. However, the exact molecular mechanism(s) behind such a role remains to be elucidated, thus the aim of this study. Experimental Approach: T47D human breast cancer cells were treated with RSVL and cell proliferation was measured
Moussa Alkhalaf
Inhibition of proliferation of T47D human breast cancer cells: alterations in progesterone receptor and p53 tumor suppressor protein.
We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effective inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24-2.4 micrograms/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels-a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR. PMID:9350037
Dinda, S; Kodali-Gali, S; Sevilla, L; Burkley, M; Hurd, C; Moudgil, V K
Effect of Medrogestone on 17beta-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.
Estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell, or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of Medrogestone (Prothil) on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) mol/l) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 h of the cell culture, Medrogestone significantly inhibits this transformation in a dose-dependent manner by 39% and 80% at 5 x 10(-8) M and 5 x 10(-5) M, respectively in T-47D cells; the effect is less intense in MCF-7 cells: 25% and 55% respectively. The IC50 values are 0.45 micromol/l in T-47D and 17.36 micromol/l in MCF-7 cells. It is concluded that the inhibition provoked by Medrogestone on the reductive 17beta-HSD activity involved in the local biosynthesis of the biologically active estrogen estradiol, may constitute a new therapeutic approach for the treatment of breast cancer. PMID:10215037
Chetrite, G S; Ebert, C; Wright, F; Philippe, J C; Pasqualini, J R
Effect of nomegestrol acetate on human estrogen sulfotransferase activity in the hormone-dependent MCF-7 and T-47D breast cancer cell lines.
Breast cancer cells possess all the enzymes involved in the last steps of estradiol (E2) bioformation, as well as in its transformation (e.g. sulfotransferases) for the conversion to estrogen sulfates (ES). As ES are biologically inactive, the formation of these conjugates is an important transformation pathway in the control of the hormone. In the present study, we explored the effect of nomegestrol acetate on the sulfotransferase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cells. After 24-h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5 x 10(-9) mol/l), it was observed that the sulfotransferase activity was present in both cell lines, since the concentrations of estrogen sulfates found were 9.40 +/- 1.10 in MCF-7 cells and 6.65 +/- 0.72 in the T-47D cells. The presence of ES was found exclusively in the culture medium, which suggests that as soon as the sulfate is biosynthesized it is secreted into the medium. Nomegestrol acetate has a stimulatory effect on sulfotransferase activity: at low doses (5 x 10(-8) and 5 x 10(-7) mol/l) this compound strongly increases the activity of this enzyme by 60.6% and 83%, respectively, in the MCF-7 cells and by 69.2% at 5 x 10(-7) mol/l in T-47D cells. At a high concentration (5 x 10(-5) mol/l) the stimulatory effect of nomegestrol acetate on the sulfotransferase activity was only 5.4% and 6.1%, respectively, in MCF-7 and T-47D cells. In conclusion, the stimulation provoked at low doses by nomegestrol acetate on the estrogen sulfotransferase activity involved in the biosynthesis of the biologically inactive estrogen sulfates in hormone-dependent breast cancer cells is an important effect of this progestin and can open attractive clinical applications. PMID:14981909
Chetrite, G�rard Samuel; Paris, Jacques; Shields-Botella, Jacqueline; Philippe, Jean-Claude; Pasqualini, Jorge Raul
Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.
In the present study, we explored the effect of the progestin medrogestone on the sulfatase and sulfotransferase activities in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. After 24 h incubation at 37 degrees C of physiological concentrations of estrone sulfate ([3H]-E1S: 5x10(-9) mol/l), it was observed that this estrogen was converted in a great proportion to E2 in both cell lines. Medrogestone significantly inhibits this transformation, at all the concentrations tested (5x10(-8) to 5x10(-5) mol/l), in both cell lines. The IC50 values were 1.93 micromol/l and 0.21 micromol/l in MCF-7 and T-47D cells, respectively. In another series of studies, after 24 h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5x10(-9) mol/l), the sulfotransferase activity was detectable in both cell lines. Estrogen sulfates (ES) are found exclusively in the culture medium, which suggests that as soon as they are formed they are excreted into the medium. Medrogestone has a biphasic effect on sulfotransferase activity in both cell lines. At low doses: 5x10(-8) and 5x10(-7) mol/l, this compound stimulates the enzyme by +73.5 and 52.7%, respectively, in MCF-7, and by 84.5 and 62.6% in T-47D cells. At high concentrations: 5x10(-6) and 5x10(-5) mol/l, medrogestone has no effect on MCF-7 cells, but inhibits the sulfotransferase activity in T-47D cells by -31.4% at 5x10(-5) mol/l. In conclusion, the inhibitory effect provoked by medrogestone on the enzyme involved in the biosynthesis of E2 (sulfatase pathway) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive ES, support a probable anti-proliferative effect of this progestin in breast tissue. Clinical applications of these findings can open new therapeutic possibilities for this disease. PMID:10529001
Chetrite, G S; Ebert, C; Wright, F; Philippe, A C; Pasqualini, J R
Effects of extremely low-frequency pulsed electromagnetic fields on morphological and biochemical properties of human breast carcinoma cells (T47D).
This study was carried out to investigate the effects of 100 and 217 Hz extremely low-frequency pulsed electromagnetic fields (ELF-PEMF) on cell proliferation, actin reorganization, and ROS generation in a human breast carcinoma cells (T47D). Cells were exposed for 24-72 h, at 100 and 217 Hz, 0.1 mT. The treatment induced a time dependent decrease in cell growth after 72 h and revealed an increase in fluorescence intensity in cytoplasm and actin aggregations around the nucleus as detected by fluorescence microscopy. The amount of actin in T47D cells increased after 48 h exposure to 100 Hz and 24 h to 217 Hz while no changes in nuclear morphology were detected. Exposing the cells to 217 Hz for 72 h caused a dramatically increase of intracellular ROS generation while with exposure to 100 Hz it remained nearly unchanged. These results suggest that exposure to ELF-PEMF (100, 217 Hz, 0.1 mT) are able inducing an increase of actin level, its migration toward nucleus but despite of these changes and dramatically increase in ROS generation the symptoms of apoptosis were not observed. Our results support the hypothesis that cell response to EMF may only be observed at certain window effects; such as frequency and intensity of EMF parameters. PMID:22676212
Sadeghipour, Razmin; Ahmadian, Shahin; Bolouri, Bahram; Pazhang, Yaghub; Shafiezadeh, Mahshid
Comparative effect of embryonic mouse fibroblasts (Balb/c-3T3) on the proliferation of hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) human breast cancer cell lines.
The effects of cellular extracts (CE) and conditioned medium (CM) of embryonic mouse BALB/c-3T3 (clone A 31) cells on the proliferation and DNA content of hormone-dependent T-47D and hormone-independent MDA-MB-231 breast cancer cell lines were explored. After 6 days of culture, CE and CM provoke an intense proliferative effect in T-47D cells which correlates with DNA content. In contrast, in the MDA-MB-231 cells a significant inhibitory effect was observed. For both CE and CM the action was dose-dependent. In the T-47D cells, the CM can abolish the inhibitory effect provoked by the potent antiestrogen ICI 164,384. It is concluded that mouse embryonic BALB/c-3T3 cells contain factors which can stimulate or inhibit the growth of human mammary cancer cells. PMID:8518407
Chetrite, G; Delalonde, L; Pasqualini, J R
99mTc-labeled sigma-receptor-binding complex: synthesis, characterization, and specific binding to human ductal breast carcinoma (T47D) cells.
sigma-Receptors have recently been shown to be expressed in a variety of human tumor cells. In an attempt to prepare 99mTc chelates that would bind to sigma-receptors and be useful for imaging sigma-receptor-positive tumors, we have synthesized and characterized a bisaminothiol (BAT) chelate appended with a sigma-receptor pharmacophore. The synthesis of target ligand VII was accomplished in three steps starting from bicyclic imidazolidino[1,2-d]dithiazapine. The labeling of the BAT ligand with 99mTc was carried out in high yields (> 80%) using stannous tartarate as a reducing agent, resulting in the target sigma-receptor-binding chelate [99mTc]BAT-EN6, III. Similarly, 99gTc chelate with ligand VII was prepared from ammonium pertechnetate by reduction with stannous tartarate. 99nTc-radiolabeled chelate was purified by reversed phase HPLC, and cell binding with human breast ductal carcinoma (T47D) was performed. A high degree of specific binding (90-97%) was obtained when sigma-receptor ligands such as halogenated phenylethylenediamines were used to determine nonspecific binding. A modest affinity dose-dependent inhibition of binding was found with BD1008, I, and 4-IPEMP, II (IC50 = 47 +/- 2 and 59 +/- 5 nM, respectively), known sigma-ligands. No specific binding was found with [99mTc]BAT, VIII [without appended sigma-pharmacophore (N-alkyl-substituted ethylenediamine)], showing that biological activity resulted from the pendent pharmacophore. 99gTc complex was found to be a potent inhibitor (Ki = 42.7 +/- 8.5 nM) of [3H]DTG binding in guinea pig brain membranes. Scatchard analysis of [99mTc]BAT-EN6 (spiked with [99gTc]BAT-EN6) binding in T47D breast cancer cells showed a saturable binding, with a Kd of 43.5 +/- 14.7 nM and a Bmax of 3121 +/- 130 fmol/(mg of protein). A biodistribution study of [99mTc]BAT-EN6 chelates in Sprague Dawley rats showed hepatic clearance, as expected. A blocking study at 4 h postinjection using 2 mumol of BD1008 with [99mTc]BAT-EN6 showed a significant decrease of radiopharmaceutical in liver (15.32 vs 22.31% ID/organ) and kidney (1.01 vs 2.21% ID/ organ), organs known to possess high concentrations of sigma-receptors. These results imply that [99mTc]BAT-EN6 binds with high affinity to sigma-receptors expressed in human breast tumor cells, and it may be useful for imaging breast cancer. PMID:9177835
John, C S; Lim, B B; Geyer, B C; Vilner, B J; Bowen, W D
Cytoprotection and regulation of heat shock proteins induced by heat shock in human breast cancer T47-D cells: role of [Ca2+]i and protein kinases.
Overexpression of heat shock protein 70 kDa alters the susceptibility of tumor cells to chemotherapeutic agents. We conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in heat shock-treated T47-D cells, a human breast cancer cell line that expresses estrogen receptors. Cells exposed to heat shock at 44 degreesC displayed increased expression of heat shock protein 72 kDa (HSP-72), glucose-regulated protein 78 kDa (GRP-78), and GRP-94 in a time-dependent manner, as shown by [35S]methionine incorporation and Western blotting experiments. The maximal rate of synthesis occurred between 2 and 4 h after heat shock. Removal of external Ca2+ inhibited the synthesis of the heat shock-induced GRP-78 but not of HSP-72 and GRP-94, whereas treatment of cells with BAPTA (a Ca2+ chelator) inhibited HSP-72 and GRP-78. Treatment with H89 (a protein kinase A inhibitor) blocked the heat shock-induced GRP-78 synthesis, whereas GF-109203X (a protein kinase C inhibitor) attenuated the heat shock-induced HSP-72 synthesis and completely blocked synthesis of GRP-78 but not of GRP-94. These results indicate that protein kinase C is involved in regulation of the heat shock-induced synthesis of HSP-72, whereas PKA and PKC are involved in the regulation of GRP-78 synthesis. Cells overexpressing HSP-72 and GRPs after heat shock displayed resistance against lethal temperature (47 degreesC for 50 min) -induced death, which was diminished after removal of external Ca2+ and treatment with GF-109203X. Heat shock increased intracellular free Ca2+ concentration ([Ca2+]i) in a temperature- and heating duration-dependent fashion, and the increase was inhibited in the absence of external [Ca2+]i and significantly reduced by pretreatment with H89 and GF-109203X. The results suggest that different pathways are involved in the induction of synthesis of HSP-72, GRP-78, and GRP-94 by heat shock. It is highly likely that only HSP-72 and GRP-78 are involved in the process of cytoprotection from the thermal injury. PMID:9806766
Kiang, J G; Gist, I D; Tsokos, G C
Effect of Org OD14 (LIVIAL) and its metabolites on human estrogen sulphotransferase activity in the hormone-dependent MCF-7 and T-47D, and the hormone-independent MDA-MB-231, breast cancer cell lines.
The concentration of estrogen sulphates (ES) is particularly high in tumoural breast tissues in post-menopausal women. It is well known that during the post-menopausal phase the levels of circulating estrogens are very low, suggesting the local production of these hormones in the breast cancer tissue itself. Breast cancer cells possess all the enzymes involved in the last steps of estradiol (E2) formation, as well as in its transformation (e.g.: sulphotransferase). As ES are not biologically active, the formation of this conjugate is an important transformation pathway in the control of E2. Consequently, it was interesting to investigate the factors which regulate the sulphotransferase activity. In the present study, we explored the effect of Org OD14; the active substance in Livial, and of its main metabolites (Org 4094, Org 30126, Org OM-38) on the estrogen sulphotransferase activity in the hormone-dependent: MCF-7, T-47D, and hormone-independent: MDA-MB-231, human breast cancer cell lines. After 24 hours incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5 x 10(-9) mol/l), it was observed that the sulphotransferase activity is detectable in MCF-7 and T-47D cells, since the concentrations of ES found were 14.90 and 17.30 pmol/mg DNA, respectively, whereas in MDA-MB-231 cells the concentration of ES found was only 2.01 pmol/mg DNA. Sulphates are found exclusively in the culture medium, which suggests that as soon as the sulphate is biosynthesized it is secreted into the medium. Org OD14, Org 30126, and Org 4094 have a biphasic effect on sulphotransferase activity in the hormone-dependent cells only. At low doses (5 x 10(-8) mol/l) these compounds stimulate this enzyme by 63, 101, and 51%, respectively in the MCF-7 cells, and by 41, 102, and 80%, respectively in the T-47D cells. No stimulatory effect was detected with Org OM-38. At high concentrations (5 x 10(-5) mol/l) Org OD14 and its three metabolites inhibit the sulphotransferase activity in MCF-7 and T-47D cells by 50-70%. In conclusion, the stimulatory effect provoked at low doses by Org OD14 and its metabolites (Org 4094, Org 30126) on the estrogen sulphotransferase involved in the biosynthesis of the inactive estrogen sulphates in estrogen-dependent breast cancer cells, can contribute to the protection of breast tissue in postmenopausal women with hormone replacement therapy. PMID:10226553
Chetrite, G S; Kloosterboer, H J; Philippe, J C; Pasqualini, J R
Proviral Structure, Chromosomal Location, and Expression of HERV-K-T47D, a Novel Human Endogenous Retrovirus Derived from T47D Particles
We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-M�sch, J. Virol. 69:6408�6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3? long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag, prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.
Seifarth, Wolfgang; Baust, Corinna; Murr, Andreas; Skladny, Heyko; Krieg-Schneider, Frank; Blusch, Jurgen; Werner, Thomas; Hehlmann, Rudiger; Leib-Mosch, Christine
Met-HGF\\/SF Mediates Growth Arrest and Differentiation in T47D Breast Cancer Cells1
Hepatocyte growth factor\\/scatter factor (HGF\\/SF) is a pluripotent growth factor that exerts mitogenic, motogenic, and morphogenic effects. To elucidate the cellular mechanisms underlying the pluripotent function of this growth factor, T47D human breast cancer cells were transfected with human hgf\\/sf. The hgf\\/sf-positive clones exhibited different levels of biologically functional HGF\\/SF expression and up-regulation of endogenous Met (HGF\\/SF receptor) expression. In
Dvora Ronen; Rom T. Altstock; Michal Firon; Leonid Mittelman; Tama Sobe; James H. Resau; George F. Vande Woude; Ilan Tsarfaty
Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites.
The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity. PMID:17919675
Spink, David C; Wu, Susan J; Spink, Barbara C; Hussain, Mirza M; Vakharia, Dilip D; Pentecost, Brian T; Kaminsky, Laurence S
Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites
The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 ?M benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17?-estradiol (E2) metabolism, whereas BKF levels greater than 1 ?M inhibited E2 metabolism. Time-course studies showed that induction of CYP1-catalyzed E2 metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays, to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.
Spink, David C.; Wu, Susan J.; Spink, Barbara C.; Hussain, Mirza M.; Vakharia, Dilip D.; Pentecost, Brian T.; Kaminsky, Laurence S.
Activity and expression of progesterone metabolizing 5?-reductase, 20?-hydroxysteroid oxidoreductase and 3?(?)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells
Background Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5?-reduced metabolites (5?-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5?-reductase (5?R) activity. Conversely, the activities of 3?-hydroxysteroid oxidoreductase (3?-HSO) and 20?-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5?-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive). Methods For the enzyme activity studies, either whole cells were incubated with [14C]progesterone for 2, 4, 8, and 24 hours, or the microsomal/cytosolic fraction was incubated for 15�60 minutes with [3H]progesterone, and the metabolites were identified and quantified. Semi-quantitative RT-PCR was employed to determine the relative levels of expression of 5?R type1 (SRD5A1), 5?R type 2 (SRD5A2), 20?-HSO (AKR1C1), 3?-HSO type 2 (AKR1C3), 3?-HSO type 3 (AKR1C2) and 3?-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control. Results The relative 5?-reductase activity, when considered as a ratio of 5?-pregnanes/4-pregnenes, was 4.21 (� 0.49) for MCF-7 cells, 6.24 (� 1.14) for MDA-MB-231 cells, 4.62 (� 0.43) for T-47D cells and 0.65 (� 0.07) for MCF-10A cells, constituting approximately 6.5-fold, 9.6-fold and 7.1 fold higher conversion to 5?-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20?-HSO and 3?-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20?-HSO activity was 8-14-fold higher and the 3?-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5?R:20?-HSO ratios were 16.9 � 32.6-fold greater and the 5?R:3?-HSO ratios were 5.2 � 10.5-fold greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. RT-PCR showed significantly higher expression of 5?R1 (p < 0.001), and lower expression of 20?-HSO (p < 0.001), 3?-HSO2 (p < 0.001), 3?-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. Conclusion The findings provide the first evidence that the 5?R activity (leading to the conversion of progesterone to the cancer promoting 5?-pregnanes) is significantly higher in the tumorigenic MCF-7, MDA-MB-231 and T-47D breast cell lines than in the nontumorigenic MCF-10A cell line. The higher 5?R activity coincides with significantly greater expression of 5?R1. On the other hand, the activities of 20?-HSO and 3?-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in activity correlate with significantly higher expression of 20?-HSO, 3?-HSO2 and 3?-HSO3 in MCF-10A cells. Changes in progesterone metabolizing enzyme expression (resulting in enzyme activity changes) may be responsible for stimulating breast cancer by increased production of tumor-promoting 5?-pregnanes and decreased production of anti-cancer 20? � and 3?-4-pregnenes.
Wiebe, John P; Lewis, Michael J
Naringenin: a partial agonist on estrogen receptor in T47D-KBluc breast cancer cells
Naringenin is present abundantly in citrus fruits and is one of the natural alternatives to synthetic estrogen, but the mechanism of how naringenin functions is not well known. Our study revealed that the relative estrogenic potency of the substances was E2 > genistein > naringenin. Naringenin (at 5 ?M) was found to repress both luciferase activity and pS2 mRNA expression, which was induced by E2 (at 0.1 ?M) or genistein (at 5 ?M). Naringenin, as well as E2 and genistein, was found to modulate the transcription of pS2 and TGF?3 in T47D-KBluc cells through an estrogen receptor-dependent mechanism. Results of our study indicated that naringenin was a weak estrogen agonist that exhibits anti-estrogenic effect in estrogen-rich states and estrogenic activity in estrogen-deficient states in T47D-KBluc breast cancer cells.
Kim, Sunzoo; Park, Tae In
Progesterone-specific stimulation of triglyceride biosynthesis in a breast cancer cell line (T-47D)
The purpose of this study was to examine the lactogenic response of human mammary cancer cell lines to hormones in vitro. Progesterone was found to stimulate the incorporation of 14C from (14C)acetate into triglycerides (TG) and to promote accumulation of TG with a fatty acid composition similar to that of human milk fat in T-47D cells. Lipid droplets were observed in larger numbers without concomitant accumulation of casein granules in cells incubated with progesterone, but secretion of lipid into the medium did not occur. An effect of progesterone on TG accumulation was detectable after 12 hr and was maximal at 72 hr. Increasing doses of progesterone (10(-9) to 10(-5) M) caused a progressive increase in TG accumulation. The presence of cortisol and/or prolactin did not alter TG formation nor the dose response of the cells to progesterone. The growth rate of T-47D cells was not altered by the presence of progesterone in the medium. Neither of the human mammary cancer cell lines, MCF-7 and HBL-100, nor the human fibroblast cell lines, 28 and 857, responded to progesterone. The data indicate that, while the normally lactogenic hormones do not stimulate milk product biosynthesis in the cell lines tested, progesterone specifically stimulated synthesis and accumulation of TG in the T-47D cells.
Judge, S.M.; Chatterton, R.T. Jr.
Quantitative Proteomics and Transcriptomics Addressing the Estrogen Receptor Subtype-mediated Effects in T47D Breast Cancer Cells Exposed to the Phytoestrogen Genistein*
The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor ? (ER?) and ? (ER?)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ER? expression (T47D-ER?), the effect of a varying intracellular ER?/ER? ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ER?-expressing T47D-ER? cells with inhibited ER? expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ER? breast cancer cells with low levels of ER? and no expression of ER?. In addition, data from our study indicate that ER?-mediated gene and protein expression counteracts ER?-mediated effects because in T47D-ER? cells expressing ER? and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ER? decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ER? cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ER?/ER? ratio) in the cells or tissue of interest.
Sotoca, Ana M.; Sollewijn Gelpke, Maarten D.; Boeren, Sjef; Strom, Anders; Gustafsson, Jan-?ke; Murk, Albertinka J.; Rietjens, Ivonne M. C. M.; Vervoort, Jacques
Growth arrest of the breast cancer cell line, T47D, by TNF alpha; cell cycle specificity and signal transduction.
The effects of tumour necrosis factor-alpha (TNF alpha) on the growth and DNA synthesis of the human breast cell line, T47D, were studied. A dose-dependent, reversible inhibition of thymidine incorporation and cell growth was observed in the range of 0.1 ng ml-1 to 100 ng ml-1 of TNF alpha. Cell viability was not impaired in any of the experiments. Flow-cytometric DNA analysis demonstrated that after 24 h exposure to TNF alpha, T47D cells accumulated in the G1 phase of the cell cycle, and were depleted in the G2/M and S phases, suggesting a block in the progression of the G1/S transition. The involvement of protein kinases (PK) and protein phosphatases in TNF alpha-induced signal transduction was also investigated. A transient and rapid 2-fold increase in total cellular protein kinase C (PKC) activity was detected after 10 min exposure to TNF alpha. To study the role of the observed PKC activation in the cytostatic effect of TNF alpha, T47D cells were exposed to the cytokine in the presence of the potent PKC inhibitor, H7. The inhibitory effect of TNF alpha on thymidine incorporation was not affected by exposure to H7 at concentrations sufficient to block the stimulation of thymidine up-take induced by the PKC agonist, phorbol-12-myristate-13-acetate (PMA). The involvement of other signalling pathways was addressed using the cyclic nucleotide-dependent PK inhibitor, H8; the calmodulin-dependent PK inhibitor, W7; and the inhibitor of protein phosphatases PP1 and PP2B, okadaic acid. Exposure of T47D cells to these enzyme inhibitors failed to antagonise the inhibition of thymidine incorporation by TNF alpha. Taken together, these results indicate that the cytostatic effect of TNF alpha on T47D cells occurs at the G1/S transition of the cell cycle, and is mediated by an intracellular pathway which does not involve the activity of protein kinases C and A, nor protein phosphatases PP1, PP2B.
Pusztai, L.; Lewis, C. E.; McGee, J. O.
17 beta-estradiol-induced increases in glucose-regulated protein 78kD and 94kD protect human breast cancer T47-D cells from thermal injury.
Heat shock alters the susceptibility of tumor cells to chemotherapeutic agents. We conducted experiments to study the regulation of expression of heat shock proteins (HSP) in 17 beta-estradiol-treated T47-D cells, a human breast cancer cell line. Cells exposed to 17 beta-estradiol for 24-48 h displayed increased expression of glucose regulated protein 78kD (GRP-78) and 94kD (GRP-94), as shown by [35S]methionine incorporation and Western blotting experiments. The increase was time (5 h to 48 h)-dependent at 1 nM and 1 microM 17 beta-estradiol. Cells overexpressing GRP-78 and -94 after treatment with 17 beta-estradiol displayed resistance against heat shock (47 degrees C for 50 min)-induced death. Removal of external Ca2+ or treatment of cells with BAPTA (a Ca2+ chelator) did not alter the synthesis of GRP-78 and -94, suggesting that the 17 beta-estradiol effect on the synthesis of GRP-78 and -94 is Ca(2+)-independent. In addition, exposure of cells to 17 beta-estradiol up to 100 microM did not increase [Ca2+]i, which further supports the view that the estrogen-induced GRPs are not regulated by [Ca2+]i. Treatment with H89 (a protein kinase A inhibitor, 1 microM, 30 min) or GF-109203X (a protein kinase C inhibitor, 1 microM, 30 min) also did not change the GRP synthesis, indicating that protein kinase A and C are not involved in regulation of GRP synthesis. PMID:9551250
Kiang, J G; Gist, I D; Tsokos, G C
Effects of Estetrol on Migration and Invasion in T47-D Breast Cancer Cells through the Actin Cytoskeleton.
Estetrol (E4) is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor (ER) modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17?-estradiol (E2) on T47-D ER+ breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, re-distribution of actin fibers toward the cell membrane was observed. However, when E4 was added to E2, an inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr(558), which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of estrogen receptor-?. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring ER modulator in the breast. PMID:24904530
Giretti, Maria Silvia; Montt Guevara, Maria Magdalena; Cecchi, Elena; Mannella, Paolo; Palla, Giulia; Spina, Stefania; Bernacchi, Guja; Di Bello, Silvia; Genazzani, Andrea Riccardo; Genazzani, Alessandro D; Simoncini, Tommaso
Effects of Estetrol on Migration and Invasion in T47-D Breast Cancer Cells through the Actin Cytoskeleton
Estetrol (E4) is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor (ER) modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17?-estradiol (E2) on T47-D ER+ breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, re-distribution of actin fibers toward the cell membrane was observed. However, when E4 was added to E2, an inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin�radixin�moesin family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr558, which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of estrogen receptor-?. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring ER modulator in the breast.
Giretti, Maria Silvia; Montt Guevara, Maria Magdalena; Cecchi, Elena; Mannella, Paolo; Palla, Giulia; Spina, Stefania; Bernacchi, Guja; Di Bello, Silvia; Genazzani, Andrea Riccardo; Genazzani, Alessandro D.; Simoncini, Tommaso
Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells.
Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials. PMID:15748827
Shields-Botella, J; Chetrite, G; Meschi, S; Pasqualini, J R
On the role of 17 alpha-estradiol and 17 beta-estradiol in the proliferation of MCF7 and T47D-A11 human breast tumor cells.
A comparative study of the proliferative effect of 17 beta-estradiol and 17 alpha-estradiol on human estrogen-sensitive cell lines was performed. When using charcoal-dextran stripped human female sera-supplemented media the administration of the hormones, 17 alpha-estradiol at 3 X 10(-10)M, and 17 beta-estradiol at 3 X 10(-11)M, resulted in a ten-fold increase in cell yield when compared with non-estrogen supplemented controls after cells were grown for periods between 10 to 14 days. No significant metabolization of 17 alpha-estradiol into 17 beta-estradiol occurred as measured by the E2 levels in the supernatants of the cell culture flasks. Increased concentrations of 17 beta-estradiol and 17 alpha-estradiol added to the media bathing C7MCF7-173 cells resulted in a triggering of a partially successful shut-off effect; this phenomenon was not observed with T47D-All cells. These results are compatible with predictions stemming from the indirect and direct negative working hypothesis for the regulation of cell proliferation. PMID:4066773
Papendorp, J T; Schatz, R W; Soto, A M; Sonnenschein, C
Extranuclear ER? is associated with regression of T47D PKC?-overexpressing, tamoxifen-resistant breast cancer
Background Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKC?)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKC? preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17?-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKC? tumor regression requires extranuclear ER? and interaction with the extracellular matrix. Methods T47D:A18/PKC? cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ER?) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ER? with caveolin-1. Results We report that although T47D:A18/PKC? cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ER? rapidly translocates to extranuclear sites during T47D:A18/PKC? tumor regression in response to both raloxifene and E2, whereas ER? is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ER? to the nucleus. T47D:A18/neo tumors that do not overexpress PKC? maintain ER? in the nucleus during tamoxifen-mediated regression. An association between ER? and caveolin-1 increases in tumors regressing in response to E2. Conclusions Extranuclear ER? plays a role in the regression of PKC?-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ER? in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKC?-expressing tumors encountered in the clinic.
Effect of embryonic mouse cells BALB/c-3T3 on the proliferation of the human mammary cancer cell line T-47D.
In the present study, we explore the effect of the cellular extracts and culture medium of the embryonic mouse cell line BALB/c-3T3 (clone A31) on the proliferation and DNA content of the human T-47D breast cancer cell line. These effects were also studied in the presence of the potent anti-estrogen ICI 164,384. All experiments were prepared in MEM medium containing 5% fetal calf serum treated with dextran charcoal, as well as the homogenization of the BALB/c-3T3 cells to obtain the cellular extract. Aliquots of cellular extracts (2%) corresponding to 2 x 10(6) cells, or culture medium (16%), are incubated with the T-47D cells. After 9 days of culture, cellular extracts and culture medium provoke an intense proliferative effect corresponding respectively to 2 and 5 times the control value of T-47D cells. These effects on cell proliferation are correlated with DNA content. Although the anti-estrogen ICI 164,384 (5 x 10(-8) M) alone decreases the proliferation of T-47D cells by half, the presence of the culture medium from the BALB/c-3T3 cells abolishes this effect and, on the contrary, increases the cell proliferation 4-fold. It is concluded that mouse embryonic cells (BALB/c-3T3) contain factor(s) which stimulate very intensively the proliferation of hormone-dependent T-47D mammary cancer cells. This factor(s) is present in both the cell and the culture medium and can antagonize the anti-proliferative effect of the anti-estrogen ICI 164,384. PMID:1562526
Chetrite, G; Pasqualini, J R
Activity and expression of progesterone metabolizing 5?-reductase, 20?-hydroxysteroid oxidoreductase and 3?(?)-hydroxysteroid oxidoreductases in tumorigenic (MCF7, MDA-MB-231, T-47D) and nontumorigenic (MCF10A) human breast cancer cells
BACKGROUND: Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5?-reduced metabolites (5?-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5?-reductase (5?R) activity. Conversely, the activities of 3?-hydroxysteroid oxidoreductase (3?-HSO) and 20?-HSO enzymes appeared to be higher in normal tissues. The
John P Wiebe; Michael J Lewis
Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells
Several pathological and disease conditions can alter the mechanical properties of the extracellular matrix (ECM). Conversely, some diseases may arise from changes in the density or rigidity of the ECM. This necessitates the use and development of in vitro models to understand how both biophysical and biochemical signals regulate complex cellular behaviors. T47D breast epithelial cells will differentiate into duct-like tubules when cultured in a floating three-dimensional (3D) collagen gel, but not a 3D collagen gel that is left attached to the culture dish. This paper details several protocols we have developed for analyzing breast cell biology in 3D matrices, including culturing cells in 3D collagen gels, immunostaining cellular structures, and performing biochemical procedures directly from cells embedded in collagen gels.
Wozniak, Michele A.
Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells
Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan) and NAP-C (N-acetyl penicillamine-chitosan) in anticancer drug delivery targeting epidermal growth factor receptor (EGFR). Doxorubicin (DOX) and antisense oligonucleotide (ASOND)-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D) were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% � 5% for ASOND and 70% � 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% � 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo.
Talaei, Fatemeh; Azizi, Ebrahim; Dinarvand, Rassoul; Atyabi, Fatemeh
Effects of interaction between estradiol-17 beta and progesterone on the proliferation of cloned breast tumor cells (MCF-7 and T47D).
We have previously reported that the proliferation of cloned MCF-7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal-dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 X 10(-11) M estradiol-17 beta to the culture media. These observations suggest that the proliferation of T47D and MCF-7 cells is regulated by a blood-borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF-7 cells were grown in DME containing 2-40% CDFHS. Progesterone, at 3 X 10(-7) M to 3 X 10(-12) M, had no effect on the yield of MCF-7 or T47D cells that were cultured in the presence or absence of estradiol-17 beta. PMID:4044660
Schatz, R W; Soto, A M; Sonnenschein, C
Isodeoxyelephantopin from Elephantopus scaber (Didancao) induces cell cycle arrest and caspase-3-mediated apoptosis in breast carcinoma T47D cells and lung carcinoma A549 cells
Background Isodeoxyelephantopin (IDOE) isolated from Elephantopus scaber L. (Didancao) is used in Chinese medicine for the treatment of some types of cancer. The anti-cancer mechanism of IDOE remains unclear. This study aims to investigate the antiproliferative activity of IDOE on breast carcinoma T47D cells and lung carcinoma A549 cells. Methods The growth inhibitory effects of IDOE on breast carcinoma T47D cells, lung carcinoma A549 cells, and normal lymphocytes were evaluated by the MTT assay. Morphological analysis of apoptosis induction was performed by acridine orange/ethidium bromide dual-staining and Hoechst 33342 nuclear staining. The cell cycle profile, caspase-3 expression, and annexin V staining were evaluated by flow cytometry. Results IDOE inhibited the growth of A549 and T47D cells in a dose- and time-dependent manner with IC50 values of 10.46 and 1.3�?g/mL, respectively. IDOE was not significantly toxic to normal lymphocytes. The cells became detached from the monolayer and rounded up, had fragmented nuclei and condensed chromatin, and the numbers of apoptotic cells increased (P?=?0.0003). IDOE-induced cell death was associated with activated caspase-3 expression followed by cell cycle arrest at G2/M phase. Conclusions IDOE inhibited the proliferation of breast cancer cells and lung carcinoma cells and induced caspase-3-mediated apoptosis and cell cycle arrest in the treated cells.
Enhancing the Effects of Low Dose Doxorubicin Treatment by the Radiation in T47D and SKBR3 Breast Cancer Cells
Purpose Breast cancer is the most common malignancy of women worldwide. Radiotherapy consists of a vital element in the treatment of breast cancer but relative side effects and different radioactive responses are limiting factors for a successful treatment. Doxorubicin has been used to treat cancers for over 30 years and is considered as the most effective drug in the treatment of breast cancer. There are also many chronic side effects that limit the amount of doxorubicin that can be administered. The combined radio-drug treatment, with low doses, can be an approach for reducing side effects from single modality treatments instead of suitable cure rates. Methods We have studied the effect of 1, 1.5, and 2 Gy doses of 9 MV X-rays along with 1 �M doxorubicin on inducing cell death, apoptosis and also p53 and PTEN gene expression in T47D and SKBR3 breast cancer cells. Results Doxorubicin treatment resulted in upregulation of radiation-induced levels of p53 and downregulation of PTEN at 1 and 1.5 Gy in T47D breast cancer cells, as well as downregulation of p53 mRNA level of expression and upregulation of PTEN mRNA level of expression in SKBR3 breast cancer cell line. In addition, doxorubicin in combination with radiation decreased the viability of breast cancer cell lines in the both cell lines. Conclusion Low doses of doxorubicin, with least cell toxicity, may be an effective treatment for breast cancer when used in conjunction with ionizing radiation.
Aghaee, Fahimeh; Baradaran, Behzad; Mesbahi, Asghar; Mohammadzadeh, Mohammad; Jafarabadi, Mohammad Asghari
Effects of Org OD14 (Livial) and its metabolites on 17 beta-hydroxysteroid dehydrogenase activity in hormone-dependent MCF-7 and T-47D breast cancer cells.
It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell, or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of Org OD14 (active substance in Livial) and its metabolites (Org 30126, Org 4094, Org OM38) on the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) M) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 hours, Org OD14 significantly inhibits this transformation in a dose-dependent manner, by 32 and 73% at 5 x 10(-7) M and 5 x 10(-5) M respectively, in T-47D cells; the effect is similar in MCF-7 cells. Among the three Org OD14 metabolites tested, Org 4094 and Org 30126 (3 alpha- and 3 beta-hydroxy metabolites) are more potent than their precursor, and Org OM-38 (4-ene isomer) is the weakest of the three steroids. The IC50 values were 0.79, 1.98, 7.12, and 22.84 microM in MCF-7 cells for Org 4094, Org 30126, Org OD14, and Org OM-38, respectively, and 4.83, 1.44, 2.03, and 35.25 microM, respectively, in T-47D cells. As Org OD14 and two of its metabolites, Org 30126 and Org 4094, also strongly decrease the conversion of estrone sulphate to estradiol in the hormone-dependent MCF-7 and T-47D breast cancer cells, it is concluded that the inhibition provoked by these steroids on the enzymes (estrone sulphatase and 17 beta-HSD) involved in the local biosynthesis of the biologically active estrogen estradiol, may reduce the risk of breast cancer in postmenopausal women during long-term hormone replacement treatment with Livial. PMID:10226552
Chetrite, G S; Kloosterboer, H J; Philippe, J C; Pasqualini, J R
Action of danazol on the conversion of estrone sulfate to estradiol and on the sulfatase activity in the MCF-7, T-47D and MDA-MB-231 human mammary cancer cells.
In the present studies the action of Danazol on the conversion of estrone sulfate (E1S) to estradiol (E2) as well as on the sulfatase activity in the MCF-7 and T-47D, hormone-dependent, and MDA-MB-231, hormone-independent, mammary cancer cell lines was explored. Using intact cells we observed that Danazol blocks very significantly the radioactivity uptake and the conversion of [3H]E1S to E2 in all the cells studied. In particular, a very strong effect (85% decrease of these parameters versus the control values) is observed in the T-47D cells. In another series of studies using cell homogenates it is observed that Danazol inhibits the sulfatase activity in all these cell lines. The effect of Danazol is dose-dependent and significant from a concentration of 1 microM. At concentrations of 8 microM E1S, 10(-5) M Danazol, the inhibition of sulfatase activity is 38% in MCF-7, 36% in MDA-MB-231, and 27% in T-47D cells. Analysis by Lineweaver-Burk plot shows that the inhibitory effect is competitive. As E1S is one of the main sources of E2 in human mammary tumors, the present data could open new possibilities for therapeutic applications in hormone-dependent breast cancer. PMID:8338787
Nguyen, B L; Ferme, I; Chetrite, G; Pasqualini, J R
Inhibition of hTERT Gene Expression by Silibinin-Loaded PLGA-PEG-Fe3O4 in T47D Breast Cancer Cell Line.
Introduction : Nowadays, using drug delivery is an essential method to improve cancer therapy through decreasing drug toxicity and increasing efficiency of treatment. Silibinin (C25H22O10), a polyphenolic flavonoid which is isolated from the milk thistle plant, has various applications in cancer therapy but it has hydrophobic structure with low water solubility and bioavailability. To increase the effect of silibinin, silibinin-loaded PLGA-PEG-Fe3O4 was prepared to determine the inhibitory effect of this nanodrug on Telomerase gene expression. Methods : The rate of silibinin loaded into PLGA-PEG-Fe3O4 was measured. Then, the cytotoxic effect of silibinin-loaded PLGA-PEG-Fe3O4 was determined by Methyl Thiazol Tetrazolium (MTT) assay. After that, inhibition of Telomerase gene expression was indicated through Real-time PCR. Results : Data analysis from MTT assay showed that silibinin-loaded PLGA-PEG-Fe3O4 had dose dependent cytotoxic effect on T47D cell line. MTT assay showed no cytotoxic effect of free PLGA-PEG-Fe3O4 on T47D breast cancer cell line. Real Time PCR analysis showed that the level of telomerase gene expression more efficiently decreased with silibinin-loaded PLGA-PEG-Fe3O4 than with free silibinin alone. Conclusion : The present study indicates that this nanodrug causes down-regulation of Telomerase gene expression in cancer cells. Therefore, PLGA-PEG-Fe3O4 could be an appropriate carrier for hydrophobic agents such as silibinin to improve their action in cancer therapy. PMID:23878789
Ebrahimnezhad, Zohreh; Zarghami, Nosratollah; Keyhani, Manoutchehr; Amirsaadat, Soumaye; Akbarzadeh, Abolfazl; Rahmati, Mohammad; Mohammad Taheri, Zohreh; Nejati-Koshki, Kazem
Effects of the activated mitogen-activated protein kinase pathway via the c-ros receptor tyrosine kinase on the T47D breast cancer cell line following alcohol exposure.
Compared to other cancers affecting women, breast cancer is significantly associated with alcohol consumption. However, the principles underlying the carcinogenesis of alcohol-induced breast cancer and the related metastatic mechanisms have yet to be established. To observe the effect of alcohol on the growth regulation in breast cancer cells, we identified differentially expressed proteins in alcohol-exposed human breast cancer T47D cells using gel-based proteomics analysis. The expression of c-ros�receptor tyrosine kinase (ROS1) was increased and activated by autophosphorylation, thereby activating mitogen- and stress-activated protein kinase�1 (MSK1) through the mitogen?activated protein kinase (MAPK) pathway; activated MSK1, in turn, phosphorylated histone�3 serine�10 (H3S10p) residues in the nucleus. The increase in H3S10 phosphorylation consequently increased the level of expression of immediate-early gene such as c-fos. This study demonstrated that when breast cancer cells are exposed to alcohol, phosphorylated ROS1 activates MSK1 via Erk1/2 in the MAPK pathway, which then induces modifications to histone residues that regulate gene expression by 14-3-3 protein recruitment, leading to a lack of control of breast cancer cell proliferation. PMID:23292247
Lee, Hyung Tae; Kim, Se Kye; Choi, Mi Ran; Park, Ji Hyun; Jung, Kyoung Hwa; Chai, Young Gyu
Molecular analysis of MEN1 expression in MCF7, T47D and MDA-MB 468 breast cancer cell lines treated with adriamycin using RT-PCR and immunocytochemistry
Background and the purpose of the study MEN1 is an important tumor suppressor gene that encodes a nuclear protein called menin. Recent data suggest that interactions between menin and other proteins have important roles in control of the cell cycle and apoptosis. In addition, estrogen receptor (ER), an important prognostic factor is differentially expressed in breast cancer cells. In this study the MEN1 gene and protein expression in MCF7, T47D and MDA-MB-468 breast cancer cell lines with different ER status following exposure to adriamycin (ADR) was investigated. Materials and methods Cytotoxicity of ADR on these cell lines was determined using MTT assay. The mRNA and protein levels were analyzed in tested cell lines using RT-PCR and immunocytochemistry (ICC) assays, respectively. Results ADR cytotoxicity was highest on MDA-MB-468 and lowest on MCF7 cells. MEN1 mRNA showed significant decrease after ADR exposure only in the MDA-MB-468 cell line. Menin protein expression was higher in MDA-MB-468 and lower in MCF7 cells. Conclusion Differential molecular responses to adriamycin were observed in cancer cell lines. Molecular data also suggest that MEN1 as a new biomarker can be used in combination with current biomarkers for prediction of response to chemotherapy.
Azizi, E.; Namazi, A.; Kaabinejadian, S.; Fouladdel, Sh.; Rezaei, P.; Ramezani, M.
Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action
Introduction��The oncoprotective role of food-derived polyphenol antioxidants has been described but the implicated mechanisms are not yet\\u000a clear. In addition to polyphenols, phenolic acids, found at high concentrations in a number of plants, possess antioxidant\\u000a action. The main phenolic acids found in foods are derivatives of 4-hydroxybenzoic acid and 4-hydroxycinnamic acid.\\u000a \\u000a \\u000a \\u000a \\u000a Methods��This work concentrates on the antiproliferative action of caffeic
Marilena Kampa; Vassilia-Ismini Alexaki; George Notas; Artemissia-Phoebe Nifli; Anastassia Nistikaki; Anastassia Hatzoglou; Efstathia Bakogeorgou; Elena Kouimtzoglou; George Blekas; Dimitrios Boskou; Achille Gravanis; Elias Castanas
A Novel Monoclonal Antibody (7B10) with Differential Reactivity between Human Mammary Carcinoma and Normal Breast1
A monoclonal antibody (7B10) which displays differential reactivity with breast carcinomas compared to benign lesions or normal breast tissue was selected by fusion of spleen cells from BALB\\/c mice immunized with the T47D human mammary carcinoma cell line. The antigen, rec ognized by 7B10 on T47D cells, appeared to be both surface and cyto plasm localized, as demonstrated by indirect
Gianfranco Pancino; Colette Charpin; Fabien Calvo; Marie-Claude Guillemin; Alberto Roseto
Evaluation of anticancer effects of newly synthesized dihydropyridine derivatives in comparison to verapamil and doxorubicin on T47D parental and resistant cell lines in vitro.
Failure of current anticancer drugs mandates screening for new compounds of synthetic or biological origin to be used in cancer therapy. Multidrug resistance (MDR) is one of the main obstacles in the chemotherapy of cancer. Efflux of cytotoxic agents mediated by P-glycoprotein (P-gp or MDR1) is believed to be an important mechanism of multidrug resistance. Therefore, we decided to investigate the antiproliferative effects of seven newly synthesized 1,4-dihydropyridine (DHP) derivatives in comparison to verapamil (VP) and doxorubicin (DOX) on human breast cancer T47D cells and its MDR1 overexpressed and moderately resistant cells (RS cells) using MTT cytotoxicity assay. We also examined the effects of these compounds on cytotoxicity of DOX in these two cell types. The cytotoxicity assays using MTT showed that most of the tested new DHP derivatives and VP at 10 microM concentration had varying levels of toxicity on both T47D and RS cells. The toxicity was mostly in the range of 10-25%. However, the cytotoxicity of these DHP derivatives, similar to VP, was significantly less than DOX when comparing IC(50) values. Furthermore, these compounds in general had relatively more cytotoxicity on T47D vs RS cells at 10-microM concentration. Among new DHPs, compounds 7a (3,5-dibenzoyl-4-(2-methylthiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine) and 7d (3,5-diacetyl-4-[2-(2-chlorophenyl)thiazol-4-yl)]-1,4-dihydro-2,6-dimethylpyridine) showed noticeable potentiation of DOX cytotoxicity (reduction of DOX IC(50)) compared to DOX alone in both cells, particularly in RS cells. This effect was similar to that of VP, a known prototype of MDR1 reversal agent. In other words, compounds 7a and 7d resensitized RS cells to DOX or reversed their resistance. Results indicate that compound 7d exerts highest effect on RS cells. Therefore, these two newly synthesized DHP derivatives, compounds 7a and 7d, are promising as potential new MDR1 reversal agents and should be further studied on other highly resistant cells due to MDR1 overexpression and with further molecular investigation. PMID:17805981
Bazargan, L; Fouladdel, S; Shafiee, A; Amini, M; Ghaffari, S M; Azizi, E
Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53
Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines
Tsing-Fen Ho; Chieh-Ju Ma; Chien-Hsing Lu; Yo-Ting Tsai; Yu-Hong Wei; Jo-Shu Chang; Jun-Kai Lai; Pin-Ju Cheuh; Chi-Tai Yeh; Pin-Chi Tang; Jinghua Tsai Chang; Jiunn-Liang Ko; Fu-Shing Liu; Hungchen E. Yen; Chia-Che Chang
Effect of tibolone (Org OD14) and its metabolites on estrone sulphatase activity in MCF-7 and T-47D mammary cancer cells.
Tibolone (Org OD14) is a synthetic steroid with weak estrogenic, progestagenic and androgenic properties which is used for the treatment of menopausal complaints. Since the compound possesses estrogenic activity, it is important to assess its effects on pathways in breast cells involved in the development of breast cancer. In the present study we compared the dose-response effect (range: 5 x 10(-8) to 5 x 10(-5) M) of Tibolone and its metabolites (Org OM38, Org 4094, and Org 30,126) on the "sulphatase" pathway of two hormone-dependent human breast cancer cells: MCF-7 and T-47D; the results were compared with Norethisterone. After 24 hours incubation with physiological concentrations of estrone sulphate (E1 S: 5 x 10(-9) M) and high doses (5 x 10(-5) and 5 x 10(-6) M) of the different compounds, the conversion of E1 S to estradiol (E2) was strongly inhibited (85-98%) with all five substances tested. When low doses (5 x 10(-8) and 5 x 10(-7) M) were used, Tibolone and the metabolites Org 4094 and Org 30,126 were still very efficient at blocking the conversion of E1S to E2 (70-90%) in both cells. This inhibitory effect is less intense with the metabolite Org OM38 (45-70%). Norethisterone was the least potent antisulphatase agent (60-20%) tested. In conclusion, this very significant inhibitory effect of Tibolone and of its metabolites Org 4094 and Org 30,126 on the enzymes involved in the biosynthesis of E2 in human breast cancer cells, points to a potential beneficial effect of Tibolone which may be of relevance in its application for the treatment of climacteric complaints. PMID:9066643
Chetrite, G; Kloosterboer, H J; Pasqualini, J R
Progestins Increase Insulin Receptor Content and Insulin Stimulation of Growth in Human Breast Carcinoma Cells1
The effects of progesterone on the growth of breast carcinoma cells are undefined. In the present study we investigated the effect of progestins on insulin receptor gene expression and insulin action in human breast cancer cells. Treatment of T47D cells with the synthetic progestin R5020 induced a time- and dose-dependent increase in insulin receptor content as measured by both ligand-binding
Vincenzo Papa; Constance C. Reese; Antonio Brunetti; Riccardo Vigneti; Peniti K. Siiteri; Ira D. Goldfine
Protein Kinase C? Expression Confers Retinoic Acid Sensitivity on MDA-MB-231 Human Breast Cancer Cells
Retinoic acid activation of retinoic acid receptor ? (RAR?) induces protein kinase C? (PKC?) expression and inhibits proliferation of the hormone-dependent T-47D breast cancer cell line. Retinoic acid has no effect on proliferation or PKC? expression in a hormone-independent, breast cancer cell line (MDA-MB-231). To test the role of PKC? in retinoic acid-induced growth arrest of human breast cancer cells
Yunhi Cho; David A. Talmage
Direct identification of proteins from T47D cells and murine brain tissue by matrix-assisted laser desorption\\/ionization post-source decay\\/collision-induced dissociation
The purpose of this study is to determine the feasibility of the direct matrix-assisted laser desorption\\/ ionization (MALDI) identification of proteins in fixed T47D breast cancer cells and murine brain tissues. The ability to identify proteins from cells and tissue may lead to biomarkers that effectively predict the onset of defined disease states, and their dynamic behavior could be an
Paul H. Pevsner; Frederick Naftolin; Dean E. Hillman; Douglas C. Miller; Ahmed Fadiel; Alexander Kogus; Arnold Stern; Herbert H. Samuels
Estradiol-Induced Regression in T47D:A18/PKC? Tumors Requires the Estrogen Receptor And Interaction with the Extracellular Matrix
Several breast cancer tumor models respond to estradiol (E2) by undergoing apoptosis, a phenomenon known to occur in clinical breast cancer. Prior to the application of tamoxifen as an endocrine therapy, high dose E2 or diethystilbesterol (DES) treatment was successfully utilized, albeit with unfavorable side effects. It is now recognized that such an approach may be a potential endocrine therapy option. We have explored the mechanism of E2-induced tumor regression in our T47D:A18/PKC? tumor model that exhibits autonomous growth, tamoxifen-resistance and E2-induced tumor regression. Fulvestrant, a selective estrogen receptor downregulator, prevents T47D:A18/PKC? E2-induced tumor growth inhibition and regression when given prior or subsequent to tumor establishment, respectively. Interestingly, E2-induced growth inhibition is only observed in vivo or when cells are grown in Matrigel but not in two-dimensional tissue culture, suggesting the requirement of the extracellular matrix (ECM). Tumor regression is accompanied by increased expression of the pro-apoptotic Fas/FasL proteins and downregulation of the pro-survival Akt pathway. Inhibition of colony formation in Matrigel by E2 is accompanied by increased expression of Fas and shRNA knockdown partially reverses colony formation inhibition. Classical ERE-regulated transcription of pS2, PR, TGF?, C3 and cathepsin D is independent of the inhibitory effects of E2. A membrane impermeable E2-BSA conjugate is capable of mediating growth inhibition, suggesting the involvement of a plasma membrane ER. We conclude that E2-induced T47D:A18/PKC? tumor regression requires participation of ER?, the ECM, Fas/FasL and Akt pathways, allowing the opportunity to explore new predictive markers and therapeutic targets.
Zhang, Yiyun; Zhao, Huiping; Asztalos, Szilard; Chisamore, Michael; Sitabkhan, Yasmin; Tonetti, Debra A.
Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration.
Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acalpha2-6GalNAcalpha1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development. PMID:15770530
Julien, Sylvain; Lagadec, Chann; Krzewinski-Recchi, Marie-Ange; Courtand, Gilles; Le Bourhis, Xuefen; Delannoy, Philippe
The effects of silver nanoparticles and doxorubicin combination on DNA structure and its antiproliferative effect against T47D and MCF7 cell lines.
The structural changes in DNA caused by the combined effects of silver nanoparticles (Ag NPs) and doxorubicin (DOX) was investigated along with their corresponding inhibitory roles in the growth of T47D and MCF7 cells. The UV-visible titration studies showed that DOX + AgNPs could form a novel complex with DNA and this interaction is in the interface between the value induced by electrostatic and intercalative binding. The values of binding constants revealed that DOX + AgNPs interact more strongly with DNA as compared to Ag NPs or DOX alone. Our CD data revealed that although Ag NPs and DOX alone could alter DNA structure, this combination leads to transition of DNA conformation to an ordered and compact molecular form so called psi-type, considering that DNA is relatively thermally stable in the condition used. Thus, we observed that DOX + AgNPs induces conformational change on DNA. The anticancer property of DOX + AgNPs by MTT assay, DAPI stain and flow cytometry analyses demonstrated that this combination can tremendously diminish proliferation of T47D and MCF7 cells compared to DOX or Ag NPs alone. Furthermore, this combination was comparatively non-toxic towards the human endometrial stem cells proliferation. Collectively, these results reveal that DOX + AgNPs could proffer a novel strategy for the development of promising and efficient chemotherapy agents. PMID:23030005
Hekmat, Azadeh; Saboury, Ali Akbar; Divsalar, Adeleh
Human estrogen sulfotransferase (hEST1) activities and its mRNA in various breast cancer cell lines. Effect of the progestin, promegestone (R-5020).
Using reverse transcriptase-polymerase chain reaction amplification it was possible to detect the presence of type 1 human estrogen sulfotransferase (hEST1) mRNA in the hormone-dependent: MCF-7 and T-47D, and hormone-independent: MDA-MB-231 and MDA-MB-468, human breast cancer cells. The expression of this mRNA is significantly higher in the MDA-MB-468 cells and a correlation of this mRNA expression with the enzymatic activity was observed. The progestin promegestone (R-5020) at a low concentration (5 x 10(-7) M) can significantly increase the estrogen sulfotransferase activity and its mRNA in the hormone-dependent MCF-7 and T-47D cells. As estrogen sulfates are biologically inactive, the stimulatory effect on sulfotransferase by promegestone may open attractive possibilities in the control of estradiol in human breast cancer. PMID:9749835
Chetrite, G; Le Nestour, E; Pasqualini, J R
Degradation of endothelial basement membrane by human breast cancer cell lines
During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of (35S)methionine-labeled and (3H)proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer.
Yee, C.; Shiu, R.P.
Effects of a progestogen on normal human breast epithelial cell apoptosis in vitro and in vivo.
Many investigators have reported cyclic proliferation of normal human breast epithelial cells. A delicate balance between proliferation and apoptosis (programmed cell death) ensures breast homeostasis. Both the follicular and luteal phases of the menstrual cycle are characterized by proliferation, whereas apoptosis occurs only at the end of the latter phase. In this study, we observed that the withdrawal of a synthetic progestin (nomegestrol acetate or NOMAC), but not continuous treatment with it, induced apoptosis of normal human breast epithelial cells in vitro and in women who applied NOMAC gel to their breasts. Furthermore, this apoptotic response was specific to normal breast cells, since withdrawal of NOMAC did not induce apoptosis of tumoral T47D cells in vitro or of fibroadenoma cells in women. These observations open up new perspectives in the prevention of hyperplasia and breast cancer. PMID:14659344
Desreux, J; Kebers, F; No�l, A; Francart, D; Van Cauwenberge, H; Heinen, V; Peyrollier, K; Thomas, J L; Bernard, A M; Paris, J; Delansorne, R; Foidart, J M
Anti-apoptotic effect of claudin-1 in tamoxifen-treated human breast cancer MCF7 cells
Background��Claudin-1 is a membrane protein of tight junctions, and is associated with the development of various cancers. However, the\\u000a significance of claudin-1 expression in cancer cells is not well understood. Here, we showed for the first time the anti-apoptotic\\u000a effect of claudin-1 in human breast cancer MCF-7 cells.\\u000a \\u000a \\u000a \\u000a \\u000a Methods��Human breast cancer MCF-7 and T47 D cells were treated with or
Harue Akasaka; Fuyuki Sato; Satoko Morohashi; Yunyan Wu; Yang Liu; Jun Kondo; Hiroki Odagiri; Kenichi Hakamada; Hiroshi Kijima
Second-generation substituted quinolines as anticancer drugs for breast cancer.
Cancer cells have reduced capacity for gap junctional inter-cellular communication (GJIC). One feasible approach to reduce growth of cancer cells is to enhance GJIC. This report shows that a second-generation substituted quinoline, PQ7, has anti-tumor effect. Scrape load/dye transfer and colony growth assays were performed to measure GJIC and tumor formation of T47D breast cancer cells. PQ7 at 500 nM induced a 16-fold increase in the GJIC in T47D cells. In addition to an increase in GJIC, a 50% decrease of colony growth was observed with 100 nM of PQ7. PQ7-treated nu/nu mice showed a 100% regression of xenograft tumor growth of T47D cells. The results show that PQ7 has a promising role in exerting anti-tumor activity in human breast cancer cells. PMID:21036704
Heiniger, Brian; Gakhar, Gunjan; Prasain, Keshar; Hua, Duy H; Nguyen, Thu A
Effect of nomegestrol acetate on estrone-sulfatase and 17beta-hydroxysteroid dehydrogenase activities in human breast cancer cells.
It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E1S: 5 x 10(-9)M), nomegestrol acetate blocked very significantly the conversion of E1S to E2. In the MCF-7 cells, using concentrations of 5 x 10(-6)M and 5 x 10(-5) M of nomegestrol acetate, the decrease of E1S to E2 was, respectively, -43% and -77%. The values were, respectively, -60% and -71% for the T-47D cells. Using E1S at 2 x 10(-6) M and nomegestrol acetate at 10(-5) M, a direct inhibitory effect on the enzyme of -36% and -18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E1: 5 x 10(-9)M) this estrogen is converted in a great proportion to E2. Nomegestrol acetate inhibits this transformation by -35% and -85% at 5 x 10(-7)M and 5 x 10(-5)M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, -48%, at 5 x 10(-5)M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17beta-HSD activities which converts E1S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E2 can open new clinical possibilities in breast cancer therapy. PMID:8918978
Chetrite, G; Paris, J; Botella, J; Pasqualini, J R
HERV-K-T47D-Related Long Terminal Repeats Mediate Polyadenylation of Cellular Transcripts
The human genome harbors thousands of long terminal repeats (LTRs) that are derived from endogenous retroviruses and contain elements able to regulate the expression of neighboring cellular genes. We have investigated the ability of human endogenous retroviral (HERV)-K LTRs to provide transcriptional processing signals for nonviral sequences. Four chimeric cDNA clones isolated from a cDNA library derived from the human
Corinna Baust; Wolfgang Seifarth; Herbert Germaier; R�diger Hehlmann; Christine Leib-M�sch
Detection of bone sialoprotein in human breast cancer tissue and cell lines at both protein and messenger ribonucleic acid levels.
The recent demonstration that bone sialoprotein (BSP) can be detected in human breast cancer tissue by immunoperoxidase suggests that this phosphoprotein is ectopically expressed by malignant mammary epithelial cells. Its detection in human breast cancer cells raises questions about its potential role(s) during breast cancer progression. Because BSP is secreted and is present in the serum, the positivity of breast cancer cells for BSP could have been the result of an uptake of the circulating phosphoprotein by the cells rather than of an intrinsic expression. We examined the expression of BSP at both the protein and mRNA levels in nine human breast cancer samples as well as in three human breast cancer cell lines (MCF-7, T47-D, and MDA-MB-231) using immunohistochemistry, flow cytometric analysis, immunoblot, and reverse-transcriptase PCR. BSP was detected at both protein and mRNA levels in human breast cancer tissue and in the three human breast cancer cell lines. Using a specific polyclonal anti-BSP antibody, we showed by both fluorescence-activated cell sorter analysis and immunohistochemistry experiments that all of the human breast cancer cell lines studied express BSP. This was localized at the cell surface and in the cytosol of the estrogen receptor-positive MCF-7 and T47-D cell lines, whereas it was detected only in the cytosol of the estrogen receptor-negative MDA-MB-231 cells. Using the same polyclonal anti-BSP antibody, we were able to identify an approximately 97-kd band on total protein extracts from the three cell lines by immunoblotting. Reverse-transcriptase PCR reactions using specific oligonucleotides performed on total RNA of nine human breast cancer biopsy samples and the three cell lines demonstrated the presence of BSP mRNA in all of the samples examined. This study is the first demonstration that human malignant breast epithelial cell lines express BSP at the protein and mRNA levels. Our study identified MCF-7, T47-D, and MDA-MB-231 cells as useful models for the examination of the molecular mechanisms involved in the ectopic expression of BSP in breast malignant lesions. PMID:8765320
Bellahc�ne, A; Antoine, N; Clausse, N; Tagliabue, E; Fisher, L W; Kerr, J M; Jar�s, P; Castronovo, V
Sodium butyrate induces P53-independent, Fas-mediated apoptosis in MCF-7 human breast cancer cells
This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells.Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF-7, MCF-7ras, T47-D, and BT-20 cells, as well as arrest in G2/M in MDA-MB-231 cells.Transient transfection of MCF-7 and T47-D cells with wild-type and antisense p53 did not modify butyrate-induced apoptosis. Pifithrin-?, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF-7 and T47-D cells treated with butyrate. These results indicate that P53 was not involved in butyrate-induced growth inhibition of breast cancer cells.Treatment of MCF-7 cells with anti-Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas-induced apoptosis, as massive apoptosis was observed rapidly when MCF-7 cells were treated with butyrate and anti-Fas agonist antibody. In addition, butyrate-induced apoptosis in MCF-7 cells was considerably reduced by anti-Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate-induced apoptosis may be mediated by Fas signalling.These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53-independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas-triggered apoptosis in MCF-7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy.
Chopin, Valerie; Toillon, Robert-Alain; Jouy, Nathalie; Bourhis, Xuefen Le
Reevaluation of the proposed autocrine proliferative function of prolactin in breast cancer.
The pituitary hormone prolactin (PRL) has been implicated in tumourigenesis. Expression of PRL and its receptor (PRLR) was reported in human breast epithelium and breast cancer cells. It was suggested that PRL may act as an autocrine/paracrine growth factor. Here, we addressed the role of locally synthesised PRL in breast cancer. We analysed the expression of PRL in human breast cancer tumours using qPCR analysis and in situ hybridization (ISH). PRL mRNA expression was very low or undetectable in the majority of samples in three cDNA arrays representing samples from 144 breast cancer patients and in 13 of 14 breast cancer cell lines when analysed by qPCR. In accordance, PRL expression did not reach detectable levels in any of the 19 human breast carcinomas or 5 cell lines, which were analysed using a validated ISH protocol. Two T47D-derived breast cancer cell lines were stably transfected with PRL-expressing constructs. Conditioned medium from the T47D/PRL clones promoted proliferation of lactogen-dependent Nb2 cells and control T47D cells. Surprisingly, the PRL-producing clones themselves displayed a lower proliferation rate as compared to the control cells. Their PRLR protein level was reduced and the cells were no longer responsive to exogenous recombinant PRL. Taken together, these data strongly indicate that autocrine PRL signalling is unlikely to be a general mechanism promoting tumour growth in breast cancer patients. PMID:24146212
Nitze, Louise Maymann; Galsgaard, Elisabeth Douglas; Din, Nanni; Lund, Vibe Luja; Rasmussen, Birgitte Bruun; Berchtold, Martin Werner; Christensen, Leif; Panina, Svetlana
Transcriptional activation of the c-myc oncogene in the T-47D mammary cancer cells by conditioned media from embryonic mouse fibroblasts (BALB/c-3T3). Effect of the antiestrogen ICI 164,384.
The effects of the conditioned medium (CM) of embryonic mouse fibroblast (BALB/c-3T3) cells (clone A31) on the mRNA of the c-myc oncogene of the hormone-dependent T-47D mammary cancer cells were explored. After 6h incubation a significant stimulation of c-myc expression was observed with a 10% v/v concentration of CM, and the effect was even higher using a CM concentration of 30% v/v. It is suggested that this action of CM could be related to the stimulatory effect on proliferation and DNA synthesis induced by the CM, as was recently demonstrated for the T-47D cells. It was also observed that the antiestrogen ICI 164,384 (5 x 10(-6) M) can inhibit the stimulatory action of CM on c-myc expression. It is concluded that the fibroblast mouse embryonic cells contain and secrete factor(s) involved in the mechanism of proliferation of human hormone-dependent mammary cancer cells. PMID:7645941
Pasqualini, J R; Chen, T A; Maloche, C; Chetrite, G; Valentin, E; Allfrey, V
Conjugated Linoleic Acid (CLA) inhibits expression of the Spot 14 (THRSP) and fatty acid synthase genes and impairs the growth of human breast cancer and liposarcoma cells
Spot 14 (THRSP, S14) is a nuclear protein involved in the regulation of genes required for fatty acid synthesis in normal and malignant mammary epithelial and adipose cells. Havartine and Bauman reported that conjugated linoleic acid (CLA) inhibits S14 gene expression in bovine mammary and mouse adipose tissues, and reduces milk fat production in cows. We hypothesized that CLA inhibits S14 gene expression in human breast cancer and liposarcoma cells, and that this will retard their growth. Exposure of T47D breast cancer cells to a mixture of CLA isomers reduced the expression of the S14 and fatty acid synthase (FAS) genes. The mixture caused a dose-related inhibition of T47D cell growth, as did pure c9, t11- and t10, c12-CLA, but not linoleic acid. Similar effects were observed in MDA-MB-231 breast cancer cells. Provision of 8 ?M palmitate fully (CLA mix, t10, c12-CLA) or partially (c9, t11-CLA) reversed the antiproliferative effect in T47D cells. CLA likewise suppressed levels of S14 and FAS mRNAs in liposarcoma cells, and caused growth inhibition that was prevented by palmitic acid. CLA did not affect the growth of nonlipogenic HeLa cells or human fibroblasts. We conclude that, as in bovine mammary and mouse adipose cells, CLA suppresses S14 and FAS gene expression in human breast cancer and liposarcoma cells. Rescue from the antiproliferative effect of CLA by palmitic acid indicates that reduced tumor lipogenesis is a major mechanism for the anticancer effects of CLA.
Donnelly, Christina; Olsen, Arne M.; Lewis, Lionel D.; Eisenberg, Burton L.; Eastman, Alan; Kinlaw, William B.
Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate.
Human breast cancer tissue contains enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol (E(2)) formation. In this tissue, E(2) can be synthesized by two main pathways: (1) sulfatase-transforms estrogen sulfates into bioactive E(2), and the (2) aromatase-converts androgens into estrogens. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. In the present study, we demonstrated in T-47D and MCF-7 human breast cancer cells that norelgestromin (NGMN) (a metabolite of norgestimate) is a potent inhibitory agent of the estrone sulfatase activity. After 24h incubation of physiological concentrations of E(1)S (5 x 10(-9)mol/l) the inhibitory effect of NGMN at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 43+/-7, 74+/-4 and 97+/-2%, respectively, in T-47D cells; 25+/-4, 57+/-5 and 96+/-2% respectively, in MCF-7 cells. Comparative studies using medroxyprogesterone acetate (MPA) showed that this progestin also has an inhibitory effect on sulfatase activity, but significantly less intense than that of NGMN. The inhibition for MPA at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 31+/-5, 47+/-3 and 61+/-3%, respectively, for T-47D cells; 6+/-3, 20+/-3 and 63+/-4%, respectively, for MCF-7 cells. In conclusion, the present data show that NGMN is a very potent inhibitory agent for sulfatase activity in the hormone-dependent breast cancer cells, resulting in decreased tissue concentration of E(2). The clinical significance of this finding remains to be elucidated. PMID:12711003
Pasqualini, Jorge R; Caubel, Patrick; Friedman, Andrew J; Philippe, Jean-Claude; Chetrite, G�rard Samuel
Genistein modulates proliferation and mitochondrial functionality in breast cancer cells depending on ERalpha/ERbeta ratio.
Breast cancer is the most common malignancy in women of developed countries. The aim of this study was to investigate whether genistein, a soy phytoestrogen, and 17?-estradiol (E2) could have effects on the cell cycle and mitochondrial function and dynamics. Three human breast cancer cell lines with different estrogen receptor alpha (ER?) and estrogen receptor beta (ER?) ratio were used: MCF-7 (high ER?/ER? ratio), T47D (low ER?/ER? ratio) and MDA-MB-231 (ER-negative). Cell proliferation, cell cycle, mitochondrial functionality, and mitochondrial dynamics parameters were analyzed. E2 and genistein treatment induced cell proliferation and apoptosis inhibition in MCF-7, but not in T47D and MDA-MB-231. Moreover, genistein treatment produced an up-regulation of ER? and a rise in cytochrome c oxidase activity in T47D cells, decreasing the ATP synthase/cytochrome c oxidase ratio. Finally, genistein treatment produced a drop in mitochondrial dynamics only in MCF-7 cells. In summary, the beneficial effects of genistein consumption depend on the ER?/ER? ratio in breast cells. Therefore, genistein treatment produces cell cycle arrest and an improvement of mitochondrial functionality in T47D cells with a low ER?/ER? ratio, but not in MCF-7 (high ER?/ER? ratio) and MDA-MB-231 (ER-negative) ones. PMID:24375531
Pons, Daniel Gabriel; Nadal-Serrano, Mercedes; Blanquer-Rossello, M Mar; Sastre-Serra, Jorge; Oliver, Jordi; Roca, Pilar
Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53.
Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status. PMID:17881028
Ho, Tsing-Fen; Ma, Chieh-Ju; Lu, Chien-Hsing; Tsai, Yo-Ting; Wei, Yu-Hong; Chang, Jo-Shu; Lai, Jun-Kai; Cheuh, Pin-Ju; Yeh, Chi-Tai; Tang, Pin-Chi; Tsai Chang, Jinghua; Ko, Jiunn-Liang; Liu, Fu-Shing; Yen, Hungchen E; Chang, Chia-Che
Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53
Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X{sub L}, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.
Ho, T.-F. [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Medical Technology, Central Taiwan University of Science and Technology, Taichung 40605, Taiwan (China); Ma, C.-J. [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Lu, C.-H. [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Tsai, Yo-Ting [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Wei, Y.-H. [Graduate School of Biotechnology and Bioinformatics, Yuan Ze University, Taoyuan, Taiwan (China); Chang, J.-S. [Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan (China); Lai, J.-K. [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China); Cheuh, Pin-Ju [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Yeh, C.-T. [Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan (China); Tang, P.-C. [Department of Animal Science, National Chung Hsing University, Taichung, Taiwan (China); Jingua, T.C.; Ko, J.-L. [Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan (China); Liu, F.-S. [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Yen, H.E. [Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China)] (and others)
Human Sprouty1 suppresses growth, migration, and invasion in human breast cancer cells.
Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Expression of human Sprouty1 (hSpry1) gene is downregulated in most breast cancer patients, implicating it as an important tumor suppressor gene. So, we hypothesized that overexpression of hSpry1 gene may suppress breast cancer cell growth, migration, and invasion. Here, we demonstrate that in breast cancer cell lines, MDA-MB-231 and T47D, transfection-induced overexpression of hSpry1 reduced cell population, proliferation, and colony formation in vitro without affecting cell apoptosis. Adhesion molecules act as both positive and negative modulators of cellular migration and invasion. Here, we found that overexpression of hSpry1 enhances the initial establishment events in breast cancer cell adhesion to type IV collagen and vitronectin. Moreover, the overexpression of hSpry1 in the highly invasive MDA-MB-231 breast cancer cells causes a significant reduction in cellular migration and invasion through Matrigel membranes. In addition, we showed that hSpry1 overexpression prevents VEGF secretion. VEGF is essential for primary tumor growth, migration, and invasion. Thus, our study provides a novel mechanism of tumor suppression activity of hSpry1. PMID:24510305
Mekkawy, Ahmed H; Pourgholami, Mohammad H; Morris, David L
Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells
Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs) by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-II, the processed form of LC3-I, was increased. Treatment with the autophagy inhibitor, 3-methyladenine (3-MA), significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.
Cao, Xuchen; Liu, Bowen; Cao, Wenfeng; Zhang, Weiran; Zhang, Fei; Zhao, Hongmeng; Meng, Ran; Zhang, Lin; Niu, Ruifang; Hao, Xishan
Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells.
Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs) by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-II, the processed form of LC3-I, was increased. Treatment with the autophagy inhibitor, 3-methyladenine (3-MA), significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control. PMID:23592903
Cao, Xuchen; Liu, Bowen; Cao, Wenfeng; Zhang, Weiran; Zhang, Fei; Zhao, Hongmeng; Meng, Ran; Zhang, Lin; Niu, Ruifang; Hao, Xishan; Zhang, Bin
Prodigiosin, the red pigment of Serratia marcescens, shows cytotoxic effects and apoptosis induction in HT-29 and T47D cancer cell lines.
In this study, a red pigment of Serratia marcescens PTCC 1111 was purified and identified for antiproliferative activities in HT-29 and T47D cancer cell lines. (1)H-NMR spectroscopy and LC/MS analysis confirmed prodigiosin structure. The antiproliferative effects of prodigiosin were determined by employing the MTT assay. The changes in cell cycle pattern were studied with 4',6-diamidino-2-phenylindole (DAPI) reagent using flow cytometry assay, and Annexin V-PI method was used for apoptotic analysis. Results of MTT assay showed that HT-29 cells were more sensitive to prodigiosin than T47D cells. Prodigiosin-treated HT-29 cells showed increase in S phase and decrease in G2/M, but treated T47D cells showed cell cycle pattern relatively similar to Roswell Park Memorial Institute medium (RPMI). Apoptotic effect of prodigiosin was higher than doxorubicin in HT-29 cells. The data reported here indicate that prodigiosin is a promising antineoplastic agent that triggers apoptosis in different cancer cell lines. PMID:21985476
Dalili, D; Fouladdel, Sh; Rastkari, N; Samadi, N; Ahmadkhaniha, R; Ardavan, A; Azizi, E
Radiosensitization in human breast carcinoma cells by thymoquinone: role of cell cycle and apoptosis.
TQ (thymoquinone), the bioactive constituent of black seed (Nigella sativa), has been shown to inhibit the growth of various human cancers both in vitro and in vivo. This study reports the radiosensitizing effect of TQ on human breast carcinoma cells (MCF7 and T47D). TQ in combination with single dose of ionizing radiation (2.5 Gy) was found to exert supra-additive cytotoxic effects on both the carcinomas as measured by cell proliferation and colony-formation assays. Annexin V binding and FACS analysis revealed the role of enhanced apoptosis and cell cycle modulation in the mechanism of TQ-mediated radiosensitization, thus supporting TQ as an adjuvant for preclinical testing in cancer chemo-radiotherapy. PMID:21557727
Velho-Pereira, Reelma; Kumar, Amit; Pandey, Badri N; Jagtap, Aarti G; Mishra, Kaushala P
Ras-association domain family 1C protein promotes breast cancer cell migration and attenuates apoptosis
Background The Ras association domain family 1 (RASSF1) gene is a Ras effector encoding two major mRNA forms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing. RASSF1A is a tumor suppressor gene. However, very little is known about the function of RASSF1C both in normal and transformed cells. Methods Gene silencing and over-expression techniques were used to modulate RASSF1C expression in human breast cancer cells. Affymetrix-microarray analysis was performed using T47D cells over-expressing RASSF1C to identify RASSF1C target genes. RT-PCR and western blot techniques were used to validate target gene expression. Cell invasion and apoptosis assays were also performed. Results In this article, we report the effects of altering RASSF1C expression in human breast cancer cells. We found that silencing RASSF1C mRNA in breast cancer cell lines (MDA-MB231 and T47D) caused a small but significant decrease in cell proliferation. Conversely, inducible over-expression of RASSF1C in breast cancer cells (MDA-MB231 and T47D) resulted in a small increase in cell proliferation. We also report on the identification of novel RASSF1C target genes. RASSF1C down-regulates several pro-apoptotic and tumor suppressor genes and up-regulates several growth promoting genes in breast cancer cells. We further show that down-regulation of caspase 3 via overexpression of RASSF1C reduces breast cancer cells' sensitivity to the apoptosis inducing agent, etoposide. Furthermore, we found that RASSF1C over-expression enhances T47D cell invasion/migration in vitro. Conclusion Together, our findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor, but instead may play a role in stimulating metastasis and survival in breast cancer cells.
B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells
Background B-cell lymphoma 6 (BCL6) protein, an evolutionarily conserved zinc finger transcription factor, showed to be highly expressed in various human cancers in addition to malignancies in the lymphoid system. This study investigated the role of BCL6 expression in breast cancer and its clinical significance in breast cancer patients. Methods Expression of BCL6 protein was assessed using in situ hybridization and immunohistochemistry in 127 breast cancer patients and 50 patients with breast benign disease as well as in breast cell lines. Expression of BCL6 was restored or knocked down in two breast cancer cell lines (MCF-7 and T47D) using BCL6 cDNA and siRNA, respectively. The phenotypic change of these breast cancer cell lines was assessed using cell viability MTT, Transwell invasion, colony formation, and flow cytometry assays and in a xenograft mice model. Luciferase reporter gene, immunoblot, and qRT-PCR were used to investigate the molecular events after manipulated BCL6 expression in breast cancer cells. Results BCL6 protein was highly expressed in breast cancer cell lines and tissue specimens and expression of BCL6 protein was associated with disease progression and poor survival of breast cancer patients. In vitro, the forced expression of BCL6 results in increased proliferation, anchorage-independent growth, migration, invasion and survival of breast cancer cell lines, whereas knockdown of BCL6 expression reduced these oncogenic properties of breast cancer cells. Moreover, forced expression of BCL6 increased tumor growth and invasiveness in a nude mouse xenograft model. At the gene level, BCL6 was a target gene of miR-339-5p. Expression of BCL6 induced expression of CXCR4 and cyclinD1 proteins. Conclusions The current study demonstrated the oncogenic property of BCL6 in breast cancer and further study could target BCL6 as a novel potential therapeutic strategy for breast cancer.
Function of RasGRP3 in the formation and progression of human breast cancer
Introduction Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer. Methods The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe� DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed. Results RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells. Conclusions Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.
Structures and Mechanisms of Antitumor Agents - Xestoquinones Uncouple Cellular Respiration and Disrupt HIF Signaling in Human Breast Tumor Cells
The organic extract of a marine sponge Petrosia alfiani selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1 � 7) including three new compounds 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1 � 7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), that possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC50 value of 0.2 ?M. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.
Du, Lin; Mahdi, Fakhri; Datta, Sandipan; Jekabsons, Mika B.; Zhou, Yu-Dong; Nagle, Dale G.
Human breast cancer cells contain a phosphoramidon-sensitive metalloproteinase which can process exogenous big endothelin-1 to endothelin-1: a proposed mitogen for human breast fibroblasts.
Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.
Patel, K. V.; Schrey, M. P.
Progesterone receptor inhibits proliferation of human breast cancer cells via induction of MAPK phosphatase 1 (MKP-1/DUSP1).
The roles of progesterone (P(4)) and of progesterone receptor (PR) in development and pathogenesis of breast cancer remain unclear. In this study, we observed that treatment of T47D breast cancer cells with progestin antagonized effects of fetal bovine serum (FBS) to stimulate cell proliferation, whereas siRNA-mediated knockdown of endogenous PR abrogated progestin-mediated anti-proliferative effects. To begin to define mechanisms for the anti-proliferative action of P(4)/PR, we considered the role of MAPK phosphatase 1 (MKP-1/DUSP1), which catalyzes dephosphorylation and inactivation of MAPKs. Progestin treatment of T47D cells rapidly induced MKP-1 expression in a PR-dependent manner. Importantly, P(4) induction of MKP-1 was associated with reduced levels of phosphorylated ERK1/2, whereas siRNA knockdown of MKP-1 blocked progestin-mediated ERK1/2 dephosphorylation and repression of FBS-induced cell proliferation. The importance of PR in MKP-1 expression was supported by findings that MKP-1 and PR mRNA levels were significantly correlated in 30 human breast cancer cell lines. By contrast, no correlation was observed with the glucocorticoid receptor, a known regulator of MKP-1 in other cell types. ChIP and luciferase reporter assay findings suggest that PR acts in a ligand-dependent manner through binding to two progesterone response elements downstream of the MKP-1 transcription start site to up-regulate MKP-1 promoter activity. PR also interacts with two Sp1 sites just downstream of the transcription start site to increase MKP-1 expression. Collectively, these findings suggest that MKP-1 is a critical mediator of anti-proliferative and anti-inflammatory actions of PR in the breast. PMID:22020934
Chen, Chien-Cheng; Hardy, Daniel B; Mendelson, Carole R
Structural effects of TiO2 nanoparticles and doxorubicin on DNA and their antiproliferative roles in T47D and MCF7 cells.
The structural changes in DNA caused by the combined effects of TiO2 nanoparticles (TiO2 NPs) and doxorubicin (DOX) were investigated along with their corresponding inhibitory roles in the growth of T47D and MCF7 cells. The UV-visible titration studies showed that DOX+ TiO2 NPs could form a novel complex with DNA. The data also reveal that the TiO2-DOX complex forms through a 1:4 stoichiometric ratio in solution. The values of binding constants reveal that DOX+TiO2 NPs interact more strongly with DNA as compared to TiO2 NPs or DOX alone. CD data show that DOX+TiO2 NPs can noticeably cause disturbance on DNA structure compared to TiO2 NPs or DOX alone, considering that DNA is relatively thermally stable in the condition used. The anticancer property of 0.3 �M DOX+ 60 �M TiO2 NPs and 0.4 �M DOX+ 670 �M TiO2 NPs by MTT assay and DAPI stain demonstrates that this combination can tremendously diminish proliferation of T47D and MCF7cells compared to DOX or TiO2 NPs alone. The UV-Vis absorption spectroscopy, flow cytometry and fluorescence microscopy experiments show much more enhancement of DOX uptake through the use of TiO2 NPs. These results reveal that DOX+TiO2 NPs could proffer a novel strategy for the development of promising and efficient chemotherapy agents. PMID:23387974
Hekmat, Azadeh; Saboury, Ali Akbar; Divsalar, Adeleh; Seyedarabi, Arefeh
Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists
There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals. Confidence in in vitro assay results is strengthe...
Bisphenol AF-Induced Endogenous Transcription Is Mediated by ER? and ERK1/2 Activation in Human Breast Cancer Cells
Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ER?, ER? and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ER? and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ER?-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ER?-positive T47D and MCF7 cells as well as overexpression of ER? in ER?-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ER? and ERK1/2 activation in human breast cancer cells.
Li, Ming; Guo, Jing; Gao, Wenhui; Yu, Jianlong; Han, Xiaoyu; Zhang, Jing; Shao, Bing
Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer
Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ? CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ? The pro-angiogenic effects of CYP4Z1 have been studied in vitro and in vivo. ? CYP4Z1 regulates expression and production of VEGF-A and TIMP-2. ? CYP4Z1-induced angiogenesis is associated with PI3K and ERK1/2 activation. ? CYP4Z1 may be an attractive target for anti-cancer therapy.
Yu, Wei [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China)] [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China)] [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China)] [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Guifang [Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China)] [Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Cai, Xiaojun [Department of Ophthalmology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China)] [Department of Ophthalmology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States)] [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Yang, Jing, E-mail: firstname.lastname@example.com [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China) [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)
Endocrine-responsive Pancreatic Carcinoma: Steroid Binding and Cytotoxicity Studies in Human Tumor Cell Lines1
We have begun to investígate the steroid responsiveness of pancreatic cancer by comparing human (MiaPaCa, Colo-357, RWP-1, RWP-2) and rodent (AR42J) pancreatic tumor cell lines with cultured estrogen recep tor-positive breast cancer cells (MCF-7, T47-D). The four human pan creatic tumors contain measurable levels of specific estradio! binding sites with dissociation constants (A\\
Chris Benz; Charlene Hollander; Becky Miller
Non-steroid receptor-mediated antiproliferative activity of styrylpyrone derivative in human breast cancer cell lines.
The antiproliferative activity of a styrylpyrone derivative (SPD) plant extract, was studied in three different human breast cancer cell lines in culture, and was compared with tamoxifen. The number of living cells was evaluated by Methylene Blue staining technique. SPD showed strong antiproliferative activity in estrogen receptor (ER) and progestin receptor (PgR) positive MCF-7 cells (EC50 = 6.30 x 10(-7) M) and receptor-negative MDA-MB-231 (EC50 = 5.62 x 10(-7) M), but it partially inhibited the high progestin receptor positive T47D cells (EC50 = 1.58 x 10(-6) M). Whereas tamoxifen, a nonsteroidal antiestrogen exhibited strong inhibition on MCF-7 cells (EC50 = 1.41 x 10(-6) M) and partial inhibition on T47D cells (EC50 = 2.5 x 10(-6) M), but did not affect the MDA-MB-231 cells in the concentration range 0.1 nM-1 microM (EC50 = 5.01 microM). At the same concentration range SPD and tamoxifen did not inhibit the proliferation of normal human liver cell line CCL 13 and normal bovine kidney MDBK; whereas adriamycin, a common chemotherapy drug for the treatment of advance cancer, caused 95% inhibition at 10(-6) M. Competitive binding studies showed SPD had no ability to inhibit the binding of [3H]estradiol and [3H]progesterone to ER and PgR, respectively but, tamoxifen exhibited affinity for ER. Therefore, it can be concluded that the antiproliferative activity of SPD was selective towards breast cancer cell lines and not mediated by ER or PgR. PMID:9673398
Pihie, A H; Stanslas, J; Din, L B
Paradoxical effect of estradiol: it can block its own bioformation in human breast cancer cells.
The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E(2)) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), aromatase, involved in the last steps of E(2) bioformation in this tissue. Quantitative data show that the 'sulfatase pathway', which transforms estrogen sulfates into the bioactive unconjugated E(2), is 100-500 times higher than the 'aromatase pathway' which converts androgens into estrogens. In this paper we explore the effect of E(2) on the sulfatase activity using two hormone-dependent human breast cancer cells: MCF-7 and T-47D. The action of E(2) on the sulfatase activity was evaluated by the conversion of estrone sulfate (E(1)S) into E(2). The cells were incubated in Minimal Essential Medium (MEM) containing 5% steroid-depleted fetal calf serum and incubated with physiological concentrations of [(3)H]E(1)S (5 x 10(-9) M) alone (control) or in the presence of E(2) (5 x 10(-10) to 5 x 10(-5) M) for 24 h at 37 degrees C. It was found that E(2) is a potent inhibitory agent of the estrone sulfatase activity in both cell lines. A low concentration of E(2): 5 x 10(-9) M decreases the sulfatase activity by 67% in MCF-7 cells and 57% in T-47D cells. More than 80% of the decrease in the formation of E(2) was obtained with the dose of 5 x 10(-7) M in both cell lines. It is concluded that this paradoxical effect of E(2) adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Preliminary results are indicated in the Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology (Quebec, Canada, 24-27 June 2000) [J. Steroid Biochem. Molec. Biol. 76 (2001) 95-104](1) and presented at the 83rd Annual Meeting of the Endocrine Society (Denver, USA, 20-23 June 2001 (abstract no. P2-615). PMID:11530280
Pasqualini, J R; Chetrite, G
Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies
A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors cross-reacts with the M/sub r/ 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with (/sup 3/H)promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. B receptors purified under these conditions are transformed and biologically active. They were maintained as undergraded 120-kDa doublets and retained both hormone and DNA binding activities. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG/sub 1/), B-64 (IgG/sub 1/), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG/sub 1/), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology.
Estes, P.A.; Suba, E.J.; Lawler-Heavner, J.; Elashry-Stowers, D.; Wei, L.L.; Toft, D.O.; Sullivan, W.P.; Horwitz, K.B.; Edwards, D.P.
Combined Low Doses of PPAR? and RXR Ligands Trigger an Intrinsic Apoptotic Pathway in Human Breast Cancer Cells
Ligand activation of peroxisome proliferator-activated receptor (PPAR)? and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPAR? ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21WAF1/Cip1. Functional experiments indicate that the nuclear factor-?B site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPAR?/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPAR? and RXR ligands may provide a therapeutic advantage in breast cancer treatment.
Bonofiglio, Daniela; Cione, Erika; Qi, Hongyan; Pingitore, Attilio; Perri, Mariarita; Catalano, Stefania; Vizza, Donatella; Panno, Maria Luisa; Genchi, Giuseppe; Fuqua, Suzanne A.W.; Ando, Sebastiano
Sulfotransferase 1A1 (SULT1A1) gene expression is regulated by members of the NFI transcription factors in human breast cancer cells
Background Sulfotransferase 1A1 (SULT1A1) gene expression is tissue specific, with little to no expression in normal breast epithelia. Expression in breast tumors has been documented, but the transcriptional regulation of SULT1A1 in human breast tissue is poorly understood. We identified Nuclear Factor I (NFI) as a transcription factor family involved in the regulation of SULT1A1 expression. Methods Transcription Factor Activation Profiling Plate Array assay was used to identify the possible transcription factors that regulate the gene expression of SULT1A1in normal breast MCF-10A cells and breast cancer ZR-75-1 cells. Expression levels of NFI-C and SULT1A1 were determined by real-time RT-PCR using total RNA isolated from 84 human liver samples. Expression levels of SULT1A1, NFI-A, NFI-B, NFI-C, and NFI-X were also determined in different human breast cancer cell lines (MCF-7, T-47D, ZR-75-1, and MDA-MB-231), in the transformed human epithelial cell line MCF-10A, and in ZR-75-1 cells that were transfected with siRNAs directed against NFI-A, NFI-B, NFI-C, or NFI-X for 48�h. The copy numbers of SULT1A1 in cell lines ZR-75-1, MCF-7, T-47D, MDA-MB-231, and MCF-10A were determined using a pre-designed Custom Plus TaqMan� Copy Number kit from Life Technologies. Results In normal human liver samples, SULT1A1 mRNA level was positively associated with NFI-C. In different human breast cancer and normal epithelial cell lines, SULT1A1 expression was positively correlated with NFI-B and NFI-C. SULT1A1 expression was decreased 41% and 61% in ZR-75-1 cells treated with siRNAs against NFI-A and NFI-C respectively. SULT1A1 gene expression was higher in cells containing more than one SULT1A1 copy numbers. Conclusions Our data suggests that SULT1A1 expression is regulated by NFI, as well as SULT1A1 copy number variation in human breast cancer cell lines. These data provide a mechanistic basis for the differential expression of SULT1A1 in different tissues and different physiological states of disease.
Metformin inhibits histone H2B monoubiquitination and downstream gene transcription in human breast cancer cells
Metformin, one of the most widely prescribed antihyperglycemic drugs, has recently received increasing attention for its potential effects with regard to cancer prevention and treatment. However, the mechanisms behind the suppression of cancer cell growth by metformin remain far from completely understood. The aim of the present study was to investigate whether metformin could regulate histone modification and its downstream gene transcription, and its potential function in inhibiting breast cancer cell proliferation. A T47D cell proliferation curve was determined by cell counting following metformin treatment with differing doses or time courses. The cell cycle was analyzed by flow cytometry with propidium iodide staining. Histone H2B monoubiquitination was evaluated by western blotting subsequent to histone extraction. The histone H2B monoubiquitination downstream gene expression level was determined by quantitative PCR. The results showed that metformin changed the cell-cycle check-point and inhibited breast cancer cell proliferation in a dose-dependent manner. AMPK was activated and histone H2B monoubiquitination and downstream gene transcription were inhibited following metformin treatment in the T47D cells. The effect of metformin on T47D cell proliferation was dependent on AMPK activity. It was concluded that metformin can suppress breast cancer cell growth by the activation of AMPK and the inhibition of histone H2B monoubiquitination and downstream gene transcription. This study reveals a novel potential mechanism of cancer cell growth suppression by metformin.
Benzene-poly-carboxylic acid complex, a novel anti-cancer agent induces apoptosis in human breast cancer cells.
Some cases of breast cancer are composed of clones of hormonal-independent growing cells, which do not respond to therapy. In the present study, the effect of Benzene-Poly-Carboxylic Acid Complex (BP-C1) on growth of human breast-cancer cells was tested. BP-C1 is a novel anti-cancer complex of benzene-poly-carboxylic acids with a very low concentration of cis-diammineplatinum (II) dichloride. Human breast cancer cells, MCF-7 and T47D, were used. Cell viability was detected by XTT assay and apoptosis was detected by Flow Cytometry and by annexin V/FITC/PI assay. Caspases were detected by western blot analysis and gene expression was measured by using the Applied Biosystems� TaqMan� Array Plates. The results showed that exposure of the cells to BP-C1 for 48 h, significantly (P<0.001) reduced cell viability, induced apoptosis and activated caspase 8 and caspace 9. Moreover, gene expression experiments indicated that BP-C1 increased the expression of pro-apoptotic genes (CASP8AP1, TNFRSF21, NFkB2, FADD, BCL10 and CASP8) and lowered the level of mRNA transcripts of inhibitory apoptotic genes (BCL2L11, BCL2L2 and XIAP. These findings may lead to the development of new therapeutic strategies for treatment of human cancer using BP-C1 analog. PMID:24523856
Fares, Fuad; Azzam, Naiel; Fares, Basem; Larsen, Stig; Lindkaer-Jensen, Steen
Benzene-Poly-Carboxylic Acid Complex, a Novel Anti-Cancer Agent Induces Apoptosis in Human Breast Cancer Cells
Some cases of breast cancer are composed of clones of hormonal-independent growing cells, which do not respond to therapy. In the present study, the effect of Benzene-Poly-Carboxylic Acid Complex (BP-C1) on growth of human breast-cancer cells was tested. BP-C1 is a novel anti-cancer complex of benzene-poly-carboxylic acids with a very low concentration of cis-diammineplatinum (II) dichloride. Human breast cancer cells, MCF-7 and T47D, were used. Cell viability was detected by XTT assay and apoptosis was detected by Flow Cytometry and by annexin V/FITC/PI assay. Caspases were detected by western blot analysis and gene expression was measured by using the Applied Biosystems� TaqMan� Array Plates. The results showed that exposure of the cells to BP-C1 for 48 h, significantly (P<0.001) reduced cell viability, induced apoptosis and activated caspase 8 and caspace 9. Moreover, gene expression experiments indicated that BP-C1 increased the expression of pro-apoptotic genes (CASP8AP1, TNFRSF21, NFkB2, FADD, BCL10 and CASP8) and lowered the level of mRNA transcripts of inhibitory apoptotic genes (BCL2L11, BCL2L2 and XIAP. These findings may lead to the development of new therapeutic strategies for treatment of human cancer using BP-C1 analog.
Fares, Fuad; Azzam, Naiel; Fares, Basem; Larsen, Stig; Lindkaer-Jensen, Steen
Alpha Cyano-4-Hydroxy-3-Methoxycinnamic Acid Inhibits Proliferation and Induces Apoptosis in Human Breast Cancer Cells
This study investigated the underlying mechanism of 4-hydroxy-3-methoxycinnamic acid (ACCA), on the growth of breast cancer cells and normal immortal epithelial cells, and compared their cytotoxic effects responses. Treatment of breast cancer cells (MCF-7, T47D, and MDA-231) with ACCA resulted in dose- and time-dependent decrease of cell proliferation, viability in colony formation assay, and programmed cell death (apoptosis) with minimal effects on non-tumoral cells. The ability of ACCA to suppress growth in cancer cells not expressing or containing defects in p53 gene indicates a lack of involvement of this critical tumor suppressor element in mediating ACCA-induced growth inhibition. Induction of apoptosis correlated with an increase in Bax protein, an established inducer of programmed cell death, and the ratio of Bax to Bcl-2, an established inhibitor of apoptosis. We also documented the ability of ACCA to inhibit the migration and invasion of MDA-231 cells with ACCA in vitro. Additionally, tumor growth of MDA-231 breast cancer cells in vivo was dramatically affected with ACCA. On the basis of its selective anticancer inhibitory activity on tumor cells, ACCA may represent a promising therapeutic drug that should be further evaluated as a chemotherapeutic agent for human breast cancer.
Hamdan, Lamia; Arrar, Zoheir; Al Muataz, Yacoub; Suleiman, Lutfi; Negrier, Claude; Mulengi, Joseph Kajima; Boukerche, Habib
Development of the Human Breast
Mammalia are so named based on the presence of the mammary gland in the breast. The mammary gland is an epidermal appendage, derived from the apocrine glands. The human breast consists of the parenchyma and stroma, originating from ectodermal and mesodermal elements, respectively. Development of the human breast is distinctive for several reasons. The human breast houses the mammary gland that produces and delivers milk through development of an extensive tree-like network of branched ducts. It is also characterized by cellular plasticity, with extensive remodeling in adulthood, a factor that increases its susceptibility to carcinogenesis. Also, breast development occurs in distinct stages via complex epithelial�mesenchymal interactions, orchestrated by signaling pathways under the regulation of systemic hormones. Congenital and acquired disorders of the breast often have a basis in development, making its study essential to understanding breast pathology.
Javed, Asma; Lteif, Aida
Aluminium and human breast diseases.
The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 � 28 ?g/l) compared with control healthy subjects (mean 131 � 10 ?g/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 ?g/l) compared with human serum (median 6 ?g/l) or human milk (median 25 ?g/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate. PMID:22099158
Darbre, P D; Pugazhendhi, D; Mannello, F
Human breasts: Unsupported hypotheses reviewed
Unlike other apes, human females� breasts develop before first pregnancy and are permanently enlarged. Evidence suggests breasts\\u000a act as signals to males but the critical data required to confirm this are lacking. These facts have led to a number of hypotheses\\u000a about the evolutionary and adaptive significance of the human breast which fall into two groups. Those that address the
T. M. Caro
The transcriptional responsiveness of LKB1 to STAT-mediated signaling is differentially modulated by prolactin in human breast cancer cells
Background Liver kinase 1 (LKB1) is an important multi-tasking protein linked with metabolic signaling, also controlling polarity and cytoskeletal rearrangements in diverse cell types including cancer cells. Prolactin (PRL) and Signal transducer and activator of transcription (STAT) proteins have been associated with breast cancer progression. The current investigation examines the effect of PRL and STAT-mediated signaling on the transcriptional regulation of LKB1 expression in human breast cancer cells. Methods MDA-MB-231, MCF-7, and T47D human breast cancer cells, and CHO-K1 cells transiently expressing the PRL receptor (long form), were treated with 100�ng/ml of PRL for 24�hours. A LKB1 promoter-luciferase construct and its truncations were used to assess transcriptional changes in response to specific siRNAs or inhibitors targeting Janus activated kinase 2 (JAK2), STAT3, and STAT5A. Real-time PCR and Western blotting were applied to quantify changes in mRNA and protein levels. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays were used to examine STAT3 and STAT5A binding to the LKB1 promoter. Results Consistent with increases in mRNA, the LKB1 promoter was up-regulated by PRL in MDA-MB-231 cells, a response that was lost upon distal promoter truncation. A putative GAS element that could provide a STAT binding site mapped to this region, and its mutation decreased PRL-responsiveness. PRL-mediated increases in promoter activity required signaling through STAT3 and STAT5A, also involving JAK2. Both STATs imparted basally repressive effects in MDA-MB-231 cells. PRL increased in vivo binding of STAT3, and more definitively, STAT5A, to the LKB1 promoter region containing the GAS site. In T47D cells, PRL down-regulated LKB1 transcriptional activity, an effect that was reversed upon culture in phenol red-free media. Interleukin 6, a cytokine activating STAT signaling in diverse cell types, also increased LKB1 mRNA levels and promoter activity in MDA-MB-231 cells. Conclusions LKB1 is differentially regulated by PRL at the level of transcription in representative human breast cancer cells. Its promoter is targeted by STAT proteins, and the cellular estrogen receptor status may affect PRL-responsiveness. The hormonal and possibly cytokine-mediated control of LKB1 expression is particularly relevant in aggressive breast cancer cells, potentially promoting survival under energetically unfavorable conditions.
Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines
Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.
Ho, T.-F. [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Medical Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Y.-T.; Chuang, S.-M.; Lin, S.-C.; Feng, B.-L. [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Lu, C.-H. [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Yu, W.-J. [Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan (China); Chang, J.-S. [Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan (China); Sustainable Environment Research Center, National Cheng Kung University, Tainan, Taiwan (China)], E-mail: email@example.com; Chang, C.-C. [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China)], E-mail: firstname.lastname@example.com
Real-Time Growth Kinetics Measuring Hormone Mimicry for ToxCast Chemicals in T-47D Human Ductal Carcinoma Cells
High-throughput screening (HTS) assays capable of profiling thousands of environmentally relevant chemicals for in vitro biological activity provide useful information on the potential for disrupting endocrine pathways. Disruption of the estrogen signaling pathway has been implic...
Effects of 60-Hz fields, estradiol and xenoestrogens on human breast cancer cells
If exposure to xenoestrogens or electromagnetic fields (EMFs) such as 60 Hz contributes to the etiology of breast cancer, it is likely that they must stimulate the growth of breast cells, damage genetic material or enhance the effects of other mitogenic or mutagenic agents (co-promotion). Therefore, the ability of xenoestrogens or exposure to 60-Hz fields to stimulate the entry of growth-arrested human breast cancer cells into the cell cycle was determined using cyclin-dependent kinase 2 (Cdk2) activity, synthesis of cyclin D1 and cdc2 activity. Exposure of estrogen receptor-positive MCF-7 or T-47D cells to estrogen and xenoestrogens (DDT and Red No.3) increased Cdk2 and cyclin B1-cdc2 activity and cyclin D1 synthesis. Exposure of breast cancer cells to 12 mG or 1 or 9 G electromagnetic fields at 60 Hz failed to stimulate Cdk2 or cyclin B1-cdc2 activity or cyclin D1 synthesis. Simultaneous co-exposure of cells to 60-Hz fields and chemical promoters did not enhance Cdk2 activation above the levels produced by the chemical promoter alone. Estrogen and xenoestrogens also stimulated binding of the estrogen receptor to the estrogen receptor element but the EMF did not. Phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of p53 and pRb105 in MCF-7 cells, but EMF exposure had no effect. DNA-damaging chemotherapeutic agents and Red Dye No. 3 were found to increase p53 site-specific DNA binding in breast cancer cells, but EMF exposure did not. These studies suggest that estrogen and xenoestrogens stimulate growth-arrested breast cancer cells to enter the growth cycle, but EMF exposure does not. Site-specific p53-DNA binding was increased in MCF-7 cells treated with DNA-damaging agents, but not by EMF exposure. EMF exposure does not appear to act as a promoter or DNA-damaging agent for human breast cancer cells in vitro. 34 refs., 10 figs.
Dees, C.; Travis, C. [Oak Ridge National Lab., TN (United States); Garrett, S. [Oak Ridge Institute for Science and Education, Oak Ridge, TN (United States); Henley, D. [Univ. of Tennessee, Knoxville (United States)
Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells.
The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role. PMID:2166713
Giacchetti, S; Gauvill�, C; de Cr�moux, P; Bertin, L; Berthon, P; Abita, J P; Cuttitta, F; Calvo, F
Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line.
We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogen-response element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A18 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ER77. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways. Images Figure 2 Figure 3 Figure 4 Figure 5
Pink, J. J.; Fritsch, M.; Bilimoria, M. M.; Assikis, V. J.; Jordan, V. C.
Immunologic analysis of human breast cancer progesterone receptors. 2. Structure, phosphorylation, and processing
The authors have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (M/sub r/ 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (M/sub r/ 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. The independence of A- and B-receptor complexes was confirmed by the fining that purified, transformed B receptors bind well to DNA-cellulose. Additional studies focused on the covalent modifications of receptors. The previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with (/sup 32/P)orthophosphate, and the B receptors were isolated by immunoprecipitation with PR-6 and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis. In both treatment states, B receptors were labeled in vivo with /sup 32/P, thus demonstrating directly that human PR are phosphoproteins. Since B receptors were labeled in the absence of hormone and also after their in vivo transformation by hormone, they appear to be substrates for two phosphorylation reactions, one in the untransformed state and another after they are tightly bound to chromatin. The second phosphorylation may account for the mobility shift of the receptors on SDS gels. On the basis of these data a model of human PR structure and subcellular receptor dynamics is presented.
Wei, L.L.; Sheridan, P.L.; Krett, N.L.; Francis, M.D.; Toft, D.O.; Edwards, D.P.; Horwitz, K.B.
Retinoids Modulate Expression of the Endocytic Partners Megalin, Cubilin, and Disabled-2 and Uptake of Vitamin D-Binding Protein in Human Mammary Cells12
The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.
Chlon, Timothy M.; Taffany, David A.; Welsh, JoEllen; Rowling, Matthew J.
Airborne particulate collected from central Taiwan induces DNA strand breaks, Poly(ADP-ribose) polymerase-1 activation, and estrogen-disrupting activity in human breast carcinoma cell lines.
The objectives of this investigation were to examine whether airborne particles induce DNA damaging and estrogen-disrupting effects and to explore the relationships between them. In this study, airborne particulate was collected at an urban, a suburban, and a rural site in central Taiwan. The organic solvent extracts of airborne particulate were examined in human MCF-7 and T47D-KBluc breast cancer cells. We observed significant increases in reactive oxygen species (ROS) generation in MCF-7 cells after treatment with the particulate extracts whereas aryl hydrocarbon receptor (AhR) antagonist blocked the particulate-induced ROS generation in cells. Further, induction of CYP1A1 protein expression was confirmed by immunoblots in cells treated with airborne particles, suggesting the roles of AhR status in mediating the particulate-induced toxicity. In addition, we observed that at non-cytotoxic concentration (?0.01�m(3) air equivalent), airborne particles induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells. These decreases were completely blocked by three types of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 cells exposed to airborne particles as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that airborne particles induce decreases in intracellular NAD(P)H and NAD(+) through PARP-1 activation mediated by formation of DNA strand breaks. Furthermore, we confirmed that with series dilution airborne particles (?10(-7)-10(-2) m(3) air equivalent) possess both estrogenic and anti-estrogenic activities as determined by the ER?-mediated reporter gene assay in human T47D-KBluc breast cancer cells. In conclusions, we confirmed that the DNA-damaging activity and estrogenicity of airborne particles varied considerably with concentration (air equivalent). Our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to airborne particles and that oxidative stress and the subsequent induction of DNA damage may, in part, contribute to airborne particle-induced carcinogenesis. PMID:23043339
Chen, Shou-Tung; Lin, Chia-Chi; Liu, Yi-Shiau; Lin, Che; Hung, Pei-Tzu; Jao, Chia-Wen; Lin, Po-Hsiung
Effects of simultaneous knockdown of HER2 and PTK6 on malignancy and tumor progression in human breast cancer cells.
Breast cancer is the most common malignancy in women of the Western world. One prominent feature of breast cancer is the co- and overexpression of HER2 and protein tyrosine kinase 6 (PTK6). According to the current clinical cancer therapy guidelines, HER2-overexpressing tumors are routinely treated with trastuzumab, a humanized monoclonal antibody targeting HER2. Approximately, 30% of HER2-overexpressing breast tumors at least initially respond to the anti-HER2 therapy, but a subgroup of these tumors develops resistance shortly after the administration of trastuzumab. A PTK6-targeted therapy does not yet exist. Here, we show for the first time that the simultaneous knockdown in vitro, compared with the single knockdown of HER2 and PTK6, in particular in the trastuzumab-resistant JIMT-1 cells, leads to a significantly decreased phosphorylation of crucial signaling proteins: mitogen-activated protein kinase 1/3 (MAPK 1/3, ERK 1/2) and p38 MAPK, and (phosphatase and tensin homologue deleted on chromosome ten) PTEN that are involved in tumorigenesis. In addition, dual knockdown strongly reduced the migration and invasion of the JIMT-1 cells. Moreover, the downregulation of HER2 and PTK6 led to an induction of p27, and the dual knockdown significantly diminished cell proliferation in JIMT-1 and T47D cells. In vivo experiments showed significantly reduced levels of tumor growth following HER2 or PTK6 knockdown. Our results indicate a novel strategy also for the treatment of trastuzumab resistance in tumors. Thus, the inhibition of these two signaling proteins may lead to a more effective control of breast cancer. PMID:23364537
Ludyga, Natalie; Anastasov, Natasa; Rosemann, Michael; Seiler, Jana; Lohmann, Nadine; Braselmann, Herbert; Mengele, Karin; Schmitt, Manfred; H�fler, Heinz; Aubele, Michaela
Role of ERRF, a Novel ER-Related Nuclear Factor, in the Growth Control of ER-Positive Human Breast Cancer Cells
Whereas estrogen�estrogen receptor ? (ER) signaling plays an important role in breast cancer growth, it is also necessary for the differentiation of normal breast epithelial cells. How this functional conversion occurs, however, remains unknown. Based on a genome-wide sequencing study that identified mutations in several breast cancer genes, we examined some of the genes for mutations, expression levels, and functional effects on cell proliferation and tumorigenesis. We present the data for C1orf64 or ER-related factor (ERRF) from 31 cell lines and 367 primary breast cancer tumors. Whereas mutation of ERRF was infrequent (1 of 79 or 1.3%), its expression was up-regulated in breast cancer, and the up-regulation was more common in lower-stage tumors. In addition, increased ERRF expression was significantly associated with ER and/or progesterone receptor (PR) positivity, which was still valid in human epidermal growth factor receptor 2 (HER2)�negative tumors. In ER-positive tumors, ERRF expression was inversely correlated with HER2 status. Furthermore, higher ERRF protein expression was significantly associated with better disease-free survival and overall survival, particularly in ER- and/or PR-positive and HER2-negative tumors (luminal A subtype). Functionally, knockdown of ERRF in two ER-positive breast cancer cell lines, T-47D and MDA-MB-361, suppressed cell growth in vitro and tumorigenesis in xenograft models. These results suggest that ERRF plays a role in estrogen-ER�mediated growth of breast cancer cells and could, thus, be a potential therapeutic target.
Su, Dan; Fu, Xiaoying; Fan, Songqing; Wu, Xiao; Wang, Xin-Xin; Fu, Liya; Dong, Xue-Yuan; Ni, Jianping Jenny; Fu, Li; Zhu, Zhengmao; Dong, Jin-Tang
Role of ERRF, a novel ER-related nuclear factor, in the growth control of ER-positive human breast cancer cells.
Whereas estrogen-estrogen receptor ? (ER) signaling plays an important role in breast cancer growth, it is also necessary for the differentiation of normal breast epithelial cells. How this functional conversion occurs, however, remains unknown. Based on a genome-wide sequencing study that identified mutations in several breast cancer genes, we examined some of the genes for mutations, expression levels, and functional effects on cell proliferation and tumorigenesis. We present the data for C1orf64 or ER-related factor (ERRF) from 31 cell lines and 367 primary breast cancer tumors. Whereas mutation of ERRF was infrequent (1 of 79 or 1.3%), its expression was up-regulated in breast cancer, and the up-regulation was more common in lower-stage tumors. In addition, increased ERRF expression was significantly associated with ER and/or progesterone receptor (PR) positivity, which was still valid in human epidermal growth factor receptor 2 (HER2)-negative tumors. In ER-positive tumors, ERRF expression was inversely correlated with HER2 status. Furthermore, higher ERRF protein expression was significantly associated with better disease-free survival and overall survival, particularly in ER- and/or PR-positive and HER2-negative tumors (luminal A subtype). Functionally, knockdown of ERRF in two ER-positive breast cancer cell lines, T-47D and MDA-MB-361, suppressed cell growth in vitro and tumorigenesis in xenograft models. These results suggest that ERRF plays a role in estrogen-ER-mediated growth of breast cancer cells and could, thus, be a potential therapeutic target. PMID:22341523
Su, Dan; Fu, Xiaoying; Fan, Songqing; Wu, Xiao; Wang, Xin-Xin; Fu, Liya; Dong, Xue-Yuan; Ni, Jianping Jenny; Fu, Li; Zhu, Zhengmao; Dong, Jin-Tang
Downregulation of cPLA2? expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway.
Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA(2)? in chemotaxis of human breast cancer cells. Inhibition of cPLA(2)? expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA(2)? expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA(2)? regulated migration. Knockdown of cPLA(2)? suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKC?, downstream of Akt, was also dampened. Knockdown of cPLA(2)? also impaired the phosphorylation of integrin ?1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA(2)? plays an important role in cancer cell chemotaxis. PMID:21600875
Tian, Gang; Wang, Xueqian; Zhang, Fei; Geng, Hua; Hou, Wei; Chen, Liwei; Guo, Hua; Zhang, Ning
Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A.
The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells. PMID:23340249
Hammer, Alan; Rider, Leah; Oladimeji, Peter; Cook, Leslie; Li, Quanwen; Mattingly, Raymond R; Diakonova, Maria
Effect of promegestone, tamoxifen, 4-hydroxytamoxifen and ICI 164,384 on the oestrone sulphatase activity of human breast cancer cells.
In the present study, we explored the effects on sulphatase activity by Promegestone (R-5020), Tamoxifen (TAM), 4-hydroxytamoxifen (4-OH-TAM) and ICI 164,384 in the T-47D hormone-dependent and MDA-MB-231 hormone-independent mammary cancer cell lines. Using homogenates of these cells it was observed that Promegestone has a significant effect on the inhibition of oestrone (E1) sulphatase activity. As this effect is competitive, it is suggested that there is a direct action of this compound on the enzyme. Tamoxifen has very little or no effect, 4-hydroxytamoxifen has an intermediate effect and ICI 164,384 is active in the enzyme inhibition, particularly with MDA-MB-231 cells. The present data could open new possibilities for human breast cancer treatment, as sulphatase is very active in the first step of the conversion of oestrone sulphate (E1S) to oestradiol (E2), and oestradiol is one of the principal carcinogenic factors in human hormone-dependent breast cancer. PMID:8352561
Chetrite, G; Varin, C; Delalonde, L; Pasqualini, J R
Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)
Introduction Na+/H+ exchanger regulatory factor 1 (NHERF1, also known as EBP50 or NHERF) is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity (LOH) at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified. Methods To confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA (shRNA) to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses. Results We found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G1-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level. Conclusion Our findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities.
Pan, Yong; Wang, Lei; Le Dai, Jia
Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer
NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction in breast cancer.
North, Paula; Kong, Amanda; Huang, Jian; Miao, Qing Robert
Essiac? and Flor-Essence? herbal tonics stimulate the in vitro growth of human breast cancer cells
People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence{reg_sign} and Essiac{reg_sign} Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence{reg_sign} and Essiac{reg_sign} for their effects on the growth of human tumor cells in culture. The effect of Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors. Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay. Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls and activated ER dependent luciferase activity in MCF-7 cells. A 10{sup -7} M concentration of ICI 870,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence{reg_sign} and Essiac{reg_sign}, but did not affect cell proliferation. Flor-Essence{reg_sign} and Essiac{reg_sign} Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action. Cancer patients and health care providers can use this information to make informed decisions about the use of these CAMs.
Kulp, K S; Montgomery, J L; McLimans, B; Latham, E R; Shattuck, D L; Klotz, D M; Bennett, L M
Synthesis of new N,N'-bis[1-aryl-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides and evaluation of their cytotoxicity against human hepatoma and breast cancer cells.
Abstract N,N'-Bis[1-aryl-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides were synthesized by the reaction of 2 mols of 1-aryl-3-(piperidine-1-yl)-1-propanone hydrochlorides with 1?mol of hydrazine hydrate. Aryl part was C6H5 (P1), 4-CH3C6H4 (P2), 4-CH3OC6H4 (P3), 4-HOC6H4 (P4), 4-ClC6H4 (P5), 3-CH3OC6H4 (P6), 4-FC6H4 (P7) and 4-BrC6H4 (P8). Except P1, all compounds were reported for the first time. The chemical structures were confirmed by UV, (1)H NMR, (13)C NMR and HRMS spectra. P1, P2, P7 and P8 against human hepatoma (Huh7) cells and P1, P2, P4, P5, P6, P7 and P8 against breast cancer (T47D) cells have shown cytotoxicity. P1, P2 and P7 had more potent cytotoxicity against Huh7 cells than the reference compound 5-FU, whereas only P2 was more potent than the 5-FU against T47D cells. Representative compound P7 inhibited the mitochondrial respiration at 144, 264 and 424?�M concentrations dose-dependantly in liver homogenates. The results suggest that P1, P2, P7 and P8 may serve as model compounds for further synthetic studies. PMID:23859151
Kucukoglu, Kaan; Gul, H Inci; Cetin-Atalay, Rengul; Baratli, Yosra; Charles, Anne-Laure; Sukuroglu, Murat; Gul, Mustafa; Geny, Bernard
The effectiveness of nano chemotherapeutic particles combined with mifepristone depends on the PR isoform ratio in preclinical models of breast cancer
There is clinical and experimental evidence suggesting that antiprogestins might be used for the treatment of selected breast cancer patients. Our aim was to evaluate the effect of albumin-bound paclitaxel (Nab-paclitaxel) and pegylated doxorubicin liposomes (PEG-LD) in combination with mifepristone (MFP) in experimental breast cancer models expressing different ratios of progesterone receptor (PR) isoforms A and B. We used two antiprogestin-responsive (PRA>PRB) and two resistant (PRA
Rojas, Paola; Lamb, Caroline; Colombo, Lucas; May, Maria; Molinolo, Alfredo; Lanari, Claudia
The effectiveness of nano chemotherapeutic particles combined with mifepristone depends on the PR isoform ratio in preclinical models of breast cancer.
There is clinical and experimental evidence suggesting that antiprogestins might be used for the treatment of selected breast cancer patients. Our aim was to evaluate the effect of albumin-bound paclitaxel (Nab-paclitaxel) and pegylated doxorubicin liposomes (PEG-LD) in combination with mifepristone (MFP) in experimental breast cancer models expressing different ratios of progesterone receptor (PR) isoforms A and B. We used two antiprogestin-responsive (PRA>PRB) and two resistant (PRA
Sequeira, Gonzalo; Vanzulli, Silvia I; Rojas, Paola; Lamb, Caroline; Colombo, Lucas; May, Maria; Molinolo, Alfredo; Lanari, Claudia
Comparative properties of the nuclear aryl hydrocarbon (Ah) receptor complex from several human cell lines
The aryl hydrocarbon (Ah) responsiveness of the T-47D, Hep G2, LS180, MCF-7, A431, C-4II and MDA-MB-231 human cancer cell lines was determined by the induction of CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity. With the exception of the MDA-MB-231 breast cancer cell line, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity in the remaining six cell lines
Xiaohong Wang; Jane S. Thomsen; Michael Santostefano; Rhonda Rosengren; Stephen Safe; Gary H. Perdew
Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues.
It is well accepted that estradiol (E2) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E1S) 'via sulfatase' is a much more likely precursor for E2 than is androstenedione 'via aromatase'. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E2 was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at -80 degrees C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45-75 mg) were incubated in 20 mM Tris-HCl, pH 7.2 with physiological concentrations of [3H]-E1S (5 x 10(-9)M) alone or in the presence of E2 (5 x 10(-5) to 5 x 10(-7) M) during 30 min or 3 h. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E1 was 3.20 +/- 0.15 and 0.42 +/- 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 +/- 1.8 and 3.5 +/- 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 x 10(-7) M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 x 10(-5) M after 30 min incubation. The values were 24% and 18% for 5 x 10(-7) M and 49% and 42% for 5 x 10(-5) M, respectively after 3h incubation. It was observed that [3H]-E1S is only converted to [3H]-E1 and not to [3H]-E2 in normal or cancerous breast tissues, which suggests a low or no 17beta-hydroxysteroid dehydrogenase (17beta-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone. PMID:17481887
Chetrite, G S; Cortes-Prieto, J-C; Philippe, J-C; Pasqualini, J R
Estradiol and estrone C-16 derivatives as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: blocking of ER+ breast cancer cell proliferation induced by estrone.
Estrogens play an important role in the development of breast cancer. Inhibiting 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1)--the enzyme responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2)--would thus allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16beta-benzyl-E2 (1) as a lead compound for developing inhibitors of the transformation of estrone (E1) into E2, we modified the benzyl group of 1 to improve its inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line T-47D, which exerts a strong endogenous 17beta-HSD1 activity. In this intact cell model, 16beta-m-carbamoylbenzyl-E2 (4m) emerged as a potent inhibitor of 17beta-HSD1 with an IC50 value of 44 nM for the transformation of [14C]-E1 (60 nM) into [14C]-E2 (24-h incubation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with 4m at a concentration of 0.5 microM induced some proliferation (38%) of T-47D estrogen-sensitive (ER+) breast cancer cells. Interestingly, when 4m (0.5 microM) was given with E1 (0.1 nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E1 after its reduction to E2 by 17beta-HSD1. Thus, in addition to generating useful structure-activity relationships for the development of 17beta-HSD1 inhibitors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E2 and consequently its proliferative effect in T-47D ER+ breast cancer cells. PMID:18035543
Laplante, Yannick; Cadot, Christine; Fournier, Michelle-Audrey; Poirier, Donald
Autocrine human GH promotes radioresistance in mammary and endometrial carcinoma cells.
Although recent advances in breast cancer treatment regimes have improved patient prognosis, resistance to breast cancer therapies, such as radiotherapy, is still a major clinical challenge. In the current study, we have investigated the role of autocrine human GH (hGH) in resistance to ionising radiation (IR)-based therapy. Cell viability and total cell number assays demonstrated that autocrine hGH promoted cell regrowth in the mammary carcinoma cell lines, MDA-MB-435S and T47D, and the endometrial carcinoma cell line, RL95-2, following treatment with IR. In addition, autocrine hGH enhanced MDA-MB-435S and T47D cell clonogenic survival following radiation exposure. The enhanced clonogenic survival afforded by autocrine hGH was mediated by JAK2 and Src kinases. Investigation into the DNA repair capacity demonstrated that autocrine hGH reduced IR-induced DNA damage in MDA-MB-435S and T47D cells. Functional antagonism of hGH increased RL95-2 sensitivity to IR in cell viability and total cell number assays, reduced clonogenic survival and enhanced the induction of DNA damage. Thus, autocrine hGH reduced sensitivity to treatment with IR in mammary and endometrial carcinoma cell lines in vitro, while functional antagonism of hGH sensitised endometrial carcinoma cells to IR. Functional antagonism of hGH, used in conjunction with radiotherapy, may therefore enhance treatment efficacy and improve the prognosis of patients with breast and endometrial cancer. PMID:22807498
Bougen, Nicola M; Steiner, Michael; Pertziger, Mikhail; Banerjee, Arindam; Brunet-Dunand, Severine E; Zhu, Tao; Lobie, Peter E; Perry, Jo K
The interrelationship between DRIM gene expression and cytogenetic and phenotypic characteristics in human breast tumor cell lines
Background In order to facilitate the identification of genes involved in the metastatic phenotype we have previously developed a pair of cell lines from the human breast carcinoma cell line MDA-MB-435, which have diametrically opposite metastatic potential in athymic mice. Differential display analysis of this model previously identified a novel gene, DRIM (down regulated in metastasis), the decreased expression of which correlated with metastatic capability. DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C. elegans, but little else is currently known about its function or pattern of expression. In a detailed analysis of the DRIM gene locus we quantitatively evaluated gene dosage and the expression of DRIM transcripts in a panel of breast cell lines of known metastatic phenotype. Results Fluorescent in situ hybridization (FISH) analyses mapped a single DRIM gene locus to human chromosome 12q23~24, a region of conserved synteny to mouse chromosome 10. We confirmed higher expression of DRIM mRNA in the non-metastatic MDA-MB-435 clone NM2C5, relative to its metastatic counterpart M4A4, but this appeared to be due to the presence of an extra copy of the DRIM gene in the cell line's genome. The other non-metastatic cell lines in the series (T47D MCF-7, SK-BR-3 and ZR-75-1) contained either 3 or 4 chromosomal copies of DRIM gene. However, the expression level of DRIM mRNA in M4A4 was found to be 2�4 fold higher than in unrelated breast cells of non-metastatic phenotype. Conclusions Whilst DRIM expression is decreased in metastatic M4A4 cells relative to its non-metastatic isogenic counterpart, neither DRIM gene dosage nor DRIM mRNA levels correlated with metastatic propensity in a series of human breast tumor cell lines examined. Collectively, these findings indicate that the expression pattern of the DRIM gene in relation to the pathogenesis of breast tumor metastasis is more complex than previously recognized.
Goodison, Steve; Viars, Carrie; Grazzini, Maren; Urquidi, Virginia
Anti-aromatase effect of resveratrol and melatonin on hormonal positive breast cancer cells co-cultured with breast adipose fibroblasts.
Targeting the estrogen pathway has been proven effective in the treatment for estrogen receptor positive breast cancer. There are currently two common groups of anti-estrogenic compounds used in the clinic; Selective Estrogen Receptor Modulators (SERMs, e.g. tamoxifen) and Selective Estrogen Enzyme Modulators (SEEMs e.g. letrozole). Among various naturally occurring, biologically active compounds, resveratrol and melatonin have been suggested to act as aromatase inhibitors, which make them potential candidates in hormonal treatment of breast cancer. Here we used a co-culture model in which we previously demonstrated that primary human breast adipose fibroblasts (BAFs) can convert testosterone to estradiol, which subsequently results in estrogen receptor-mediated breast cancer T47D cell proliferation. In the presence of testosterone in this model, we examined the effect of letrozole, resveratrol and melatonin on cell proliferation, estradiol (E2) production and gene expression of CYP19A1, pS2 and Ki-67. Both melatonin and resveratrol were found to be aromatase inhibitors in this co-culture system, albeit at different concentrations. Our co-culture model did not provide any indications that melatonin is also a selective estrogen receptor modulator. In the T47D-BAF co-culture, a melatonin concentration of 20nM and resveratrol concentration of 20?M have an aromatase inhibitory effect as potent as 20nM letrozole, which is a clinically used anti-aromatase drug in breast cancer treatment. The SEEM mechanism of action of especially melatonin clearly offers potential advantages for breast cancer treatment. PMID:24929094
Chottanapund, Suthat; Van Duursen, M B M; Navasumrit, Panida; Hunsonti, Potchanee; Timtavorn, Supatchaya; Ruchirawat, Mathuros; Van den Berg, Martin
Novel retinoic acid metabolism blocking agents have potent inhibitory activities on human breast cancer cells and tumour growth
Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER ?ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumuor xenografts. The ER +ve cell lines were more sensitive (IC50 values between 3.0 and 609?nM) to the RAMBAs than the ER ?ve MDA-MB-231 cell line (IC50=5.6�24.0??M). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-? (ER-?). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.
Patel, J B; Mehta, J; Belosay, A; Sabnis, G; Khandelwal, A; Brodie, A M H; Soprano, D R; Njar, V C O
Microbiota of Human Breast Tissue
In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly reaching the ducts from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analyzed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria was detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactation. The principal phylum was Proteobacteria. The most abundant taxa in the Canadian samples were Bacillus (11.4%), Acinetobacter (10.0%), Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), Comamonadaceae (5.7%), Gammaproteobacteria (5.0%), and Prevotella (5.0%). In the Irish samples the most abundant taxa were Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%), and Pseudomonas (5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined.
Urbaniak, Camilla; Cummins, Joanne; Brackstone, Muriel; Macklaim, Jean M.; Gloor, Gregory B.; Baban, Chwanrow K.; Scott, Leslie; O'Hanlon, Deidre M.; Burton, Jeremy P.; Francis, Kevin P.; Tangney, Mark
Bisphenol-A-induced inactivation of the p53 axis underlying deregulation of proliferation kinetics, and cell death in non-malignant human breast epithelial cells
Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway�accompanied by dose-dependent evasion of apoptosis and increased proliferation�in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21WAF1 and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ER? (Estrogen Receptor): ER? ratio�an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures�another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth�a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.
Dairkee, Shanaz H.
Detection of circulating breast cancer cells using photoacoustic flow cytometry
NASA Astrophysics Data System (ADS)
According to the American Cancer Society, more than 200,000 new cases of breast cancer are expected to be diagnosed this year. Moreover, about 40,000 women died from breast cancer last year alone. As breast cancer progresses in an individual, it can transform from a localized state to a metastatic one with multiple tumors distributed through the body, not necessarily contained within the breast. Metastasis is the spread of cancer through the body by circulating tumor cells (CTCs) which can be found in the blood and lymph of the diagnosed patient. Diagnosis of a metastatic state by the discovery of a secondary tumor can often come too late and hence, significantly reduce the patient's chance of survival. There is a current need for a CTC detection method which would diagnose metastasis before the secondary tumor occurs or reaches a size resolvable by current imaging systems. Since earlier detection would improve prognosis, this study proposes a method of labeling of breast cancer cells for detection with a photoacoustic flow cytometry system as a model for CTC detection in human blood. Gold nanoparticles and fluorescent polystyrene nanoparticles are proposed as contrast agents for T47D, the breast cancer cell line of choice. The labeling, photoacoustic detection limit, and sensitivity are first characterized and then applied to a study to show detection from human blood.
Bhattacharyya, Kiran
Mechanisms of drug sensitization to TRA-8, an agonistic death receptor 5 antibody, involve modulation of the intrinsic apoptotic pathway in human breast cancer cells
TRA-8, a monoclonal antibody to death receptor 5 induces apoptosis in various cancer cells; however the degree of sensitivity varies from highly sensitive to resistant. We have previously shown resistance to TRA-8 can be reversed using chemotherapeutic agents, but the mechanism underlying this sensitization was not fully understood. Here, we examined the combination of TRA-8 with doxorubicin or bortezomib in breast cancer cells. In TRA-8 resistant BT-474 and T47D cells, both chemotherapy agents synergistically sensitized cells to TRA-8 cytotoxicity with enhanced activation of apoptosis demonstrated by cleavage of caspases and PARP, reduced Bid, increased pro-apoptotic Bcl-2 proteins, and increased mitochondrial membrane depolarization. Doxorubicin or bortezomib combined with TRA-8 also reduced Bcl-XL and XIAP in treated cells. Furthermore, targeting these proteins with pharmacological modulators, AT-101, BH3I-2? and AT-406, produced sensitization to TRA-8. TRA-8 combined with AT-101 or BH3I-2?, inhibitors of anti-apoptotic Bcl-2 proteins, produced synergistic cytotoxicity against ZR-75-1, BT-474, and T47D cells. The IAP targeting compound, AT-406, was synergistic with TRA-8 in BT-474 cells and to a lesser extent T47D cells. Activation of the intrinsic apoptotic pathway was a common mechanism associated with sensitization of TRA-8 resistant breast cancer cell lines. Collectively, these studies show that the Bcl-2 and IAP families of proteins are involved in TRA-8 and chemotherapy resistance via their modulation of the intrinsic apoptotic pathway. Targeting these proteins with novel agents sensitized TRA-8 resistant breast cancer cells, suggesting this approach may represent a potent therapeutic strategy in the treatment of breast cancer.
Amm, Hope M.; Zhou, Tong; Steg, Adam D.; Kuo, Huichien; Li, Yufeng; Buchsbaum, Donald J.
Characterization of Sigma Receptor Mediated Apoptosis in Breast Tumor Cells.
National Technical Information Service (NTIS)
We have shown that chronic exposure of tumor cells to sigma-2 agonists produces apoptosis and that these compounds potentiate effects of anti- neoplastic agents. To determine if this effect is mediated by ceramides, breast tumors (MCF-7/Adr-, T47D) were i...
K. W. Crawford
The role of YY1 in reduced HP1? gene expression in invasive human breast cancer cells
Introduction Heterochromatin protein 1 (HP1) associates with chromatin by binding to histone H3 and contributes to gene silencing. There are three isoforms of HP1 in mammals: HP1?, ?, and ?. Studies have shown that the level of HP1? is reduced in invasive human breast cancer cell lines such as MDA-MB-231 and HS578T compared with non-invasive cell lines such as MCF7 and T47D. It is hypothesized that reduced HP1? expression may lead to impaired epigenetic silencing of genes that are important in the acquisition of an invasive phenotype. We set out to determine whether reduced expression of HP1? in invasive breast cancer cell lines occurs at the level of transcription. Methods We used transient transfection assays to investigate the mechanism of differential transcriptional activity of the human HP1? gene promoter in different cell lines. Mutational analysis of putative transcription factor binding sites in an HP1? gene reporter construct was performed to identify transcription factors responsible for the differential activity. SiRNA-mediated knockdown and chromatin immunoprecipitation experiments were performed to determine the role of a specific transcription factor in regulating the HP1? gene. Results The transcription factor yin yang 1 (YY1) was found to play a role in differential transcriptional activity of the HP1? gene. Examination of the YY1 protein and mRNA levels revealed that both were reduced in the invasive cell line HS578T compared with MCF7 cells. YY1 knockdown in MCF7 cells resulted in a decreased level of HP1? mRNA, indicating that YY1 positively regulates HP1? expression. Chromatin immunoprecipitation experiments verified YY1 occupancy at the HP1? gene promoter in MCF7 cells but not HS578T cells. Overexpression of YY1 in HS578T cells decreased cell migration in a manner independent of HP1? overexpression. Conclusions Our data suggests that a reduction of YY1 expression in breast cancer cells could contribute to the acquisition of an invasive phenotype through increased cell migration as well as by reduced expression of HP1?.
Lieberthal, Jason G; Kaminsky, Marissa; Parkhurst, Christopher N; Tanese, Naoko
Effects of biosurfactants on the viability and proliferation of human breast cancer cells
Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3�T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24�h exposure to 0.05�g�l-1 surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15�g�l-1 BioEG for 48�h decreased cancer cells� viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.
Effects of biosurfactants on the viability and proliferation of human breast cancer cells.
Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3�T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24�h exposure to 0.05�g�l(-1) surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15�g�l(-1) BioEG for 48�h decreased cancer cells' viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein. PMID:24949273
Duarte, Cristina; Gudi�a, Eduardo J; Lima, Cristovao F; Rodrigues, Ligia R
The Human Cell Surfaceome of Breast Tumors
Introduction. Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. Methods. We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. Results. We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. Conclusions. The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors.
da Cunha, Julia Pinheiro Chagas; Galante, Pedro Alexandre Favoretto; de Souza, Jorge Estefano Santana; Pieprzyk, Martin; Carraro, Dirce Maria; Old, Lloyd J.; Camargo, Anamaria Aranha; de Souza, Sandro Jose
Membrane-initiated estradiol signaling of epithelial-mesenchymal transition-associated mechanisms through regulation of tight junctions in human breast cancer cells.
Tumor cells utilize inappropriate epithelial-mesenchymal transition (EMT) mechanisms during the invasive process. It is becoming increasingly clear that estradiol (E2) induces breast cancer cell progression and enhances EMT; however, the mechanisms associated with this are unclear. We investigated the role of E2 on the expression and intracellular localization of the tight junction (TJ)-associated proteins, zonula occluden 1 (ZO-1), ZO-1-associated nucleic acid binding (ZONAB), and occludin, on the activation of c-Src and human epidermal growth factor receptor 2 (HER2) expression and cellular migration in the estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. We demonstrated that 1�nM E2 elicits c-Src activation after 15�min. The p-Src/ZO-1 complex led to ZO-1 and ZONAB disruption at the TJ and increased expression of HER2 mRNAs. These changes correlate with decreased expression of the epithelial markers occludin and CRB3 and increased synthesis of N-cadherin. This led to increased MCF-7 cell migration induced by E2, even in the presence of a cell proliferation inhibitor. Incubation with ICI 182,780 (Fulvestrant), an ER antagonist, precluded the effects of E2 on c-Src phosphorylation, p-Src/ZO-1 complex formation, ZO-1/ZONAB nuclear translocation, and migration of MCF-7 cells. Our findings suggest that E2 promotes TJ disruption during tumor progression and increases cell motility. We propose a novel pathway where estrogens promote EMT-associated mechanisms that possibly lead to metastasis. PMID:24771004
Jim�nez-Salazar, Javier E; Posadas-Rodr�guez, Pedro; Lazzarini-Lechuga, Roberto C; Luna-L�pez, Armando; Zentella-Dehesa, Alejandro; G�mez-Quiroz, Luis E; K�nigsberg, Mina; Dom�nguez-G�mez, Guadalupe; Dami�n-Matsumura, Pablo
Discovery of estrogen receptor ? target genes and response elements in breast tumor cells
Background Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. Results Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. Conclusions Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.
Lin, Chin-Yo; Strom, Anders; Vega, Vinsensius Berlian; Li Kong, Say; Li Yeo, Ai; Thomsen, Jane S; Chan, Wan Ching; Doray, Balraj; Bangarusamy, Dhinoth K; Ramasamy, Adaikalavan; Vergara, Liza A; Tang, Suisheng; Chong, Allen; Bajic, Vladimir B; Miller, Lance D; Gustafsson, Jan-Ake; Liu, Edison T
Inhibition of tumor promoting signals by activation of SSTR2 and opioid receptors in human breast cancer cells
Background Somatostatin receptors (SSTRs) and opioid receptors (ORs) belong to the superfamily of G-protein coupled receptors and function as negative regulators of cell proliferation in breast cancer. In the present study, we determined the changes in SSTR subtype 2 (SSTR2) and ?, ? and ?-ORs expression, signaling cascades and apoptosis in three different breast cancer cells namely MCF-7, MDA-MB231 and T47D. Methods Immunocytochemistry and western blot analysis were employed to study the colocalization and changes in MAPKs (ERK1/2 and p38), cell survival pathway (PI3K/AKT) and tumor suppressor proteins (PTEN and p53) in breast cancer cell lines. The nature of cell death upon activation of SSTR2 or OR was analysed using flow cytometry analysis. Results The activation of SSTR2 and ORs modulate MAPKs (ERK1/2 and p38) in cell dependent and possibly estrogen receptor (ER) dependent manner. The activation of tumor suppressor proteins phosphatase and tensin homolog (PTEN) and p53 antagonized the PI3K/AKT cell survival pathway. Flow cytometry analyses reveal increased necrosis as opposed to apoptosis in MCF-7 and T47D cells when compared to ER negative MDA-MB231 cells. Furthermore, receptor and agonist dependent expression of ORs in SSTR2 immunoprecipitate suggest that SSTR2 and ORs might interact as heterodimers and inhibit epidermal growth factor receptor phosphorylation. Conclusion Taken together, findings indicate a new role for SSTR2/ORs in modulation of signaling pathways involved in cancer progression and provide novel therapeutic approaches in breast cancer treatment.
Epigenetic Effects of Human Breast Milk
A current aim of nutrigenetics is to personalize nutritional practices according to genetic variations that influence the way of digestion and metabolism of nutrients introduced with the diet. Nutritional epigenetics concerns knowledge about the effects of nutrients on gene expression. Nutrition in early life or in critical periods of development, may have a role in modulating gene expression, and, therefore, have later effects on health. Human breast milk is well-known for its ability in preventing several acute and chronic diseases. Indeed, breastfed children may have lower risk of neonatal necrotizing enterocolitis, infectious diseases, and also of non-communicable diseases, such as obesity and related-disorders. Beneficial effects of human breast milk on health may be associated in part with its peculiar components, possible also via epigenetic processes. This paper discusses about presumed epigenetic effects of human breast milk and components. While evidence suggests that a direct relationship may exist of some components of human breast milk with epigenetic changes, the mechanisms involved are still unclear. Studies have to be conducted to clarify the actual role of human breast milk on genetic expression, in particular when linked to the risk of non-communicable diseases, to potentially benefit the infant�s health and his later life.
Verduci, Elvira; Banderali, Giuseppe; Barberi, Salvatore; Radaelli, Giovanni; Lops, Alessandra; Betti, Federica; Riva, Enrica; Giovannini, Marcello
Laurenditerpenol, a new diterpene from the tropical marine alga Laurenciaintricata that potently inhibits HIF-1 mediated hypoxic signaling in breast tumor cells.
The degree of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) promotes tumor cell adaptation and survival under hypoxic conditions. Therefore, specific HIF-1 inhibitors represent an important new class of potential tumor-selective therapeutic agents. A T47D human breast tumor cell-based reporter assay was used to examine extracts of plants and marine organisms for inhibitors of HIF-1 activation. Bioassay-guided fractionation of the lipid extract of the red alga Laurencia intricata yielded a structurally novel diterpene, laurenditerpenol (1). The structure of 1 was determined spectroscopically. The relative configurations of the substituents of each ring system were assigned on the basis of NOESY correlations. The absolute configuration of position C-1 was determined by the modified Mosher ester procedure (directly in NMR tubes). Compound 1 potently inhibited hypoxia-activated HIF-1 (IC50: 0.4 microM) and hypoxia-induced VEGF (a potent angiogenic factor) in T47D cells. Compound 1 selectively inhibits HIF-1 activation by hypoxia but not iron chelator-induced activation. Further, 1 suppresses tumor cell survival under hypoxic conditions without affecting normoxic cell growth. Compound 1 inhibits HIF-1 by blocking the induction of the oxygen-regulated HIF-1alpha protein. Mitochondrial respiration studies revealed that 1 suppresses oxygen consumption. PMID:15620241
Mohammed, Kaleem A; Hossain, Chowdhury Faiz; Zhang, Lei; Bruick, Richard K; Zhou, Yu-Dong; Nagle, Dale G
The Breast Cancer Susceptibility Gene BRCA1 Regulates Progesterone Receptor Signaling in Mammary Epithelial Cells
The progesterone receptor (PR) plays roles in normal mammary development and breast cancer formation, where it may exert both stimulatory and inhibitory actions. Previously, the breast cancer susceptibility gene product BRCA1 was found to interact with and inhibit the transcriptional activity of estrogen receptor-?. In this study, we found that exogenous wild-type BRCA1 inhibited the activity of the PR in transient transfection assays utilizing a mouse mammary tumor virus-Luc reporter. Wild-type BRCA1 inhibited the activity of endogenous PR in human breast cancer cells (T47D and MCF-7) and inhibited the activity of exogenous PR-A, PR-B, and [PR-A plus PR-B] isoforms. On the other hand, knockdown of endogenous BRCA1 using small interfering RNA enhanced the progesterone-stimulated activity of the PR by about 4-fold. We documented an in vivo association of the endogenous BRCA1 with PR isoforms A and B and a direct in vitro interaction between BRCA1 and PR, which was partially mapped. Whereas down-regulation of the coactivator p300 contributes to the BRCA1-mediated repression of estrogen receptor-?, this mechanism does not contribute to inhibition of PR activity, because exogenous p300 did not rescue the BRCA1 repression of PR activity. The BRCA1-PR interaction has functional consequences. Thus, we showed that BRCA1 inhibits the expression of various endogenous progesterone-responsive genes and inhibits progesterone-stimulated proliferation of T47D cells. Finally, exogenous progesterone caused an exaggerated proliferative response in the mammary glands of mice harboring a mammary-targeted conditional deletion of the full-length isoform of Brca1. These findings suggest that BRCA1 regulates the activity of progesterone, a major hormone of pregnancy that may also participate in mammary carcinogenesis.
Ma, Yongxian; Katiyar, Pragati; Jones, Laundette P.; Fan, Saijun; Zhang, Yiyu; Furth, Priscilla A.; Rosen, Eliot M.
The PIKfyve�ArPIKfyve�Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines
Highlights: �We assess PAS complex proteins and phosphoinositide levels in breast cancer cells. �Sac3 and ArPIKfyve are markedly elevated in triple-negative breast cancer cells. �Sac3 silencing inhibits proliferation in triple-negative breast cancer cell lines. �Phosphoinositide profiles are altered in breast cancer cells. �This is the first evidence linking high Sac3 with breast cancer cell proliferation. -- Abstract: The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve�ArPIKfyve�Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P�PtdIns(3,5)P{sub 2} synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P�PtdIns(3,5)P{sub 2} conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P{sub 2} in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or indirectly the homeostasis of these lipids in TNBC. Together, our results uncover an unexpected role for Sac3 phosphatase in TNBC cell proliferation. Database analyses, discussed herein, reinforce the involvement of Sac3 in breast cancer pathogenesis.
Ikonomov, Ognian C., E-mail: firstname.lastname@example.com; Filios, Catherine, E-mail: email@example.com; Sbrissa, Diego, E-mail: firstname.lastname@example.com; Chen, Xuequn, E-mail: firstname.lastname@example.com; Shisheva, Assia, E-mail: email@example.com
Modulation of Androgen and Progesterone Receptors by Phytochemicals in Breast Cancer Cell Lines
We have used a tissue culture system based on breast carcinoma cell lines to investigate a large number of naturally occurring compounds and beverages for steroid hormone agonist and antagonist activity. The cell lines used, T-47D and BT-474, produce prostate specific antigen (PSA) upon stimulation with androgens, progestins, glucocorticoids and mineralocorticoids. This biomarker is secreted and can be measured in
Rachel S. Rosenberg; Linda Grass; David J. A. Jenkins; Cyril W. C. Kendall; Eleftherios P. Diamandis
Therapeutic Implications of Progesterone Receptor-Mediated Regulation of Cell Cycle in Breast Cancer.
National Technical Information Service (NTIS)
Since the 2007 summary report, we have made significant progress in elucidating the novel mechanisms by which PR regulates expression of E2F1, a key regulator of cell cycle progression, in T47D breast cancer cells. In addition to a direct regulatory pathw...
H. Wade
Transformation of estrone and estradiol in hormone-dependent and hormone-independent human breast cancer cells. Effects of the antiestrogen ICI 164,384, danazol, and promegestone (R-5020).
Using different hormone-dependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231, Hs-578S, MDA-MB-436) human breast cancer cells, the interconversion estrone (E1)<-->estradiol (E2) was explored. The data show very clearly that in the hormone-dependent cells the tendency is to form E2 after incubation with E1, whereas after incubation with E2 most of this estrogen remains unchanged. In the hormone-independent cells, in contrast most of E1 remains E1, while E2 is converted into E1. The tendency of the reductive<-->oxidative direction is supported by the analysis of estrogens in the culture medium. To explore the possible action of different drugs on the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity, it was observed that the potent antiestrogen ICI 164,384 inhibits the conversion of E1 to E2, while a lesser effect is observed with Danazol and only weak inhibition is obtained with the progestagen Promegestone (R-5020). It is concluded that the orientation of 17 beta-HSD activity for the interconversion E1<-->E2 in hormone-dependent and -independent cells is related to the hormonal status of the cells. PMID:7647331
Nguyen, B L; Chetrite, G; Pasqualini, J R
17?-estradiol regulates giant vesicle formation via estrogen receptor-alpha in human breast cancer cells.
A significant proportion of the genes regulated by 17-beta-estradiol (E2) via estrogen receptor alpha (ER?) have roles in vesicle trafficking in breast cancer. Intracellular vesicle trafficking and extracellular vesicles have important roles in tumourigenesis. Here we report the discovery of giant (3-42?m) intracellular and extracellular vesicles (GVs) and the role of E2 on vesicle formation in breast cancer (BC) cell lines using three independent live cell imaging techniques. Large diameter vesicles, GVs were also identified in a patient-derived xenograft BC model, and in invasive breast carcinoma tissue. ER?-positive (MCF-7 and T47D) BC cell lines demonstrated a significant increase in GV formation after stimulation with E2 which was reversed by tamoxifen. ER?-negative (MDA-MB-231 and MDA-MB-468) BC cell lines produced GVs independently of E2 and tamoxifen. These results indicate the existence of both intracellular and extracellular vesicles with considerably larger dimensions than generally recognised with BC cells and suggest that the GVs are regulated by E2 via ER? in ER?-positive BC but by E2-independent mechanisms in ER-ve BC. PMID:24931391
Wright, Paul K; Jones, Sarah Bowen; Ardern, Nicholas; Ward, Rebecca; Clarke, Robert B; Sotgia, Federica; Lisanti, Michael P; Landberg, Goran; Lamb, Rebecca
17?-estradiol regulates giant vesicle formation via estrogen receptor-alpha in human breast cancer cells
A significant proportion of the genes regulated by 17-beta-estradiol (E2) via estrogen receptor alpha (ER?) have roles in vesicle trafficking in breast cancer. Intracellular vesicle trafficking and extracellular vesicles have important roles in tumourigenesis. Here we report the discovery of giant (3-42?m) intracellular and extracellular vesicles (GVs) and the role of E2 on vesicle formation in breast cancer (BC) cell lines using three independent live cell imaging techniques. Large diameter vesicles, GVs were also identified in a patient-derived xenograft BC model, and in invasive breast carcinoma tissue. ER?-positive (MCF-7 and T47D) BC cell lines demonstrated a significant increase in GV formation after stimulation with E2 which was reversed by tamoxifen. ER?-negative (MDA-MB-231 and MDA-MB-468) BC cell lines produced GVs independently of E2 and tamoxifen. These results indicate the existence of both intracellular and extracellular vesicles with considerably larger dimensions than generally recognised with BC cells and suggest that the GVs are regulated by E2 via ER? in ER?-positive BC but by E2-independent mechanisms in ER-ve BC.
Wright, Paul K; Jones, Sarah Bowen; Ardern, Nicholas; Ward, Rebecca; Clarke, Robert B; Sotgia, Federica; Lisanti, Michael P; Landberg, Goran; Lamb, Rebecca
Retinol Esterification in Human Breast Cancer Cells.
National Technical Information Service (NTIS)
This study was designed to identify the target genes of retinoids in the normal human mammary epithelial cells (HMEC) and breast cancer cells. In our early studies, we found that the IL-1Beta gene was a responsive gene to retinoic acid (RA) and its expres...
L. Liu
Telomere fusions in early human breast carcinoma
Several lines of evidence suggest that defects in telomere maintenance play a significant role in the initiation of genomic instability during carcinogenesis. Although the general concept of defective telomere maintenance initiating genomic instability has been acknowledged, there remains a critical gap in the direct evidence of telomere dysfunction in human solid tumors. To address this topic, we devised a multiplex PCR-based assay, termed TAR (telomere-associated repeat) fusion PCR, to detect and analyze chromosome end-to-end associations (telomere fusions) within human breast tumor tissue. Using TAR fusion PCR, we found that human breast lesions, but not normal breast tissues from healthy volunteers, contained telomere fusions. Telomere fusions were detected at similar frequencies during early ductal carcinoma in situ and in the later invasive ductal carcinoma stage. Our results provide direct evidence that telomere fusions are present in human breast tumor tissue and suggest that telomere dysfunction may be an important component of the genomic instability observed in this cancer. Development of this robust method that allows identification of these genetic aberrations (telomere fusions) is anticipated to be a valuable tool for dissecting mechanisms of telomere dysfunction.
Tanaka, Hiromi; Abe, Satoshi; Huda, Nazmul; Tu, LiRen; Beam, Matthew J.; Grimes, Brenda; Gilley, David
Steroid hormone regulation of prostate-specific antigen gene expression in breast cancer
We have recently reported that about 30-40% of female breast tumours produce prostate-specific antigen (PSA) and that PSA production is associated with the presence of oestrogen (ER) and progesterone (PR) receptors. We have now developed a tissue culture system to study the regulation of the PSA gene in breast cancer. The breast carcinoma cell line T-47D produces PSA when stimulated
N Zarghami; L Grass; EP Diamandis
ExtraNuclear Signalling of Estrogen Receptor to Breast Cancer Cytoskeletal Remodelling, Migration and Invasion
BackgroundEstrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted.Methodology\\/Principal FindingsIn estrogen receptor (ER) positive T47-D breast cancer
Maria Silvia Giretti; Xiao-Dong Fu; Giovanni de Rosa; Ivana Sarotto; Chiara Baldacci; Silvia Garibaldi; Paolo Mannella; Nicoletta Biglia; Piero Sismondi; Andrea Riccardo Genazzani; Tommaso Simoncini; Neil Hotchin
Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells
Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized
Ouardia Ait-Mohamed; Valentine Battisti; V�ronique Joliot; Lauriane Fritsch; Julien Pontis; Souhila Medjkane; Catherine Redeuilh; Aazdine Lamouri; Christine Fahy; Mohamed Rholam; Djebbar Atmani; Slimane Ait-Si-Ali; Pranela Rameshwar
Anti-breast cancer agents, quinolines, targeting gap junction.
Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). The process of cancer formation causes a disruption in cell homeostasis, affecting the ability to respond to extracellular signals, as well as triggering some intracellular events which alter gap junctional intercellular communication (GJIC). Previous research has shown that the first two generations of substituted quinolines have anti-cancer effects in human breast cancer cells. This report presents the synthesis and bioactivities of third generation substituted quinolines. Scrape load/dye transfer studies showed that 100 nM of PQ15, a third generation substituted quinoline, causes a 4.5-fold increase of gap junction activity in T47D breast cancer cells. Furthermore, a significant decrease of cell proliferation and viability was observed in the presence of 200 nM PQ15 compared to control. The expression of ?-survivin was reduced to <40% in the treatment of 200 nM PQ15 compared to solvent alone. Alpha-survivin expression is upregulated in human cancers and associated with resistance to chemotherapy, suggesting that ?-survivin prolongs the survival of cancer cells. Thus, it has been shown that substituted quinolines stimulate gap junction activity, decrease alpha survivin expression, and subsequently inhibit cancer cell growth. Our findings demonstrate that PQ15 has a promising role in exerting anti-cancer activity in human breast cancer cells. PMID:21801150
Bernzweig, Julie; Heiniger, Brian; Prasain, Keshar; Lu, Jianyu; Hua, Duy H; Nguyen, Thu A
Anti-breast Cancer Agents, Quinolines, Targeting Gap Junction
Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). The process of cancer formation causes a disruption in cell homeostasis, affecting the ability to respond to extracellular signals, as well as triggering some intracellular events which alter gap junctional intercellular communication (GJIC). Previous research has shown that the first two generations of substituted quinolines have anti-cancer effects in human breast cancer cells. This report presents the synthesis and bioactivities of third generation substituted quinolines. Scrape load/dye transfer studies showed that 100 nM of PQ15, a third generation substituted quinoline, causes a 4.5-fold increase of gap junction activity in T47D breast cancer cells. Furthermore, a significant decrease of cell proliferation and viability was observed in the presence of 200 nM PQ15 compared to control. The expression of ?-survivin was reduced to <18% in the treatment of 100 nM PQ15 compared to control without treatment or solvent. Alpha-survivin expression is upregulated in human cancers and associated with resistance to chemotherapy, suggesting that ?-survivin prolongs the survival of cancer cells. Thus, it has been shown that substituted quinolines stimulate gap junction activity, decrease alpha survivin expression, and subsequently inhibit cancer cell growth. Our findings demonstrate that PQ15 has a promising role in exerting anti-cancer activity in human breast cancer cells.
Bernzweig, Julie; Heiniger, Brian; Prasain, Keshar; Lu, Jianyu; Hua, Duy H.; Nguyen, Thu A.
Conjugation of Monoclonal Antibodies to Super Paramagnetic Iron Oxide Nanoparticles for Detection of her2/neu Antigen on Breast Cancer Cell Lines
Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide (SPIO) nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20�g antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques.
Shamsipour, Fereshteh; Zarnani, Amir Hassan; Ghods, Roya; Chamankhah, Mahmood; Forouzesh, Flora; Vafaei, Sedigheh; Bayat, Ali Ahmad; Akhondi, Mohammad Mehdi; Ali Oghabian, Mohammad; Jeddi-Tehrani, Mahmood
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...
The molecular landscape of the normal human breast - defining normal
A key approach in understanding how breast cancer can occur is to determine the regulatory pathways at play in the normal breast and to identify precisely the normal developmental mechanisms subverted during early breast cancer progression. Using normal human breast tissue samples, Pardo and colleagues have identified the gene targets and pathways displaying fluctuating expression as a consequence of the menstrual cycle. Detailed characterization of how the human breast functions in its normal state, and how this may be perturbed at its earliest point, will provide a critical step toward the prevention of breast cancer.
Dydrogesterone (Duphaston) and its 20-dihydro-derivative as selective estrogen enzyme modulators in human breast cancer cell lines. Effect on sulfatase and on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity.
Estradiol (E2) is one of the main factors which control the growth and evolution of breast cancer. Consequently, to block the formation of E2 inside cancer cells has been an important target in recent years. Breast cancer cells possess all the enzymatic systems (e.g. sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase [17beta-HSD]) involved in the conversion of estrogen precursors into E2. Sulfotransferase, which converts estrogen to its sulfate, is also present in this tumoral tissue. Duphaston is a synthetic progestogen with properties similar to the natural progesterone. In the present study we examined the effect of Duphaston and its 20-dihydro-metabolite on the sulfatase and 17beta-HSD activities in MCF-7 and T-47D breast cancer cells. The cells were incubated with estrone sulfate (E1S) (5x10(-9)M) in the absence or presence of Duphaston or its 20-dihydro-metabolite (5x10(-5) to 5x10(-9)M) for 24h at 37 degrees C. In another series of experiments, estrone (E1) (5x10(-9)M) was incubated with T-47D cells in the absence or presence of the two progestogens (5x10(-5) to 5x10(-9)M) for 24h at 37 degrees C. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. Duphaston and its 20-dihydro-metabolite, at concentrations of 5x10(-7) and 5x10(-5)M, inhibited the conversion of E1S to E2 by 14% and 63%, 65% and 74%, respectively, in MCF-7 cells; the values were 15% and 48% and 31% and 51%, respectively, in T-47D cells. In another series of experiments it was observed that, after 24-h incubation, E1 (5x10(-9)M) was converted in a great proportion to E2 in the T-47D cells and that this transformation was significantly inhibited by Duphaston and its 20-dihydro-metabolite. The IC50 value, corresponding to 50% of the inhibition in the conversion of 1 to E2, was 9x10(-6)M for 20-dihydro-metabolite in this cell line. It was concluded that the progestogen Duphaston and its 20-dihydro-metabolite are potent inhibitory agents on sulfatase and 17beta-HSD activities in breast cancer cells. Duphaston is a progestogen with properties similar to the endogenous progesterone. The data open interesting perspectives to study the biological responses of these progestogens in clinical trials of patients with breast cancer. PMID:15274306
Chetrite, G�rard Samuel; Thole, Hubert H; Philippe, Jean-Claude; Pasqualini, Jorge Raul
TNF{alpha} acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-{kappa}B-dependent pathways
Tumor necrosis factor {alpha} (TNF{alpha}) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNF{alpha}, the participation of TNF{alpha} receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNF{alpha} induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappaB (NF-{kappa}B) transcriptional activation. A TNF{alpha}-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-{kappa}B transcriptional activation and cell proliferation, just like wild-type TNF{alpha}, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNF{alpha} signaling and biological effect. Moreover, in vivo TNF{alpha} administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-{kappa}B activity, Bay 11-7082, resulted in regression of TNF{alpha}-promoted tumor. Bay 11-7082 blocked TNF{alpha} capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-x{sub L}in vivo and in vitro. Our results reveal evidence for TNF{alpha} as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNF{alpha} antagonists and NF-{kappa}B pharmacological inhibitors in established breast cancer treatment.
Rivas, Martin A.; Carnevale, Romina P.; Proietti, Cecilia J.; Rosemblit, Cinthia; Beguelin, Wendy; Salatino, Mariana; Charreau, Eduardo H. [Laboratory of Molecular Mechanisms of Carcinogenesis, Instituto de Biologia y Medicina Experimental, CONICET, Vuelta de Obligado 2490, Buenos Aires, C1428ADN (Argentina); Frahm, Isabel [Servicio de Patologia, Sanatorio Mater Dei, Buenos Aires (Argentina); Sapia, Sandra [Laboratorio de Patologia, Fundaleu, Buenos Aires (Argentina); Brouckaert, Peter [Department of Molecular Biomedical Research, VIB and Ghent University, Ghent (Belgium); Elizalde, Patricia V. [Laboratory of Molecular Mechanisms of Carcinogenesis, Instituto de Biologia y Medicina Experimental, CONICET, Vuelta de Obligado 2490, Buenos Aires, C1428ADN (Argentina); Schillaci, Roxana [Laboratory of Molecular Mechanisms of Carcinogenesis, Instituto de Biologia y Medicina Experimental, CONICET, Vuelta de Obligado 2490, Buenos Aires, C1428ADN (Argentina)], E-mail: firstname.lastname@example.com
Ectopic human chorionic gonadotropin in breast carcinoma
Summary Immunoreactive human chorionic gonadotropin (hCG) was found in 9 of 65 surgically removed malignant breast tumors. Concentrations ranged from 5 to greater than 500 mIU hCG\\/g tumor. hCG was measured by a ?-chain specific radioimmunoassay. In further study of these specimens, an immunoperoxidase staining technique was used to stain for hCG in formalin-fixed sections. The hCG was shown to
A. Castro; P. Buschbaum; M. Nadji; W. Voigt; S. Tabei; A. Morales
Osteolytic Sterol in Human Breast Cancer
Eleven of twelve human breast cancers contained a lipid which increased urinary 45Ca and 40Ca excretion of 45Ca-labeled, parathyroidectomized rats receiving a low Ca diet. The lipid has mobility on thin-layer chromatography and gas-liquid chromatography close to, but not identical with, that of 7-dehydrocholesterol. Authentic 7-dehydrocholesterol has osteolytic activity similar to that of the extracted sterol. Fluorescence and Lieberman-Burchard reactions
Gilbert S. Gordan; Theodore J. Cantino; Linda Erhardt; James Hansen; Warren Lubich
Method for imaging breast tumors using labeled monoclonal anti-human breast cancer antibodies
US Patent & Trademark Office Database
Hybridomas producing monoclonal antibodies suitable for imaging and diagnosis of human breast tumors and such monoclonal antibodies are claimed. The monoclonals are characterized by breast tumor binding range, breast cancer cell line range, and selectivity. Immunoimaging agents comprising the monoclonal antibody and a detectable label, either directly or indirectly conjugated to the antibody are claimed. Methods for imaging breast tumors using the immunoimaging agents are described and claimed.
TReP-132 Is a Novel Progesterone Receptor Coactivator Required for the Inhibition of Breast Cancer Cell Growth and Enhancement of Differentiation by Progesterone
The sex steroid progesterone is essential for the proliferation and differentiation of the mammary gland epithelium during pregnancy. In relation to this, in vitro studies using breast carcinoma T47D cells have demonstrated a biphasic progesterone response, consisting of an initial proliferative burst followed by a sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate
Florence Gizard; Romain Robillard; Barbara Gross; Olivier Barbier; Francoise Revillion; Jean-Philippe Peyrat; Gerard Torpier; Dean W. Hum; Bart Staels
Terminal Differentiation of Human Breast Cancer through PPAR?
We have previously demonstrated that PPAR? stimulates the terminal differentiation of adipocyte precursors when activated by synthetic ligands, such as the antidiabetic thiazolidinedione (TZD) drugs. We show here that PPAR? is expressed at significant levels in human primary and metastatic breast adenocarcinomas. Ligand activation of this receptor in cultured breast cancer cells causes extensive lipid accumulation, changes in breast epithelial
Elisabetta Mueller; Pasha Sarraf; Peter Tontonoz; Ronald M. Evans; Katherine J. Martin; Ming Zhang; Christopher Fletcher; Samuel Singer; Bruce M. Spiegelman
Involvement of a Human Endogenous Retrovirus in Breast Cancer.
National Technical Information Service (NTIS)
We are testing the hypothesis that human mammary tumor virus (HMTV), a human endogenous retrovirus (HERV) closely related to mouse mammary tumor virus (MMTV), is etiologically involved in a subset of human breast cancers. We continue to collect blood and ...
R. F. Garry
Crosstalk between PKC? and Notch-4 in endocrine-resistant breast cancer cells
The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor ? (ER?)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase C? (PKC?) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKC? overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKC?-overexpressing breast cancer cells.Analysis of published microarray data from ER?+ breast carcinomas shows that PKC? expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKC?-overexpressing, TAM-resistant T47D model, PKC? selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKC?-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKC?-expressing T47D cells. In PKC?-overexpressing T47D xenografts, an orally active ?-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKC? overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKC?- and Notch-4-overexpressing, endocrine-resistant breast cancers.
Yun, J; Pannuti, A; Espinoza, I; Zhu, H; Hicks, C; Zhu, X; Caskey, M; Rizzo, P; D'Souza, G; Backus, K; Denning, M F; Coon, J; Sun, M; Bresnick, E H; Osipo, C; Wu, J; Strack, P R; Tonetti, D A; Miele, L
Modeling the interaction of binary and ternary mixtures of estradiol with bisphenol A and bisphenol A F in an in vitro estrogen mediated transcriptional activation assay (T47D-KBluc).
Humans are concurrently exposed to xenoestrogens and to physiological levels of endogenous estrogens. Endogenous estrogen levels vary from low levels in infants to high levels during pregnancy and in young women. However, few studies have addressed how xenoestrogens interact with...
Control of sulfatase activity by nomegestrol acetate in normal and cancerous human breast tissues.
Nomegestrol acetate (NOMAC), a 17alpha-hydroxy-nor-progesterone derivative (17alpha-acetoxy-6-methyl-19-nor-4,6-pregnadiene-3,20-dione, the active substance in Lutenyl), is a potent and useful clinical synthetic progestin for the treatment of menopausal complaints and is under current development for oral contraception. Previous studies in this laboratory demonstrated that NOMAC can block sulfatase and 17beta-hydroxysteroid dehydrogenase, the enzymes involved in the biosynthesis and transformation of estradiol (E2) in hormone-dependent MCF-7 and T-47D breast cancer cells. In the present study, the effect of NOMAC on sulfatase activity using total breast cancer tissue, compared to the effect in normal breast tissue, was explored. Slices of tumoral or normal breast tissues (45-65 mg) were incubated in buffer (20 mM Tris-HCl, pH 7.2) with physiological concentrations of [3H]-estrone sulfate (5x10(-9) M), alone or in the presence of nomegestrol acetate (5x10(-5) - 5x10(-7) - 5x10(-9) M), for 4 h at 37 degrees C. Estrone sulfate (E1S), estrone (E1) and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [3H]- E1S was only converted to [3H]- E1 and not to [3H]- E2, in normal or cancerous breast tissues, which suggests a low or no 17beta-HSD activity under these experimental conditions. The sulfatase activity was more intense with breast cancer tissue than normal tissue, since the concentrations of E1 were 42.5 +/- 3.4 and 27.2 +/- 2.5 pg/mg tissue, respectively. NOMAC, at the concentration of 5x10(-5) M, inhibited this conversion by 49.2% and 40.8% in cancerous and normal breast tissues, respectively. The sulfatase inhibition at low concentration (5x10(-7) M) was 32.5% and 22.8%, respectively. It is concluded that sulfatase activity is almost twice as potent in cancerous breast tissues than in normal tissues. Nomegestrol acetate is a strong anti-sulfatase agent, in particular with cancerous breast tissues. The inhibition of estrone sulfatase activity by NOMAC in total normal or cancerous breast tissues can open attractive perspectives for future clinical trials. PMID:16080533
Chetrite, G�rard Samuel; Thomas, Jean-Louis; Shields-Botella, Jaqueline; Cortes-Prieto, Joaquin; Philippe, Jean-Claude; Pasqualini, Jorge Raul
A monoclonal antibody to the human c-erbB3 protein stimulates the anchorage-independent growth of breast cancer cell lines.
The c-erbB3 protein is a member of the type I growth factor receptor family. It has a widespread pattern of expression in normal tissues and is overexpressed in about 20% of breast cancers. We have raised a specific monoclonal antibody, called SGP1, against the extracellular domain of c-erbB3 which recognises the native form of the protein. The monoclonal antibody was found to modestly but significantly stimulate the anchorage-independent cloning efficiency of the breast tumour cell lines BT483 and T47D, both of which express the c-erbB3 protein. No effect was observed on 293 cells lacking expression, nor did a control isotype-matched antibody promote the growth of any of the cells tested. These results suggest that the c-erbB3 protein may normally act as a growth factor receptor. Images Figure 1 Figure 2 Figure 3 Figure 6
Rajkumar, T.; Gullick, W. J.
Pesticides and polychlorinated biphenyl residues in human breast lipids and their relation to breast cancer
The etiology of human breast cancer is unknown; accepted risk factors, e.g., menstrual, reproductive, and family histories, are implicated in less than half of all cases. Various halogenated hydrocarbons - acting as either co-carcinogens or promoting agents - which are derived from the environment and are concentrated in human fatty stores, may also play a role in breast cancer risk.
F. Jr. Falck; A. Jr. Ricci; P. Deckers; M. S. Wolff; J. Godbold
Prolactin Inhibits Expression of the Proto-oncogene BCL6 in Breast Cancer through a Stat5a Dependent Mechanism
Stat5a mediates prolactin-induced differentiation of mammary epithelia, and loss of Stat5 signaling in human breast cancer is associated with undifferentiated histology and poor prognosis. The transcriptional repressor BCL6 shares DNA target sequences with Stat5, disrupts differentiation of breast epithelia, is down-regulated during lactation, and upregulated in poorly differentiated breast cancer. Here we identify prolactin as a potent suppressor of BCL6 protein expression in human breast cancer through a mechanism that requires Stat5a, but not prolactin-activated Stat5b, MEK-ERK, or PI3K-AKT pathways. Prolactin rapidly suppressed BCL6 mRNA in T47D, MCF7, ZR75.1 and SKBr3 breast cancer cell lines, followed by prolonged reduction of BCL6 protein levels within 3h. Prolactin suppression of BCL6 was enhanced by overexpression of Stat5a but not Stat5b, was mimicked by constitutively active Stat5a, but did not require the transactivation domain of Stat5a. Stat5 chromatin immunoprecipitation demonstrated physical interaction with a BCL6 gene regulatory region, and BCL6 transcript repression required histone deacetylase activity based on sensitivity to trichostatin A. Functionally, BCL6 overexpression disrupted prolactin induction of Stat5 reporter genes. Prolactin suppression of BCL6 was extended to xenotransplant tumors in nude mice in vivo and to freshly isolated human breast cancer explants ex vivo. Quantitative immunohistochemistry revealed elevated BCL6 in high grade and metastatic breast cancer compared to DCIS and nonmalignant breast, and cellular BCL6 protein levels correlated negatively with nuclear Stat5a (r=?0.52; P<0.001) but not with Stat5b. Loss of prolactin-Stat5a signaling and concomitant upregulation of BCL6 may represent a regulatory switch facilitating undifferentiated histology and poor prognosis of breast cancer.
Tran, Thai H.; Utama, Fransiscus E.; Lin, Justin; Yang, Ning; Sjolund, Ashley B.; Ryder, Amy; Johnson, Kevin J.; Neilson, Lynn M.; Liu, Chengbao; Brill, Kristin L.; Rosenberg, Anne L.; Witkiewicz, Agnieszka K.; Rui, Hallgeir
Histological and biological evolution of human premalignant breast disease
Most human invasive breast cancers (IBCs) appear to develop over long periods of time from certain pre-existing benign lesions. Of the many types of benign lesions in the human breast, only a few appear to have significant premalignant potential. The best characterized of these include atypical hyperplasias and in situ carcinomas and both categories are probably well on along the
D C Allred; S K Mohsin; SAW Fuqua
Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines.
Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1-29)NH(2) re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1-29)NH(2) and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor. PMID:18506184
Barabutis, N; Schally, A V
Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines
Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1�29)NH2 re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1�29)NH2 and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.
Barabutis, N; Schally, A V
Beta Human Chorionic Gonadotropin - Induction of Apoptosis in Breast Cancer.
National Technical Information Service (NTIS)
Agents that induce apoptosis in breast cancer cells have great potential to facilitate chemotherapeutic intervention and improve patient outcomes. In this study the effects of injecting purified human chorionic gonadotropin (hCG) directly into human breas...
K. J. Cullen
Gene Targeting in Normal Human Breast Epithelial Cells.
National Technical Information Service (NTIS)
This exploration grant was to test if it is possible to achieve efficient homologous recombination and gene targeting in immortalized but otherwise normal human breast epithelial cells. Although gene targeting has been achieved in somatic human cells usin...
A. M. Thorburn
Role of p53 in Human Breast Cancer.
National Technical Information Service (NTIS)
The principal objectives are to: (1) Transfect p53 mutants commonly observed in human breast cancer into normal human mammary epithelial cells obtained from different donors and isolate clones; (2) Characterize the clones for extension of lifespan and imm...
J. W. Shay
Growth Factor Receptor-Directed Therapy in Human Breast Cancer.
National Technical Information Service (NTIS)
Overexpression of HER-2 growth factor receptor in human breast cancer is associated with poor prognosis and disease progression. We have targeted these receptor pathways for therapeutic intervention, using a humanized monoclonal antibody to HER-2 receptor...
R. J. Pietras
p42/p44 MAPK-mediated Stat3Ser727 phosphorylation is required for progestin-induced full activation of Stat3 and breast cancer growth.
Stat3 is a signaling node for multiple oncogenic pathways and is therefore frequently active in breast cancer. As experimental and clinical evidence reveals that progestins are key players in controlling mammary gland tumorigenesis, we studied Stat3 participation in this event. We have previously shown that progestins induce Stat3Tyr705 phosphorylation and its transcriptional activation in breast cancer cells. In this study, we demonstrate that progestins also induce Stat3 phosphorylation at Ser727 residue, which occurs via activation of c-Src/p42/p44 MAPK pathways in murine progestin-dependent C4HD cells and in T-47D cells. Expression of a Stat3S727A vector, which carries a serine-to-alanine substitution at codon 727, shows that Stat3Ser727 phosphorylation is required for full transcriptional activation of cyclin D1 gene expression by progestins and for in vivo Stat3 recruitment on cyclin D1 promoter. Transfection of Stat3S727A in murine and human breast cancer cells abolished progestin-induced in vitro and in vivo growth. Moreover, we found a positive correlation between progesterone receptor expression and nuclear localization of Stat3Ser727 phosphorylation in breast cancer biopsies. These data highlight Stat3 phosphorylation in Ser727 residue as a nongenomic action by progestins, necessary to promote breast cancer growth. PMID:23329648
Tkach, Mercedes; Rosemblit, Cinthia; Rivas, Mart�n A; Proietti, Cecilia J; D�az Flaqu�, Mar�a Celeste; Mercogliano, Mar�a Florencia; Beguelin, Wendy; Maronna, Esteban; Guzm�n, Pablo; Gercovich, Felipe G; Deza, Ernesto Gil; Elizalde, Patricia V; Schillaci, Roxana
Integrin activation controls metastasis in human breast cancer
NASA Astrophysics Data System (ADS)
Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin v3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin v3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated ?v?3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant ?v?3D723R, but not ?v?3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin ?v?3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.
Felding-Habermann, Brunhilde; O'Toole, Timothy E.; Smith, Jeffrey W.; Fransvea, Emilia; Ruggeri, Zaverio M.; Ginsberg, Mark H.; Hughes, Paul E.; Pampori, Nisar; Shattil, Sanford J.; Saven, Alan; Mueller, Barbara M.
Human papilloma virus is associated with breast cancer
Background: There is increasing evidence that high-risk human papilloma virus (HPV) is involved in cancers in addition to cervical cancer. For example, it is generally accepted that HPV has a role in a significant proportion of head and neck tumours, and it has long been hypothesised that hormone dependent oncogenic viruses, such as HPV may have causal roles in some human breast cancers. A number of reports have identified HPV DNA in breast tissue and breast cancer specimens, but these rely on standard polymerase chain reaction (PCR), which is criticised for its propensity for contamination. Methods: We have used two different technologies, in situ and standard PCR (with sequencing), and histology based on light microscopy. Results: We unambiguously demonstrate the presence of high-risk HPV in the cells of breast cancer specimens and breast cancer cell lines. In addition, we also show that the oncogenic characteristics of HPV associated breast cancer are very similar to HPV-associated cervical cancer. Specifically, that putative koilocytes are present in some HPV associated breast cancers. Interpretation: The above observations indicate a likely causal role for high-risk HPV in human breast cancer and offer the possibility of primary prevention of some breast cancers by vaccination against HPV.
Heng, B; Glenn, W K; Ye, Y; Tran, B; Delprado, W; Lutze-Mann, L; Whitaker, N J; Lawson, J S
Extra-Nuclear Signalling of Estrogen Receptor to Breast Cancer Cytoskeletal Remodelling, Migration and Invasion
Background Estrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted. Methodology/Principal Findings In estrogen receptor (ER) positive T47-D breast cancer cells ER activation with 17?-estradiol induces rapid and dynamic actin cytoskeleton remodeling with the formation of specialized cell membrane structures like ruffles and pseudopodia. These effects depend on the rapid recruitment of the actin-binding protein moesin. Moesin activation by estradiol depends on the interaction of ER? with the G protein G?13, which results in the recruitment of the small GTPase RhoA and in the subsequent activation of its downstream effector Rho-associated kinase-2 (ROCK-2). ROCK-2 is responsible for moesin phosphorylation. The G?13/RhoA/ROCK/moesin cascade is necessary for the cytoskeletal remodeling and for the enhancement of breast cancer cell horizontal migration and invasion of three-dimensional matrices induced by estrogen. In addition, human samples of normal breast tissue, fibroadenomas and invasive ductal carcinomas show that the expression of wild-type moesin as well as of its active form is deranged in cancers, with increased protein amounts and a loss of association with the cell membrane. Conclusions/Significance These results provide an original mechanism through which estrogen can facilitate breast cancer local and distant progression, identifying the extra-nuclear G?13/RhoA/ROCK/moesin signaling cascade as a target of ER? in breast cancer cells. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.
Giretti, Maria Silvia; Fu, Xiao-Dong; De Rosa, Giovanni; Sarotto, Ivana; Baldacci, Chiara; Garibaldi, Silvia; Mannella, Paolo; Biglia, Nicoletta; Sismondi, Piero; Genazzani, Andrea Riccardo; Simoncini, Tommaso
Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-? and survivin
Introduction Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer. Methods To investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-? (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER). Results AGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown. Conclusions Transiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.
Antiproliferative Effects of Cucurbitacin B in Breast Cancer Cells: Down-Regulation of the c-Myc/hTERT/Telomerase Pathway and Obstruction of the Cell Cycle
Naturally occurring cucurbitacins have been shown to have anticancer, antimicrobial and anti-inflammatory activities. In this study, we determined the effects of cucurbitacin B extracted from the Thai herb Trichosanthes cucumerina L. on telomerase regulation in three human breast cancer cell lines (T47D, SKBR-3, and MCF-7) and a mammary epithelium cell line (HBL-100). Cell viability after treatment with cucurbitacin B, which is an active ingredient of this herb, was assessed. Telomeric Repeat Amplification Protocol (TRAP) assays and RT-PCR (qualitative and realtime) were performed to investigate activity of telomerase as well as expression of human telomerase reverse transcriptase (hTERT) and c-Myc. The c-Myc protein level was also determined in SKBR-3 and HBL-100 cells. Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells. The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100. Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells.
Duangmano, Suwit; Dakeng, Sumana; Jiratchariyakul, Weena; Suksamrarn, Apichart; Smith, Duncan R.; Patmasiriwat, Pimpicha
Antiproliferative effects of cucurbitacin B in breast cancer cells: down-regulation of the c-Myc/hTERT/telomerase pathway and obstruction of the cell cycle.
Naturally occurring cucurbitacins have been shown to have anticancer, antimicrobial and anti-inflammatory activities. In this study, we determined the effects of cucurbitacin B extracted from the Thai herb Trichosanthes cucumerina L. on telomerase regulation in three human breast cancer cell lines (T47D, SKBR-3, and MCF-7) and a mammary epithelium cell line (HBL-100). Cell viability after treatment with cucurbitacin B, which is an active ingredient of this herb, was assessed. Telomeric Repeat Amplification Protocol (TRAP) assays and RT-PCR (qualitative and realtime) were performed to investigate activity of telomerase as well as expression of human telomerase reverse transcriptase (hTERT) and c-Myc. The c-Myc protein level was also determined in SKBR-3 and HBL-100 cells. Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells. The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100. Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells. PMID:21614210
Duangmano, Suwit; Dakeng, Sumana; Jiratchariyakul, Weena; Suksamrarn, Apichart; Smith, Duncan R; Patmasiriwat, Pimpicha
Cacospongionolide and Scalaradial, Two Marine Sesterterpenoids as Potent Apoptosis-Inducing Factors in Human Carcinoma Cell Lines
Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-?B subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.
Malik, Shoaib Ahmad; Iodice, Carmine; De Rosa, Salvatore; Maiuri, Maria Chiara; Carnuccio, Rosa
Human breast tissue disposition and bioactivity of limonene in women with early stage breast cancer
Limonene is a bioactive food component found in citrus peel oil that has demonstrated chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited forty-three women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for 2 � 6 weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean=41.3 ?g/g tissue) while the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P=0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase 3 expression. No significant changes in serum leptin, adiponectin, TGF-?1, IGFBP-3 and IL-6 levels were observed following limonene intervention. There was a small but statistically significant post-intervention increase in IGF-1 levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell cycle arrest and reduced cell proliferation. Further placebo-controlled clinical trials and translational research are warranted to establish limonene�s role for breast cancer prevention or treatment.
Miller, Jessica A.; Lang, Julie E.; Ley, Michele; Nagle, Ray; Hsu, Chiu-Hsieh; Thompson, Patricia A; Cordova, Catherine; Waer, Amy; Chow, H.-H. Sherry
Integrin activation controls metastasis in human breast cancer
Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin v3
Brunhilde Felding-Habermann; Timothy E. O'Toole; Jeffrey W. Smith; Emilia Fransvea; Zaverio M. Ruggeri; Mark H. Ginsberg; Paul E. Hughes; Nisar Pampori; Sanford J. Shattil; Alan Saven; Barbara M. Mueller
Epigenetic Regulation of Gelsolin Expression in Human Breast Cancer Cells
Gelsolin is a multifunctional, actin-binding protein that is greatly decreased in many transformed cell lines and tumor tissues, including breast cancers. Downregulation of gelsolin RNA occurs in most breast cancers of rats, mice, and humans, but gross mutations of the gelsolin gene have not been found. Here we demonstrate by PCR and RT-PCR analysis that there are no point mutations
Lawrence M. Mielnicki; Angela M. Ying; Karen L. Head; Harold L. Asch; Bonnie. B. Asch
Human Breast Cancer Cell/Tissue Bank and Database.
National Technical Information Service (NTIS)
The final year of the University of Michigan Human Breast Cell/Tissue Bank and Data base has been dedicated to providing investigators from around the country and internationally with the cells and tissues we have banked and with new breast cancer cell li...
S. Ethier
Role of AKT2 in Human Breast Cancer.
National Technical Information Service (NTIS)
Frequent alterations of AKT2 oncogene have been frequently detected in human malignancies, including breast carcinoma (1, 2). To better understand the role of AKT2 in breast carcinogenesis, we have isolated a novel protein, APBP, that binds to and is phos...
Z. Q. Yuan J. Q. Cheng
Role of cdc25 Phosphatases in Human Breast Cancer.
National Technical Information Service (NTIS)
A summary is presented of research performed during the second year of a project to determine the role of Cdc25 phosphatases in human breast cancer. The research involves three specific aims. In the first aim, the role of Cdc25B in breast cancer prolifera...
J. J. Manfredi
Allopurinol and oxypurinol in human breast milk
To pregnant or breast feeding women drugs should be given with caution. We report the case of a 5-week-old breast-fed infant whose mother was taking 300 mg allopurinol\\/day for 4 weeks. Allopurinol and oxypurinol were detected by HPLC in maternal plasma and breast milk with a method first described here. In infant's plasma taken 2 h after breast feeding oxypurinol
I. Kamilli; U. Gresser
Is human cytomegalovirus associated with breast cancer progression?
Background It has been hypothesized that human cytomegalovirus (HCMV) may be associated with breast cancer progression. However, the role of HCMV infection in breast cancer remains controversial. We aimed to assess whether HCMV genes (UL122 and UL83) could be detected in breast carcinomas and reinvestigated their possible association with breast cancer progression. DNA from paraffin-embedded tissues was analyzed by real-time PCR. We investigated 20 fibroadenomas and 27 primary breast carcinomas (stages II, III, and IV). Findings Two carcinomas were positive for HCMV, one was positive for two TaqMan viral detection probes, and one was positive for a sole TaqMan viral detection probe (UL83), whereas the remainder of the samples was negative. Conclusions Samples studied showed no association between HCMV infection and breast cancer progression.
Characterization of Gene Expression in Human Breast Tumor Endothelium.
National Technical Information Service (NTIS)
Angiogenesis is the growth of new capillary blood vessels, and is a critical component of solid tumor growth. We characterized molecular changes between human breast tumor vessels and normal vessels to identify genes that may serve as therapeutic targets....
N. Klauber-DeMore
Mathematical modeling the pathway of human breast cancer.
In order to understand the mechanism of human breast cancer we use the growth rates of clonal expansion of intermediate cells and mutation rates as parameters and build two-six stage models to fit the age-specific incidence of breast cancers in the surveillance, epidemiology, and end results (SEER) registry. We propose four types of different mechanisms for the human breast cancer and test those mechanisms by Chi-square test. Our results suggest that loss of functions of instability genes is an early event in the tumorigenesis, which is useful for early diagnosis of breast cancer. The clonal expansion of intermediate cells must depend on the hormone expression level of females, which implies that it may be effective for females to receive hormone blocking therapy for breast cancer before their menopause. PMID:24680645
Zhang, Xinan; Fang, Yile; Zhao, Yingdong; Zheng, Weiming
TNF-? enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B
Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-? on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-? exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-?B. Importantly, activation of NF-?B was
M F Rubio; S Werbajh; E G A Cafferata; A Quaglino; G P Col�; I M Nojek; E C Kordon; V E Nahmod; M A Costas
Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblasts
Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT-483). Antibodies to the adhesion molecules CD44, E-cadherin, ICAM-1, and integrin chains a2, a3, a4, a5, a6, av, a1, a3 and a7 failed to inhibit breast cancer cell
Sonia Saad; Linda J Bendall; Alexander James; David J Gottlieb; Kenneth F Bradstock
Possible DNA Viral Factors of Human Breast Cancer
Viruses are considered to be one of the high-risk factors closely related to human breast cancer. However, different studies of viruses in breast cancer present conflicting results and some of these works remain in dispute. DNA viruses, such as specific types of human papillomaviruses (HPV), Epstein�Barr virus (EBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), and human herpes virus type 8 (HHV-8), have emerged as causal factors of some human cancers. These respective exogenous viruses and the possibility of multiple viral factors are discussed in this review.
Hsu, Chun-Ru; Lu, Tsong-Ming; Chin, Lengsu William; Yang, Chi-Chiang
Antitumor Activity of the Novel Human Breast Cancer Growth Inhibitor
A novel human tumor growth inhibitor was identified by differential cDNA sequencing. The predicted amino acid sequence of this tumor suppressing factor has a significant sequence homology to mouse main mary-derived growth inhibitor and thus was named mammary-derived growth inhibitor-relatedgene (MRG).MRGwas found to be expressedin normal and benign human breast tissues but not in breast carcinomas. In situ hybridization analysis
Y. EricShi; Jian Ni; Lily Xing; Mei Zhang; Jiyou Li; Bharat B. Aggarwal; Anthony Meager; Reiner Gentz; J. LI
[Human milk--some recent aspects of breast feeding].
New data on the quality and quantity of protein and nor-protein nitrogen in human milk are discussed in the first part of this review. The second part presents a short review of current knowledge on immunologically important components of human milk (secretory IgA, Lactoferrin, ligands for folic acid and vitamine B-12. Lysozyme, cells, induction of breast milk flora in the intestine). There are very good reasons to enhance breast feeding also in developed countries. PMID:368420
Plenert, W
Expression of membrane transporters and metabolic enzymes involved in estrone-3-sulphate disposition in human breast tumour tissues.
Two-thirds of newly diagnosed hormone-dependent (HR+) breast cancers are detected in post-menopausal patients where estrone-3-sulphate (E3S) is the predominant source for tumour estradiol. Understanding intra-tumoral fate of E3S would facilitate in the identification of novel molecular targets for HR�+�post-menopausal breast cancer patients. Hence this study investigates the clinical expression of (i) organic anion-transporting polypeptides (OATPs), (ii) multidrug resistance protein (MRP-1), breast cancer resistance proteins (BCRP), and (iii) sulphatase (STS), 17?-hydroxysteroid dehydrogenase (17?-HSD-1), involved in E3S uptake, efflux and metabolism, respectively. Fluorescent and brightfield images of stained tumour sections (n�=�40) were acquired at 4� and 20� magnification, respectively. Marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analysed and was reported as �% area (ImageJ software). Tumour, stroma and non-tumour tissue areas were also quantified (Inform software), and the ratio of optical intensity per histologic area was reported as �% area/tumour, �% area/stroma and �% area/non-tumour. Functional role of OATPs and STS was further investigated in HR+ (MCF-7, T47-D, ZR-75) and HR-(MDA-MB-231) cells by transport studies conducted in the presence or absence of specific inhibitors. Amongst all the transporters and enzymes, OATPs and STS have significantly (p�<�0.0001) higher expression in HR+ tumour sections with highest target signals obtained from the tumour regions of the tissues. Specific OATP-mediated E3S uptake and STS-mediated metabolism were also observed in all HR+ breast cancer cells. These observations suggest the potential of OATPs as novel molecular targets for HR+ breast cancers. PMID:24831777
Banerjee, Nilasha; Miller, Naomi; Allen, Christine; Bendayan, Reina
Biocompatibility of Fe(3)O(4) nanoparticles evaluated by in vitro cytotoxicity assays using normal, glia and breast cancer cells.
In order to reveal the biocompatibility of Fe(3)O(4) nanoparticles and bipolar surfactant tetramethylammonium 11-aminoundecanoate cytotoxicity tests were performed as a function of concentration from low (0.1 microg ml(-1)) to higher concentration (100 microg ml(-1)) using various human glia, human breast cancer and normal cell lines. Cytotoxicity tests for human glia (D54MG, G9T, SF126, U87, U251, U373), human breast cancer (MB157, SKBR3, T47D) and normal (H184B5F5/M10, WI-38, SVGp12) cell lines exhibited almost nontoxicity and reveal biocompatibility of Fe(3)O(4) nanoparticles in the concentration range of 0.1-10 microg ml(-1), while accountable cytotoxicity can be seen at 100 microg ml(-1). The results of our studies suggest that Fe(3)O(4) nanoparticles coated with bipolar surfactant tetramethylammonium 11-aminoundecanoate are biocompatible and promising for bio-applications such as drug delivery, magnetic resonance imaging and magnetic hyperthermia. PMID:20090199
Ankamwar, B; Lai, T C; Huang, J H; Liu, R S; Hsiao, M; Chen, C H; Hwu, Y K
Immunohistochemical localization of apelin in human normal breast and breast carcinoma.
The peptide apelin is a high-affinity ligand for the G-protein coupled receptor APJ. Apelin/APJ signaling plays important roles in blood pressure regulation, body fluid homeostasis, and cardiovascular development. More recently, it has been recognized that apelin/APJ signaling may also be involved in tumor angiogenesis. Studies in experimental animals have shown that apelin is abundantly secreted in the milk, and the mammary gland contains high level of pre-proapelin mRNAs and apelin protein. High level of apelin mRNA is expressed in cultured human breast carcinoma cell line (Hs 578T). However, the status of apelin expression and localization in human breast carcinoma has not been studied. In the present study immunohistochemistry was performed to investigate the expression and localization of apelin in normal human breast tissue and breast carcinoma. Cytoplasmic apelin immunoreactivity was detected in the ductal and lobular epithelial cells and vascular endothelial cells of the normal breast tissue. The myoepithelial cells were negative. The malignant tumor cells of invasive ductal or lobular carcinoma also expressed similar level of immunoreactive apelin. The fuctional significance of apelin expression in normal nonlactating breast and breast carcinoma warrants further investigation. PMID:17823846
Wang, Zhiqin; Greeley, George H; Qiu, Suimin
Studies of human breast cancer metastasis using nude mice
Athymic nude mice have been used in recent years to study the biology of human tumors and to assess therapeutic responses in vivo rather than just in vitro. Some human tumors metastasize in nude mice, providing model systems for analyzing various aspects of the metastatic phenotype of human neoplasms. For breast carcinomas, however, the tumor-take rate of surgical specimens is
Janet E. Price; Ruo Dan Zhang
Bovine Leukemia Virus DNA in Human Breast Tissue
Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease.
Shen, Hua Min; Jensen, Hanne M.; Choi, K. Yeon; Sun, Dejun; Nuovo, Gerard
Bovine leukemia virus DNA in human breast tissue.
Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease. PMID:24750974
Buehring, Gertrude Case; Shen, Hua Min; Jensen, Hanne M; Choi, K Yeon; Sun, Dejun; Nuovo, Gerard
Cytokines in human breast cyst fluid
Gross cystic breast disease is a common benign disorder in which palpable cysts occur in the breast and are normally treated by aspiration of the contents. The cysts are classified as either Type 1, containing a high level of potassium ions and a low level of sodium ions, or as Type 2, with low potassium and high sodium ion concentrations.
David C. Parish; Margaret W. Ghilchik; Joanna M. Day; James Eaton; Atul Purohit; Michael J. Reed
c-erbB-2/c-erbA co-amplification indicative of lymph node metastasis, and c-myc amplification of high tumour grade, in human breast carcinoma.
A panel of 73 samples, including 52 primary breast carcinomas, 10 normal breast tissues and 11 axillary lymph nodes, has been analysed for the presence of amplifications and gross structural alterations, in the oncogenes c-erbB-2, c-erbA, c-myc, N-myc, c-mos and c-Ha-ras. The tumours were also classified, graded and staged histopathologically and their DNA ploidy (42 samples) was determined by flow cytometry. Three breast cancer cell lines (MCF7, ZR-75-1 and T47D) were also included in the study. Amplification of c-erbB-2 was detected in 28% of the tumours, of which 91% had an increased steady-state level of c-erbB-2 mRNA. Amplification of c-erbA was found in 23% of tumours and was always associated with the amplification of c-erbB-2. Ten out of 12 (83%) tumours which had c-erbB-2 and c-erbA co-amplification had metastasised to axillary lymph nodes (P less than 0.006). However, the human thymidine kinase gene, which is present at the same chromosomal location as these two oncogenes (17q21-22), was amplified in only tw tumours. Amplification of c-myc was detected in 21% of the tumours studied, of which 82% (P less than 0.005) were of histopathological grade 3 and none were of grade 1. Flow cytometry showed that 90% (P less than 0.01) of the analysed tumours with c-erbB-2 and c-erbA co-amplification, and 70% (P less than 0.1) of those with c-myc amplification were DNA aneuploid. This study demonstrates the potential value of c-myc amplification in the assessment of the tumour grade, rather than metastatic potential; and of the co-amplification of c-erbB-2 and c-erbA as a strong indicator of metastatic potential, rather than tumour grade. Images Figure 1 Figure 2 Figure 3
Tavassoli, M.; Quirke, P.; Farzaneh, F.; Lock, N. J.; Mayne, L. V.; Kirkham, N.
Progestin modulates the lipid profile and sensitivity of breast cancer cells to docetaxel.
Progestins induce lipid accumulation in progesterone receptor (PR)-positive breast cancer cells. We speculated that progestin-induced alterations in lipid biology confer resistance to chemotherapy. To examine the biology of lipid loaded breast cancer cells, we used a model of progestin-induced lipid synthesis. T47D (PR-positive) and MDA-MB-231 (PR-negative) cell lines were used to study progestin response. Oil red O staining of T47D cells treated with progestin showed lipid droplet formation was PR dependent, glucose dependent and reduced sensitivity to docetaxel. This protection was not observed in PR-negative MDA-MB-231 cells. Progestin treatment induced stearoyl CoA desaturase-1 (SCD-1) enzyme expression and chemical inhibition of SCD-1 diminished lipid droplets and cell viability, suggesting the importance of lipid stores in cancer cell survival. Gas chromatography/mass spectroscopy analysis of phospholipids from progestin-treated T47D cells revealed an increase in unsaturated fatty acids, with oleic acid as most abundant. Cells surviving docetaxel treatment also contained more oleic acid in phospholipids, suggesting altered membrane fluidity as a potential mechanism of chemoresistance mediated in part by SCD-1. Lastly, intact docetaxel molecules were present within progestin induced lipid droplets, suggesting a protective quenching effect of intracellular lipid droplets. Our studies suggest the metabolic adaptations produced by progestin provide novel metabolic targets for future combinatorial therapies for progestin-responsive breast cancers. PMID:22922095
Schlaepfer, Isabel R; Hitz, Carolyn A; Gij�n, Miguel A; Bergman, Bryan C; Eckel, Robert H; Jacobsen, Britta M
Human breast duct anatomy, the �sick lobe� hypothesis and intraductal approaches to breast cancer
SummaryIntroduction��Information about central and peripheral duct anatomy is a requirement for developing intraductal approaches to human breast cancer, but remains sparse. This study looks at the acquisition and digital modelling of data describing breast duct branching from thick (�subgross�) sections using data structures from the neurosciences, and at high-throughput imaging of duct anatomy in the nipple.Methods��The branching of a large
James J. Going; Timothy J. Mohun
Posttranscriptional regulation of the breast cancer susceptibility gene BRCA1 by the RNA binding protein HuR.
BRCA1 is a breast cancer susceptibility gene that is down-regulated in a significant proportion of sporadic breast cancers. BRCA1 is posttranscriptionally regulated by RNA-binding proteins, the identities of which are unknown. HuR is an RNA binding protein implicated in posttranscriptional regulation of many genes and is overexpressed in sporadic breast cancer. To investigate the possibility that these two molecules are functionally linked in breast cancer, we performed bioinformatic analysis of the BRCA1 3' untranslated region (UTR), RNA-protein assays with the HuR protein and the BRCA1 3'UTR, and immunohistochemical analysis of a cohort of breast tumors using antibodies against BRCA1 and HuR. Here, we describe the identification of two predicted HuR-binding sites in the BRCA1 3'UTR, one of which binds specifically to HuR. We also show that this interaction is disrupted by single nucleotide substitutions in the BRCA1 3'UTR and that endogenous HuR protein associates with BRCA1 transcripts in T47D and MCF7 breast cancer cells. Expression of ectopic HuR results in a significant decrease in BRCA1 protein expression and also BRCA1 3'UTR activity. Immunohistochemical analysis revealed that although BRCA1 and HuR expression were associated with some clinicopathologic features of the tumors, there was no statistically significant correlation between BRCA1 and HuR protein expression. These results identify the first posttranscriptional protein regulator of BRCA1 and have implications for understanding BRCA1 regulation in human breast cancer. PMID:19010922
Saunus, Jodi M; French, Juliet D; Edwards, Stacey L; Beveridge, Dianne J; Hatchell, Esme C; Wagner, Sarah A; Stein, Sandra R; Davidson, Andrew; Simpson, Kaylene J; Francis, Glenn D; Leedman, Peter J; Brown, Melissa A
Risk assessment of triclosan [Irgasan �] in human breast milk
Triclosan is an established bacteriostatic compound widely used in topical and dental preparations. Its pharmacokinetics and toxicology have been extensively studied in humans and animals. It is known to be absorbed from the gastrointestinal tract and across the skin.A recent report noted its occurrence in human breast milk and this has now been further investigated. Sixty two unselected samples of
A. D. Dayan
Synthesis, in vitro pharmacologic characterization, and preclinical evaluation of N-[2-(1'-piperidinyl)ethyl]-3-[125I]iodo-4-methoxybenzamide (P[125I]MBA) for imaging breast cancer.
The goal of this study was to investigate the potential use of a radioiodinated benzamide, N-[2-(1'-piperidinyl)ethyl]-3-iodo[125I]-4-methoxybenzamide (P[125I]MBA), a sigma receptor binding radioligand for imaging breast cancer. The chemical and radiochemical syntheses of PIMBA are described. The pharmacological evaluation of PIMBA was carried out for sigma-1 and sigma-2 receptor sites. The in vivo pharmacokinetics of the radioiodinated benzamide were determined in rats and comparison of P[125I]MBA with Tc-99m sestamibi were made in a rat mammary tumor model. Sigma-1 affinity (Ki) for PIMBA in guinea pig brain membranes using [3H](+)pentazocine was found to be 11.82 +/- 0.68 nM, whereas sigma-2 affinity in rat liver using [3H]DTG (1,3-o-di-tolylguanidine) was 206 +/- 11 nM. Sites in guinea pig brain membranes labeled by P[125I]MBA showed high affinity for haloperidol, (+)-pentazocine, BD1008, and PIMBA (Ki = 4.87 +/- 1.49, 8.81 +/- 1.97, 0.057 +/- 0.005, 46.9 +/- 1.8 nM, respectively). Competition binding studies were carried out in human ductal breast carcinoma cells (T47D). A dose-dependent inhibition of specific binding was observed with several sigma ligands. Ki values for the inhibition of P[125I]MBA binding in T47D cells for haloperidol, N-[2-(1'-piperidinyl)]ethyl]4-iodobenzamide (IPAB), N-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-IBP), and PIMBA were found to be 1.30 +/- 0.07, 13 +/- 1.5, 5.19 +/- 2.3, 1.06 +/- 0.5 nM, respectively. The in vitro binding data in guinea pig brain membranes and breast cancer cells confirmed binding to sigma sites. The saturation binding of P[125I]MBA in T47D cells as studied by Scatchard analysis showed saturable binding, with a Kd = 94 +/- 7 nM and a Bmax = 2035 +/- 305 fmol/mg of proteins. Biodistribution studies in Sprague-Dawley rats showed a rapid clearance of P[125I]MBA from the normal organs. The potential of PIMBA in imaging breast cancer was evaluated in Lewis rats bearing syngeneic RMT breast cancers, a cancer that closely mimics human breast cancer histology. At 1 h postinjection, tumor uptake for P[125I]MBA and Tc-99m sestamibi were found to be 0.35 +/- 0.01 and 0.32 +/- 0.01% injected dose/organ (%ID/g), respectively. The %ID/g for liver, kidneys, and heart were 2, 11, and 20 times lower, respectively, for P[125I]MBA as compared with Tc-sestamibi. Slightly higher uptake of P[125I]MBA in tumors (than Tc-sestamibi) and a low nontarget organ uptake warrants further studies of this and other sigma receptor ligands for their use as breast cancer imaging agents. PMID:10382840
John, C S; Bowen, W D; Fisher, S J; Lim, B B; Geyer, B C; Vilner, B J; Wahl, R L
Clinicopathological significance of PTPN12 expression in human breast cancer.
Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a recently identified tumor suppressor gene (TSG) that is frequently compromised in human triple-negative breast cancer. In the present study, we investigated the expression of PTPN12 protein by patients with breast cancer in a Chinese population and the relationship between PTPN12 expression levels and patient clinicopathological features and prognosis. Additionally, we explored the underlying down-regulation mechanism from the perspective of an epigenetic alteration. We examined PTPN12 mRNA expression in five breast cancer cell lines using semi-quantitative reverse-transcription PCR, and detected PTPN12 protein expression using immunohistochemistry in 150 primary invasive breast cancer cases and paired adjacent non-tumor tissues. Methylation-specific PCR was performed to analyze the promoter CpG island methylation status of PTPN12. PTPN12 was significantly down-regulated in breast cancer cases (48/150) compared to adjacent noncancerous tissues (17/150; P < 0.05). Furthermore, low expression of PTPN12 showed a significant positive correlation with tumor size (P = 0.047), lymph node metastasis (P = 0.001), distant metastasis (P = 0.009), histological grade (P = 0.012), and survival time (P = 0.019). Additionally, promoter CpG island hypermethylation occurs more frequently in breast cancer cases and breast cancer cell lines with low PTPN12 expression. Our findings suggest that PTPN12 is potentially a methylation-silenced TSG for breast cancer that may play an important role in breast carcinogenesis and could potentially serve as an independent prognostic factor for invasive breast cancer patients. PMID:23044628
Xunyi, Yuan; Zhentao, Yuan; Dandan, Jiang; Funian, Li
Regulatory mechanisms for abnormal expression of the human breast cancer specific gene 1 in breast cancer cells
Breast cancer-specific gene 1 (BCSG1), also referred as synuclein ?, was originally isolated from a human breast cancer cDNA library and the protein is mainly localized to presynaptic terminals\\u000a in the nervous system. BCSG1 is not expressed in normal or benign breast lesions, but expressed at an extremely high level in the vast majority of the\\u000a advanced staged breast carcinomas
Aiping Lu; Qing Li; Jingwen Liu
Phosphorylation of ErbB4 on Tyrosine 1056 Is Critical for ErbB4 Coupling to Inhibition of Colony Formation by Human Mammary Cell Lines
In many studies, ErbB4 expression in breast tumor samples correlates with a favorable patient prognosis. Similarly, ErbB4 signaling is coupled to cellular differentiation and growth arrest in a variety of model systems. However, in some studies, ErbB4 expression in breast tumor samples correlates with poor outcome. Likewise, studies using some human mammary tumor cell lines suggest that ErbB4 is coupled to malignant phenotypes. Thus, the roles that ErbB4 plays in human breast cancer are still poorly defined. Here we demonstrate that a constitutively active ErbB4 mutant (ErbB4-Q646C) inhibits colony formation on plastic by two human mammary tumor cell lines (SKBR3 and MCF7) and by the MCF10A immortalized human mammary cell line, but does not inhibit colony formation by the MDA-MB-453 and T47D human mammary tumor cell lines. ErbB4 kinase activity is necessary for ErbB4 function and phosphorylation of ErbB4 Tyr1056 is necessary and appears to be sufficient for ErbB4 function. The inhibition of colony formation by MCF10A cells is accompanied by growth arrest but not cell death. These data suggest that ErbB4 behaves as a mammary tumor suppressor and that loss of ErbB4 coupling to growth arrest may be an important event in mammary tumorigenesis.
Pitfield, Sarah E.; Bryant, Ianthe; Penington, Desi J.; Park, Gar; Riese, David J.
The Marine-Derived Sipholenol A-4-O-3?,4?-Dichlorobenzoate Inhibits Breast Cancer Growth and Motility in Vitro and in Vivo through the Suppression of Brk and FAK Signaling
Sipholenol A is a natural sipholane triterpenoid isolated from the Red Sea sponge, Callyspongia siphonella. Previous studies showed the antimigratory and antiproliferative activities of the semisynthetic sipholenol A esters against breast cancer cell lines. This study investigated the effects of sipholenol A-4-O-3?,4?-dichlorobenzoate (SPA) on the growth, migration and invasion of diverse human breast cancer cells. Results showed that SPA inhibited the growth of the human breast cancer cells, MDA-MB-231, MCF-7, BT-474 and T-47D, in a dose-dependent manner. Immunofluorescent analysis showed that SPA significantly reduced Ki-67-positive cells in MDA-MB-231 cells. Flow cytometry and Western blot analyses revealed that SPA treatment suppressed MDA-MB-231 cell growth by inducing cell cycle arrest at the G1 phase. In addition, SPA suppressed breast cancer cell migration, invasion and decreased Brk and FAK activation in a dose-dependent manner. Molecular docking study suggested a perfect fitting at the FAK�s FERM domain, inhibiting the main autophosphorylation site, Y397, which was further confirmed by Western blot analysis. Most known small molecule FAK inhibitors target the kinase domain, creating several off-target side effects. The in vivo studies showed that SPA treatment suppressed breast tumor growth and Ki-67, CD31, p-Brk and p-FAK expression in orthotopic breast cancer in nude mice. In conclusion, SPA inhibited the growth, invasion and migration of breast cancer cells possibly via deactivating Brk and FAK signaling, suggesting good potential for therapeutic use to control invasive breast cancer.
Akl, Mohamed R.; Foudah, Ahmed I.; Ebrahim, Hassan Y.; Meyer, Sharon A.; Sayed, Khalid A. El
CHL1 is involved in human breast tumorigenesis and progression
Highlights: �CHL1 is down-regulation in breast cancer tissues. �Down-regulation of CHL1 is related to high grade. �Overexpression of CHL1 inhibits breast cancer cell proliferation and invasion in vitro. �CHL1 deficiency induces breast cancer cell proliferation and invasion both in vitro and in vivo. -- Abstract: Neural cell adhesion molecules (CAM) play important roles in the development and regeneration of the nervous system. The L1 family of CAMs is comprised of L1, Close Homolog of L1 (CHL1, L1CAM2), NrCAM, and Neurofascin, which are structurally related trans-membrane proteins in vertebrates. Although the L1CAM has been demonstrated play important role in carcinogenesis and progression, the function of CHL1 in human breast cancer is limited. Here, we found that CHL1 is down-regulated in human breast cancer and related to lower grade. Furthermore, overexpression of CHL1 suppresses proliferation and invasion in MDA-MB-231 cells and knockdown of CHL1 expression results in increased proliferation and invasion in MCF7 cells in vitro. Finally, CHL1 deficiency promotes tumor formation in vivo. Our results may provide a strategy for blocking breast carcinogenesis and progression.
He, Li-Hong [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China) [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Ma, Qin [Department of Oncology, The General Hospital of Tianjin Medical University, Tianjin (China)] [Department of Oncology, The General Hospital of Tianjin Medical University, Tianjin (China); Shi, Ye-Hui [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China) [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Ge, Jie; Zhao, Hong-Meng [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China) [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Li, Shu-Fen [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China) [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Tong, Zhong-Sheng, E-mail: email@example.com [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China) [Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China)
Estradiol as an anti-aromatase agent in human breast cancer cells.
Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments. PMID:16413774
Pasqualini, J R; Chetrite, G S
Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways
Introduction Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. Methods We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. Results High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-?) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers. Conclusions Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-? and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.
Excretion of cefprozil into human breast milk.
The excretion of cefprozil into breast milk in nine healthy, lactating female subjects was investigated. Each subject received a single 1,000-mg oral dose of cefprozil consisting of cis and trans isomers in an approximately 90:10 ratio. Serial blood, urine, and breast milk samples were collected and analyzed for the concentrations of the cis and trans isomers by a specific high-pressure liquid chromatography-UV assay. The mean pharmacokinetic parameters for both isomers were essentially the same. The mean peak concentrations in plasma for the cis isomer were 14.8 micrograms/ml, and the area under the concentration curve was 54.8 micrograms.h/ml. The mean values of elimination half-life, renal clearance, and urinary excretion for the cis isomer were 1.69 h, 164 ml/min, and 60%, respectively. The mean concentrations in milk of the cis isomer over a 24-h period ranged from 0.25 to 3.36 micrograms/ml, with the maximum concentration appearing at 6 h after dosing. The average maximum concentration in milk of the trans isomer was less than 0.26 micrograms/ml. The concentrations of the trans isomer in plasma and in breast milk were about 1/10 of those for the cis isomer. Less than 0.3% of the dose was excreted in breast milk for both isomers of cefprozil. Even if one assumes that the concentration of cefprozil in milk remains constant at 3.36 micrograms/ml (the highest concentration of cefprozil observed in breast milk), an infant ingesting an average of 800 ml of milk per day will be exposed to a maximum amount of about 3 mg of cefprozil per day. This value represents about 0.3% of the maternal dose. Low excretion of cefprozil in breast milk and the excellent safety profile of cefprozil suggest that this cephalosporin may be administered to nursing mothers when indicated.
Shyu, W C; Shah, V R; Campbell, D A; Venitz, J; Jaganathan, V; Pittman, K A; Wilber, R B; Barbhaiya, R H
T Cell Coinhibition and Immunotherapy in Human Breast Cancer
Costimulation and coinhibition generated by the B7 family and their receptor CD28 family have key roles in regulating T lymphocyte activation and tolerance. These pathways are very attractive therapeutic targets for human cancers including breast cancer. Gene polymorphisms of B7x (B7-H4/B7S1), PD-1 (CD279), and CTLA-4 (CD152) are associated with increased risk of developing breast cancer although the underlying mechanisms are unclear. In human breast cancer microenvironment, up-regulation of coinhibitory B7/CD28 members B7x, B7-H3 (CD276), and PD-L1 (B7-H1/CD274) on tumor cells as well as PD-1 and PD-L1 on tumor-infiltrating immune cells are emerging as immune evasion pathways. Chemotherapy can affect the expression of these molecules, and therefore may dampen the immune response against breast cancer. Immunotherapy targeting T cell coinhibition as monotherapy or combined with standard therapies are in early stages of clinical development, but hold great promise for treatment of human breast cancer.
Janakiram, Murali; Abadi, Yael M.; Sparano, Joseph A.; Zang, Xingxing
Silibinin Inhibits Wnt/?-catenin Signaling by Suppressing Wnt Co-receptor LRP6 Expression in Human Prostate and Breast Cancer Cells
Silibinin is a natural compound isolated from milk thistle seed extracts, and has traditionally been used as a hepatoprotectant. A number of studies have also established the cancer therapeutic and chemopreventive role of silibinin in both in vitro and in vivo models. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for the Wnt/?-catenin pathway and represents a promising target for cancer prevention and therapy. In the present study, we found that silibinin was able to repress endogenous LRP6 expression and block Wnt3A-induced LRP6 phosphorylation and Wnt/?-catenin signaling activation in HEK293 cells. Importantly, silibinin was also able to suppress endogenous LRP6 expression and phosphorylation and block Wnt/?-catenin signaling in prostate cancer PC-3 and DU-145 cells and breast cancer MDA-MB-231 and T-47D cells. Mechanistically, silibinin inhibited LRP6 promoter activity and decreased LRP6 mRNA levels in prostate and breast cancer cells. Finally, we demonstrated that silibinin displayed anticancer activity with IC50 values comparable to those shown to suppress LRP6 expression and Wnt/?-catenin signaling activities in prostate and breast cancer cells. Our data indicate that silibinin is a novel small molecule Wnt/?-catenin signaling inhibitor by suppressing Wnt co-receptor LRP6 expression at the transcription level, and that the anti-cancer activity of silibinin is associated with its inhibitory effect on Wnt/LRP6 signaling.
Lu, Wenyan; Lin, Cuihong; King, Taj D.; Chen, Honghong; Reynolds, Robert C.; Li, Yonghe
Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line
Context: Marine algae produce different secondary metabolites with a wide range of biological activities. Many studies have been achieved on the screening of biological effects of marine organisms and a lot of active compounds were isolated and characterized. Aims: In an attempt to find cytotoxic compound of hexane fraction, isolation, identification, and cytotoxicity of active compound of this fraction were performed. Materials and Methods: In this study, total methanolic (70%) extract and partition fractions of hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and MeOH�H2O of Sargassum angustifolium, Chondria dasyphylla, and Ulva flexuosa, collected from coastlines of the Persian Gulf in south of Iran, were studied against colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D), and Swiss mouse embryo fibroblast (NIH 3T3) cell lines by MTT assay. Statistical Analysis Used: IC50 (median growth inhibitory concentration) values were calculated by Sigmaplot (10) software. Results: Hexane fraction of Chondria dasyphylla (IC50 82.26 � 4.09 ?g/ml) and MeOH-H2O fraction of Ulva flexuosa (IC50 116.92 � 8.58 ?g/ml) showed cytotoxic activity against proliferation of T47D cells. Hexane fraction of Sargassum angustifolium was also observed for cytotoxicity against T47D and HT-29 cell lines (IC50 166.42 � 26.7 and 190.24 � 52.8 ?g/ml), respectively. An investigation of a component from the hexane fraction of Sargassum angustifolium yielded a steroidal metabolite, fucosterol, with cytotoxicity in T47D and HT29 (IC50 27.94 � 9.3 and 70.41 � 7.5 ?g/ml). Conclusions: These results indicated that fucosterol, the most abundant phytosterol in brown algae, is responsible for cytotoxic effect of this extract against breast and colon carcinoma cell lines.
Khanavi, Mahnaz; Gheidarloo, Razieh; Sadati, Nargess; Ardekani, Mohammad Reza Shams; Nabavi, Seyed Mohammad Bagher; Tavajohi, Shohreh; Ostad, Seyed Nasser
Human Breast Cancer Cell/Tissue Bank and Database.
National Technical Information Service (NTIS)
During the first year of existence of the University of Michigan Human Breast Cell/Tissue Bank and Data base, we have accomplished many of the goals that were originally set forth in our Statement of Work. We have developed and implemented a system for pr...
S. Ethier
Role of Basic Fibroblast Growth Factor in Human Breast Cancer.
National Technical Information Service (NTIS)
We found that basic fibroblast growth factor (bFGF), a mitogen, inhibits human breast cancer cell lines. In MCF-7 cells, bFGF binds high-affinity tyrosine kinase FGF receptors (FGFR), activates MAP kinase, induces higher levels of G(1) cyclins D1 and E an...
R. Wieder
A comparative study of genome-wide SNP, CGH microarray and protein expression analysis to explore genotypic and phenotypic mechanisms of acquired antiestrogen resistance in breast cancer
Allelic imbalance is a common feature of many malignancies. We have measured allelic imbalance in genomic DNA from the breast\\u000a cancer cell lines T47D, MDA-MB-231, two antiestrogen sensitive (MCF7N and MCF7L) and two resistant MCF7 cell lines (MMU2 and LCC9) using single nucleotide polymorphism (SNP) oligonucleotide microarrays.\\u000a DNA from MCF7L and MMU2 cells was also analysed by comparative genome hybridisation
Neil Johnson; Valerie Speirs; Nicola J. Curtin; Andrew G. Hall
Breast tumor specific mutation in GATA3 affects physiological mechanisms regulating transcription factor turnover
Background The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-? (ER?)-positive breast tumors in which it participates with ER? and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus. Methods Immunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ER? agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Results GATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ER? and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells. Conclusions We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.
Galectin-3 and L1 retrotransposons in human breast carcinomas
Galectin-3 is a galactoside binding protein found at elevated levels in a wide variety of neoplastic cells and thought to be involved in cognitive cellular interactions during transformation and metastasis. Previously, we have shown that introduction of human galectin-3 (Mr 31,000) cDNA into the human breast cancer cells BT-549 which are galectin-3 null and non-tumorigenic in nude mice resulted in
Pratima Nangia-Makker; Rebecca Sarvis; Daniel W. Visscher; Juliet Bailey-Penrod; Avraham Raz; Fazlul H. Sarkar
Possibilities of a viral etiology for human breast cancer
Previous studies related mouse mammary tumor virus (MMTV) to human breast cancer. However, the presence of human endogenous\\u000a retroviruses (HERs) confounded these results. We selected a 660-bp sequence of the MMTVenv gene with low homology to HER (or any other known gene) and searched for a sequence homologous to it, using the polymerase\\u000a chain reaction (PCR). The 660-bp sequence was
Beatriz G.-T. Pogo; James F. Holland
Chromosomal abnormalities in human breast cancer
This review emphasizes cytogenetic changes and DNA analyses by Southern blot in primary breast tumors, rather than metastases, established cell lines, and pleural effusions. The data suggests that the most frequently altered chromosomes and chromosome regions are 1p, 1q, 2q, 3p, 5, 6q, 8p, 8q, 11p, 11q, 12, 13q, 14q, 16, 17p, and 17q. Changes on 8q, 11p, 11q, 13q,
Wendy M. Mars; Grady F. Saunders
Propofol elimination in human breast milk
Background\\/Aims: Lactating women having operations under general anesthesia are advised to pump and discard their milk for 24h after the procedure. We determined the kinetics of propofol elimination in breast milk to ascertain its safety after propofol administration.Methods: Three lactating women were studied after giving IRB-approved written informed consent. Patients were premedicated with midazolam 5 min before induction of anesthesia
M. J. Avram; M. Nitsun; J. W. Szokol; J. Saleh; G. S. Murphy; J. S. Vender; K. Raikoff
Two monoclonal antibodies identify antigens preferentially expressed on normal human breast cells versus breast cancer cells.
In order to obtain antibodies with specificity toward normal mammary epithelial antigenic determinants, we immunized BALB/c mice with normal milk cells and screened the hybridomas against an undifferentiated breast cancer cell line H466B, peripheral blood lymphocytes and normal fibroblasts. Two hybridomas were generated, which produced BA6 (IgG1) and CA4 (IgM) monoclonal antibodies (MAbs). These MAbs did not react with 5 breast cancer cell lines. In cryostat sections of normal human breast tissue, BA6 was reactive with 6/6 and CA4 was reactive with 12/13 specimens both showing an apical staining of epithelial cells. Conversely staining of malignant cells in breast cancer biopsies was observed in 4/33 specimens with BA6 and in 4/19 specimens with CA4. Computerized image analysis (SAMBA) of immunostained sections showed homogeneous distribution of staining, with a high percentage of stained cell surfaces in normal breast (mean percentages of positive surfaces : BA6 : 75% and CA4 : 82%) while, in malignant samples, staining was heterogeneous, with a mean percentage of positive surface of 25% for BA6 and 12% for CA4. Both MAbs reacted strongly with human milk fat globule membranes (HMFGM) and skimmed milk. FPLC size exclusion chromatography of skimmed milk showed that CA4 and BA6 reactive materials eluted in distinct peaks in high molecular weight ranges. Electrophoretic separation of HMFGM followed by CA4 staining detected a high molecular weight reactive band (Mr 380-600 kDa). CA4 and BA6 reactivity was reduced by protease treatment of the antigen but was not affected by neuraminidase digestion, by methanol extraction or by Na-metaperiodate oxidation. After perchloric acid treatment of HMFGM, BA6 activity was lost while the CA4 activity was found in the soluble fraction. The results reported suggest that the two MAbs identify two distinct novel epitopes of normal breast cells. PMID:1714877
Pancino, G; Mortada, M H; Charpin, C; Osinaga, E; De Cremoux, P; Betaille, B; Gobert, M G; Calvo, F; Roseto, A
Ocular input for human melatonin regulation: relevance to breast cancer
NASA Technical Reports Server (NTRS)
The impact of breast cancer on women across the world has been extensive and severe. As prevalence of breast cancer is greatest in industrialized regions, exposure to light at night has been proposed as a potential risk factor. This theory is supported by the epidemiological observations of decreased breast cancer in blind women and increased breast cancer in women who do shift-work. In addition, human, animal and in vitro studies which have investigated the melatonin-cancer dynamic indicate an apparent relationship between light, melatonin and cancer, albeit complex. Recent developments in understanding melatonin regulation by light in humans are examined, with particular attention to factors that contribute to the sensitivity of the light-induced melatonin suppression response. Specifically, the role of spectral characteristics of light is addressed, and recent relevant action spectrum studies in humans and other mammalian species are discussed. Across five action spectra for circadian and other non-visual responses, a peak sensitivity between 446-484 nm was identified. Under highly controlled exposure circumstances, less than 1 lux of monochromatic light elicited a significant suppression of nocturnal melatonin. In view of the possible link between light exposure, melatonin suppression and cancer risk, it is important to continue to identify the basic related ocular physiology. Visual performance, rather than circadian function, has been the primary focus of architectural lighting systems. It is now necessary to reevaluate lighting strategies, with consideration of circadian influences, in an effort to maximize physiological homeostasis and health.
Glickman, Gena; Levin, Robert; Brainard, George C.
An early history of human breast cancer: West meets East.
Cancer has been increasingly recognized as a global issue. This is especially true in countries like China, where cancer incidence has increased likely because of changes in environment and lifestyle. However, cancer is not a modern disease; early cases have been recorded in ancient medical books in the West and in China. Here, we provide a brief history of cancer, focusing on cancer of the breast, and review the etymology of ai, the Chinese character for cancer. Notable findings from both Western and Chinese traditional medicine are presented to give an overview of the most important, early contributors to our evolving understanding of human breast cancer. We also discuss the earliest historical documents to record patients with breast cancer. PMID:23958056
Yan, Shou-He
An early history of human breast cancer: West meets East
Cancer has been increasingly recognized as a global issue. This is especially true in countries like China, where cancer incidence has increased likely because of changes in environment and lifestyle. However, cancer is not a modern disease; early cases have been recorded in ancient medical books in the West and in China. Here, we provide a brief history of cancer, focusing on cancer of the breast, and review the etymology of ai, the Chinese character for cancer. Notable findings from both Western and Chinese traditional medicine are presented to give an overview of the most important, early contributors to our evolving understanding of human breast cancer. We also discuss the earliest historical documents to record patients with breast cancer.
Yan, Shou-He
Quantitation of HERV-K env gene expression and splicing in human breast cancer
Human endogenous retroviruses (HERVs) comprise up to 8% of the human genome. In previous studies, we demonstrated that type 1 HERV-K envelope (env) transcripts are expressed in most human breast cancers, but not in normal breast tissues. In the current study, we report that type 2 HERV-K env transcripts are also present in human breast cancers. By real-time RT�PCR, the
Feng Wang-Johanning; Andra R Frost; Bixi Jian; Lidia Epp; Danielle W Lu; Gary L Johanning
Characterization of Novel Breast Cancer Specific Gene, BCSG1, in Human Breast Cancer Progression (97Breast).
National Technical Information Service (NTIS)
We recently identified and cloned a novel breast cancer-specific gene BCSGl by direct differential cDNA sequencing. BCSG1 has a great sequence homology with Alzheimer disease (AD)-related neurotic protein synuclein, and thus was also named as synuclein ga...
Y. Liu
Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ZBP1
Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell�cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, ?-actin, ?-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell�cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.
Gu, Wei; Katz, Zachary; Wu, Bin; Park, Hye Yoon; Li, Deling; Lin, Stanley; Wells, Amber L.; Singer, Robert H.
GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast.
17?-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor ?. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology. PMID:24718936
Scaling, Allison L; Prossnitz, Eric R; Hathaway, Helen J
Effect of Decapeptyl (a GnRH analogue) and of transforming growth factor-alpha (TGF-alpha), in the presence of heparin, on the sulfatase activity of human breast cancer cells.
The effects of the polypeptide Decapeptyl (a gonadotropin-releasing hormone (GnRH) agonist analogue) and of transforming growth factor-alpha (TGF-alpha), on estrone sulfate-sulfatase activities in the homogenates of various breast cancer cell lines were studied in the presence of heparin. In hormone-dependent MCF-7 breast cancer cells, Decapeptyl can inhibit sulfatase activity, and this effect is significantly augmented in the presence of heparin. In the other hormone-dependent T-47D breast cancer cell line, the decrease of sulfatase activity was only significant when Decapeptyl was associated with heparin. No significant effect on sulfatase activity elicited by heparin, Decapeptyl or a mixture of both was found in the hormone-independent MDA-MB-231 breast cancer cells. TGF-alpha stimulates sulfatase activity in the MDA-MB-231 cells but has no effect in the MCF-7 cells; in contrast, TGF-alpha combined with heparin provokes a decrease of the sulfatase activity in both cell lines. It is concluded that the sulfatase activity in some types of breast cancer cell can be inhibited by heparin combined with the polypeptides Decapeptyl or TGF-alpha. PMID:7748810
Chetrite, G; Blumberg-Tick, J; Pasqualini, J R
Downregulation of ER60 protease inhibits cellular proliferation by inducing G1/S arrest in breast cancer cells in vitro.
ER60 protease, a 58-kDa molecular chaperone in the endoplasmic reticulum, is involved in glycoprotein synthesis. ER60 protease has been reported to be differentially expressed in various cancers including breast carcinoma. This study explored the relationship of ER60 protease with cell proliferation in breast cancer in vitro. ER60 protease expression was first determined in a panel of breast cell lines by real-time RT-PCR and Western blot analysis and found to be most abundantly expressed in T47D breast cancer cells. The ER60 protease gene was then successfully knocked down in T47D breast cancer cells using two different sequences of small-interfering RNA. The silencing efficiencies of siER-1 and siER-2 at 48-hr post-transfection were found to be >80% at the mRNA level with concomitant downregulation of the ER60 protease protein by >60% when compared with control T47D breast cancer cells. Downregulation of ER60 protease was also associated with inhibition of cell proliferation when assessed by the AlamarBlue assay. Cell cycle analysis performed on the siER-1- and siER-2-transfected cells, revealed an increase in G1 phase population and a decrease in the S and G2/M phase populations compared with control cells, implicating G1/S cell cycle arrest. It would appear that ER60 protease is involved in breast tumorigenesis and could therefore be a prospective target for cancer therapeutics. PMID:22266712
Lwin, Zin-Mar; Yip, George Wai-Cheong; Chew, Fook-Tim; Bay, Boon-Huat
Human Breast Fibroblasts Inhibit Growth of the MCF10AT Xenograft Model of Proliferative Breast Disease
Stromal fibroblasts are important for normal breast homeostasis and regulation of epithelial growth; however, this regulatory function is altered during carcinogenesis. To study the role of fibroblasts in the development of breast cancer, fibroblasts derived from normal breast (NAFs) were incorporated into the MCF10AT xenograft model of progressive proliferative breast disease. The persistence of human NAFs in xenografts was established by intracellular labeling and tyramide-coupled fluorescent in situ hybridization. Overall, the number of MCF10AT epithelial structures was decreased, and the rate of epithelial cell apoptosis was increased in xenografts containing NAFs. However, these changes were primarily in low-grade epithelial structures, corresponding to normal or mildly hyperplastic ductal epithelium. The level and rate of apoptosis of high-grade epithelial structures, corresponding to in situ and invasive carcinoma, were not consistently altered by NAFs. In addition, there was variability in the growth-inhibitory capacity of NAFs derived from different individuals. NAFs induced changes in the morphology of high-grade MCF10AT structures and in xenograft stroma, including the composition of extracellular matrix, and increased angiogenesis and lymphocytic infiltration. These findings imply that NAFs can inhibit the growth of normal and hyperplastic epi-thelium but are less able to regulate the more transformed epithelial cells that arise during carcino-genesis.
Sadlonova, Andrea; Mukherjee, Shibani; Bowe, Damon B.; Gault, Sandra R.; Dumas, Nicole A.; Van Tine, Brian A.; Frolova, Natalya; Page, Grier P.; Welch, Danny R.; Novak, Lea; Frost, Andra R.
Human Adrenal Androgens: Regulation of Biosynthesis and Role in Estrogen-Responsive Breast Cancer in a Mouse Model.
National Technical Information Service (NTIS)
These experiments investigate a mouse model for the biosynthesis of the human adrenal androgens (dehydroepiandrosterone, DHEA, and its sulfate, DHEAS) and the role of these steroids in human breast cancer growth. An androgen-dependent human breast cancer ...
P. J. Hornsby
Proliferation and differentiation in the human breast during pregnancy
Using multiple immunofluorescence labelling on human breast tissues obtained and freshly frozen at the 12th, 15th, and 18th weeks of pregnancy, we have shown that markers of mammary functional differentiation, milk proteins (?-casein and ?-casein), are synthesised by actively cycling (Ki67 positive) as well as non-cycling (Ki67 negative) cells. These results demonstrate that functional differentiation\\/maturation does not coincide with loss
Rami Suzuki; Amanda J. Atherton; Michael J. O'Hare; Catherine Clarke; Alan Entwistle; Sunil R. Lakhani
Phorbol Esters Induce Multidrug Resistance in Human Breast Cancer Cells
Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood. Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C. When we assayed human breast cancer cell lines for protein kinase C activity, we found that enzyme activity was 7-fold higher in the multidrug-resistant
Robert L. Fine; Jitendra Patel; Bruce A. Chabner
Phorbol esters induce multidrug resistance in human breast cancer cells
Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood. Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C. When the authors assayed human breast cancer cell lines for protein kinase C activity, they found that enzyme activity was 7-fold higher in the
R. L. Fine; J. Patel; B. A. Chabner
FT-Raman spectroscopy study of human breast tissue
NASA Astrophysics Data System (ADS)
Optical spectroscopy has been extensively studied as a potential in vivo diagnostic tool to provide information about the chemical and morphologic structure of tissue. Raman Spectroscpy is an inelastic scattering process that can provide a wealth of spectral features that can be related to the specific molecular structure of the sample. This article reports results of an in vitro study of the FT-Raman human breast tissue spectra. An Nd:YAG laser at 1064nm was used as the excitation source in the FT-Raman Spectrometer. The neoplastic human breast samples, both Fibroadenoma and ICD, were obtained during therapeutical routine medical procedures required by the primary disease, and the non-diseased human tissue was obtained in plastic surgery. No sample preparation was needed for the FT-Raman spectra collection. The FT-Raman spectra were recorded from normal, benign (Fibroadenomas) and malignant (IDC-Intraductal Carcinoma) samples, adding up 51 different areas. The main spectral differences of a typical FT-Raman spectra of a Normal (Non-diseased), Fibroadenoma, and Infiltrating Ductal Carcinoma (IDC) breast tissue at the interval of 600 to 1800cm-1, which may differentiate diagnostically the sample, were found in the bands of 1230 to 1295cm-1, 1440 to 1460 cm-1 and 1650 to 1680 cm-1, assigned to the vibrational bands of the carbohydrate-amide III, proteins and lipids, and carbohydrate-amide I, respectively.
Bitar Carter, Renata A.; Martin, Airton A.; Netto, Mario M.; Soares, Fernando A.
Analyzing the regulation of metabolic pathways in human breast cancer
Background Tumor therapy mainly attacks the metabolism to interfere the tumor's anabolism and signaling of proliferative second messengers. However, the metabolic demands of different cancers are very heterogeneous and depend on their origin of tissue, age, gender and other clinical parameters. We investigated tumor specific regulation in the metabolism of breast cancer. Methods For this, we mapped gene expression data from microarrays onto the corresponding enzymes and their metabolic reaction network. We used Haar Wavelet transforms on optimally arranged grid representations of metabolic pathways as a pattern recognition method to detect orchestrated regulation of neighboring enzymes in the network. Significant combined expression patterns were used to select metabolic pathways showing shifted regulation of the aggressive tumors. Results Besides up-regulation for energy production and nucleotide anabolism, we found an interesting cellular switch in the interplay of biosynthesis of steroids and bile acids. The biosynthesis of steroids was up-regulated for estrogen synthesis which is needed for proliferative signaling in breast cancer. In turn, the decomposition of steroid precursors was blocked by down-regulation of the bile acid pathway. Conclusion We applied an intelligent pattern recognition method for analyzing the regulation of metabolism and elucidated substantial regulation of human breast cancer at the interplay of cholesterol biosynthesis and bile acid metabolism pointing to specific breast cancer treatment.
HPLC determination of tramadol in human breast milk.
Tramadol is a centrally acting analgesic used for prevention and treatment of moderate to severe pain. It is estimated that 0.1% of the administered dose passes into breast milk causing potentially unwanted effects in nursing babies. Pharmacokinetically, breast milk is supposed to be a separate compartment into which the drug is excreted-mainly by passive diffusion. Due to a complex composition of breast milk, a suitable sample preparation procedure is needed with a subsequent chromatographic analysis for drug determination. Among several sample cleanup procedures tested we chose the liquid-liquid extraction procedure using n-hexane as an organic phase with back extraction into aqueous phase since it was considered the most suitable and the most compatible with the subsequent HPLC analysis. The precision and the reproducibility of the method were improved approximately two times by using metoprolol as an internal standard thus making the method also more robust with regard to a variable composition of milk samples. These characteristics, together with low detection limit and short analysis time, proved that the developed method is suitable for monitoring of tramadol in human breast milk. PMID:12899994
Kmetec, Vojko; Roskar, Robert
Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction
Summary Oncogenic human papillomavirus (HPV) types 16 and 18 commonly associated with cervical cancer are found in many epithelial malignancies at extra-genital sites including breast. The transforming gene products of HPV have also been shown to immortalize breast epithelial cellsin vitro. But the findings of HPV DNA in breast carcinoma are found to be contradictory. In the present study fine
V. Gopalkrishna; U. R. Singh; P. Sodhani; J. K. Sharma; S. T. Hedau; A. K. Mandal; B. C. Das
Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history
Background: Recent studies have revealed a possible role for the human papillomavirus (HPV) in the pathogenesis of breast cancer. In this study, patients having both a history of invasive cervical cancer and breast cancer as second primary cancer were selected for enrolment in a study of breast carcinomas for the presence of HPV. Methods: Paraffin-embedded tissue from cervical cancer, pelvic
Andreas Widschwendter; Thomas Brunhuber; Annemarie Wiedemair; Elisabeth Mueller-Holzner; Christian Marth
Src family kinases and HER2 interactions in human breast cancer cell growth and survival
Evidence from murine fibroblast models and human breast cancer cells indicates that c-Src and human EGF receptor (HER1) synergize to enhance neoplastic growth of mammary epithelial cells. To investigate whether interactions between c-Src and other HER family members may also play a role in breast tumor progression, we characterized 13 human breast carcinoma cell lines and 13 tumor samples for
Allison P Belsches-Jablonski; Jacqueline S Biscardi; Dena R Peavy; David A Tice; Davis A Romney; Sarah J Parsons
A Human Breast Cell Model of Preinvasive to Invasive Transition
A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur �spontaneously� in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA�based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context.
Rizki, Aylin; Weaver, Valerie M.; Lee, Sun-Young; Rozenberg, Gabriela I.; Chin, Koei; Myers, Connie A.; Bascom, Jamie L.; Mott, Joni D.; Semeiks, Jeremy R.; Grate, Leslie R.; Mian, I. Saira; Borowsky, Alexander D.; Jensen, Roy A.; Idowu, Michael O.; Chen, Fanqing; Chen, David J.; Petersen, Ole W.; Gray, Joe W.; Bissell, Mina J.
Human papillomavirus type 33 dna in breast cancer in Chinese
Background��The association between human papillomavirus (HPV) and anogenital tumors, especially cervical cancer, is well documented.\\u000a However, it remains unclear whether there is also a correlation between HPV infection and human breast cancer.\\u000a \\u000a \\u000a \\u000a Methods��We used PCR and Southern blot hybridization to analyze HPV-related DNA specimens from 32 cases of invasive ductal carcinoma\\u000a operated upon in the Shanghai region of China.\\u000a \\u000a \\u000a \\u000a Results��DNA
Yingyan Yu; Tadaoki Morimoto; Mitsunori Sasa; Kuniyasu Okazaki; Yosuke Harada; Tsutomu Fujiwara; Yasuo Irie; Ei-ichi Takahashi; Akira Tanigami; Keisuke Izumi
Coactivation of janus tyrosine kinase (Jak)1 positively modulates prolactin-Jak2 signaling in breast cancer: recruitment of ERK and signal transducer and activator of transcription (Stat)3 and enhancement of Akt and Stat5a/b pathways.
Prolactin (PRL) receptors (PRLRs) have been considered selective activators of Janus tyrosine kinase (Jak)2 but not Jak1, Jak3, or Tyk2. We now report marked PRL-induced tyrosine phosphorylation of Jak1, in addition to Jak2, in a series of human breast cancer cell lines, including T47D, MCF7, and SKBR3. In contrast, PRL did not activate Jak1 in immortalized, noncancerous breast epithelial lines HC11, MCF10A, ME16C, and HBL-100, or in CWR22Rv1 prostate cancer cells or MDA-MB-231 breast cancer cells. However, introduction of exogenous PRLR into MCF10A, ME16C, or MDA-MB-231 cells reconstituted both PRL-Jak1 and PRL-Jak2 signals. In vitro kinase assays verified that PRL stimulated enzymatic activity of Jak1 in T47D cells, and PRL activated Jak1 and Jak2 with indistinguishable time and dose kinetics. Relative Jak2 deficiency did not cause PRLR activation of Jak1, because overexpression of Jak2 did not interfere with PRL activation of Jak1. Instead, PRL activated Jak1 through a Jak2-dependent mechanism, based on disruption of PRL activation of Jak1 after Jak2 suppression by 1) lentiviral delivery of Jak2 short hairpin RNA, 2) adenoviral delivery of dominant-negative Jak2, and 3) AG490 pharmacological inhibition. Finally, suppression of Jak1 by lentiviral delivery of Jak1 short hairpin RNA blocked PRL activation of ERK and signal transducer and activator of transcription (Stat)3 and suppressed PRL activation of Jak2, Stat5a, Stat5b, and Akt, as well as tyrosine phosphorylation of PRLR. The data suggest that PRL activation of Jak1 represents a novel, Jak2-dependent mechanism that may serve as a regulatory switch leading to PRL activation of ERK and Stat3 pathways, while also serving to enhance PRL-induced Stat5a/b and Akt signaling. PMID:17550976
Neilson, Lynn M; Zhu, Jianquong; Xie, Jianwu; Malabarba, M Grazia; Sakamoto, Kazuhito; Wagner, Kay-Uwe; Kirken, Robert A; Rui, Hallgeir
A human breast cell model of pre-invasive to invasive transition
A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from pre-invasive to invasive phenotype as it may occur 'spontaneously' in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue
Mina J Bissell; Aylin Rizki; Valerie M. Weaver; Sun-Young Lee; Gabriela I. Rozenberg; Koei Chin; Connie A. Myers; Jamie L. Bascom; Joni D. Mott; Jeremy R. Semeiks; Leslie R. Grate; I. Saira Mian; Alexander D. Borowsky; Roy A. Jensen; Michael O. Idowu; Fanqing Chen; David J. Chen; Ole W. Petersen; Joe W. Gray
Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.
We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.
Backstrom, Malin; Link, Thomas; Olson, Fredrik J; Karlsson, Hasse; Graham, Rosalind; Picco, Gianfranco; Burchell, Joy; Taylor-Papadimitriou, Joyce; Noll, Thomas; Hansson, Gunnar C
Low dentin matrix protein 1 expression correlates with skeletal metastases development in breast cancer patients and enhances cell migratory capacity in vitro.
Small integrin-binding ligand N-linked glycoproteins (SIBLINGs) constitute a family of extracellular matrix proteins involved in bone homeostasis. Their pattern of expression has been primarily reported in bone and tooth and, more recently, in several cancer types. Dentin matrix protein 1 (DMP1), a SIBLING family member, expression was investigated by immunohistochemistry in a retrospective series of 148 primary human breast cancers. Correlations between DMP1 expression levels in the tumors and clinicopathologic features, bone metastases development and relapse of the disease were examined. DMP1 was expressed by 63.5% of the breast tumors analyzed. Significant inverse associations were found between DMP1 expression levels and the size and grade of the tumors (both, P < 0.0001). High DMP1 expression levels in the primary breast lesions were associated with a lower risk of subsequent development of skeletal metastases (P = 0.009). Patients with tumors expressing high levels of DMP1 had a significantly higher disease-free survival rate than those with low DMP1-expressing tumors (P = 0.0062). When DMP1 expression was examined in breast cancer cell lines, we found that non invasive MCF-7 and T47-D cells expressed higher levels than highly invasive MDA-MB-231 and Hs578T cells. Moreover, the specific inhibition of DMP1 expression in MCF-7 cells using siRNAs promoted significantly their migratory capability. Our data implicate for the first time DMP1 expression in breast cancer progression and bone metastases development. PMID:17136477
Bucciarelli, E; Sidoni, A; Bellezza, G; Cavaliere, A; Brachelente, G; Costa, G; Chaplet, M; Castronovo, V; Bellahc�ne, A
Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis
Background The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. Results A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. Conclusions Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines. The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.
BORIS (CTCFL) Is Not Expressed in Most Human Breast Cell Lines and High Grade Breast Carcinomas
BORIS (CTCFL) is the only known paralog of the versatile regulatory protein CTCF, a multifunctional DNA binding protein that mediates distinct gene regulatory functions involved in cell growth, differentiation, and apoptosis. Unlike CTCF, the expression of BORIS is normally restricted to specific cells in testes (the only cells where CTCF is not expressed), where it may play a role in reprogramming the methylation pattern of male germ line DNA. Frequent amplification of the 20q13.2 region, which contains the BORIS gene, and expression of BORIS transcripts in diverse human tumors and cell lines have led to the hypothesis that aberrant expression of BORIS may play a role in tumorigenesis by interfering with CTCF functions. However, recent studies using more quantitative methods indicate low frequency of BORIS expression in melanoma, ovarian, prostate, and bladder carcinomas. To investigate the relationship between chromosome 20q13 amplification and BORIS mRNA levels within breast cancer cell lines and tissues, we developed a quantitative RT-PCR assay to measure the levels of BORIS mRNA. Endpoint RT-PCR assays were also used to investigate the possible expression of alternatively spliced variants. Using multiple primer sets and controls, we found that neither mature BORIS transcripts nor spliced variants are commonly expressed at detectable levels in malignant breast cells or tissues, although endogenous BORIS transcripts can be induced in MCF-7 cells following 5-aza-2?-deoxycytidine treatment. In conclusion, in most breast cancer cells, endogenous BORIS is unlikely to be expressed at sufficient levels to interfere with CTCF functions. Thus it is improbable that aberrant BORIS expression plays a role in most human breast cancers.
Hines, William C.; Bazarov, Alexey V.; Mukhopadhyay, Rituparna; Yaswen, Paul
Gene expression profiles of human breast cancer progression.
Although distinct pathological stages of breast cancer have been described, the molecular differences among these stages are largely unknown. Here, through the combined use of laser capture microdissection and DNA microarrays, we have generated in situ gene expression profiles of the premalignant, preinvasive, and invasive stages of human breast cancer. Our data reveal extensive similarities at the transcriptome level among the distinct stages of progression and suggest that gene expression alterations conferring the potential for invasive growth are already present in the preinvasive stages. In contrast to tumor stage, different tumor grades are associated with distinct gene expression signatures. Furthermore, a subset of genes associated with high tumor grade is quantitatively correlated with the transition from preinvasive to invasive growth. PMID:12714683
Ma, Xiao-Jun; Salunga, Ranelle; Tuggle, J Todd; Gaudet, Justin; Enright, Edward; McQuary, Philip; Payette, Terry; Pistone, Maria; Stecker, Kimberly; Zhang, Brian M; Zhou, Yi-Xiong; Varnholt, Heike; Smith, Barbara; Gadd, Michelle; Chatfield, Erica; Kessler, Jessica; Baer, Thomas M; Erlander, Mark G; Sgroi, Dennis C
Desmoplastic breast carcinoma as a source of human myofibroblasts.
Primary cell cultures of human myofibroblasts can be obtained from desmoplastic breast carcinoma, a tissue rich in these cells. Explants of infiltrating breast carcinoma (scirrhous type) give rise to outgrowths of cells which are predominantly (greater than 90%) myofibroblasts. These cells retain their identifying morphologic characteristics in cell culture by both ultrastructural and immunocytochemical criteria: the presence of elongated cells with longitudinally oriented thin filaments peripherally condensed to form dense zones and abundant rough endoplasmic reticulum; cytoplasmic immunoreactivity for myosin and Type V collagen. These myofibroblasts continue to divide and retain their morphologic characteristics for at least 14 days. These findings allow for further in vitro studies concerning both the role and regulation of the myofibroblast in the desmoplastic response. Images Figure 1 Figure 2 Figure 3 Figure 4
Barsky, S. H.; Green, W. R.; Grotendorst, G. R.; Liotta, L. A.
IMP3 expression is associated with epithelial-mesenchymal transition in breast cancer.
IMP3 plays an important role in tumor invasion and metastasis, to which epithelial to mesenchymal transition (EMT) also contributes. The purpose of this study was to investigate whether IMP3 can regulate invasion and metastasis through EMT in breast cancers. The protein expression levels of IMP3 and EMT markers were analyzed by immunohistochemistry in 180 paraffin-embedded human breast tissue samples. There was an inverse correlation of IMP3 with E-cadherin protein expression (P = 0.042). IMP3 expression directly correlated with both Slug (P = 0.004) and vimentin (P < 0.001). Changes in E-cadherin, vimentin, and Slug mRNA and protein levels were examined by quantitative real-time reverse polymerase chain reaction (qRT-PCR) and western blotting. Overexpression of IMP3 reduced the expression of E-cadherin and upregulated Slug and vimentin in transfected cells. In contrast, knocking down IMP3 had the opposite expression of the three proteins. Ribo-immunoprecipitation qPCR revealed that IMP3 binds Slug mRNA directly. In a transwell assay, overexpression of Slug rescued the cell migration and invasion caused by silencing IMP3 in MDA-MB-231 cells. On the other hand, knockdown of Slug in T47D-IMP3 cells could also have the opposite change. Our results strengthen the association of IMP3 with the regulation of EMT. Slug is a functional target of IMP3. IMP3 could therefore promote invasion and migration through the EMT in breast cancer cells. PMID:25031719
Su, Peng; Hu, Jing; Zhang, Hui; Li, Weiwei; Jia, Ming; Zhang, Xiaofang; Wu, Xiaojuan; Cheng, Hongxia; Xiang, Lei; Zhou, Gengyin
IMP3 expression is associated with epithelial-mesenchymal transition in breast cancer
IMP3 plays an important role in tumor invasion and metastasis, to which epithelial to mesenchymal transition (EMT) also contributes. The purpose of this study was to investigate whether IMP3 can regulate invasion and metastasis through EMT in breast cancers. The protein expression levels of IMP3 and EMT markers were analyzed by immunohistochemistry in 180 paraffin-embedded human breast tissue samples. There was an inverse correlation of IMP3 with E-cadherin protein expression (P = 0.042). IMP3 expression directly correlated with both Slug (P = 0.004) and vimentin (P < 0.001). Changes in E-cadherin, vimentin, and Slug mRNA and protein levels were examined by quantitative real-time reverse polymerase chain reaction (qRT-PCR) and western blotting. Overexpression of IMP3 reduced the expression of E-cadherin and upregulated Slug and vimentin in transfected cells. In contrast, knocking down IMP3 had the opposite expression of the three proteins. Ribo-immunoprecipitation qPCR revealed that IMP3 binds Slug mRNA directly. In a transwell assay, overexpression of Slug rescued the cell migration and invasion caused by silencing IMP3 in MDA-MB-231 cells. On the other hand, knockdown of Slug in T47D-IMP3 cells could also have the opposite change. Our results strengthen the association of IMP3 with the regulation of EMT. Slug is a functional target of IMP3. IMP3 could therefore promote invasion and migration through the EMT in breast cancer cells.
Su, Peng; Hu, Jing; Zhang, Hui; Li, Weiwei; Jia, Ming; Zhang, Xiaofang; Wu, Xiaojuan; Cheng, Hongxia; Xiang, Lei; Zhou, Gengyin
Crosstalk between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and the vitamin D receptor (VDR) in human breast cancer cells: PPAR{gamma} binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} mediated transactivation
Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding partner, RXR{alpha}.
Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R. [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States)] [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States); Knethen, Andreas von [Institute of Biochemistry, Johann Wolfgang Goethe University, Frankfurt (Germany)] [Institute of Biochemistry, Johann Wolfgang Goethe University, Frankfurt (Germany); Choubey, Divaker [Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box 670056, Cincinnati, OH 45267 (United States)] [Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box 670056, Cincinnati, OH 45267 (United States); Mehta, Rajendra G., E-mail: email@example.com [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States)
A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo
Osteopontin (OPN) has been implicated as an important mediator of breast cancer progression and metastasis and has been investigated\\u000a for use as a potential therapeutic target in the treatment of breast cancer. However, the in vivo antitumor effect of anti-OPN\\u000a antibodies on breast cancer has not been reported. In this study, a mouse anti-human OPN antibody (1A12) was humanized by
Jianxin Dai; Bohua Li; Jinping Shi; Ling Peng; Dapeng Zhang; Weizhu Qian; Sheng Hou; Lei Zhao; Jie Gao; Zhiguo Cao; Jian Zhao; Hao Wang; Yajun Guo
A metastatic orthotopic-transplant nude-mouse model of human patient breast cancer.
We report here the development of an orthotopic-transplant model of human patient breast cancer in nude mice. Histologically-intact patient breast tumor tissue was transplanted as intact tissue to the mammary fat pad of nude mice where the tumor tissue grew extensively and metastasized to the lung. This is the first orthotopic-transplant metastatic model of human breast cancer. The potential clinical and basic-science uses of the model are discussed. PMID:8352558
Fu, X; Le, P; Hoffman, R M
Seprase, a membrane-bound protease, alleviates the serum growth requirement of human breast cancer cells
Seprase is a cell surface serine protease that is expressed to high levels by infiltrating ductal carcinomas of the breast\\u000a but its function in malignancy is unknown. MDA-MB-435 (WT435) and MDA-MB-436 (WT436) human breast cancer cells express high\\u000a levels of seprase as do the carcinoma cells in tumors of human breast cancer patients. To investigate its role in the pathobiology
Johnna D. Goodman; Tricia L. Rozypal; Thomas Kelly
Insulin Receptor Expression and Function in Human Breast Cancer Cell Lines1
We have previously reported Chat insulin receptor expression is in creased in human breast cancer specimens (V. Papa et al., J. Clin. Invest., 85:1503-1510, 1990). In the present study, in order to further understand the role of the insulin receptor in breast cancer, insulin receptor expression and function were characterized in three human breast cancer cell lines, MCF-7, ZR-75-1, and
Giovanni Milazzo; Giuseppe Damante; Chin Sung; Martha R. Stampfer; Ira D. Goldfine; Antonino Belfiore
Optical high resolution cross section imaging of a human breast model using independent component analysis
NASA Astrophysics Data System (ADS)
Optical imaging using independent component analysis (OPTICA) is enhanced to provide a high resolution cross section imaging of objects in a turbid medium by a backprojection technique. The performance is demonstrated by imaging a human breast model made of ex vivo human breast tissues. Cancerous site of 5mm size is detected at the midplane of the 33mm thick breast model. The reconstructed cross section image compares favorably with pathology findings.
Xu, M.; Alrubaiee, M.; Gayen, S. K.; Savage, H.; Alfano, R. R.
Suppression of human corneal epithelial proliferation with breast carcinoma immunotoxin.
We examined the effects of the immunotoxin 260F9 Mab-recombinant ricin A (developed against human breast carcinoma) on proliferating and confluent human corneal epithelium (HCE) cells in vitro. HCE cells derived from explants of discarded human donor corneoscleral rims were established as proliferating and confluent cell cultures, and were exposed continuously for 7 days to immunotoxin. Final cell counts at day 7, and thymidine uptake measured at days 1 and 7 postexposure, showed > 95% suppression of proliferating cells at an immunotoxin concentration of 10 ng/ml, with confluent HCE cells relatively unaffected. This immunotoxin may prove useful in treatment of proliferative ocular epithelial diseases such as epithelial downgrowth or squamous cell carcinoma of the ocular surface. PMID:8306659
Fulcher, S; Foulks, G N; Wilkerson, M; Cobo, L M; Houston, L L; Hatchell, D
The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation
Anandamide was the first brain metabolite shown to act as a ligand of �central� CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 ?M and 83�92% maximal inhibition at 5�10 ?M. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 ?M anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55,940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1�0.5 ?M) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.
De Petrocellis, Luciano; Melck, Dominique; Palmisano, Antonella; Bisogno, Tiziana; Laezza, Chiara; Bifulco, Maurizio; Di Marzo, Vincenzo
Relaxin Downregulates the Calcium Binding Protein S100A4 in MDA-MB-231 Human Breast Cancer Cells
Expressed in the human breast and in human breast cancer tissues, the heterodimeric peptide hormone relaxin is involved in extracellular matrix turnover. To investigate the role of relaxin in estrogen receptor-alpha negative human breast cancer cells, we established transfectants of the human MDA-MB-231 breast cancer cell line stably overexpressing H2-relaxin (MDA-MB-231\\/pIRES-EGFP-H2). These transfectants produced and secreted functional relaxin. Our investigations
Yvonne Radestock; Cuong Hoang-Vu; Sabine Hombach-Klonisch
Mouse Model of Human Breast Cancer Initiated by a Fusion Oncogene.
National Technical Information Service (NTIS)
In this study, we generated a novel mouse model of human breast cancer based on a recurrent chromosomal translocation that produces the TEL- NTRK3 fusion oncogene, as the initiating mutation in human secretory breast carcinoma. In this model, we created a...
S. H. Orkin
American Ginseng in the Prevention and Treatment of Human Breast Cancer.
National Technical Information Service (NTIS)
This study is examining the effects of American ginseng on human breast cancer cell proliferation in vitro and in vivo. An extract of American ginseng was shown to significantly decrease MCF-7 and MDA-MB-231 human breast cancer cell proliferation in vitro...
L. Murphy
Genetically Targeted Radiotherapy Utilizing the Human Sodium Iodide Symporter in Human Breast Carcinoma Cells.
National Technical Information Service (NTIS)
The purpose of this proposal was to examine the efficiency of NIS mediated genetically targeted radiotherapy as a possible non-invasive therapeutic treatment in human breast carcinoma. SK-Br-3 cells were transfected with hNlS plasmid to develop stable NIS...
K. Krager F. E. Domann
Expression of preprotachykinin-I (PPT-I), neurokinin-1 (NK1) and neurokinin-2 (NK2) in breast cancer cells improves tumor cell survival in bone marrow in the early stage of metastasis
Objective��To study the potential relationship between the expression of PPT-I, NK-1, NK2 and the development of breast cancer cells\\u000a in bone marrow stroma and to provide evidence of potential molecular mechanisms of bone metastasis in early stage of breast\\u000a cancer patients.\\u000a \\u000a \\u000a \\u000a Methods��The cocultures of breast cancer cell line T-47D and marrow-derived mesenchymal stem cells (MSC) were established with equal\\u000a numbers.
Huilai Zhang; Huaqing Wang; Pengfei Liu; Zhi Yao; Xishan Hao
Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells
Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 �g/ml to 12.5 �g/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 �g/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.
Ait-Mohamed, Ouardia; Battisti, Valentine; Joliot, Veronique; Fritsch, Lauriane; Pontis, Julien; Medjkane, Souhila; Redeuilh, Catherine; Lamouri, Aazdine; Fahy, Christine; Rholam, Mohamed; Atmani, Djebbar; Ait-Si-Ali, Slimane
Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts
INTRODUCTION: Accumulating evidence suggests that fibroblasts play a pivotal role in promoting the growth of breast cancer cells. The objective of the present study was to characterize and validate an in vitro model of the interaction between small numbers of human breast cancer cells and human fibroblasts. METHODS: We measured the clonogenic growth of small numbers of human breast cancer
Michael Samoszuk; Jenny Tan; Guillaume Chorn
Estrogen Induces Vav1 Expression in Human Breast Cancer Cells
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17?-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be ? form, not ?. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ER? might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.
Du, Ming-juan; Chen, Xiang-dong; Zhou, Xiao-li; Wan, Ya-juan; Lan, Bei; Zhang, Cui-zhu; Cao, Youjia
Quercetin: synergistic action with carboxyamidotriazole in human breast carcinoma cells.
Quercetin, a plant flavonoid, blocks signal transduction pathways by inhibiting 1-phosphatidylinositol 4-kinase (EC 184.108.40.206, PI kinase) and 1-phosphatidylinositol 4-phosphate 5-kinase (EC 22.214.171.124, PIP kinase), resulting in a reduction of inositol 1,4,5-trisphosphate (IP3) concentration which decreases the release of calcium from intracellular sources. Carboxyamidotriazole (CAI), a novel anticancer agent, inhibits calcium entry into cells. Because both drugs reduce cytosolic calcium levels, we tested the action of quercetin and CAI in human carcinoma cells. Human breast carcinoma MDA-MB-435 cells were grown in minimum essential medium with 10% fetal bovine serum. In growth inhibition assay the IC50s for quercetin and CAI were 55 and 4.8 microM, respectively; in clonogenic assay, 28 and 1.4 microM, respectively. When quercetin and CAI were added to the cultures, synergism was observed in isobolograms in growth inhibition and clonogenic assays. In growth inhibition assay, the best combination was 20 microM quercetin with 4 microM CAI; in clonogenic assay, 30 microM quercetin with 1.2 microM CAI. Since these drugs are in phase I trials the synergistic action of quercetin and CAI may be of interest in clinical trials for breast carcinoma. PMID:7674820
Yeh, Y A; Herenyiova, M; Weber, G
The use of human acellular dermal matrices in irradiated breast reconstruction.
This article examines the effects of radiation on prosthetic breast reconstruction when human dermal allograft is used in the reconstruction. A brief review of radiation terminology and techniques as applied to the breast is given, followed by a review of the effects of radiation on wound healing in human tissue. The effects of radiation on prosthetic breast reconstruction before the advent of dermal allografting are reviewed. The addition of dermal allograft in reconstruction has led to a reduced number of complications. An algorithm for surgical treatment of irradiated prosthetic breast reconstructions is presented, with a discussion of the authors technique. PMID:22482356
Topol, Bruce M
Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA.
The evaluation of disseminated epithelial tumor cells in breast cancer patients has generated considerable interest due to its potential association with disease recurrence. Our work was performed to analyze the usefulness of 5 mucin genes expression (MUC2, MUC3, MUC5B, MUC6 and MUC7), using RT-PCR assays, to detect disseminated cancer cells in patients with operable breast cancer. The highest frequencies of positive RT-PCR tests in breast tumor extracts were observed for MUC5B (7/15) and MUC7 (5/12). The best specificity, negative results on all peripheral blood mononuclear (PBMN) cell samples from healthy donors, were shown for MUC2, MUC5B and MUC6 RT-PCR assays. Thus, we selected MUC5B as a target gene for further evaluation. Using a nested RT-PCR, MUC5B mRNA transcripts were detected in 16/31 primary breast tumors (but not in 36 samples of normal PBMN cells) and in the human MCF-7 breast cancer cell line but not in BT20, MDA, T47D and ZR-75 breast cancer cell lines, indicating that MUC5B mRNA is expressed in a population of breast cancer cells. Using this method, 9/46 patients (19.5%) who underwent curative surgery showed positive MUC5B mRNA in bone marrow aspirates obtained prior to surgery, including 5/24 patients (20.8%) with stage I or II breast cancer, without histopathologic lymph node involvement. These results indicate that MUC5B mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cell dissemination. A comparative evaluation between MUC5B mRNA, cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA in all bone marrow aspirates suggests a putative complementation for molecular detection of disseminated carcinoma cells. Considering that breast cancer is characterized by a great phenotypic heterogeneity, the use of multimarker approach could contribute to tumor cell detection in bone marrow and blood. PMID:12478674
Berois, Nora; Varangot, Mario; S��ora, Cecilia; Zarantonelli, Leticia; Pressa, Carlos; Lavi�a, Ra�l; Rodr�guez, Jos� Luis; Delgado, Fernando; Porchet, Nicole; Aubert, Jean-Pierre; Osinaga, Eduardo
Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening
Introduction Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells. Methods We conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors. Results The primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity. Conclusions These data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.
Screening and functional analysis of a differential protein profile of human breast cancer
To improve the understanding of the enriched functions of proteins and to identify potential biomarkers in human breast cancer, the present study constructed a differentially expressed protein profile by screening immunohistochemistry maps of human breast cancer proteins. A total of 1,688 proteins were found to be differentially expressed in human breast cancer, including 773 upregulated and 915 downregulated proteins. Of these proteins, secreted and membrane proteins were screened and clustered, and more enriched biological functions and pathways were presented in the upregulated protein profiles. Furthermore, altered serum levels of peroxiredoxin (PRDX)2, PRDX6, cathepsin (CTS)B and CTSD were detected by ELISA assay. The present study provides a novel global mapping of potential breast cancer biomarkers that could be used as background to identify the altered pathways in human breast cancer, as well as potential cancer targets.
Human breast biomonitoring and environmental chemicals: use of breast tissues and fluids in breast cancer etiologic research
Extensive research indicates that the etiology of breast cancer is complex and multifactorial and may include environmental risk factors. Breast cancer etiology and exposure to xenobiotic compounds, diet, electromagnetic fields, and lifestyle have been the subject of numerous scientific inquiries, but research has yielded inconsistent results. Biomonitoring has been used to explore associations between breast cancer and levels of environmental
Judy S Lakind; Amy A Wilkins; Michael N Bates
Prolactin antagonist-endostatin fusion protein as a targeted dual-functional therapeutic agent for breast cancer.
In previous studies (Chen, W. Y. et al., Clin. Cancer Res., 5:3583-3593, 1999; Chen, N Y. et al., Int. J. Oncol., 20:813-818, 2002), we have demonstrated the ability of the human prolactin (hPRL) antagonist, G129R, to inhibit human breast cancer cell proliferation in vitro and to slow the growth rate of tumors in mice. We further revealed that the possible mechanisms of G129R antitumor effects act through the induction of apoptosis via the regulation of bcl-2 gene expression. It has been established that to sustain tumor growth, it is necessary for the development of a network of blood vessels to bring in nutrients, a process called angiogenesis. The disruption of angiogenesis has been proven to be an effective strategy to cause regression of certain tumors. One of the best-studied angiogenesis inhibitors is endostatin, which acts through the inhibition of endothelial cells. In this study, we combine the anti-breast tumor effects of G129R and the antiangiogenic effects of endostatin by creating a novel fusion protein (G129R-endostatin) specifically for breast cancer therapy. The data presented here demonstrated that this novel fusion protein was able to bind to the PRL receptor (PRLR) on T-47D human breast cancer cells and inhibit the signal transduction induced by PRL. At the same time, G129R-endostatin inhibited human umbilical vein endothelial cell (HUVEC) proliferation and disrupted the formation of endothelial tube structures with potency similar to that of endostatin. More importantly, the therapeutic efficacy of G129R-endostatin was confirmed using a mouse breast cancer cell line 4T1 in vivo. G129R-endostatin has a significantly prolonged serum half-life as compared with that of G129R or endostatin alone, and exhibited greater tumor inhibitory effects than G129R and endostatin individually or in combination. Taken together, these data demonstrate the dual therapeutic effects of G129R-endostatin, and suggests that this fusion protein has great promise as a novel anti-breast cancer agent. PMID:12839947
Beck, Michael T; Chen, Nian Y; Franek, Karl J; Chen, Wen Y
Absence of primary cilia in cell cycle-arrested human breast cancer cells.
Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki-67, primary cilia were present only in 20-40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indicate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells. PMID:24330390
Nobutani, Kentaro; Shimono, Yohei; Yoshida, Midori; Mizutani, Kiyohito; Minami, Akihiro; Kono, Seishi; Mukohara, Toru; Yamasaki, Takashi; Itoh, Tomoo; Takao, Shintaro; Minami, Hironobu; Azuma, Takeshi; Takai, Yoshimi
Radiosensitization effects of berberine on human breast cancer cells.
Berberine, an isoquinoline derivative alkaloid, has recently been shown to have antitumor activity. The present study aimed to investigate the effects of the concomitant administration of berberine and radiation on breast cancer. The effects of berberine on the radiosensitivity of MCF-7 and MDA-MB-468 cells were evaluated by using cell clonogenic assays. Cells pre-treated with berberine or dimethyl sulfoxide (DMSO) for 24 h were irradiated using a Faxitron Cabinet X-ray System to deliver the indicated doses (0, 1, 2, 3 and 4 Gy). Changes in cell cycle distribution were determined by flow cytometry. ?-H2AX foci were detected by immunofluorescence staining. The levels of Ku70, Ku86 and RAD51 proteins were evaluated by western blot analysis. We observed that berberine increased the MCF-7 and MDA-MB-468 cell radiosensitivity with cell clonogenic assays. the radiation-induced G2/M cell cycle delay was reduced in the MCF-7 cells pre-teated with berberine. Berberine pre-treatment prolonged the persistence of DNA double-strand breaks in the MCF-7 cell line. In comparison with the control cells, the protein levels of RAD51 were decreased in the MCF-7 and MDA-MB-468 cells treated with berberine, and in the cells pre-treated with 15 �M berberine for 24 h, the level of RAD51 protein decreased significantly at the indicated time-points (0, 2, 6 and 24 h) following X-ray exposure. In conclusion, berberine sensitizes human breast cancer cells to ionizing radiation by inducing cell cycle arrest and the downregulation of the homologous recombination repair protein, RAD51. Berberine may be a promising radiosensitizer for the treatment of breast cancer. PMID:22895634
Wang, Jing; Liu, Qiao; Yang, Qifeng
Loss of heterozygosity of the L-myc oncogene in human breast tumors
Recent studies suggest that loss of heterozygosity may play an important role in various human neoplasia. Cytogenetic abnormalities detected in primary breast tumors led us to examine breast tumor DNAs for deletions. In the present study, we demonstrate, using restriction fragment length polymorphism (RFLP) analysis at the L-myc proto-oncogene (chromosome 1p32), a frequent loss of heterozygosity in primary breast tumor
Ivan Bieche; Marie-Helene Champeme; Giorgio Merlo; Christian-Jacques Larsen; Robert Callahan; Rosette Lidereau
The human DEK oncogene stimulates ?-catenin signaling, invasion and mammosphere formation in breast cancer
Breast cancer is a major cause of cancer-related deaths in American women; therefore, the identification of novel breast cancer-related molecules for the discovery of new markers and drug targets remains essential. The human DEK gene, which encodes a chromatin-binding protein and DNA topology regulator, is upregulated in many types of cancer. DEK has been implicated as an oncogene in breast
L M Privette Vinnedge; R McClaine; P K Wagh; K A Wikenheiser-Brokamp; S E Waltz; S I Wells
Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes
Molecular subtypes of breast cancer with relevant bio- logical and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal- like subtypes. To investigate the ability of mass spec- trometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we per- formed a SELDI-TOF MS-based protein profiling of hu- man breast cell lines (BCLs). Triton-soluble proteins from
Anthony Goncalves; Emmanuelle Charafe-Jauffret; Francois Bertucci; Stephane Audebert; Yves Toiron; Benjamin Esterni; Florence Monville; Carole Tarpin; Jocelyne Jacquemier; Gilles Houvenaeghel; Christian Chabannon; Jean-Marc Extra; Patrice Viens; Jean-Paul Borg; D. Birnbaum
The candidate tumor suppressor CST6 alters the gene expression profile of human breast carcinoma cells: Down-regulation of the potent mitogenic, motogenic, and angiogenic factor autotaxin
We recently coined CST6 as a novel candidate tumor suppressor gene for breast cancer. CST6 indeed is expressed in the normal human breast epithelium, but little or not at all in breast carcinomas and breast cancer cell lines. Moreover, ectopic expression of CST6 in human breast cancer cells suppressed cell proliferation, migration, invasion, and orthotopic tumor growth. To obtain insights
Jin Song; Chunfa Jie; Paula Polk; Ravi Shridhar; Timothy Clair; Jun Zhang; Lijia Yin; Daniel. Keppler
MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype
BACKGROUND: MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression. RESULTS: Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and
Cherie Blenkiron; Leonard D Goldstein; Natalie P Thorne; Inmaculada Spiteri; Suet-Feung Chin; Mark J Dunning; Nuno L Barbosa-Morais; Andrew E Teschendorff; Andrew R Green; Ian O Ellis; Simon Tavar�; Carlos Caldas; Eric A Miska
Comprehensive copy number profiles of breast cancer cell model genomes
Introduction Breast cancer is the most commonly diagnosed cancer in women worldwide and consequently has been extensively investigated in terms of histopathology, immunochemistry and familial history. Advances in genome-wide approaches have contributed to molecular classification with respect to genomic changes and their subsequent effects on gene expression. Cell lines have provided a renewable resource that is readily used as model systems for breast cancer cell biology. A thorough characterization of their genomes to identify regions of segmental DNA loss (potential tumor-suppressor-containing loci) and gain (potential oncogenic loci) would greatly facilitate the interpretation of biological data derived from such cells. In this study we characterized the genomes of seven of the most commonly used breast cancer model cell lines at unprecedented resolution using a newly developed whole-genome tiling path genomic DNA array. Methods Breast cancer model cell lines MCF-7, BT-474, MDA-MB-231, T47D, SK-BR-3, UACC-893 and ZR-75-30 were investigated for genomic alterations with the submegabase-resolution tiling array (SMRT) array comparative genomic hybridization (CGH) platform. SMRT array CGH provides tiling coverage of the human genome permitting break-point detection at about 80 kilobases resolution. Two novel discrete alterations identified by array CGH were verified by fluorescence in situ hybridization. Results Whole-genome tiling path array CGH analysis identified novel high-level alterations and fine-mapped previously reported regions yielding candidate genes. In brief, 75 high-level gains and 48 losses were observed and their respective boundaries were documented. Complex alterations involving multiple levels of change were observed on chromosome arms 1p, 8q, 9p, 11q, 15q, 17q and 20q. Furthermore, alignment of whole-genome profiles enabled simultaneous assessment of copy number status of multiple components of the same biological pathway. Investigation of about 60 loci containing genes associated with the epidermal growth factor family (epidermal growth factor receptor, HER2, HER3 and HER4) revealed that all seven cell lines harbor copy number changes to multiple genes in these pathways. Conclusion The intrinsic genetic differences between these cell lines will influence their biologic and pharmacologic response as an experimental model. Knowledge of segmental changes in these genomes deduced from our study will facilitate the interpretation of biological data derived from such cells.
Shadeo, Ashleen; Lam, Wan L
A mathematical model of drug transport in human breast cancer.
A mathematical model of drug transport in tissue has been developed on the basis of a clinical study of patients with breast cancer, treated with the drug doxorubicin and of drug transport experiments using cultured human breast cancer cells. The clinical study revealed doxorubicin gradients in tumor islets of densely packed cancer cells. The mathematical model allows simultaneous drug transport through the cellular network (transcellular pathway), through the intercellular interstitium (paracellular pathway), and across the boundary between the two networks. The effective diffusion coefficient of the interstitial network is found to be much higher than that of the cellular network, in spite of the fact that the interstitium thickness is only 20-40 nm. The model simulations can be made to fit the results of the clinical study. A long-continued simulation (40 days) of drug transport into a spherical islet with a radius of 150 microm, after a bolus injection of doxorubicin, reveals that the maximum average drug concentration at the islet centre is only reached after 224 h, while it decreases by a factor 15 from the boundary to the centre of the islet. The area under the curve in a plot of the average drug concentration versus time only decreases by 10% from the boundary to the centre of the islet. PMID:10625582
Lankelma, J; Fern�ndez Luque, R; Dekker, H; Schinkel, W; Pinedo, H M
Detection of dicofol and related pesticides in human breast milk from China, Korea and Japan.
Previously, we demonstrated that the concentrations of DDTs were greater in breast milk collected from Chinese mothers than from Japanese and Korean mothers. To investigate dicofol as a possible source of the DDTs in human breast milk, we collected breast milk samples from 2007 to 2009 in China (Beijing), Korea (Seoul, Busan) and Japan (Sendai, Takarazuka and Takayama). Using these breast milk samples, we quantified the concentrations of dichlorobenzophenone, a pyrolysis product of dicofol (simply referred to as dicofol hereafter), dichlorodiphenyltrichloroethane and its metabolites (DDTs) using GC-MS. Overall, 12 of 14 pooled breast milk samples from 210 mothers contained detectable levels of dicofol (>0.1 ng g?� lipid). The geometric mean concentration of dicofol in the Japanese breast milk samples was 0.3 ng g?� lipid and significantly lower than that in Chinese (9.6 ng g?� lipid) or Korean breast milk samples (1.9 ng g?� lipid) (p<0.05 for each). Furthermore, the ?DDT levels in breast milk from China were 10-fold higher than those from Korea and Japan. The present results strongly suggest the presence of extensive emission sources of both dicofol and DDTs in China. However, exposure to dicofol cannot explain the large exposure of Chinese mothers to DDTs because of the trace levels of dicofol in the ?DDTs. In the present study, dicofol was confirmed to be detectable in human breast milk. This is the first report to identify dicofol in human samples. PMID:21051069
Fujii, Yukiko; Haraguchi, Koichi; Harada, Kouji H; Hitomi, Toshiaki; Inoue, Kayoko; Itoh, Yoshiko; Watanabe, Takao; Takenaka, Katsunobu; Uehara, Shigeki; Yang, Hye-Ran; Kim, Min-Young; Moon, Chan-Seok; Kim, Hae-Sook; Wang, Peiyu; Liu, Aiping; Hung, Nguyen Ngoc; Koizumi, Akio
S14 protein in breast cancer cells: Direct evidence of regulation by SREBP-1c, superinduction with progestin, and effects on cell growth
Most breast cancers exhibit brisk lipogenesis, and require it for growth. S14 is a lipogenesis-related nuclear protein that is overexpressed in most breast cancers. Sterol response element-binding protein-1c (SREBP-1c) is required for induction of lipogenesis-related genes, including S14 and fatty acid synthase (FAS), in hepatocytes, and correlation of SREBP-1c and FAS expression suggested that SREBP-1c drives lipogenesis in tumors as well. We directly tested the hypothesis that SREBP-1c drives S14 expression and mediates lipogenic effects of progestin in T47D breast cancer cells. Dominant-negative SREBP-1c inhibited induction of S14 and FAS mRNAs by progestin, while active SREBP-1c induced without hormone and superinduced in its presence. Changes in S14 mRNA were reflected in protein levels. A lag time and lack of progestin response elements indicated that S14 and FAS gene activation by progestin is indirect. Knockdown of S14 reduced, whereas overexpression stimulated, T47D cell growth, while nonlipogenic MCF10a mammary epithelial cells were not growth-inhibited. These data directly demonstrate that SREBP-1c drives S14 gene expression in breast cancer cells, and progestin magnifies that effect via an indirect mechanism. This supports the prediction, based on S14 gene amplification and overexpression in breast tumors, that S14 augments breast cancer cell growth and survival.
Martel, Peter M. [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States); Norris Cotton Cancer Center, Dartmouth Medical School (United States); Bingham, Chad M. [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States); Norris Cotton Cancer Center, Dartmouth Medical School (United States); McGraw, Charles J. [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States); Norris Cotton Cancer Center, Dartmouth Medical School (United States); Baker, Christina L. [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States); Norris Cotton Cancer Center, Dartmouth Medical School (United States); Morganelli, Peter M. [Department of Microbiology and Immunology, Dartmouth Medical School (United States); Norris Cotton Cancer Center, Dartmouth Medical School (United States); Meng, Marie Louise [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States); Norris Cotton Cancer Center, Dartmouth Medical School (United States); Armstrong, Jessica M. [Norris Cotton Cancer Center, Dartmouth Medical School (United States); Department of Physiology, Dartmouth Medical School (United States); Moncur, Joel T. [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States); Kinlaw, William B. [Department of Medicine, Division of Endocrinology, Dartmouth Medical School (United States) and Norris Cotton Cancer Center, Dartmouth Medical School (United States)]. E-mail: firstname.lastname@example.com
Overexpression of WWP1 is associated with the estrogen receptor and insulin-like growth factor receptor 1 in breast carcinoma.
WWP1, a HECT type E3 ubiquitin ligase frequently amplified and overexpressed in breast cancer, has the potential to become a useful clinical biomarker and therapeutic target in breast cancer. Here, we performed immunohistochemical staining in formalin-fixed and paraffin-embedded tissue sections from 187 cases of primary invasive mammary carcinoma [137 ductal carcinomas (IDC) and 50 lobular carcinomas (ILC)] by using a monoclonal anti-WWP1 antibody. The normal breast epithelium and adjacent benign epithelium are essentially negative for WWP1. Cytoplasmic WWP1 immunoreactivity was observed in 76/187 (40.6%) tumors and showed a positive correlation with ERalpha (p = 0.05) and IGF-1R proteins (p = 0.001) in this cohort. The positive correlations between WWP1 and ER/IGF-1R were also observed in a panel of 12 breast cancer cell lines by Western blot. Interestingly, the ER levels are decreased when WWP1 is silenced in ER positive MCF7 and T47D breast cancer cell lines. Finally, WWP1 ablation collectively inhibits cell proliferation with tamoxifen in MCF7 and T47D, as measured by (3)H-thymidine incorporation assays. These findings suggest that WWP1 may play an important role in ER positive breast cancer. PMID:19267401
Chen, Ceshi; Zhou, Zhongmei; Sheehan, Christine E; Slodkowska, Elzbieta; Sheehan, Christopher B; Boguniewicz, Ann; Ross, Jeffrey S
Role of CCL5 in invasion, proliferation and proportion of CD44+/CD24- phenotype of MCF-7 cells and correlation of CCL5 and CCR5 expression with breast cancer progression.
This study was undertaken to observe the effects and possible mechanism of CC chemokine ligand 5 (CCL5) on invasion, proliferation and percentage of CD44+/CD24- subpopulation of human breast cancer line MCF-7 and to investigate the correlation of expression levels of CCL5 and its receptors with the progression of breast cancer. We used real-time RT-PCR to detect the expression levels of CCL5 and its receptors CCR5, CCR1 and CCR3 in 36 breast cancer specimens of different TNM stage and their corresponding normal breast tissue. CCL5 expression and invasive ability of four human breast cancer cell lines MCF-7, SK-BR-3, T-47D and MDA-MB-231 were analyzed by real-time RT-PCR and cell invasion assay, respectively. Effects of recombinant human CCL5 (rhCCL5) on cell proliferation and percentage of the CD44+/CD24- subpopulation in MCF-7 cells were analyzed respectively by MTT assay and flow cytometry. We also used cell invasion assay to detect the invasive ability of both CD44+/CD24- and CD44+/CD24+ subpopulations of MCF-7 cells treated with rhCCL5 and/or CCR5 monoclonal antibody. Our results revealed that CCL5 and CCR5 expression were higher in breast cancer tissue than those in their corresponding normal tissue and breast cancer tissue with higher TNM stage contained more CCL5 mRNA. In addition, CCR5 expression and invasive ability of CD44+/CD24- subpopulation were higher than those of CD44+/CD24+ subpopulation of MCF-7 cells. Moreover, treatment of rhCCL5 increased the proportion of CD44+/CD24- cells and the proliferation of MCF-7 cells. Induction of rhCCL5 increased the cell invasive ability of both CD44+/CD24- and CD44+/CD24+ cells, which could be partially antagonized by CCR5 monoclonal antibody. Collectively, our data show that CCL5 increased the proportion of CD44+/CD24- subpopulation and induced invasion and proliferation of MCF-7 cells, and expression of CCL5 and CCR5 in breast cancer tissue was positively correlated with breast cancer progression. PMID:19288016
Zhang, Yimin; Yao, Feng; Yao, Xiaoli; Yi, Changhong; Tan, Cheng; Wei, Lei; Sun, Shengrong
Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells
Highlights: ? Fulvestrant radiosensitizes MCF-7 cells. ? Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ? Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy � fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 � 0.013 vs. 0.622 � 0.029 @2 Gy, 0.599 � 0.045 vs. 0.475 � 0.054 @4 Gy, and 0.472 � 0.021 vs. 0.380 � 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT increases breast cancer cell radiosensitivity compared with radiation alone. These findings have salient implications for designing clinical trials using fulvestrant and radiation therapy.
Wang, Jing, E-mail: email@example.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China) [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Yang, Qifeng, E-mail: email@example.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China)] [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Haffty, Bruce G., E-mail: email@example.com [Department of Radiation Oncology, UMDNJ-Robert Wood Johnson School of Medicine, Cancer Institute of New Jersey, NB (United States); Li, Xiaoyan, E-mail: firstname.lastname@example.com [Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China)] [Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Moran, Meena S., E-mail: email@example.com [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States)
Isolation of Fully Human Antagonistic RON Antibodies Showing Efficient Block of Downstream Signaling and Cell Migration.
RON belongs to the c-MET family of receptor tyrosine kinases. As its well-known family member MET, RON and its ligand macrophage-stimulating protein have been implicated in the progression and metastasis of tumors and have been shown to be overexpressed in cancer. We generated and tested a large number of human monoclonal antibodies (mAbs) against human RON. Our screening yielded three high-affinity antibodies that efficiently block ligand-dependent intracellular AKT and MAPK signaling. This effect correlates with the strong reduction of ligand-activated migration of T47D breast cancer cell line. By cross-competition experiments, we showed that the antagonistic antibodies fall into three distinct epitope regions of the RON extracellular Sema domain. Notably, no inhibition of tumor growth was observed in different epithelial tumor xenografts in nude mice with any of the antibodies. These results suggest that distinct properties beside ligand antagonism are required for anti-RON mAbs to exert antitumor effects in vivo. PMID:21286376
Gunes, Zeynep; Zucconi, Adriana; Cioce, Mario; Meola, Annalisa; Pezzanera, Monica; Acali, Stefano; Zampaglione, Immacolata; De Pratti, Valeria; Bova, Luca; Talamo, Fabio; Demartis, Anna; Monaci, Paolo; La Monica, Nicola; Ciliberto, Gennaro; Vitelli, Alessandra
Multiplexed ion beam imaging of human breast tumors.
Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics. PMID:24584119
Angelo, Michael; Bendall, Sean C; Finck, Rachel; Hale, Matthew B; Hitzman, Chuck; Borowsky, Alexander D; Levenson, Richard M; Lowe, John B; Liu, Scot D; Zhao, Shuchun; Natkunam, Yasodha; Nolan, Garry P
Phosphoinositide 3-kinase and INPP4B in human breast cancer.
The PI3K/Akt signaling pathway is frequently increased in many human cancers, including breast cancer. Recent studies have identified INPP4B, which inhibits PI3K signaling, as an emerging tumor suppressor in breast cancer. This short review discusses these issues and the possibility that INPP4B is an important regulator in many cancers. PMID:23551093
Bertucci, Micka C; Mitchell, Christina A
Clonal analysis of benign and malignant human breast tumors by means of polymerase chain reaction
Clonal analysis was conduced on a variety of benign and malignant human breast tumors using the method based on restriction fragment length polymorphism (RFLP) of the X 120 chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. Breast carcinoma was shown to be monoclonal in origin, consistent with a somatic mutational theory. Precancerous lesions such as atypical
Shinzaburo Noguchi; Tomohiko Aihara; Hiroki Koyama; Kazuyoshi Motomura; Hideo Inaji; Shingi Imaoka
GnRH analogs reduce invasiveness of human breast cancer cells
Objective��Bone, besides lung and liver, is one of the most preferential metastatic target sites for breast cancers. Although the precise molecular mechanisms underlying this preference need to be elucidated, it appears that bone microenvironments possess unique biological features that enable circulating cancer cells to home, survive and proliferate, and destroy bone. The majority of human breast cancers and in addition
Julia von Alten; Stefanie Fister; Hiltrud Schulz; Volker Viereck; Karl-Heinz Frosch; G�nter Emons; Carsten Gr�ndker
Effect of Estrogen on Progression of Human Proliferative Breast Cancer Disease in a Xenograft Model.
National Technical Information Service (NTIS)
We have utilized the T24-lla-ras transfected MCPi0A xenograft model of early human breast cancer progression to a) determine whether the observed epidemiologic link between estrogen and increased risk of breast cancer indeed reflect a direct growth promot...
P. V. Shekhar
Huntsman Cancer Institute researchers discover a new way to model human breast cancer:
Researchers from Huntsman Cancer Institute (HCI) at the University of Utah have discovered a new way to model human breast cancer that could lead to new tools for predicting which breast cancers will spread and new ways to test drugs that may stop its spread.
Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk
The activity of the aromatase enzyme, which converts androgens into oestrogens and has a major role in regulating oestrogen levels in the breast, is thought to be a contributing factor in the development of breast cancer. We undertook this study to assess the role of constitutional genetic variation in the human aromatase gene (Cyp19) in the development of this disease.
N Siegelmann-Danieli
Examination of a Newly Discovered human Retrovirus, XMRV, in Breast Cancer.
National Technical Information Service (NTIS)
The main objective of this research project is to test the hypothesis that a newly discovered retrovirus, XMRV, is involved in the development of human breast cancer. To accomplish this, we have examined XMRV infection in breast cancer tissue in compariso...
F. K. Yoshimura
Exploring the stem cell and non-stem cell constituents of human breast milk.
The immense potency of nutritional components of human breast milk and importance of breastfeeding is known worldwide. Recent researches had identified stem cells as integral component of human breast milk. Nevertheless, there is little proof of evidence on the stem cell constituents of breast milk. It is imperative to explore the cellular constituents of human breast milk, including of stem cells, to open new avenue in child's development and regeneration. Thus, we aimed at identifying the cellular constituents of human breast milk by phenotypic characterisation of diverse cell surface markers of hematopoietic stem cells (CD 34, CD 133, CD 117), mesenchymal stem cells (CD 90, CD 105, CD 73), myoepithelial cells (CD 29, CD 44), Immune cells (CD 209, CD 86, CD 83, CD 14, CD 13, HLADR, CD 45), as well as cell adhesion molecules (CD 31, CD 54, CD 166, CD 106, CD 49d), and other markers (ABCG2, CD140b) using flowcytometry. We found a lower expression of CD 34 (13.07���2.0�%), CD 90 (7.79���0.8�%) and CD 73 (2.19���0.41�%), indicating scanty hematopoietic and mesenchymal stem cell population in human breast milk. On contrary, myoepithelial progenitors, cell adhesion molecules, immune cells and growth factors were identified as the major constituents of breast milk. Overall, this study illuminates the benefits of breast feeding as breast milk encompasses heterogeneous cellular components that benefits child's growth, immunity and development. However, further research on these constituents of human breast milk will widen their applicability in treatment of neonatal disorders. PMID:22940915
Indumathi, S; Dhanasekaran, M; Rajkumar, J S; Sudarsanam, D
Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer
Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells, not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.
Perez-Pinera, Pablo [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Chang, Y. [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Astudillo, A. [Hospital Universitario Central de Asturias, Oviedo (Spain); Mortimer, J. [Moore's Cancer Center, University of California San Diego, San Diego, CA (United States); Deuel, T.F. [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)]. E-mail: email@example.com
Development of Anatomically Realistic Numerical Breast Phantoms with Accurate Dielectric Properties for Modeling Microwave Interactions with the Human Breast
Computational electromagnetics models of microwave interactions with the human breast serve as an invaluable tool for exploring the feasibility of new technologies and improving design concepts related to microwave breast cancer detection and treatment. In this paper we report the development of a collection of anatomically realistic 3D numerical breast phantoms of varying shape, size, and radiographic density which can be readily used in FDTD computational electromagnetics models. The phantoms are derived from T1-weighted magnetic resonance images (MRIs) of prone patients. Each MRI is transformed into a uniform grid of dielectric properties using several steps. First, the structure of each phantom is identified by applying image processing techniques to the MRI. Next, the voxel intensities of the MRI are converted to frequency-dependent and tissue-dependent dielectric properties of normal breast tissues via a piecewise-linear map. The dielectric properties of normal breast tissue are taken from the recently completed large-scale experimental study of normal breast tissue dielectric properties conducted by the Universities of Wisconsin and Calgary. The comprehensive collection of numerical phantoms is made available to the scientific community through an online repository.
Zastrow, Earl; Davis, Shakti K.; Lazebnik, Mariya; Kelcz, Frederick; Van Veen, Barry D.; Hagness, Susan C.
CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells
Highlights: �The effects of elevated CITED2 on ER function in breast cancer cells are examined. �CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. �CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.
Lau, Wen Min; Doucet, Michele; Huang, David; Weber, Kristy L.; Kominsky, Scott L., E-mail: firstname.lastname@example.com
Gene Expression Analysis in Human Breast Cancer Associated Blood Vessels
Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (5�72 fold) in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC) of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of potentially novel anti-angiogenic targets that are likley to be, but not exclusivley, relevant to breast cancer.
Jones, Dylan T.; Lechertier, Tanguy; Mitter, Richard; Herbert, John M. J.; Bicknell, Roy; Jones, J. Louise; Li, Ji-Liang; Buffa, Francesca; Harris, Adrian L.; Hodivala-Dilke, Kairbaan
Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells
There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells.
Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly
The breast\\/nipple\\/areola complex and human sexuality
The male or female breast\\/nipple\\/areola complex arises from a common mammary stem cell and develops similarly in the foetus and during infancy. At puberty the male's breasts remain rudimentary but the female's develop further, mainly through oestrogen and progesterone stimulation, and become more sensitive. Female breasts serve both nutritive and sexual functions, unlike other primates they develop at puberty before
Roy J. Levin
MYB suppresses differentiation and apoptosis of human breast cancer cells
INTRODUCTION: MYB is highly expressed in estrogen receptor positive (ER + ve) breast tumours and tumour cell lines. We recently demonstrated that MYB is essential for the proliferation of ER + ve breast cancer cells, and have now investigated its role in mammary epithelial differentiation. METHODS: MCF-7 breast cancer cells were treated with sodium butyrate, vitamin E succinate or 12-O-tetradecanoylphorbol-13-acetate
Yvette Drabsch; Robert G Ramsay; Thomas J Gonda
Methotrexate polyglutamate synthesis by cultured human breast cancer cells.
We studied the conversion of methotrexate to poly-gamma-glutamyl derivatives by cultured human breast cancer cells. After incubation with 2 micro M [3',5',9-3H]methotrexate, MCF-7 cells were washed free of extracellular drug and were boiled to lyse cells and to release drug bound to dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 188.8.131.52). The supernatant fraction was chromatographed on Sephadex G-15 to separate parent drug from polyglutamate forms. These cells rapidly and quantitatively converted methotrexate to polyglutamates, such that after 24 hr of incubation, 70 +/- (SEM) 3% of intracellular methotrexate existed as polyglutamates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase indicated that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. These studies demonstrate that methotrexate polyglutamates are readily formed in human tumor cells and bind to dihydrofolate reductase. Because these forms of the drug may be selectively retained within the cell, they may be important determinants of the duration of action and, ultimately, the cytotoxicity of methotrexate in human solid tumors.
Schilsky, R L; Bailey, B D; Chabner, B A
IKK? inhibitor in combination with bortezomib induces cytotoxicity in breast cancer cells
Bortezomib is a proteasome inhibitor with remarkable clinical antitumor activity in multiple myeloma (MM) and is under evaluation in clinical trials in various types of cancer including breast cancer. Although the initial rationale for its use in cancer treatment was the inhibition of NF-?B activity by blocking proteasomal degradation of I?B?, direct evidence indicating inhibition of constitutive NF-?B activity by bortezomib in tumor cells in patients has not yet been reported. Moreover, recent studies have shown that bortezomib activates constitutive NF-?B activity via stimulating the canonical pathway in MM cells. In this study, we first examined protein expression of I?B? after bortezomib treatment. We observed that bortezomib upregulated the phosphorylation and downregulated I?B? protein expression in a dose- and time-dependent manner in MCF7 and T47D cells, associated with phosphorylation of IKK?. Since I?B? is an inhibitor of nuclear translocation of NF-?B, we further examined alteration of NF-?B activity by bortezomib. Importantly, bortezomib significantly upregulates NF-?B activity in both MCF7 and T47D in a dose-dependent fashion, demonstrated by electrophoretic mobility shift analysis (EMSA). Furthermore, immunocytochemical analysis confirmed enhanced nuclear translocation of p65 NF-?B (RelA) by bortezomib treatment. Supershift assay showed supershifted bands by anti-p65 and -p50 antibodies. Taken together, these results indicate that bortezomib activates the canonical NF-?B pathway in both cell lines. Finally, we demonstrated that IKK? inhibitor enhanced cytotoxicity, associated with inhibition of NF-?B activity induced by bortezomib in MCF7 and T47D breast cancer cells.
Effects of Physiological Levels of the Green Tea Extract Epigallocatechin-3-Gallate on Breast Cancer Cells
Physiological concentrations of the green tea extract epigallocatechin-3-gallate (EGCG) caused growth inhibition in estrogen receptor ? (ER?)-positive MCF7 cells that was associated with down-regulation of the ER? and reduced insulin-like growth factor binding protein-2 abundance and increased protein abundance of the tumor suppressor genes p53/p21. In contrast to MCF7 cells that have wt p53, EGCG alone did not change cell proliferation or death significantly in another ER?-positive cell line T47D that possesses mutant p53. EGCG increased ER? protein levels and as a consequence, the cells responded significantly better to an ER? antagonist tamoxifen (TAM) in the presence of EGCG. EGCG significantly increased cell death in an ER?-negative cell line, MDA-MB-231 that also possesses mutant p53. EGCG significantly increased the ER? and insulin-like growth factor-I receptor levels and thereby enhanced the sensitivities of the cells to TAM and a blocking antibody targeting the insulin-like growth factor-1 receptor (?IR3). In contrast to MCF7, T47D and MDA-MB-231 breast cancer cells that exhibited significant changes in key molecules involved in breast growth and survival upon treatment with physiological levels of EGCG, the growth, survival, and levels of these proteins in non-malignant breast epithelial cells, MCF10A cells, were not affected.
Zeng, Li; Holly, Jeff M. P.; Perks, Claire M.
Cloning and expression of full-length cDNA encoding human vitamin D receptor
Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3� noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream
A. R. Baker; D. P. McDonnell; M. Hughes; T. M. Crisp; D. J. Mangelsdorf; M. R. Haussler; J. W. Pike; J. Shine; B. W. OMalley
Human chorionic gonadotropin decreases human breast cancer cell proliferation and promotes differentiation.
Human chorionic gonadotropin (hCG) is a glycoprotein produced by placental trophoblasts. Previous studies indicated that hCG could be responsible for the pregnancy-induced protection against breast cancer in women. It is reported that hCG decreases proliferation and invasion of breast cancer MCF-7 cells. Our research also demonstrates that hCG can reduce the proliferation of MCF-7 cells by downregulating the expression of proliferation markers, proliferating cell nuclear antigen (PCNA), and proliferation-related Ki-67 antigen (Ki-67). Interestingly, we find here that hCG elevates the state of cellular differentiation, as characterized by the upregulation of differentiation markers, ?-casein, cytokeratin-18 (CK-18), and E-cadherin. Inhibition of hCG secretion or luteinizing hormone/hCG receptors (LH/hCGRs) synthesis can weaken the effect of hCG on the induction of cell differentiation. Furthermore, hCG can suppress the expression of estrogen receptor alpha. hCG activated receptor-mediated cyclic adenosine monophosphate/protein kinase A signaling pathway. These findings indicated that a protective effect of hCG against breast cancer may be associated with its growth inhibitory and differentiation induction function in breast cancer cells. � 2014 IUBMB Life, 66(5):352-360, 2014. PMID:24753159
Liao, Xing-Hua; Wang, Yue; Wang, Nan; Yan, Ting-Bao; Xing, Wen-Jing; Zheng, Li; Zhao, Dong-Wei; Li, Yan-Qi; Liu, Long-Yue; Sun, Xue-Guang; Hu, Peng; Zhang, Tong-Cun
BreastDefend(TM) prevents breast-to-lung cancer metastases in an orthotopic animal model of triple-negative human breast cancer
We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of MDA-MB-231 invasive human breast cancer cells in vitro. In the present study, we evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. Oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastric gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67% (control) to 20% (BD) (p<0.05) and the number of metastases from 2.8 (0.0, 48.0) in the control group to 0.0 (0.0, 14.2) in the BD treatment group (p<0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the downregulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers.
BreastDefend� prevents breast-to-lung cancer metastases in an orthotopic animal model of triple-negative human breast cancer.
We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of MDA-MB-231 invasive human breast cancer cells in vitro. In the present study, we evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. Oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastric gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67% (control) to 20% (BD) (p<0.05) and the number of metastases from 2.8 (0.0, 48.0) in the control group to 0.0 (0.0, 14.2) in the BD treatment group (p<0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the downregulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers. PMID:22842551
Jiang, Jiahua; Thyagarajan-Sahu, Anita; Loganathan, Jagadish; Eliaz, Isaac; Terry, Colin; Sandusky, George E; Sliva, Daniel
Boron-Based 4-Hydroxytamoxifen Bioisosteres for Treatment of de Novo Tamoxifen Resistant Breast Cancer
Tamoxifen remains the first line therapy for estrogen receptor positive (ER+) breast cancer. However, polymorphisms of the gene encoding P450 2D6 could result in no protein expression or no CYP2D6 enzymatic activity and may significantly reduce the benefit of the hormone therapy. To address this issue, we designed and synthesized three 4-hydroxytamoxifen bioisosteres utilizing a boron-aryl carbon bond that can be oxidized under physiological conditions to yield 4-hydroxytamoxifen. We show that the bioisosteres inhibit the growth of two ER+ breast cancer cell lines, MCF-7 and T47D, with potencies comparable to or greater than that of 4-hydroxytamoxifen. We further demonstrate that after incubation with breast cancer cells, the majority of the bioisosteres has been converted to 4-hydroxytamoxifen. Our study suggests that boron-based 4-hydroxytamoxifen bioisosteres may be an effective therapeutic remedy for intrinsic tamoxifen resistance in breast cancer patients deficient in CYP2D6 metabolism.
Sequence dependent potentiation of gemcitabine by flavopiridol in human breast cancer cells
Summary Purpose. Flavopiridol is a novel cyclin dependent kinase (cdk) inhibitor currently in Phase I and II clinical trials. We investigated the interaction between flavopiridol and gemcitabine in two human breast cancer cell lines.
Shadan Ali; Basil F. El-Rayes; Olivia Aranha; Fazlul H. Sarkar; Philip A. Philip
Effect of COX-2 Inhibitors on the Aromatase Gene (CYP19) Expression in Human Breast Cancer.
National Technical Information Service (NTIS)
Aromatase (CYP19) is responsible for estrogen biosynthesis, and CYP- 19 and cyclooxygenase-2 (COX-2) are both overexpressed in human breast cancers. Prostaglandin activates the CYP19 promotor and increases gene expression therefore we hypothesized that ce...
C. L. Shapiro
Oncostatin-M promotes phenotypic changes associated with mesenchymal and stem cell-like differentiation in breast cancer.
Cancer stem cell (CSC) biology and the epithelial-to-mesenchymal transition (EMT) are thought to be mechanistically linked and may be key components of cancer development and progression. However, stimuli that induce EMT and CSC-like features ('stemness') are poorly defined. We and others have shown that the inflammatory cytokine oncostatin-M (OSM) mediates phenotypic changes in breast cancer that are consistent with EMT and dedifferentiation, including enhanced migration and loss of hormone receptors. In this study, we have expanded on these prior observations to determine whether OSM is a cell-extrinsic driver of EMT and/or stemness. OSM stimulation of the luminal breast cancer cell lines MCF7 and T47D induced EMT features including loss of membranous E-cadherin and induction of snail and slug expression. OSM treatment markedly enhanced the formation of mammospheres (up to 20-fold, P<0.001), which displayed high expression of the pluripotency factor SOX2. The proportion of cells with a CD44(high)CD24(-/low) phenotype was similarly increased by OSM (P<0.001). OSM-induced mammosphere formation and CD44(high)CD24(-/low) induction was dependent on PI3K signalling. In silico analysis of human breast tumours (from a publicly available data set, n=322) confirmed that co-expression of a PI3K transcriptional signature, but not MAPK or STAT3 signatures, was necessary to detect an association between OSMR and poor prognosis. Assessment of a second in silico data set (n=241 breast tumours) confirmed a significant relationship between OSMR, markers of EMT and CSCs, and chemotherapy resistance. Direct analysis of mRNA expression by RT-PCR in a third cohort (n=72 breast tumours) demonstrated that high expression of OSM is associated positively with indicators of EMT (SNAI1, P<0.001) and stemness (SOX2, P<0.05). Our data suggest for the first time that OSM may promote a clinically relevant EMT/CSC-like phenotype in human breast cancer via a PI3K-dependent mechanism. PMID:23584474
West, N R; Murray, J I; Watson, P H
Endothelin messenger RNA and receptors are differentially expressed in cultured human breast epithelial and stromal cells.
Paracrine regulation is implicit in the biosynthesis and secretion of milk in the breast. An important determinant for this regulation in vivo is proximate cellular location as exemplified by stromal and epithelial cells in breast tissue. Cultured human breast epithelial cells exhibited low constitutive expression of mRNA for endothelin which was enhanced 20-fold after prolactin stimulation. Human breast stromal cells did not express measurable levels of endothelin mRNA under similar conditions. In a similar differential manner, the stimulated release of immunoreactive endothelin into medium overlay was observed only for breast epithelial and not stromal cells. Specific cell-surface receptors for endothelin and biochemical responsiveness to the peptide were observed only in the stromal cells. Images
Baley, P A; Resink, T J; Eppenberger, U; Hahn, A W
IL-6-induced epithelial-mesenchymal transition promotes the generation of breast cancer stem-like cells analogous to mammosphere cultures.
Recently, the inflammatory cytokine IL-6 has been reported as a potent inducer of epithelial-mesenchymal transition (EMT) in breast cancer cells with an epithelial phenotype. Furthermore, EMT induces stem cell features in normal and transformed mammary cells. We explored whether IL-6-induced EMT promoted the generation of breast cancer stem-like cells (BrCSCs) in epithelial-like breast cancer cells, and whether the cytokines EGF and bFGF, analogous to IL-6, per se induced epithelial-mesenchymal transition, resulting in the enrichment of BrCSCs in mammosphere cultures. Herein, we provide evidence that IL-6 is capable of generating CD44+ cells with stem-like properties through induction of the EMT in the epithelial-like T47D breast cancer cells. We also show that mammosphere cultures of epithelial-like breast cancer cells, T47D, MCF7, ZR-75-1 and MDA-MB-453 cells, consistently generated stem-like cancer cells solely as a result of the EGF and bFGF cytokines in the mammosphere media mediating EMT. This finding demonstrated the link between the inflammatory cytokine IL-6 and BrCSCs and identified an important mechanism for the enrichment of BrCSCs in mammosphere cultures. Thus, EMT appears to be a critical mechanism for the induction of cancer cells with stem-like properties, and EMT of non-stem cancer cells could be a source of CSCs. PMID:22134360
Xie, Guozhu; Yao, Qiwei; Liu, Ying; Du, Shasha; Liu, Aihua; Guo, Zhaoze; Sun, Aimin; Ruan, Jian; Chen, Longhua; Ye, Changsheng; Yuan, Yawei
Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans
We present three-dimensional (3D) in vivo images of human breast cancer based on fluorescence diffuse optical tomography (FDOT). To our knowledge, this work represents the first reported 3D fluorescence tomography of human breast cancer in vivo. In our protocol, the fluorophore Indocyanine Green (ICG) is injected intravenously. Fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using
Alper Corlu; Regine Choe; Turgut Durduran; Mark A. Rosen; Martin Schweiger; Simon R. Arridge; Mitchell D. Schnall; Arjun G. Yodh
Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status
Potential antiproliferative effects of tocotrienols, the major vitamin E component in palm oil, were investigated on the growth\\u000a of both estrogen-responsive (ER+) MCF7 human breast cancer cells and estrogen-unresponsive (ER-) MDA-MD-231 human breast cancer\\u000a cells, and effects were compared with those of ?-tocopherol (?T). The tocotrienol-rich fraction (TRF) of palm oil inhibited\\u000a growth of MCF7 cells in both the presence
Kalanithi Nesaretnam; Ruth Stephen; Ray Dils; Philippa Darbre
Targeting of Rac GTPases blocks the spread of intact human breast cancer
High expression of Rac small GTPases in invasive breast ductal carcinoma is associated with poor prognosis, but its therapeutic value in human cancers is not clear. The aim of the current study was to determine the response of human primary breast cancers to Rac-based drug treatments ex vivo. Three-dimensional organotypic cultures were used to assess candidate therapeutic avenues in invasive breast cancers. Uniquely, in these primary cultures, the tumour is not disaggregated, with both epithelial and mesenchymal components maintained within a three-dimensional matrix of type I collagen. EHT 1864, a small molecule inhibitor of Rac GTPases, prevents spread of breast cancers in this setting, and also reduces proliferation at the invading edge. Rac1+ epithelial cells in breast tumours also contain high levels of the phosphorylated form of the transcription factor STAT3. The small molecule Stattic inhibits activation of STAT3 and induces effects similar to those seen with EHT 1864. Pan-Rac inhibition of proliferation precedes down-regulation of STAT3 activity, defining it as the last step in Rac activation during human breast cancer invasion. Our data highlights the potential use of Rac and STAT3 inhibition in treatment of invasive human breast cancer and the benefit of studying novel cancer treatments using three-dimensional primary tumour tissue explant cultures.
Katz, Elad; Sims, Andrew H.; Sproul, Duncan; Caldwell, Helen; Dixon, J. Michael; Meehan, Richard R.; Harrison, David J.
Tissue Specific DNA Methylation in Normal Human Breast Epithelium and in Breast Cancer
Cancer is a heterogeneous and tissue-specific disease. Thus, the tissue of origin reflects on the natural history of the disease and dictates the therapeutic approach. It is suggested that tissue differentiation, mediated mostly by epigenetic modifications, could guide tissue-specific susceptibility and protective mechanisms against cancer. Here we studied breast specific methylation in purified normal epithelium and its reflection in breast cancers. We established genome wide methylation profiles of various normal epithelial tissues and identified 110 genes that were differentially methylated in normal breast epithelium. A number of these genes also showed methylation alterations in breast cancers. We elaborated on one of them, TRIM29 (ATDC), and showed that its promoter was hypo-methylated in normal breast epithelium and heavily methylated in other normal epithelial tissues. Moreover, in breast carcinomas methylation increased and expression decreased whereas the reverse was noted for multiple other carcinomas. Interestingly, TRIM29 regulation in breast tumors clustered according to the PAM50 classification. Thus, it was repressed in the estrogen receptor positive tumors, particularly in the more proliferative luminal B subtype. This goes in line with previous reports indicating tumor suppressive activity of TRIM29 in estrogen receptor positive luminal breast cells in contrast to oncogenic function in pancreatic and lung cancers. Overall, these findings emphasize the linkage between breast specific epigenetic regulation and tissue specificity of cancer.
Avraham, Ayelet; Cho, Sean Soonweng; Uhlmann, Ronit; Polak, Mia Leonov; Sandbank, Judith; Karni, Tami; Pappo, Itzhak; Halperin, Ruvit; Vaknin, Zvi; Sella, Avishay; Sukumar, Saraswati; Evron, Ella
Human Progesterone Receptor Displays Cell Cycle-Dependent Changes in Transcriptional Activity
The human progesterone receptor (PR) contains multiple Ser-Pro phosphorylation sites that are potential substrates for cyclin-dependent kinases, suggesting that PR activity might be regulated during the cell cycle. Using T47D breast cancer cells stably transfected with an mouse mammary tumor virus (MMTV) chloramphenicol acetyltransferase reporter (Cat0) synchronized in different phases of the cell cycle, we found that PR function and phosphorylation is remarkably cell cycle dependent, with the highest activity in S phase. Although PR expression was reduced in the G2/M phase, the activity per molecule of receptor was markedly reduced in both G1 and G2/M phases compared to the results seen with the S phase of the cell cycle. Although PR is recruited to the MMTV promoter equivalently in the G1 and S phases, recruitment of SRC-1, SRC-3, and, consequently, CBP is reduced in G1 phase despite comparable expression levels of SRC-1 and SRC-3. In G2/M phase, site-specific phosphorylation of PR at Ser162 and at Ser294, a site previously reported to be critical for transcriptional activity and receptor turnover, was abolished. Treatment with the histone deacetylase inhibitor trichostatin A elevated G1 and G2/M activity to that of the S phase, indicating that the failure to recruit sufficient levels of active histone acetyltransferase is the primary defect in PR-mediated transactivation.
Narayanan, Ramesh; Edwards, Dean P.; Weigel, Nancy L.
Human-Compatible Animal Models for Preclinical Research on Hormones in Breast Cancer.
National Technical Information Service (NTIS)
Our most significant findings during the second year of work included the following: (1) The PRL-humanized mice synthesize and secrete hPRL that is fully biologically active at the human PRL receptor as evidenced by activation of STAT5 in human breast can...
K. A. Gregerson
Human Leukocyte Antigen-G (HLA-G) Polymorphism and Expression in Breast Cancer Patients
Human leukocyte antigen-G (HLA-G) is known to be implicated in a tumor-driven immune escape mechanism in malignancies. The purpose of this study was to investigate HLA-G polymorphism and expression in breast cancer. HLA-G alleles were determined by direct DNA sequencing procedures from blood samples of 80 breast cancer patients and 80 healthy controls. Soluble HLA-G (sHLA-G) was measured by enzyme-linked immunosorbent assay (ELISA) from serum specimens. HLA-G expression in breast cancer lesions was also analyzed by immunohistochemistry staining. The presence of HLA-G 3? untranslated region (UTR) 14-bp sequence was analyzed and found to be associated with reduced risk of breast cancer susceptibility based on HLA-G expression in tissues (P?=?0.0407). Levels of sHLA-G were higher in the breast cancer group (median 117.2 U/mL) compared to the control group (median 10.1 U/mL, P<0.001). The area under the receiver operating characteristic curve (AU-ROC) values of sHLA-G for differentiating breast cancer from normal controls and for detecting metastasis from other stages of breast cancer were 0.89 and 0.79, respectively. HLA-G polymorphism and expression may be involved in breast carcinogenesis and sHLA-G concentrations could be used as a diagnostic marker for detecting breast cancer.
Jeong, Seri; Park, Seho; Park, Byeong-Woo; Park, Younhee; Kwon, Oh-Joong; Kim, Hyon-Suk
Gadd45a levels in human breast cancer are hormone receptor dependent
Background Gadd45a is a member of the Gadd45 family of genes that are known stress sensors. Gadd45a has been shown to serve as an effector in oncogenic stress in breast carcinogenesis in murine models. The present study was aimed at clarifying the expression of Gadd45a in human breast cancer and its correlation with clinicopathologic features. Methods The expression levels of Gadd45a in breast tissue samples of female breast surgery cases were examined by immunohistochemistry (IHC) using a Gadd45a antibody. Percent staining was determined and statistical analyses were applied to determine prognostic correlations. Results 56 female breast surgery cases were studied: Normal (11), Luminal A (9), Luminal B (11), HER2+ (10), Triple Negative (15). There was a highly significant difference in percent Gadd45a staining between groups [Mean]: Normal 16.3%; Luminal A 65.3%; Luminal B 80.7%; HER2+ 40.5%; TN 32%, P?0.001, ANOVA. Gadd45a IHC levels for Normal cases found 82% negative/low. Luminal A breast cancer cases were found to be 67% high. Luminal B breast cancers were 100% high. Her2+ cases were 50% negative/low. Triple Negative cases were 67% negative/low. This difference in distribution of Gadd45a levels across breast cancer receptor subtypes was significant, P?=?0.0009. Conclusions Gadd45a levels are significantly associated with hormone receptor status in human breast cancer. Normal breast tissue displays low Gadd45a levels. High Gadd45a levels are associated with Luminal A and Luminal B subtypes. Absence of hormone receptors in Triple Negative subtype is associated with Negative/Low levels of Gadd45a. Further studies are indicated to elucidate the role of Gadd45a in breast cancer as a potential prognosticator or target for treatment.
Role of ferritin alterations in human breast cancer cells
Breast cancer is the most common malignancy in women. Successful treatment of breast cancer relies on a better understanding\\u000a of the molecular mechanisms involved in breast cancer initiation and progression. Recent studies have suggested a crucial\\u000a role of perturbations in ferritin levels and tightly associated with this, the deregulation of intracellular iron homeostasis;\\u000a however, the underlying molecular mechanisms for the
Svitlana I. Shpyleva; Volodymyr P. Tryndyak; Olga Kovalchuk; Athena Starlard-Davenport; Vasyl� F. Chekhun; Frederick A. Beland; Igor P. Pogribny
Decreased expression of human D-glucuronyl C5-epimerase in breast cancer.
D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes in proteoglycan biosynthesis. However, nothing is known about expression and activity of the protein in cancer. In this study, we investigated GLCE expression in human breast cancer using multipex RT-PCR, QRT-PCR and Western-blot assays. In total, 21 patients without malignancy and 74 patients with breast tumor were investigated. The obtained data showed that in 82-84% of human breast tumors there is either downregulation or loss of D-glucuronyl C5-epimerase mRNA expression and significant decrease of the protein content. In most cases (77%), GLCE expression was decreased also in the normal-appearing tissue surrounding the tumor node but the protein amount was comparable to normal breast tissue. These findings represent the first data about involvement of human D-glucuronyl C5-epimerase in malignant transformation. PMID:17985344
Grigorieva, Elvira; Eshchenko, Tatiana; Rykova, Valentina I; Chernakov, Alexei; Zabarovsky, Eugene; Sidorov, Sergei V
RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis
Breast cancer occur both in hereditary and sporadic forms, and the later one comprises an overwhelming majority of breast cancer cases among women. Numerical and structural alterations involving chromosome 8, with loss of short arm (8p) and gain of long arm (8q), are frequently observed in breast cancer cells and tissues. In this study, we show that most of the human breast tumor cell lines examined display an over representation of 8q24, a chromosomal locus RecQL4 is regionally mapped to, and consequently, a markedly elevated level of RecQL4 expression. An increased RecQL4 mRNA level was also observed in a majority of clinical breast tumor samples (38/43) examined. shRNA-mediated RecQL4 suppression in MDA-MB453 breast cancer cells not only significantly inhibit the in vitro clonogenic survival and in vivo tumorigenicity. Further studies demonstrate that RecQL4 physically interacts with a major survival factor-survivin and its protein level affects survivin expression. Although loss of RecQL4 function due to gene mutations causally linked to occurrence of human RTS with features of premature aging and cancer predisposition, our studies provide the evidence that overexpression of RecQL4 due to gene amplification play a critical role in human breast tumor progression.
Chi, Zhenfen; Liu, Jing; Guo, Dan; Lu, Xuemei; Hei, Tom K.; Balajee, Adayabalam S.; Zhao, Yongliang
Inappropriate production of collagen and prolyl hydroxylase by human breast cancer cells in vivo.
Thirty-two scirrhous cancers of breast have been examined to determine the origin of the collagen stroma in these tumours. Employing two immunohistochemical techniques it has been shown that the malignant epithelial cells in 30 of these tumours contain not only collagen but also prolyl hydroxylase, a key enzyme in collagen biosynthesis. Neither this enzyme nor collagen was detectable in the spindle cells in the stroma of these tumours. Neither the epithelium in normal breast, that in fibrocystic disease and in fibroadenomata, nor the malignant epithelium in two medullary cancers of breast contained either collagen or prolyl hydroxylase. These results strongly suggest that the malignant epithelium of scirrhous breast cancers produces its own collagen stroma and that the scirrhous reaction in these tumours is not a host response to tumour invasion. The production of collagen and prolyl hydroxylase by breast cancer cells (of the scirrhous type) therefore represents another example of inappropriate protein production by a human tumour. PMID:169865
Al-Adnani, M S; Kirrane, J A; McGee, J O
Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors
Genomic DNA copy number alterations are key genetic events in the development and progression of human cancers. Here we report a genome-wide microarray comparative genomic hybridization (array CGH) analysis of DNA copy number variation in a series of primary human breast tumors. We have profiled DNA copy number alteration across 6,691 mapped human genes, in 44 predominantly advanced, primary breast
Jonathan R. Pollack; Therese S�rlie; Charles M. Perou; Christian A. Rees; Stefanie S. Jeffrey; Per E. Lonning; Robert Tibshirani; David Botstein; Anne-Lise B�rresen-Dale; Patrick O. Brown
Cloning of Novel Oncogenes Involved in Human Breast Cancer.
National Technical Information Service (NTIS)
While the aberrant overexpression of HER family receptors is known to be involved in breast cancer, the complex nature of the genetic events that trigger the development of breast cancer remain to be identified. The authors have developed, refined, and ap...
C. J. Der
Estrogen Receptor Alpha in Human Breast Cancer: Occurrence and Significance
Estrogens have long been recognized as being important for stimulating the growth of a large proportion of breast cancers. Now it is recognized that estrogen action is mediated by two receptors, and the presence of estrogen receptor a (ERa)3 correlates with better prognosis and the likelihood of response to hormonal therapy. Over half of all breast cancers overexpress ERa and
Simak Ali; R. Charles Coombes
Phorbol esters induce multidrug resistance in human breast cancer cells
Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood. Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C. When the authors assayed human breast cancer cell lines for protein kinase C activity, they found that enzyme activity was 7-fold higher in the multidrug-resistance cancer cells compared with the control, sensitive parent cells. Exposure of drug-sensitive cells to the phorbol ester phorbol 12,13-dibutyate (P(BtO)/sub 2/) led to an increase in protein kinase C activity and induced a drug-resistance phenotype, whereas exposure of drug-resistant cells to P(BtO)/sub 2/ further increased drug resistance. In sensitive cells, this increased resistance was accomplished by a 3.5-fold increased phosphorylation of a 20-kDa particulate protein and a 35-40% decreased intracellular accumulation of doxorubicin and vincristine. P(BtO)/sub 2/ induced resistance to agents involved in the multidrug-resistant phenotype (doxorubicin and vincristine) but did not affect sensitivity to an unrelated alkylating agent (melphalan). The increased resistance was partially or fully reversible by the calcium channel blocker verapamil and by the calmodulin-antagonist trifluoperazine. These data suggest that stimulation of protein kinase C playus a role in the drug-transport changes in multidrug-resistant cells. This may occur through modulation of an efflux pump by protein phosphorylation.
Fine, R.L.; Patel, J.; Chabner, B.A.
Growth characteristics and metastatic properties of human breast cancer xenografts in immunodeficient mice.
We evaluated the growth and metastatic potential of two human breast cancer cell lines and 16 patient-derived biopsy specimens, representing the most common histological types of breast carcinomas, upon subcutaneous implantation into severe combined immunodeficient (SCID) mice. The method of engraftment we used, based on implantation of intact tissue specimens and complete immunosuppression of the host, provided an easier system to grow human breast carcinoma specimens in mouse models and resulted in a 50% success rate of tumor take. No correlation was found between growth in SCID mice and pathological diagnosis, grading, or estrogen/progesterone receptor expression by the tumor biopsy specimen. Serial passage of the tumor fragments in SCID mice resulted in increased metastasis rates and more rapid emergence of a palpable tumor mass. A tumor from a patient with infiltrating ductal carcinoma, which grew aggressively and metastasized in 100% of the female SCID mice, was also successfully engrafted in 100% of nonobese diabetic (NOD)/SCID female mice, but systemic spread was minimal. Fragments of the same tumor grew in only 33% of male SCID mice with very limited metastases. A strong correlation (r = 0.997) was observed between tumor burden and the presence of soluble (serum) interleukin-2 receptor, a marker associated with a subset of human breast tumors. All together, these data indicate the usefulness of SCID/human breast tumor xenografts for measuring tumor progression and evaluating novel therapeutic approaches to breast cancer. Images Figure 1 Figure 2 Figure 3 Figure 5
Visonneau, S.; Cesano, A.; Torosian, M. H.; Miller, E. J.; Santoli, D.
Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion
Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (?0 cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and ?0 epithelial cells. The focal proteins in these networks include (i) FN1 (fibronectin) and (ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 3 of 6 members of peroxiredoxin whose expression were altered in ?0 epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect chromosomal stability. Consistent with above finding our study revealed an increase in DNA double strand breaks and unique chromosomal rearrangements in ?0 breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that (i) claudin-1 and 7 were indeed downregulated in ?0 breast epithelial cells, (ii) downregulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and (iii) claudin-1 and -7 were also downregulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells.
Kulawiec, Mariola; Safina, Alfiya; Desouki, Mohamed Mokhtar; Still, Ivan; Matsui, Sei-Ichi; Bakin, Andrei; Singh, Keshav K.
Persistent Pesticides in Human Breast Milk and Cryptorchidism
Introduction Prenatal exposure to some pesticides can adversely affect male reproductive health in animals. We investigated a possible human association between maternal exposure to 27 organochlorine compounds used as pesticides and cryptorchidism among male children. Design Within a prospective birth cohort, we performed a case�control study; 62 milk samples from mothers of cryptorchid boys and 68 from mothers of healthy boys were selected. Milk was collected as individual pools between 1 and 3 months postpartum and analyzed for 27 organochlorine pesticides. Results Eight organochlorine pesticides were measurable in all samples (medians; nanograms per gram lipid) for cases/controls: 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p?-DDE): 97.3/83.8; ?-hexachlorocyclohexane (?-HCH): 13.6/12.3; hexachlorobenzene (HCB): 10.6/8.8; ? -endosulfan: 7.0/6.7; oxychlordane: 4.5/4.1; 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p?-DDT): 4.6/4.0; dieldrin: 4.1/3.1; cis-heptachloroepoxide (cis-HE): 2.5/2.2. Five compounds [octachlorostyrene (OCS); pentachlorobenzene, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p?-DDD); o,p?-DDT; mirex] were measurable in most samples (detection rates 90.8�99.2%) but in lower concentrations. For methoxychlor, cis-chlordane, pentachloroanisole (PCA), ? -HCH, 1,1-dichloro-2-(2-chlorophenyl)-2,2(4-chlorophenyl)ethane, trans-chlordane, ? -HCH, and o,p?-DDE, both concentrations and detection rates were low (26.5�71.5%). Heptachlor, HCH (?, ? ), aldrin, ?-endosulfan and trans-heptachloroepoxide were detected at negligible concentrations and low detection rates and were not analyzed further. Seventeen of 21 organochlorine pesticides [p,p?-DDT, p,p?-DDE, p,p?-DDD, o,p?-DDT, HCH (? , ?, ? ), HCB, PCA, ? -endosulfan, cis-HE, chlordane (cis-, trans-) oxychlordane, methoxychlor, OCS, and dieldrin] were measured in higher median concentrations in case milk than in control milk. Apart from trans-chlordane (p = 0.012), there were no significant differences between cryptorchid and healthy boys for individual chemicals. However, combined statistical analysis of the eight most abundant persistent pesticides showed that pesticide levels in breast milk were significantly higher in boys with cryptorchidism (p = 0.032). Conclusion The association between congenital cryptorchidism and some persistent pesticides in breast milk as a proxy for maternal exposure suggests that testicular descent in the fetus may be adversely affected.
Damgaard, Ida N.; Skakkebaek, Niels E.; Toppari, Jorma; Virtanen, Helena E.; Shen, Heqing; Schramm, Karl-Werner; Petersen, J?rgen H.; Jensen, Tina K.; Main, Katharina M.
Fibroblast activation protein expression by stromal cells and tumor-associated macrophages in human breast cancer.
Fibroblast activation protein (FAP) has long been known to be expressed in the stroma of breast cancer. However, very little is known if the magnitude of FAP expression within the stroma may have a prognostic value and reflect the heterogeneous biology of the tumor cell. An earlier study had suggested that stromal FAP expression in breast cancer was inversely proportional to prognosis. We, therefore, hypothesized that stromal FAP expression may correlate with clinicopathologic variables and may serve as an adjunct prognostic factor in breast cancer. We evaluated the expression of FAP in a panel of breast cancer tissues (n = 52) using a combination of immunostain analyses at the tissue and single-cell level using freshly frozen or freshly digested human breast tumor samples, respectively. Our results showed that FAP expression was abundantly expressed in the stroma across all breast cancer subtypes without significant correlation with clinicopathologic factors. We further identified a subset of FAP-positive (or FAP(+)) stromal cells that also expressed CD45, a pan-leukocyte marker. Using freshly dissociated human breast tumor specimens (n = 5), we demonstrated that some of these FAP(+)CD45(+) cells were CD11b(+)CD14(+)MHC-II(+), indicating that they were likely tumor-associated macrophages (TAMs). Although FAP(+)CD45(+) cells have been demonstrated in the mouse tumor stroma, our results demonstrating that human breast TAMs expressed FAP were novel and suggested that existing and future FAP-directed therapy may have dual-therapeutic benefits targeting both stromal mesenchymal cells and immune cells such as TAMs. More work is needed to explore the role of FAP as a potential targetable molecule in breast cancer treatment. PMID:24074532
Tchou, Julia; Zhang, Paul J; Bi, Yingtao; Satija, Celine; Marjumdar, Rajrupa; Stephen, Tom L; Lo, Albert; Chen, Haiying; Mies, Carolyn; June, Carl H; Conejo-Garcia, Jose; Pur�, Ellen
Detection and genotyping of human papillomavirus in breast cancer tissues from Iraqi patients.
Studies have suggested a possible link between breast cancer pathogenesis and human papillomavirus (HPV) infection. This study in Iraq used in situ hybridization to detect the frequency and genotyping of HPV in tissue specimens from 129 patients diagnosed with malignant breast cancer, 24 with benign breast tumours and 20 healthy controls. In the breast cancer group, cocktail HPV genotypes were detected in 60 (46.5%) archived tissue blocks. Of these, genotypes 16 (55.5%), 18 (58.4%), 31 (65.0%) and 33 (26.6%) were detected. Mixed HPV genotypes 16 + 18, 16 + 18 + 31, 16 + 18 + 33, 18 + 33, 16 + 31 and 18 + 31 were found in 5.0%, 25.0%, 8.3%, 7.7%, 10.0% and 13.3% of cancer cases respectively. Only 3 benign breast tumour tissues (12.5%) and none of the healthy breast tissue specimens were HPV-DNA-positive. The detection of high-oncogenic HPV genotypes in patients with breast cancer supports the hypothesis of an etiologic role for the virus in breast cancer development. PMID:24960513
Ali, S H M; Al-Alwan, N A S; Al-Alwany, S H M
MMTV mouse models and the diagnostic values of MMTV-like sequences in human breast cancer
Mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-driven transgenic mice are excellent models for breast cancer as they allow for the targeted expression of various oncogenes and growth factors in neoplastic transformation of mammary glands. Numerous MMTV-LTR-driven transgenic mouse models of breast cancer have been created in the past three decades, including MMTV-neu/ErbB2, cyclin D1, cyclin E, Ras, Myc, int-1 and c-rel. These transgenic mice develop mammary tumors with different latency, histology and invasiveness, reflecting the oncogenic pathways activated by the transgene. Recently, homologous sequences of the env gene of MMTV have been identified in approximately 40% of human breast cancers, but not in normal breast or other types of cancers, suggesting possible involvement of mammary tumor virus in human breast carcinogenesis. Accumulating evidence demonstrates the association of MMTV provirus with progesterone receptor, p53 mutations and advanced-stage breast cancer. Thus, the detection of MMTV-like sequences may have diagnostic value to predict the clinical outcome of breast cancer patients.
Taneja, Pankaj; Frazier, Donna P; Kendig, Robert D; Maglic, Dejan; Sugiyama, Takayuki; Kai, Fumitake; Taneja, Neetu K; Inoue, Kazushi
Koilocytes indicate a role for human papilloma virus in breast cancer
Background: High-risk human papilloma viruses (HPVs) are candidates as causal viruses in breast cancer. The scientific challenge is to determine whether HPVs are causal and not merely passengers or parasites. Studies of HPV-related koilocytes in breast cancer offer an opportunity to address this crucial issue. Koilocytes are epithelial cells characterised by perinuclear haloes surrounding condensed nuclei and are commonly present in cervical intraepithelial neoplasia. Koilocytosis is accepted as pathognomonic (characteristic of a particular disease) of HPV infection. The aim of this investigation is to determine whether putative koilocytes in normal and malignant breast tissues are because of HPV infection. Methods: Archival formalin-fixed normal and malignant breast specimens were investigated by histology, in situ PCR with confirmation of the findings by standard PCR and sequencing of the products, plus immunohistochemistry to identify HPV E6 oncoproteins. Results: human papilloma virus-associated koilocytes were present in normal breast skin and lobules and in the breast skin and cancer tissue of patients with ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDCs). Interpretation: As koilocytes are known to be the precursors of some HPV-associated cervical cancer, it follows that HPVs may be causally associated with breast cancer.
Lawson, J S; Glenn, W K; Heng, B; Ye, Y; Tran, B; Lutze-Mann, L; Whitaker, N J
Flaxseed inhibits metastasis and decreases extracellular vascular endothelial growth factor in human breast cancer xenografts.
Angiogenesis is important in tumor growth, progression and metastatic dissemination. Vascular endothelial growth factor (VEGF) is one key factor in promotion of breast cancer angiogenesis. VEGFs are bioactive in the extracellular space where they become available to the endothelial cells. Phytoestrogens such as lignans have been shown to alter breast cancer incidence and be cancer-protective in rats. We show that supplementation of 10% flaxseed, the richest source of mammalian lignans, to nude mice with established human breast tumors reduced tumor growth and metastasis. Moreover, flaxseed decreased extracellular levels of VEGF, which may be one mechanistic explanation to the decreased tumor growth and metastasis. PMID:12142076
Dabrosin, Charlotta; Chen, Jianmin; Wang, Linda; Thompson, Lilian U
Recurrent Breast Abscesses due to Corynebacterium kroppenstedtii, a Human Pathogen Uncommon in Caucasian Women.
Background. Corynebacterium kroppenstedtii (Ck) was first described in 1998 from human sputum. Contrary to what is observed in ethnic groups such as Maori, Ck is rarely isolated from breast abscesses and granulomatous mastitis in Caucasian women. Case Presentation. We herein report a case of recurrent breast abscesses in a 46-year-old Caucasian woman. Conclusion. In the case of recurrent breast abscesses, even in Caucasian women, the possible involvement of Ck should be investigated. The current lack of such investigations, probably due to the difficulty to detect Ck, may cause the underestimation of such an aetiology. PMID:23008788
Le Fl�che-Mat�os, Anne; Berthet, Nicolas; Lomprez, Fabienne; Arnoux, Yolande; Le Guern, Anne-Sophie; Leclercq, India; Burgui�re, Ana Maria; Manuguerra, Jean-Claude
Recurrent Breast Abscesses due to Corynebacterium kroppenstedtii, a Human Pathogen Uncommon in Caucasian Women
Background. Corynebacterium kroppenstedtii (Ck) was first described in 1998 from human sputum. Contrary to what is observed in ethnic groups such as Maori, Ck is rarely isolated from breast abscesses and granulomatous mastitis in Caucasian women. Case Presentation. We herein report a case of recurrent breast abscesses in a 46-year-old Caucasian woman. Conclusion. In the case of recurrent breast abscesses, even in Caucasian women, the possible involvement of Ck should be investigated. The current lack of such investigations, probably due to the difficulty to detect Ck, may cause the underestimation of such an aetiology.
Le Fleche-Mateos, Anne; Berthet, Nicolas; Lomprez, Fabienne; Arnoux, Yolande; Le Guern, Anne-Sophie; Leclercq, India; Burguiere, Ana Maria; Manuguerra, Jean-Claude
Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer.
We previously observed that the HERV type K (HERV-K) envelope (env) protein was expressed in the majority of human breast tumors from a U.S. cohort of women from Texas. We also made the preliminary observation that the expression of HERV-K env transcripts was associated with markers of disease progression. In this follow-up study, env protein expression was evaluated immunohistochemically in an additional 195 paraffin-embedded breast tumors from a second U.S. patient cohort (Baltimore, Maryland) and in 110 tumors from Chinese patients. Moreover, we compared env transcript expression between fresh-frozen normal and cancerous breast tissues. We observed that while env mRNA and protein expression was undetectable in normal breast tissue and in a subset of uninvolved normal-appearing tissue adjacent to the tumor epithelium, it was overexpressed in most tumors. Furthermore, env expression was associated with breast cancer progression. In Baltimore cohort women, HERV-K tumor positivity was significantly associated with disease stage and lymph node metastasis. In Chinese women, HERV-K env positivity was significantly associated with tumor size, TNM stage, and lymph node metastases, which is consistent with the observations in the U.S. cohort. We also found that Chinese breast cancer patients with a high expression of HERV-K had a decreased overall survival compared with patients who had either a moderate or low HERV-K expression in their tumors (P = 0.049, ?(2) log rank test). In conclusion, the HERV-K env gene is expressed in the majority of breast cancers from U.S. or Chinese women but not in normal breast tissue. High expression of HERV-K env protein in breast cancer patients is associated with markers of disease progression and poor disease outcome, indicating that HERV-K env protein is a novel candidate prognostic marker for breast cancer. PMID:22593804
Zhao, Jing; Rycaj, Kiera; Geng, Shanshan; Li, Ming; Plummer, Joshua B; Yin, Bingnan; Liu, Hong; Xu, Xu; Zhang, Yinchun; Yan, Yanfang; Glynn, Sharon A; Dorsey, Tiffany H; Ambs, Stefan; Johanning, Gary L; Gu, Lin; Wang-Johanning, Feng
The T61 human breast cancer xenograft: An experimental model of estrogen therapy of breast cancer
Summary Endocrine therapy is one of the principal treatment modalities of breast cancer, both in an adjuvant setting and in advanced disease. The T61 breast cancer xenograft described here provides an experimental model of the effects of estrogen treatment at a molecular level. T61 is an estrogen receptor positive tumor which was originally derived from a T1N0M0 invasive ductal cancer
Nils Briinner I; Mogens Spang-Thomsen; Kevin Cullen
Novel Aza-resveratrol analogs: synthesis, characterization and anticancer activity against breast cancer cell lines.
Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-? was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future. PMID:23273518
Siddiqui, Areej; Dandawate, Prasad; Rub, Rukhsana; Padhye, Subhash; Aphale, Shama; Moghe, Alpana; Jagyasi, Amrit; Venkateswara Swamy, K; Singh, Bhupendra; Chatterjee, Anwesha; Ronghe, Amruta; Bhat, Hari K