text:
Gefitinib targets EGFR dimerization and ERK1/2 phosphorylation to inhibit pleural mesothelioma cell proliferation.
ABSTRACT Altered EGFR activity is a causal factor for human tumor development, including malignant pleural mesotheliomas. The aim of the present study was the evaluation of the effects of Gefitinib on EGF-induced mesothelioma cell proliferation and the intracellular mechanisms involved. Cell proliferation, DNA synthesis and apoptosis were measured by MTT, thymidine incorporation and FACS analysis; EGFR, ERK1/2 and Akt expression and phosphorylation by Western blot, whereas receptor sites were analyzed by binding studies. Gefitinib inhibited EGF-induced proliferation in two mesothelioma cell lines, derived from pleural effusion (IST-Mes2) or tumor biopsy (ZL55). The treatment with Gefitinib induced cell cycle arrest in both cell lines, while apoptosis was observed only for high concentrations and prolonged drug exposure. EGF-dependent mesothelioma cell proliferation was mediated by EGFR and ERK1/2 phosphorylation, while Akt was not affected. Gefitinib inhibited both EGFR and ERK1/2 activation, being maximal at drug concentrations that induce cytostatic effects, suggesting that the proapoptotic activity of Gefitinib is independent from EGFR inhibition. Gefitinib treatment increased EGFR Bmax, possibly through membrane stabilization of inactive receptor dimers that we show to be induced by the drug also in the absence of EGF. EGFR activation of ERK1/2 represents a key pathway for pleural mesothelioma cell proliferation. Low concentrations of Gefitinib cause mesothelioma cell cycle arrest through the blockade of EGFR activity while high concentrations induce apoptosis. Finally, we propose that the formation of inactive EGFR dimers may contribute to the antitumoral activity of Gefitinib.
- SourceAvailable from: link.springer.com
Article: Significant increase in hEGF uptake is correlated with formation of EGFR dimers induced by the EGFR tyrosine kinase inhibitor gefitinib.[Show abstract] [Hide abstract]
ABSTRACT: PURPOSE: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) is approved for cancer treatment. We investigated whether gefitinib treatment can enhance human EGF (hEGF) uptake in vitro, thereby increasing the potential of hEGF as a vehicle for EGFR-targeted therapy. METHODS: Western blotting was used to detect the effect of gefitinib on EGFR signaling. Different EGFR-expressing tumor cells (SCC-1, 22B, A549, and HT-29) were pretreated with gefitinib, and then with (125)I-hEGF or (125)I-Vectibix (an anti-EGFR monoclonal antibody). Cell-associated activity was then measured. A cross-linking assay detected increased EGFR dimer formation in gefitinib-treated cells. RESULTS: Total EGFR levels were not changed, but EGFR phosphorylation was reduced in cells pretreated with gefitinib. Gefitinib mediated formation of EGFR dimers; binding of (125)I-hEGF to cells pretreated with gefitinib significantly increased. In contrast, binding of (125)I-Vectibix to tumor cells did not increase. Although total EGFR levels did not increase, binding of hEGF to EGFR + tumors was significantly enhanced after gefitinib treatment, because of increased hEGF binding to gefitinib-induced EGFR dimers. CONCLUSION: These results suggest that hEGF could enhance EGFR-targeting when used with gefitinib.Cancer Chemotherapy and Pharmacology 06/2013; · 2.80 Impact Factor
Article: Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures[Show abstract] [Hide abstract]
ABSTRACT: Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.Cell cycle (Georgetown, Tex.) 02/2013; 12(3):491-500. · 5.24 Impact Factor
Article: Cytomegalovirus-induced salivary gland pathology: resistance to Kinase inhibitors of the upregulated host cell EGFR/ERK pathway is associated with CMV-dependent stromal overexpression of IL-6 and fibronectin.[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: Recently we identified a relationship between human cytomegalovirus (hCMV) and human salivary gland (SG) mucoepidermoid carcinoma (MEC) in over 90% of cases; tumorigenesis in these cases uniformly correlated with active hCMV protein expression and an upregulation of the EGFR [rightwards arrow] ERK pathway. Our previously characterized, novel mouse organ culture model of mouse CMV (mCMV)-induced tumorigenesis displays a number of histologic and molecular characteristics similar to human MEC. METHODS: Newborn mouse submandibular glands (SMGs) were incubated with 1 x 105 PFU/ml of lacZ-tagged mCMV RM427+ on day 0 for 24 hours and then cultured in virus-free media for a total of 6 or 12 days with or without EGFR/ERK inhibitors and/or aciclovir. SMGs were collected for histology, immunolocalization (pERK, FN, IL-6), viral distribution, or Western blot analysis (pERK). RESULTS: Here we report: (1) mouse SMG tumors soon exhibit an acquired resistance to EGFR/ERK pathway kinase inhibitors, alone or in combination; (2) long term tumor regression can only be sustained by concurrent inhibitor and antiviral treatment; (3) CMV-dependent, kinase inhibitor resistance is associated with overexpression of fibronectin and IL-6 proteins in abnormal stromal cells. CONCLUSIONS: Acquired resistance to kinase inhibitors is dependent upon CMV dysregulation of alternative pathways with downstream effectors common with the targeted pathway, a phenomenon with important therapeutic implications for human MEC of salivary glands.Herpesviridae. 01/2013; 4(1):1.
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176 Current Cancer Drug Targets, 2010, 10, 176-191
1568-0096/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
Gefitinib Targets EGFR Dimerization and ERK1/2 Phosphorylation to
Inhibit Pleural Mesothelioma Cell Proliferation
R.E. Favoni*,1, A. Pattarozzi4, M. Lo Casto2, F. Barbieri4, M. Gatti4, L. Paleari#,3, A. Bajetto4,
C. Porcile4, G. Gaudino§,5, L. Mutti6, G. Corte1,4 and T. Florio4
1Department of Translational Oncology, Gene Transfer Laboratory, 2Department of Translational Oncology, Molecular
Oncology & Angiogenesis Laboratory, 3Department of Advanced Diagnostic Technologies, Lung Cancer Unit, National
Cancer Institute (Istituto Nazionale per la Ricerca sul Cancro), Largo R. Benzi, 10 - 16132 Genoa, Italy; 4Department
of Oncology, Biology and Genetics, Section of Pharmacology, University of Genoa, Viale Benedetto XV, 2 - 16132
Genoa, Italy; 5DISCAFF Department and DFBC Center University of Piemonte Orientale "A. Avogadro" - 28100 No-
vara, Italy; 6Department of General Medicine & Laboratory of Clinical Oncology Vercelli Hospital - 13100 Vercelli,
Abstract: Altered EGFR activity is a causal factor for human tumor development, including malignant pleural mesothe-
liomas. The aim of the present study was the evaluation of the effects of Gefitinib on EGF-induced mesothelioma cell pro-
liferation and the intracellular mechanisms involved. Cell proliferation, DNA synthesis and apoptosis were measured by
MTT, thymidine incorporation and FACS analysis; EGFR, ERK1/2 and Akt expression and phosphorylation by Western
blot, whereas receptor sites were analyzed by binding studies. Gefitinib inhibited EGF-induced proliferation in two meso-
thelioma cell lines, derived from pleural effusion (IST-Mes2) or tumor biopsy (ZL55). The treatment with Gefitinib in-
duced cell cycle arrest in both cell lines, while apoptosis was observed only for high concentrations and prolonged drug
exposure. EGF-dependent mesothelioma cell proliferation was mediated by EGFR and ERK1/2 phosphorylation, while
Akt was not affected. Gefitinib inhibited both EGFR and ERK1/2 activation, being maximal at drug concentrations that
induce cytostatic effects, suggesting that the proapoptotic activity of Gefitinib is independent from EGFR inhibition. Ge-
fitinib treatment increased EGFR Bmax, possibly through membrane stabilization of inactive receptor dimers that we
show to be induced by the drug also in the absence of EGF. EGFR activation of ERK1/2 represents a key pathway for
pleural mesothelioma cell proliferation. Low concentrations of Gefitinib cause mesothelioma cell cycle arrest through the
blockade of EGFR activity while high concentrations induce apoptosis. Finally, we propose that the formation of inactive
EGFR dimers may contribute to the antitumoral activity of Gefitinib.
Keywords: Human malignant pleural mesothelioma cells, EGFR targeting, Gefitinib, preclinical chemotherapy.
Human malignant pleural mesothelioma (hMPM) is a
biologically aggressive neoplasm arising from the mesothe-
lial cells lining the pleura [1]. Approximately 80% of
hMPMs is associated with chronic environmental asbestos
fiber exposure and other potential early stage co-factors, like
radiations or genetic predisposition [2]. Because most of the
patients are not suitable for radical surgery [3] or radiother-
apy [4], systemic chemotherapy is the only useful option.
Unfortunately, all chemotherapy phase II trials using single
or combination agent schedules reported poor response rates,
without significant impact on survival [5]. A meta-analysis
of studies between 1965 and 2001 found cis-platinum the
most active drug used as monotherapy [6]. Moreover, a
number of other antineoplastic agents (doxorubicin, vinorel-
bine, ifosfamide, methotrexate, gemcitabine, paclitaxel, pe-
metrexed and raltitrexed) also showed cytotoxic activity in
*Address correspondence to this author at the Department of Oncologia
Traslazionale, Lab. Trasferimento Genico, Istituto Nazionale per la Ricerca
sul Cancro (IST-Nord, B3, Rm18), Largo Rosanna Benzi, 10 - 16132
Genova, Italy; Tel: +39-10-5737.316; Fax: +39-10-5737.473;
E-mail: firstname.lastname@example.com
Present addresses: #Department of Advanced Diagnostic Technologies,
Functional Genomics Unit, National Cancer Institute, Genoa, Italy
§CRCH, Univ. of Hawaii, 651 Ilalo Street, Honolulu, HI, 96813, USA
mesotheliomas, used as single-agent treatment. At present,
the combination of folate inhibitors (either pemetrexed or
raltitrexed) and cis-platinum is considered the front line
chemotherapy for hMPM patients [7]. Two recent studies
showed that pemetrexed and gemcitabine have a synergistic
effect on hMPM cell lines in vitro [8] and that imatinib-
mesylate enhances the effect of gemcitabine in human ma-
lignant mesothelioma xenografts [9]. Nevertheless, remain-
ing hMPM a still unsolved therapeutic problem, novel trans-
lational approaches aimed to discover innovative treatments,
are emerging from the disclosure of the biochemical charac-
teristics and the elucidation of the cellular and molecular
basis of hMPM cell transformation.
In preclinical studies, pharmacological interferences with
the activity of several autocrine/paracrine growth factors,
such as EGF, TGF?, IGFs, VEGF, PDGF and their receptors
[10-15], opened novel possible therapeutic strategies for
many tumor histotypes. Particularly, EGF and its receptor
(EGFR), found to be over-expressed in human malignant
mesotheliomas especially in the epithelial histotype [10],
represent a promising therapeutic molecular target. EGFR is
a 170 KDa single-chain membrane-spanning glycoprotein
constituted by an extracellular ligand-binding domain, a
transmembrane region and an intracellular portion with in-
trinsic tyrosine kinase (TK) activity [16]. EGFR mediates
multiple cellular functions including proliferation, migration,
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Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 177
apoptosis and differentiation [16]. Upon ligand binding, the
receptor undergoes a transition from an inactive monomeric
to an active homo- and/or heterodimeric form, characterized
by the trans-phosphorylation of intra-cytoplasmic tyrosine
residues, leading to the increase of the intrinsic TK activity
and to recruitment of signaling proteins [17]. EGFR, whose
signaling is tightly controlled in normal cells, promotes on-
cogenic activation of several cytoplasmatic signaling path-
ways when it is overexpressed, and the degree of overex-
pression has been shown to be related to tumorigenicity in
different tissues. In addition, an abnormal activity of TKs, or
of key components of their intracellular signaling pathways,
could result in cancer development [18]. One strategy for
blocking EGFR signaling is the use of small, orally bioavail-
able molecules able to penetrate large tumors and selectively
inhibit TK activity. Gefitinib (ZD1839, IressaTM) is an
anilino-quinazoline derivative that selectively and competi-
tively inhibits the Mg-ATP binding within EGFR, resulting
in the suppression of TK activity and, consequently, of
EGFR phosphorylation, EGF-dependent signaling (MAPK
activation) and cell proliferation [19-21]. Wakeling et al.
first reported that Gefitinib caused tumor regression in vitro
[22]; later, it was demonstrated that tumor degeneration oc-
curred in vitro regardless the degree of EGFR overexpres-
sion [20, 23]. In vitro, Gefitinib resulted as effective, or even
more effective, against hMPM cell lines as it is against the
non-small cell lung cancer cell line A549 [24], suggesting
that the compound may be a therapeutic option for patients
with hMPM. However, subsequent studies showed that dif-
ferent TK inhibitors, including Gefitinib, when used in single
treatment modality are not effective or have only limited
activity [25].
The present study was aimed to a better elucidation of the
molecular mechanisms underlying Gefitinib effects on the
EGF-dependent tumorigenic signaling in hMPM cells. Two
previously characterized hMPM cells lines (IST-Mes2 and
ZL55) [26, 27], studied here are highly EGFR expressing
and were identified as chemosensitive and chemoresistant,
respectively, toword a panel of the so-called second genera-
tion antiblastic compounds (alchylants, antimetabolites,
apoptosis-inducers), acting via totally different mechanisms
[28]. Here, we show that Gefitinib, hampering ERK1/2 acti-
vation induced by EGF, affects at different extent the prolif-
eration of both cell lines in a concentration- and time-
dependent manner. Our results suggest that this TK inhibitor
may act not only by competing with the receptor ATP bind-
ing, but also by inducing a conformational change from
monomeric to aberrant dimeric structure, leading to a bio-
logically inactive ligand-independent receptor. These results
may provide the rationale for the possible application of Ge-
fitinib as a novel and effective therapy of hMPM.
Drugs and Reagents
The 4-anilino-quinazoline EGFRTK inhibitor Gefitinib
(ZD1839, IressaTM, C22H24ClFN4O3, MW = 446.9), provided
by AstraZeneca S.p.A. (Milano, Italy), was diluted as 10 mM
stock solution, in DMSO, aliquoted and stored at -20°C [29].
When appropriate, the same dilution of the same vehicle was
added in the respective control samples. Lyophilized recom-
binant human EGF (rhEGF) was purchased by PeproTech
(London, UK), reconstituted in ddH2O and stored, in work-
ing aliquots of 50 μg/100 μl, at -20°C. Human recombinant
(3-[125I]-iodotyrosyl) EGF, aqueous solution (s.a. > 750
Ci/mmol), was from GE Healthcare (Milano, Italy). MTT [3
- (4, 5 - dimethylthiazol - 2yl) - 2, 5 - diphenyltetrazolium
bromide)] and bovine serum albumin, fraction V, were from
Sigma-Aldrich (Milano, Italy). DMEM, foetal bovine serum
(FBS), phosphate buffered saline w/o Ca++/Mg++ (PBS), L-
glutamine and penicillin/streptomycin were supplied by
Euroclone (Pavia, Itlay). HBSS w/o Ca++/Mg++ was from
Gibco-Invitrogen (Milano, Italy). Complete mini protease
inhibitor cocktail tablets was from Roche Diagnostics GmbH
(Mannheim, Germany). Antibodies anti-EGFR and anti-
phospho-EGFR (Tyr1173); antibody against HER2 (Neu,
CB11 sc-52349), kindly provided by Dr. S. Salvi (IST -
Genova), and antibody against HER3 (C17, sc-285), kindly
provided by Dr. A. Alama (IST - Genova), were from Santa
Cruz Biotechnology (Santa Cruz, CA, USA); antibodies
against ERK1/2 and phospho-ERK1/2, Akt and phospho-Akt
(Ser473) were purchased by Cell Signaling Technology
(Danvers, MA, USA). BS3 (Bis[sulfosuccini-midyl] suber-
ate) crosslinker agent was purchased by Thermo Fisher Sci-
entific Inc. (Rockford, IL, USA).
Cell Lines and Culture Conditions
hMPM cell lines were derived from pleural effusion
(IST-Mes2) and tumor biopsy (ZL55) from asbestos ex-
posed, pharmacologically untreated, male patients, 65 and 52
years old, respectively. IST-Mes2 were established in our
laboratory in 1996 [26] whereas ZL55, established as con-
tinuous in vitro culture in 1992, were kindly provided by Dr.
B. Wegemann and Prof. R.A. Stahel (University of Zurich,
Switzerland) [27]. Both cell lines were maintained in culture
in Dulbecco's Modified Eagle's Medium High Glucose
(DMEM) supplemented with 5% heat-inactivated FBS
(EuroClone), glutamine (2 mM) and antibiotics (0.02 U/ml
penicillin and 0.02 mg/ml streptomycin) at 37°C in 95:5%
air-CO2 ratio atmosphere.
Colorimetric MTT Assay: Short and Long-Term Con-
centration-Response Curves and Cell Growth Recovery
Cell viability was evaluated by quantifying spectropho-
tometrically the reduction of MTT salt to formazan crystals,
by mitochondrial dehydrogenases. Five thousands cells/well
were plated, in triplicate, in 96-well plates, in DMEM 5%
heat-inactivated FBS. After 24 h incubation medium was
replaced by fresh medium containing the drugs or the respec-
tive vehicle. In the last 4 h of treatment, 50 μl of 2 mg/ml
MTT solution in phosphate buffered saline (PBS) was added,
avoiding direct light, to the medium (250 μl total volume),
then medium was sucked-off and 150 μl of DMSO added.
Plates were incubated 5 min at r.t. in the dark and, within 10
min, ODs were read in a spectrophotometer at 540 nm [30].
In order to determine the cell growth recovery, 3 days-
treated cells were incubated with drug-free fresh medium for
3 additional days. MTT assay was finally carried out as pre-
viously described.
Page 3
178 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
Detection of Drug-Induced Apoptosis and Necrosis by
FITC-Annexin V/PI Double Staining Flow Cytometry
and Cell Cycle Analysis
Fluorescence-activated cell sorting (FACS) analysis was
performed using a double staining Annexin V-FITC (fluo-
rescein isothiocyanate)/propidium iodide (PI) apoptosis de-
tection kit (FITC-labelled recombinant human Annexin V,
Bender-MedSystems, Vienna, Austria). Briefly, 1.5 x 105
cells were plated in 35 mm Petri dishes in whole culture me-
dium. After 24 h, Gefitinib was added to cell cultures for
different times; supernatants and monolayers were collected
and volume brought to 2 ml with PBS. Pellets obtained after
centrifugation for 5 min at 1400 r.p.m. were resuspended in
195 μl of binding buffer (BB: DMEM, 40 mM Hepes, 0.1%
BSA). FITC-Annexin V was added to a final concentration
of 1 mg/ml (5 μl), mixed and incubated for 10 min at r.t., in
the dark. Suspensions were washed once with 1.5 ml PBS
and centrifuged. Pellets were resuspended in 190 μl BB and
10 μl PI were then added to reach a final concentration of 1
mg/ml. Samples, maintained on ice, were measured within
15 min with a DAKO CyAnADP fluorescence-activated cell
sorter (FACS).
Analysis of cell cycle progression of asynchronously
growing cells and measurements of diploid (2N) DNA con-
tent were performed resuspending treated and untreated cells
in 150 μl of 0.2% Triton-X 100 solution plus 150 μl of a 50
mg/ml ddH2O stock solution of the staining PI, on ice in the
dark, before FACS examination.
Cell Lysate Preparation and Protein Quantification
Untreated, exponentially growing cell cultures were
plated in 100 mm Petri dish at 6 x 105 cells in DMEM 5%
FBS to reach 80% confluence (96 h) followed by 72 h se-
rum-starvation (DMEM 0.1% FBS). Monolayers were then
treated with Gefitinib for 30 min and EGF was added during
the last 10 min of incubation [29]. At the end of incubation
cells were washed once with D-PBS w/o Ca++/Mg++ on ice,
and 150 μl of lysis buffer (Tris-HCl 20mM, NaCl 137 mM,
EDTA-Na2 2mM, glycerol 10%, NP40 0.1%, containing
protease inhibitor cocktail [Complete mini, Roche], PMSF
1mM and Na3VO4 1mM) were added to each dish. After 10
min on ice, cell monolayers were manually scraped, lysates
were harvested and centrifuged at 5000 r.p.m., 4°C for 10
min to remove debris; supernatant were collected and total
proteins concentration measured by Bradford assay (BioRad
Laboratories); samples were immediately used for SDS-
PAGE separation of proteins or stored at -20°C. ERK1/2
activation kinetic was monitored, after 72 h serum-
starvation, adding EGF (40 ng/ml) for 2-120 minutes before
the lysis of cell monolayers, as described above.
SDS-PAGE/Western Blot for EGFR, pEGFR, ERK1/2,
pERK1/2, Akt, pAkt Peptides
Proteins (80 μg for EGFR, 20 μg for ERK and Akt
evaluation) were diluted in equal volume of 2x reducing
sample buffer (2% SDS, 62.5 mM Tris-HCl pH 6.8, 0.01%
bromphenol blue, 1.43 mM 2?-mercaptoethanol and 0.1%
glycerol), size-fractionated on 10% SDS-polyacrylamide
gels, transferred on polyvinylidene difluoride (PVDF) mem-
brane (BioRad Laboratories) and probed with specific anti-
bodies [31]. Detection of immunocomplexes was performed
using the enhanced chemiluminescence system (GE
[3H]-thymidine Incorporation Assay
DNA synthesis was measured by means of the [3H]-
thymidine uptake assay, as reported [32]. 2 x 104 cells/well
were plated in 24-well plates, serum-starved for 72 h and
then treated with EGF for 24 h; in the last 4 h cells were
pulsed with 1 μCi/ml of [3H]-thymidine (GE Healthcare).
Then cells were trypsinized, extracted in 10% trichloroacetic
acid (TCA), and filtered under vacuum through fiberglass
filters (Millipore Co., Bedford, MA). Filters were sequen-
tially washed, under vacuum, with 10 and 5% TCA and 95%
ethanol. TCA-insoluble fraction was measured in a scintilla-
tion counter. When indicated, cultures were pre-treated with
PD98059 (1 and 10 μM, for 10 min) or Wortmannin (1 and
10 μM, for 10 min).
Cells were seeded at low density (3 x 104/well/400 μl
complete 5% FBS DMEM) on glass chamber-slides (Lab-
Teck NUNC 8 wells # 177402). After 24 h, monolayers were
serum-starved for 72 h and treated for 5, 10, 60 and 120 min
with EGF (40 ng/ml), alone or after treatment with 1x and 2x
[IC50] Gefitinib. After 2 washes with PBS, cells were fixed
for 10 min in 4% paraformaldehyde (PFA). Cells were then
incubated with mouse anti-ERK1/2 (Santa Cruz) or rabbit
anti phospho-ERK1/2 (Cell Signaling) antibodies (1:100, for
1h at r.t.). After three washes with PBS cells were incubated
with secondary Alexafluor 488 or Alexafluor 568-conjugated
anti mouse and anti rabbit IgG secondary antibodies (Invi-
trogen), both diluted 1:100. Cells were washed three times
with PBS and Hoechst 33258 (Sigma) was used for nuclear
staining. After three washes with PBS cells were mounted in
Mowiol and analyzed with a DM2500 microscope (Leica
Microsystems, Wetzlar, Germany) equipped with a
DFC350FX digital camera (Leica Microsystems) [33].
Radioreceptor Assay With [125I]-labeled EGF/Unlabeled
EGF Competitive Binding to EGFR
1.5 x 105 tumor cells were plated in 400 μl/well DMEM
5% FBS in 24-well plates in duplicate, incubated at 37°C,
5% CO2 and allowed to attach o.n. Cell monolayers were
washed once with 1 ml/well BB for additional 60 min and
then co-incubated with [125I]-EGF (average 2 x 104 cpm in
200 μl/well of BB) and increasing concentrations of unla-
beled peptide (0.195 to 50 ng/ml for standard curve + 400
ng/ml excess for determination of non-specific binding) in
200 μl/well BB (total volume/well = 400 μl BB). A second
set of wells was treated in parallel with the same amounts of
labeled/unlabeled ligand and simultaneously co-incubated
with 0.5-1x [IC50] of Gefitinib. Plates were then incubated
for 2 h at 4°C on a shaker with gentle agitation. After bind-
ing, medium was removed from each well and cell monolay-
ers washed 3 times with 0.5 ml/well ice-cold Washing Buffer
(HBSS + 0.1% BSA). Cells were lysed by incubating the
monolayers for 30 min at r.t. with 0.75 ml of Solubilizing
Page 4
Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 179
Solution (20 mM Hepes, 1% Triton X-100, 10% glycerol in
ddH2O). Solubilized material (0.6 ml) was measured in a ?-
counter (Packard, Cobra II Autogamma) to quantitate the
released radioactivity of the bound ligand [30]. Specific
binding was calculated after subtraction of non-specific
binding (excess unlabeled EGF).
Cross-Linking Experiments
2x105 IST-Mes2 cells, plated in 60 mm Petri dishes, were
serum-starved for 72 h with 0.1% FBS DMEM. Monolayers
were exposed to Gefitinib (40 μM) and/or rhEGF (100
ng/ml) and then washed twice with cold PBS. 1.5 ml of 3
mM solution of BS3 [bis(sulfosuccinimidyl) suberate], a wa-
ter-soluble cross-linker, in cold PBS were added to each
Petri dish and cells maintained on ice for 30 min [34]. The
reaction was stopped by adding 250 mM (final concentra-
tion) of glycine in PBS for 5 min at 4°C. After washing with
1x cold PBS, monolayers were lysed with 150 μl/Petri of
lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Triton-X100, 2.5 mM Na-
pyrophosphate, 1 mM ?-glycerol phosphate, 1 mM Na3VO4,
1 mg/ml leupeptin, 1 mM PMSF) for 20 min, on ice [34].
Cells were then scraped and the suspension centrifuged at
10000 x g for 10 min at 4°C. Supernatants were collected
and proteins separated by SDS-PAGE on 6% gel, blotted
onto PVDF and Western blot analysis performed as de-
scribed before.
Statistical Analysis
Unless otherwise specified, all cell growth experiments
were performed in triplicate and repeated at least three times.
Competitive binding experiments were performed in dupli-
cate and repeated twice. SDS-PAGE/WB experiments were
repeated a couple of times. Data are expressed as mean ±
S.E. mean values and statistical significance was assessed by
analysis of variance with a Two-way ANOVA: p values less
than or equal to 0.05 were considered statistically significant.
[IC50] values were obtained using Prism 4 software program
for Windows (GraphPad Software, Inc., San Diego, CA,
USA). Scatchard analysis as well as Bmax, KD and sites/cell
number of binding assays have been calculated by means of
Microsoft Office Excel 2007 software.
ErbB Family Receptor Members Expression and Activity
in hMPM Cells
A panel of six hMPM cell lines, derived from previously
therapeutically untreated patients and the SV40-transfected
mesothelial cells Met5A [2], were screened for the expres-
sion of the EGF receptors ErbB-1 (EGFR or HER1), ErbB-2
(HER2/c-neu) and ErbB-3 (HER3) expression. By Western
blot analysis, different levels of EGFR have been detected in
all cell lines: from low (Met5A) and intermediate (SPC212,
ZL34 and MPP89), to the relatively high in pleural effusion-
derived IST-Mes1, IST-Mes2 and tumor biopsy-derived
ZL55 cells, all belonging to the epithelial histological sub-
type, Fig. (1A). The proliferation rate of all serum-starved
hMPM cell lines resulted to be significantly increased by
three days incubation with rhEGF, in a concentration-
dependent manner (10-80 ng/ml) (data not shown). Moreo-
ver, IST-Mes2 and ZL55 cells, treated with 40 ng/ml of
rhEGF (10 min, after 72 h of serum-starvation) showed a
marked autophosphorylation of EGFR, Fig. (1B), indicating
the expression of a physiologically active EGFR in both cell
lines. Similar results were obtained in all the hMPM cell
lines examined (data not shown). Finally, we show that both
IST-Mes2 and ZL55 cell lines do not express either HER2 or
HER3 receptor, Fig. (1C) and (1D), respectively. This find-
ing allows to exclude that the HER2/HER3 receptors may
participate in the EGF-induced signal transduction.
Fig. (1). Basal and EGF-activated EGFR expression in human ma-
lignant pleural mesothelioma (hMPM) cell lines. (A): expression of
p170 KDa EGFR by a panel of seven hMPM cell lines. Human
ovarian tumor A2780 and estrogen-independent breast adenocarci-
noma MDA-MB468 cell lines, lacking and overexpressing EGFR,
respectively, have been used as negative [C-] and positive [C+]
controls. (B): serum-starved IST-Mes2 and ZL55 hMPM cell lines
treated for 10 min with 40 ng/ml rhEGF showed the appearance of
the phospho-EGFR tyrosine kinase (pEGFRTK) signal, indicating
that the expressed EGFR is functional. In C and D, the lack of
HER2 and HER3 expression, respectively, in IST-Mes2 and ZL55
cells is shown. Three human breast cancer cell lines SKBR-3 and
MCF-7 (HER2 and HER3 expressing, respectively) and MDA-
MB468 (HER2 lacking) have been used as positive and negative
controls. EGFR and pEGFRTK proteins were evaluated by Western
Blot analysis on total cell lysates using specific targeted antibodies.
IST-Mes2 and ZL55 cell lines, the most EGFR expressing, have
been the two hMPM in vitro models selected to carry out the entire
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180 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
From these preliminary results, IST-Mes2 and ZL55 cell
lines were chosen as models of hMPM, suspension or solid
origin, respectively, to test their in vitro sensitivity to Gefit-
Gefitinib Long-Term Treatment Inhibits Cell Prolifera-
tion in Dose-Dependent Manner
The ability of Gefitinib to inhibit hMPM cell growth was
examined in vitro on IST-Mes2 and ZL55 cell lines. A rela-
tively short-term drug treatment (24 - 48 h) did not reveal a
statistically significant effect on both cell lines and the [IC50]
was not reached (Table 1). On the contrary, after long-term
drug exposure (from 72 h on), Gefitinib caused a concentra-
tion- and time-dependent cell growth inhibition, as shown in
Fig. (2A) and (2B). [IC50] values (mean ± S.E. mean; n = 3
independent determinations) ranged from 17.3 to 22.6 μM
for IST-Mes2 and from 7.6 to 12.9 μM for ZL55. The time-
related, rounded up [IC50] values (μM) for Gefitinib were: 19
at 72 h, 18 at 96 h, 23 at 120 h and 17 at 144 h for IST-Mes2
and 13 at 72 and 96 h, 10 at 120 h and 8 at 144 h for ZL55
(Table 1). Comparing [IC50] values at the correspondent
times of drug-exposure, we show that ZL55 cells possess a
higher chemosensitivity to Gefitinib than IST-Mes2 (concen-
trations required to reach 50% of growth inhibition at 96,
120 and 144 h are 28%, 57% and 53% respectively lower in
ZL55 vs. IST-Mes2).
Gefitinib [IC50] values reached at 96 h for IST-Mes2 (?
20 μM) and at 120 h for ZL55 (? 10 μM) or the relative frac-
tions (0.2x) or multiple (2x) were selected as the concentra-
tions to be tested in the following experiments, because of
the best interassay uniformity among the three determina-
tions performed. In parallel experiments, exponential cell
growth was monitored throughout the entire period of drug
exposure (72 to 144 h), as shown in the insets to Fig. (2A
and 2B).
Cell Growth Recovery After Gefitinib Inhibitory Effect
In order to verify whether the growth inhibition caused
by Gefitinib was related to cell death, we evaluated the per-
sistence of living cells upon drug removal in experiments of
cell growth recovery, carried out on IST-Mes2 and ZL55 cell
lines. The treatment of IST-Mes2 with Gefitinib for 72 h led
to a concentration-dependent growth inhibition up to almost
80% at 40 μM, Fig. (3A). Conversely, after additional 72 h
of culture in fresh medium in the absence of the drug, these
cells showed a sustained recovery of proliferation, up to the
concentration of 20 μM. At higher Gefitinib concentrations,
the recovery was only partial and non statistically signifi-
The proliferation of ZL55 cells was recovered after drug
removal in a similar profile than IST-Mes2 cells, with the
Table 1. Gefitinib [IC50] on IST-Mes2 and ZL55 hMPM Cell
IST-Mes2 ZL55 t
(μM ± S.E. mean)
19.0 ± 1.7
18.4 ± 1.5
22.6 ± 6.2
17.3 ± 0.6
12.9 ± 0.1
12.7 ± 1.2
9.6 ± 0.8
7.6 ± 0.3
Gefitinib concentrations inhibiting 50% of cell proliferation ([IC50] ± S.E. mean)
calculated after short (S.T.) and long term (L.T.) treatment with 5-30 μM (IST-Mes2)
and 1-30 μM (ZL55) assays are listed. [IC50]s selected to carry out all the experimental
approaches on IST-Mes2 and ZL55 cell lines and specific times at which they have
been reached are also indicated (bold faces); these values have been chosen for all
experiments on the basis of the best interassay data consistency. N.R. = [IC50] not
Fig. (2). Concentration-dependent growth inhibition in hMPM IST-Mes2 A) and ZL55 B) cells in response to Gefitinib, assessed by MTT
assay. Long term treatment for 72 h (closed circles), 96 h (closed squares), 120 h (closed triangles) and 144 h (open circles) shows the con-
centration- and time-dependent drug sensitivity of both cell lines to Gefitinib. Drug concentrations were used in the range of 5-30 μM for
IST-Mes2 and 1-30 μM for ZL55. The [IC50] values, as obtained by means of GraphPad Prism 4TM software, ranged from 17.3 to 22.6 μM
for IST-Mes2 and from 7.6 to 12.9 μM for ZL55. Values are reported as percent of cell growth inhibition vs. untreated cells; each curve rep-
resents the mean of three experiments, each point performed in triplicate. Bars symbolize the standard errors of the mean (± S.E. mean) rang-
ing between 0.35 and 2.80 for IST-Mes2 and between 0.99 and 5.01 for ZL55 cell lines; asterisks [*] and points framed on the curves indi-
cate p < 0.05. Insets represent the proliferation progression of untreated cells measured by MTT assay at each selected time of drug activity
evaluation as described above.
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Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 181
growth recovery observed for Gefitinib concentrations up to
10 μM Fig. (3B). Thus, in both cell lines Gefitinib exerted a
concentration-dependent growth inhibitory effect that was
cytostatic up to the drug [IC50] and started to become cyto-
toxic at higher concentrations.
Fig. (3). Cell growth recovery of hMPM cell lines after Gefitinib
exposure of IST-Mes2 A) and ZL55 B) cell lines treated for 72 h
with increasing concentrations of Gefitinib (grey columns) followed
by 72 h culture in drug-free medium (closed columns). Untreated
cells (open column) and untreated cells + fresh drug-free medium
(closed column) are represented; arrows point 1x and 2x drug
[IC50]. Gefitinib induces a cytostatic effect up to the [IC50] values
(20 and 10 μM, respectively); at 2x [IC50] growth inhibition levels
were around 80% and the recovery was very small for IST-Mes2
cells, whereas growth inhibition reached 65% and there was essen-
tially no recovery for ZL55 cell line. Histograms are the mean ±
S.E. mean, ranging between 0.51 and 0.87 for IST-Mes2 and be-
tween 3.28 and 20.57 for ZL55 cell lines, of two independent ex-
periments performed in triplicate by MTT assay.
Early and Late Apoptosis in IST-Mes2 and ZL55 Cells
Upon Gefitinib Exposure
We determined the percentage of viable or dead cells, ei-
ther apoptotic or necrotic, and evaluated whether Gefitinib
may affect cell survival, by phosphatidylserine recognition
on the extracellular surface and Propidium Iodide (PI) DNA
uptake. For this purpose, Annexin V-FITC/PI stained cells
were analysed in a fluorescence-activated cell sorter (FACS).
In Fig. (4) are reported the results of flow cytometry experi-
ments on IST-Mes2 (A) and ZL55 (B), after their exposure
to Gefitinib at the pre-determined 1x (20 and 10 μM, respec-
tively) and 2x (40 and 20 μM, respectively) [IC50] for 12-72
h. After 1x [IC50] Gefitinib treatment, in IST-Mes2 cells the
percentage of total apoptotic population, constituted by
early-mid (FITC+/PI-) plus late (double positive FITC+/PI+)
state (R4 and R2 quadrants of the cytogram, respectively),
increased gradually, in time-dependent fashion, from about
6-7% at 12-24 h, to over 8% at 48 h, to the 19% at 72 h
(+99%, +146%, +177% and +363% R4+R2 increase of
treated vs. untreated cells, respectively), Fig. (4A). Con-
versely, in ZL55 cell line Gefitinib 1x [IC50] did not exert
significant pro-apoptotic activity at any drug concentration
and time of treatment (range 0.68 - 0.94%), Fig. (4B). Dou-
ble [IC50] concentrations induced a highly significant in-
crease in apoptotic cells (R2 + R4) in IST-Mes2 cell line
compared to 1x [IC50] (about 2 folds at 12-48 h and 4.5 folds
after 72 h exposure, Fig. (4A). ZL55 cells did not show any
significant variation in R2+R4 at the shorter times, whereas
at the longest time of treatment (72 h) a significant amount
of apoptotic cells was observed, although less than 5% of
which represented cells in early apoptotic phase, Fig. (4B).
Long term experiments, up to 144 h drug-exposure, revealed
no further increase in cellular apoptotic state. Cell cycle
analysis closely correlates with Annexin V binding data,
showing a progressive, time-dependent shifting of Gefitinib-
treated cells from S to the resting G0/G1 phase and to a final
recruitment in early to late apoptosis (cells in sub G0/G1
phase), after 72 h drug, of 14 and 73%, at 1x and 2x [IC50]
respectively, for IST-MES2 and 19 and 80%, at 1x and 2x
[IC50] respectively, for ZL55 (see the values inside the cy-
tograms relative to 72 h Gefitinib treatment in Fig. 4A and
Gefitinib Suppresses EGF-Induced Phosphorylation of
EGFR and Hinders the Transcription/Proliferative
ERK1/2 Pathway in IST-Mes2 and ZL55 Cell Lines
The specificity of the inhibition of IST-Mes2 cell prolif-
eration by Gefitinib was investigated evaluating the effect of
the drug on EGF-induced EGFR phosphorylation, as well as
on the phosphorylation status of other downstream effectors
such as ERK1/2-MAPK and Akt. The expression of EGFR,
ERK1/2 and Akt was determined by immunoblot analysis
and the basal phosphorylation in unstimulated cells was neg-
ligible for both IST-Mes2, Fig. (5A) and (6A) lane 1 and
ZL55, Fig. (5B) and (6B) lane 1. Under serum starvation
culture conditions (72 h) and exposure to EGF (40 ng/ml for
10 min), EGFR and ERK1/2 phosphorylation was induced in
both cell lines, Fig. (5A), lane 5; Fig. (6A), lane 3; Fig. (5B),
lane 5; Fig. (6B), lane 3). Moreover, exposure of the cells to
different Gefitinib concentrations, related to the respective
[IC50] (4-20-40 and 2-10-20 μM for IST-Mes2 and ZL55,
respectively) for 30 min, before and during the incubation
with EGF, reverted the phosphorylation of EGFR, Fig. (5A),
lanes 6 to 8; Fig. (5B), lanes 6 to 8 and significantly reduced
ERK1/2 activation, Fig. (6A), lane 4; Fig. (6B), lane 4. Fi-
nally, Gefitinib administered alone to IST-Mes2 and ZL55,
under the same experimental conditions for 30 min, did not
affect the EGFR and ERK1/2 phosphorylation, Fig.
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182 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
Fig. (4). Flow cytometry analysis of IST-Mes2 and ZL55 hMPM cell lines. Cytograms of FITC-annexin V/PI flow cytometry after time-
course with and without Gefitinib 20 ?M (1x [IC50]) and 40 ?M (2x [IC50]) on IST-Mes2 A) and 10 ?M (1x [IC50]) and 20 ?M (2x [IC50]) on
ZL55 B) cell lines are represented. The lower left quadrants (R3) of each panel represent intact living cells (FITC-Annexin V/PI negative);
R1 contain the non-viable, necrotic cells (PI uptake positive); R2 show the late apoptotic (FITC-Annexin V positive) and necrotic cells (PI
uptake positive) whereas the lower right quadrants (R4) represent the cells in the early to mid apoptotic state (FITC-Annexin V positive and
PI negative). The most significant among six-eight experiments for each cell line is shown. Histograms at the bottom of cytograms indicate
the percentage of the total selected cell population detected in the early to mid (R4 subset, close section of the columns) and late (R2 subset,
open section of the columns), as well as in the total R2+R4 (sum of close + open sections of the columns) apoptotic state in untreated and
drug-treated samples at various time intervals (12 to 72 h). Values inside the cytograms relative to 72h of Gefitinib treatment represent the
percentage of cell distribution among the cell cycle phases. In IST-Mes2 cells, A), the percentage of early + late apoptotic cells shifted from
6-7% at early time (12-24h) to 19% at the longest time (72h) after 1x [IC50] drug treatment, whereas, double [IC50] drug concentration in-
duced a consistent increase in total apoptotic (early + late) cell number, ranging from 2 to 4.5 fold of 1x [IC50] values at 12-72 h drug expo-
sure, respectively. These data show that the apoptotic phenomenon is both concentration and time-dependent in IST-Mes2 cell line. In ZL55
cells, B) any substantial variation in early + late apoptotic cells occurred after short times of incubation with 1x [IC50] of Gefitinib; on the
contrary, at 2x [IC50] and 72h drug exposure a significant increase in total apoptotic cells was detected, 95% of which belonging to the late
apoptotic cells. These data demonstrate that in ZL55 cells, Gefitinib does not induce apoptosis neither in presence of high drug concentrations
nor after prolonged times of drug exposure.
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Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 183
Fig. (5). Expression and phosphorylation status of EGFR in IST-Mes2 (A) and in ZL55 (B) cell lines. Basal level EGFR (lanes 1); EGF (40
ng/ml) treatment (lanes 5); EGF treatment in combination with Gefitinib (40, 20, 4 μM for IST-Mes2 and 20, 10, 2 μM for ZL55) (lanes 6, 7,
8), and treatment with different Gefitinib concentrations alone (lanes 2, 3, 4) are represented. After 72h starvation, basal EGFR phosphoryla-
tion is abolished in both cell lines but it is induced after rhEGF treatment. When Gefitinib is added to cells in the presence of rhEGF, EGFR
phosphorylation is significantly inhibited. EGFR activation is visualized by immunoblot analysis using anti EGFR and anti phospho-
EGFRTyr1173 antibodies. On the left side of Western blot panels arrows indicate the p170 KDa EGFR/pEGFR. The histograms represent the
pEGFR/EGFR ratio as % of controls evaluated by densitometry of three independent experiments. Bars represent mean ± S.E. mean; ** = p <
0.01 vs. untreated cells (lane 1); oo = p < 0.01 vs EGF-stimulated cells (lane 5).
Fig. (6). Expression and phosphorylation status of EGFR downstream effectors ERK1/2-MAPK and Akt, in IST-Mes2 (A) and in ZL55 (B)
cells. Panels A) and B), show, after the same treatments described in Fig. (5), the levels of total and phosphorylated ERK1/2 (top) and Akt
(bottom). EGF activation of ERK1/2 was reverted by Gefitinib treatment. On the contrary, in both cell lines the downstream effector Akt was
not phosphorylated after rhEGF treatment. PDGF, selected as positive control, triggered the activation of Akt. ERK1/2 and Akt activation was
visualized by immunoblot analysis using anti phospho-ERK1/2 and phosphoS473-Akt targeted antibodies. On the left vertical axis arrows indi-
cate the p44/42 KDa ERK1/2/pERK1/2 and p56 KDa Akt/pAkt molecular weight positions.
(5A), lanes 2 to 4; Fig. (5B), lanes 2 to 4; Fig. (6A), lane 2;
Fig. (6B), lane 2. No Akt activation, after EGF treatment was
detected in both hMPM cell lines, Fig. (6A), lanes 3, 4; Fig.
(6B), lanes 3, 4 while it occurs after PDGF treatment, used
as positive control, Fig. (6A), lanes 5, 6. The lack of Akt
activation upon EGF treatment correlated with the independ-
ence from this pathway of the EGF-dependent hMPM cell
proliferation; in fact, in [3H]-thymidine uptake experiments,
EGF-induced cell proliferation was not affected by pretreat-
ment with Wortmannin, a furanosteroid, specific inhibitor of
PI-3K. Conversely, the role of ERK1/2 in such effects was
confirmed by the complete abolishment of EGF proliferative
activity by the aminomethoxyflavone PD98059, a potent
selective inhibitor of MEK activation, Fig. (7).
Kinetics of ERK1/2 Activation and its Subcellular Time-
Dependent Compartmentalization
The different response of the two cell lines to Gefitinib
(cell cycle arrest or apoptosis) prompted us to evaluate
whether differences in the kinetics or subcellular localization
of ERK1/2 occurred after EGF treatment. In fact, it is now
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184 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
well known that differences in the timing of ERK1/2 activa-
tion or in its nuclear localization affect the final output of TK
receptor activation [35, 36].
Serum-starved cells were exposed to rhEGF (40 ng/ml
for 2-120 min) and total cell lysates were analyzed by West-
ern blot using both anti ERK1/2 and anti pERK1/2 antibod-
ies. ERK1/2 phosphorylation was induced after 2 min of
rhEGF stimulation, reached the maximal activation at 5-10
min and started to decline up to 40 min. Unexpectedly, after
1 h of treatment ERK1/2 phosphorylation started to increase
again reaching a second peak after 120 min. The same kinet-
ics trend was observed in both cell lines, Fig. (8 A/B).
By immunofluorescence staining, we monitored ERK1/2-
pERK1/2 intracellular localization, distribution and nuclear
translocation upon 5, 10, 60 and 120 min of rhEGF stimula-
tion, as percentage of immunopositive cells, Fig. (9 A/B) and
Suppl. Fig. (1). In untreated IST-Mes2 cells, phospho-
ERK1/2 immunoreactivity was mainly confined to the cyto-
plasmatic area (0 min); after treatment with 40 ng/ml rhEGF,
a significant amount of immunoreactivity translocated into
the nucleus reaching the maximal intensity after 10 min
(about 70% of the cells). After longer exposure times (60-
120 min) nuclear phospho-ERK1/2 decreased but persisted
in the nucleus in higher percentage of cells than in controls.
In the presence of low Gefitinib concentration (20 μM), a
striking reduction of nuclear translocation of pERK1/2 was
observed, Fig. (9A) that was further increased with the
higher Gefitinib concentration (40 μM). ZL55, Fig. (9B),
have a higher basal percentage of cells showing phospho-
ERK1/2 in the nucleus (about 35%) than IST-Mes2 cells and
a higher rate of nuclear translocation of phospho-ERK1/2, in
response to rhEGF, that reached the 100% of immunoposi-
tive cells after 10 min of treatment. This pattern of response
was also observed after Gefitinib treatment in which, in spite
of a significant reduction, still an important localization of
phospho-ERK1/2 into the nucleus was maintained.
Fig. (7). Role of ERK1/2 and Akt in IST-Mes2 cell proliferation.
[3H]-thymidine uptake experiments were carried out after PD98059
and Wortmannin pretreatment to abolish ERK1/2 or Akt activity,
respectively. These data indicate the central role of ERK1/2 and the
independency from Akt activation for EGF-induced hMPM cell
proliferation. In the figure are reported, as % of the increase above
controls (untreated cells), the effects of rhEGF treatment (40 ng/ml)
on IST-Mes2 cells, alone or in the presence of 10 min pretreatment
with 1 and 10 μM Wortmannin or PD98059. Columns W and PD
reports the effects of 1μM Wortmannin and 1μM PD98059 alone,
respectively. After exposure to the PI-3K inhibitor Wortmannin,
cells retain a growing state whereas the MEK inhibitor PD98059
totally abolishes the rhEGF dependent cell proliferation. Bars indi-
cate the average values of two independent experiments, performed
in quadruplicate, ± S.E. mean; ** = p < 0.01 vs. rhEGF alone.
Fig. (8). ERK1/2 activation kinetics in IST-Mes2 (A) and ZL55 (B) hMPMP cell lines after rhEGF (40 ng/ml) treatment. After serum-
starvation for 72 h, cells were treated with the growth factor for sequential times ranging from 2 to 120 min and total cell lysates were ana-
lyzed by Western blot using anti ERK1/2 and anti pERK1/2 antibodies. Time zero represents baseline ERK1/2 activity in untreated cells. In
bold are represented the times at which the maximal ERK1/2 activation has been reached (5-10 min). The position of ERK1/ERK2 (p44/p42
MAPKs) are indicated on the left side of Western blot panels. Histograms represent densitometric analysis values of the each single band as
detetcted by immunoblot and reported as percentage of its own basal point. Bars represent the average values of three independent experi-
ments ± S.E. mean ; * = p < 0.05; ** = p < 0.01 vs. untreated cells (lane 0).
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Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 185
Altogether these data indicate that Gefitinib is able to
block EGF-induced cytoplasm => nucleus translocation of
Exposure to Gefitinib Up-Regulates EGF Receptor Site
Number in IST-Mes2 and ZL55 Cell Lines
EGF competitive binding assay was performed to charac-
terize the EGFR status of hMPM cells and the possible ef-
fects of Gefitinib treatment. Both cell lines displayed similar
levels of EGF binding sites (Table 2). Scatchard analysis of
[125I]-EGF binding to untreated IST-Mes2 and ZL55 cells
resulted in a best fit according to the one-affinity model,
with one single class of binding sites totally in the high-
affinity state (KD = 0.15 and 0.19 nM for IST-Mes2 and
ZL55, respectively) (Table 2 and Fig. 10).
The interference of Gefitinib in the EGF competitive
binding to its receptor was tested on both hMPM cells. Using
a standardized procedure, cells were treated simultaneously
with Gefitinib and [125I]-labelled EGF, in the presence of
increasing concentrations of unlabeled peptide (0.195-12.5
ng/ml). The estimation of non-specific binding was per-
formed by adding a high concentration of cold rhEGF (400
ng/ml) that completely displaced (>95%) radiolabeled EGF
binding. Gefitinib, at concentrations corresponding to 0.5-1x
[IC50], caused a dramatic increase of radioligand specific
binding, with the calculated number of sites/cell increasing
of 150 and 230% in IST-Mes2 and ZL55 cell lines, respec-
tively, Table 2 and Fig. (10A) and Fig. (10B) insets. Fur-
thermore, the up-regulation of EGF binding was in direct
relationship with the increasing drug concentrations used
(tested from 0.0001x to 2x [IC50]) (data not shown). Assays
carried out on cell monolayers serum-starved for 48 h,
showed a dramatic amplification of the EGF binding vs. cells
kept in serum-containing medium (Table 2), presumably
because of the presence of a higher number of unoccupied
monomeric receptors available to be bound. In order to for-
tify these findings further experimental conditions have been
selected: administration of Gefitinib before the la-
beled/unlabeled ligands for competitive binding at 4°C, fol-
lowed by 1, 48 and 72 h incubation at 37°C, caused a pro-
gressive decrease of EGF binding which was inversely pro-
portional to the period of incubation with the drug before the
competitive binding (Table 2). As expected, Gefitinib treat-
ment, performed after EGF binding, did not cause any varia-
tion in receptor level comparing untreated and treated cells
(Table 2).
Fig. (9). Subcellular localization of phospho ERK1/2 in IST-Mes2 (A) and ZL55 (B) cells and effects of different Gefitinib concentrations.
Data are obtained from immunofluorescence experiments, as depicted in Supplementary Fig. (1). In the graphs are reported the % of immuno
positive vs. total cell counted from three independent microscopic fields. The percentage of cells immunopositive in the cytosol are reported
by the white bars while the immunopositivity in the nucleus is depicted by the black bars. Cells were treated with 40 ng/ml rhEGF for 5, 10,
60 and 120 min in the absence or presence of 1x [IC50] (20 and 10 μM for IST-Mes2 and ZL55, respectively) and 2x [IC50] (40 and 20 μM
for IST-Mes2 and ZL55, respectively) for 30 min. The data, representing the average results of two independent experiments, are reported as
mean percentage ± S.E. mean.
Bolded times on the horizontal axis are those at which the maximal nuclear peak accumulation has been detected. Letters (B, C, D, E) on top
of the columns are referred to the couples of pictures showed in the Supplementary Fig. (1).
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186 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
Gefitinib-Mediated Formation of EGFR Dimer Com-
plexes Evaluated by Chemical Cross-Linking in Intact
It has been shown that EGFR dimerization is not induced
only by the specific ligand but also can take place after
treatment of cells with quinazoline derivatives in the absence
of the growth factor [37]. We demonstrated that this phe-
nomenon applies also to IST-Mes2 cells after Gefitinib
treatment. In particular we show that EGFR dimer formation
can be detected in Western blot experiments, after membrane
protein cross-linking, following administration of Gefitinib.
The monomeric form of EGFR (molecular mass 170 KDa)
was present in both untreated and drug-treated cells but after
rhEGF (100 ng/ml) or Gefitinib (40 μM) treatment, as well
as in cells exposed concomitantly to both compounds, a
Table 2. Radioreceptor Assay on Human Malignant Pleural Mesothelioma Cell Lines Treated with Gefitinib (rhEGF Competitor)
- Scatchard Analysis Data
Drug (μ μM) Protocol Bmax (pM) KD (nM) Sites x 10 4/Cell
Cell Line
0.5x [IC50]
A) IST-Mes2 Sit 0.15 0.13
B) IST-Mes2 Gefitinib (9.0) Sit 4 27 53 0.09 0.10 13 66 +408
C) IST-Mes2 Gefitinib (9.0) Set 4 22 67 0.08 0.13 8 28 +250
D) IST-Mes2 Gefitinib (9.0) Set 4 33 49 0.12 0.11 10 15 + 50
E) IST-Mes2 Gefitinib (9.0) Set 4 39 39 0.18 0.10 8 9 + 12
F) IST-Mes2
1x [IC50]
Radioreceptor assay of IST-Mes2 and ZL55 cell lines with EGF competitor. Scatchard analysis data: Bmax, KD and sites/cell, basal and after treatment with ZD1839 are indicated.
(Sit = Simultaneous drug treatment; Set = Sequential drug treatment; C = Control, T = drug-treated; Bmax = maximum binding capacity; KD = dissociation constant; T/?C % =
treated/untreated sites/cell ratio. A = standard Sit protocol (drug + binding 2h 4°C); B = Sit protocol with pre-starvation for 48h with 0.1% FBS; C = Set protocol: drug 1h 4°C ->
wash -> 1h 37°C in binding buffer -> binding 2h 4°C; D = drug 2h 4°C -> wash -> 48h 37°C in binding buffer -> binding 2h 4°C; E = as for D with 72h 37°C in binding buffer
before binding 2h 4°C; F = Set protocol: 1h 37°C -> binding 2h 4°C -> wash -> 1h 37°C -> drug 2h 4°C. Average values of Bmax, KD and sites/cell of two experiments are indi-
cated. These data indicate that, after drug treatment, binding affinity (Kd) of EGFR remained constant whereas their capacity (Bmax) and number of sites/cell varied tangibly.
Fig. (10). Scatchard plot of [125I]-labelled EGF binding to IST-Mes2 and ZL55 cell lines. Untreated (open squares) and Gefitinib-treated
(closed squares) IST-Mes2 (A) and ZL55 (B) are drawn. Insets represent competitive inhibition of [125I]-EGF binding to EGFR by unlabeled
EGF expressed in cpm of radioligand vs. increasing concentrations of unlabelled ligand (left) and in % of untreated (right). Values of max
binding capacity (Bmax), dissociation constant (KD) and n° sites/cells of untreated and treated cells, average of two distinct standard Sit pro-
tocol experiments [see A) IST-Mes2 and A) ZL55 of Table 2], are also indicated in the small windows on the curves. The observed shift in
the number of binding sites (11 to 27 x 104 for IST-Mes2 and 8 to 26 x 104 for ZL55) is closely related to the presence of Gefitinib. Moreo-
ver, it appears also to be concentration-dependent as from other experiments using a progressive 0.0001x to 2x [IC50] range of drug concen-
trations. Radioreceptor assays were carried out with cell drug treatment simultaneously (Sit) or sequentially (Set) to the labeled/unlabelled
competition at 4°C for 2 h as indicated in the Methods section.
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Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 187
marked dimerization of EGFR (approximately 360 KDa im-
munoreactive bands) was evidenced, Fig. (11A), and quanti-
fied by densitometry in Fig. (11B).
Interestingly, dimers formed upon Gefitinib treatment
were not active both in the presence and in the absence of
rhEGF, as demonstrated by the failure of tyrosine phos-
phorylation of the receptor in Western blotting experiments
using phospho-specific EGFR antibody, Fig. (11C). This
experimental approach confirms that Gefitinib is able to pro-
voke by itself the EGFR dimerization process, event usually
attributed only to the physiologic ligand-receptor interaction.
Despite the incidence of hMPM is expected to continu-
ously increase for at least two decades, detection and treat-
ment of this cancer still remain very difficult tasks. The
overall outlook for the management of this neoplasm has
improved with the emergence of new-designed chemothera-
pies. Several late generations of better targeted cytostatic
compounds, alone or in combination, are currently under
evaluation at preclinical level and in patients with advanced
disease. Some of them are able to extend the overall survival
of patients, therefore foreseeing important therapeutical pro-
gress. Interference with the activity of growth factor recep-
tors and/or with the activated intracellular signal transduc-
tion pathways represents the main strategy presently pursued
to develop novel selective antitumor drugs [38, 39]. Over the
last ten years, experimental evidence has demonstrated the
key role of EGFR autocrine activation in a variety of human
cancers. Thus, the blockade of EGFR is now one of the tar-
get most frequently considered for the development of novel
biopharmacological approaches in translational cancer re-
search [40].
High levels of expression of EGFR have been detected in
approximately 70% of hMPM [24]. Therefore, in this study
we analyzed the effects of the inhibition of EGFR activity on
IST-Mes2 and ZL55 human epithelial mesothelioma cell
survival. We demonstrate that hMPM cell exposure to Gefit-
inib, a low molecular weight, ATP competitor, acting as po-
tent and selective EGFR inhibitor, induces inhibition of cell
proliferation, in a concentration-dependent manner. ZL55
cells are more sensitive to Gefitinib as compared to IST-
Mes2, since the drug induced growth arrest with an [IC50]
about 30 to 50% lower than that observed in IST-Mes-2
cells, accordingly to the time of treatment. Importantly, a
highly significant effect of Gefitinib was also identified in
ZL55 cells that were previously showed to be relatively
chemoresistant to other cytotoxic drugs, including cis-
platinum, taxanes, gemcitabine, anti-topoisomerase I, fen-
retinide [28]. The inhibitory effect of Gefitinib on cell prolif-
eration, at low concentrations, was mainly cytostatic, as
shown by the cell growth recovery assay: in this experimen-
tal approach, drug withdrawal from the culture medium
caused the retrieving of cell proliferation. This effect was
well-related with biochemical experiments, in which low
concentrations of Gefitinib completely abolished EGFR
phosphorylation and the down-stream activation of ERK1/2.
However, high drug concentrations (> [IC50]) caused cell
death, as shown by the absence of growth recovery after re-
Fig. (11). Chemical cross-linking of IST-Mes2 cells to evaluate EGFR dimerization and phosphorylation after EGF and/or Gefitinib treat-
ment. EGFR dimers formation was evaluated after 10 min cells exposure to 100 ng/ml of rhEGF in the presence or absence of 40 μM Gefit-
inib pretreatment (30 min) and immunoblot analysis, using anti EGFR (A) and anti phospho EGFR (C) antibodies. Arrows between panels A
and C indicate the molecular masses of ? 170 and ? 360 KDa of EGFR monomer and dimer, respectively. On the left side (panel A) molecu-
lar weight markers are represented. (B) Histogram indicates the monomeric (close square)/dimerized (open square) % ratio evaluated by
densitometric analysis of the bands.
Page 13
188 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
moval of Gefitinib from the culture medium. This dose-
related behaviour was confirmed by both apoptosis and cell
cycle analysis: using these approaches we show that Gefit-
inib treatment provokes a time- and concentration-dependent
induction of apoptosis with a maximum at 72 h for both cell
lines for concentrations higher than [IC50], although IST-
Mes2 cells were more prone to undertake the apoptotic
pathway, as compared to ZL55. In consideration that EGFR
phosphorylation is totally abolished for Gefitinib concentra-
tions that do not affect cell viability, we can hypothesize
that, while the cytostatic effect is related to the interaction of
the drug with EGFR, the cytotoxic effect could be related to
additional unidentified mechanisms activated by high drug
concentrations. Moreover, the inhibition of EGF-induced
ERK1/2 phosophorylation represented in our cell model the
main intracellular mechanisms of the antiproliferative effects
observed after Gefitinib treatment. Since it was reported that
the kinetics and the cellular localization of ERK1/2 may de-
termine the final biological response after its activation, we
evaluated whether these parameters may be related to the
highest propension of IST-Mes2 to undergo apoptosis than
ZL55. While we did not identify differences in the ERK1/2
activation kinetics, since both cell lines showed a rapid acti-
vation of the MAP kinase followed by a second peak of ac-
tivity after 2 h of treatment with rhEGF, a more striking dif-
ference was observed in the cytosol-nucleus translocation of
the active isoform of ERK1/2 that was much more higher in
ZL55 cells even in the presence of Gefitinib. Even if we
cannot provide definitive proofs in support, these results
suggest that the more persistent localization of phospho-
ERK1/2 in the nucleus of ZL55 somehow protect these cells
from apoptosis. Further studies are required to confirm this
Interestingly, in both cell lines, EGF-dependent prolifera-
tion relied mainly on ERK1/2 activation, while Akt did not
seem to be involved. In fact, we observed a lack of inhibition
of cell proliferation in the presence of Wortmannin and the
absence of Akt activation upon EGF treatment.
[125I]-EGF binding assay on hMPM cells was performed
to shed light on possible mechanisms of Gefitinib effects. In
both cell lines, when radiolabeled EGF was added to cell
monolayers simultaneously with Gefitinib, an unexpected 2-
3 fold increase in the number of sites/cell was observed. This
phenomenon was dependent on the drug concentration.
Scatchard analysis identified a single class of high affinity
EGFR and KD values were not significantly changed after
drug treatment.
The known mechanism of action of Gefitinib is the com-
petitive inhibition of ATP binding at the catalytic site of
EGFR tyrosine kinase [41]. However, several preclinical
studies on breast and ovarian carcinomas demonstrated that
Gefitinib causes a complete and long lasting inhibition of
EGFR phosphorylation, an event that was proposed to be
also dependent on the sequestration of inactive drug-receptor
complexes [37, 42]. It is known that growth factors induce
transphosphorylation of their receptors mainly by promoting
the association of monomeric receptors into active dimers. It
was hypothesized that receptor dimerization could be also
induced by interaction of anilino-quinazolines at the ATP-
binding site even in the absence of ligand binding [43]. Thus,
we surmised that Gefitinib-induced formation of inactive
dimers could be responsible for the apparent increase in
EGFR binding sites. In fact, we confirmed a significant in-
crease in EGFR dimerization after Gefitinib treatment, with
the dimers stabilized in the non-phosphorylated (inactive)
status even in the presence of EGF. Thus, the stabilization in
the membrane of inactive dimers may represent another way
by which Gefitinib may impair EGFR activity. Similar re-
sults were previously reported by Lichtner et al. [44] that
showed a 2-3 fold increase of EGF binding in the presence
of quinazolines (Gefitinib and PD153035) in human epider-
moid A431 cells that correlated with EGFR dimer formation
even in the absence of ligand. As far as the mechanism re-
sponsible for the increase in EGF binding sites is concerned,
we can hypothesize that either the Gefitinib-dependent
EGFR dimerization can unmask new binding sites in the
inactive receptors or it allows EGF binding to spare recep-
tors sequestered in membrane compartments that prevent
ligand binding in untreated cells. In any case, the final result
is that less active EGFR molecules are available for EGF
Interestingly, this behaviour seems to be more specific
for quinazolines than for other dianilinophthalimide deriva-
tives [44]. It was observed that quinazolinic drugs not only
cause the sequestration of EGFR into inactive dimers [44]
but also trap the ligand into those complexes, thus reducing
the ligand available for the binding to still functional recep-
tors. A similar mechanism of inhibition by Gefitinib was also
identified in a series of breast carcinoma cell lines [37], in
which Gefitinib directly promotes the formation of inactive
EGFR-based homodimers or heterodimers (EGFR/HER2,
EGFR/HER3). Moreover, because a relationship between
Gefitinib and Erlotinib (a similar compound) also with
HER3 receptor have been shown [45], we verified the ex-
pression, by IST-Mes2 and ZL55 cell lines, of other mem-
bers of the HER receptor family (HER2 and HER3) poten-
tially able to form heterodimers with EGFR. Being both
HER2 and HER3 not expressed in hMPM cell lines used we
can conclude that the observed dimers are EGFR homodi-
However, although our and previous studies [44] inde-
pendently demonstrated that the formation of not functional
EGFR dimers is correlated to a dramatic increase in the bind-
ing of [125I]-EGF, an exact molecular mechanism linking
both events is still not identified.
Scientific literature is still lacking of information regard-
ing the behavior of EGF binding to its receptor in presence
of Gefitinib in hMPM in vitro models. To our knowledge no
other in vitro studies, after that of Janne et al. [24], on
hMPM cell lines treated with anti-EGFRTK compounds
have been reported. In that case the authors demonstrate that
Gefitinib is as effective or more effective in vitro against
hMPM as it was against N-SCLC A549 cells; this suggests
that Gefitinib may be a worth optional treatment for patients
with hMPM. Our in vitro study supports these conclusions
and identifies the activation of ERK1/2 as one of the intra-
cellular pathways critical for hMPM cell proliferation. Re-
markably, the results derived from the biological rather the
biochemical approaches on IST-Mes2 and ZL55 cell lines
are not completely overlapping: we can reasonably deduce
Page 14
Gefitinib Targets EGFR Dimerization Current Cancer Drug Targets, 2010, Vol. 10, No. 2 189
that this could be due to the cells origin from patients having
a different, but unfortunately unknown, clinical anamnesis.
In summary, the main goal of the present study was the
assessment of the antiproliferative activity of Gefitinib on
human malignant pleural mesothelioma cells, in vitro. Find-
ings also provide a rationale for the evaluation of anticancer
activity of this compound, either alone or in combination
with traditional cytotoxic antineoplastic agents, in cancer
patients with epithelial, fibrous and/or mixed tumors ex-
pressing or overexpressing functional EGFR.
We also tried to shed light on the molecular machinery
regulated by Gefitinib and structurally-related molecules.
Particularly, we considered the events at EGF binding level
and the subsequent biochemical modifications switched on.
We propose that, in the presence of Gefitinib, EGFRs are
sequestered in inactive dimers at membrane level, preventing
the activation of down-stream effectors, and, in particular, of
the ERK1/2 MAP kinase pathway, Fig. (12). We believe that
a better characterization of the molecular mechanisms of
drugs such as Gefitinib, may help to explore their use as
combination therapy with compounds acting with different
pharmacological mechanisms to, ultimately, leads to the de-
velopment of more efficient anticancer therapeutics.
Authors are grateful to AstraZeneca (Milano, Italy) for
providing us with Gefitinib. Authors thank prof. Maria Cris-
tina Mingari, head of the Immunology Section, dr. Alessan-
dro Poggi, head of the Molecular Oncology and Angiogene-
sis Section and Dr. Michela Paganuzzi, head of the Clinical
Fig. (12). Schematic representation of molecular mechanism of EGF-induced regulation of cell cycle progression and proposed mechanisms
of action of Gefitinib, as detected in vitro in two asbestos induced-hMPM cell models. In normal cells, extracellular signal (triggered by EGF
and several homologous ligands) interacts with the specific domain of a tyrosine kinase transmembrane receptor (EGFRTK) (A) thereby
leading to its dimerization and promoting, by autophosphorylation of intrinsic tyrosine residues located in its cytoplasmic C-terminal tail, its
activation (B). EGFR could also undergo heterodimerization with other HER subfamily members (HER2, HER3). However, these receptors
were not detected in the cell lines analyzed and thus do not participate in the EGF-induced signaling. In physiological conditions EGFR sig-
naling is tightly controlled but in pathologic situations, when generally EGFR is overexpressed, EGF promotes oncogenic activation of sev-
eral intracellular signaling pathways. EGFRTK, following the "ERK1/2 route", stimulates by sequential phosphorylations several down-
stream effectors, in turn Ras, Raf, MEK1/MEK2 and, finally, ERK1/ERK2. Phospho-ERK1/2 translates into the nucleus (C) where subse-
quent gene transcription leads to the activation of several transcription factors involved in metabolism, cytoskeletal proteins regulation and
cell proliferation. In other contexts EGFRTK is the starting point for activation, via PI-3K, also of the "Akt route" which is in charge of vas-
cular permeability, apoptosis suppression, endothelial cell survival, cell cycle progression and other proteins phosphorylation (D). Using
cells pretreated with Wortmannin (E), a PI-3K inhibitor, we demonstrated that, in our cell models, EGF-dependent proliferation does not
require the Akt pathway but, on the other hand, in the presence of the MEK inhibitor PD98059 (F) to block ERK1/2 activation induced by
EGF caused a complete growth arrest. Gefitinib treatment exerts a dual inhibitory effect on EGF-induced proliferation: the block of TK
phosphorylation by occupying the ATP binding site pocket on the tyrosine kinase (G) and the stabilization of inactive EGFR homodimers
even in absence of EGF (H); as a result Gefitinib treatment hinders the EGF-dependent activation of ERK1/2 pathway, which is dominant for
cell survival and proliferation (I).
Page 15
190 Current Cancer Drug Targets, 2010, Vol. 10, No. 2 Favoni et al.
Pathology Section of the National Cancer Institute of Genoa
- Italy, for the assistance and advice in cell cycle analysis
and for the opportunity they gave us to use the Packard In-
strument Cobra II ?-counter for the evaluation of radiorecep-
tor experiments.
BS3 = bis(sulfosuccinimidyl) suberate
EGFR = epidermal growth factor receptor
= extracellular signal-regulated kinase 1 and 2
= fluorescence-activated cell sorter
FITC = fluorescein isothiocyanate
HER2 and 3 = human epidermal growth factor receptor 2
and 3
hMPM = human malignant pleural mesothelioma
= insulin-like growth factors
= mitogen-activated protein kinase
MTT = 3-(4,5-dimethylthiazol-2yl)-2,5
yltetrazolium bromide)
= phosphorus
= platelet-derived growth factor
PI = propidium iodide
= phosphoinositide 3-kinase
= recombinant human epidermal growth fac-
= transforming growth factor beta
= tyrosine kinase
VEGF = vascular endothelial growth factor
Supplementary material is available on the publishers
Web site along with the published article.
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text:
Fashion yes or what were they thinking?
Find a Conversation
Fashion yes or what were they thinking?
|Tue, 08-02-2011 - 11:30am
Okay, so I can maybe see this as a swimsuit top, you would have to be careful what you wore it under as a bra, though. Here is the information on it:
• This dazzling bra is fit for va-va-voom vixens, femme fatales and sultry senoritas
• Features adjustable spaghetti straps
• Bead and sequin construction
• Underwire
• Lightly padded cups
• Double hook and eye closure adjustable to three sizes
By 4 Now Fashions
|Thu, 08-04-2011 - 9:07pm
I suppose it could be worn under a sheer top, and/or the type of top or dress

subset: commoncrawl


text:
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Medicare Part B: The $1,200 question
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The End Is Near ... again
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text: Items 13 to 24 of 37 total Previous 1 / 2 / 3 / 4 Next View: 12 / 24 / 48 Works awesome in Picotope By Ben on 7/18/2014 Ben would recommend this product to a friend I have this ATO in my picotope and it always keeps the water level within 1/4" in the tank. Pump is quiet and I've never experienced the beeping issues that other's have talked about. My no hassle procedure for a water change is: -Turn off ATO -Remove water -Replace water to level where I want it -Turn ATO on Done, no alarms or anything like that - going on 6 months with this unit. Durring setup you love it but then... By shawn on 7/2/2014 shawn wouldn't recommend this product to a friend The safety features sound great but seem to also cause headaches. It is set with a 48 hr no fill alarm that isn't mentioned in the booklet. After setup if it's not activated to fill within the 48 hrs the alatm goes of with blue light. The book says the blue light means misalignment or the lense needs to be cleaned. So you clean it and check alignment, reset it by pulling out power plug. 48 hrs it goes off again. If your tank doesn't evaporate that quickly I dont see a way around this repetitive annoying alarm. It could be a very nice little ATO but appears to be to smart for my setup. Same problem . Beeping all times By Not good ato on 6/22/2014 Not good ato wouldn't recommend this product to a friend Beeps all times , not very accurate . Won't recomend Use with float switch for backup By pleco on 6/21/2014 pleco would recommend this product to a friend It works well most of the time, but should only be used with a back-up float switch to turn it off in case of high water level. Product worked flawlessly for me for the last two weeks until this morning when it malfunctioned and overfilled the sump, emptying the 10G reservoir over the course of one hour. Best ATO ever By Nickbruh on 6/4/2014 Nickbruh would recommend this product to a friend I have a 15 gallon rimless cube with sps and Los and designer zoas I was a big beleive in toppin off manually everyday ... Mad was I dumb with this ato I mean it comes with piping , a pump, I mean literally everything and was so dan easy to setup literally don't let the 149$ price tag fool you, this thing is fool proof I've been in the game for 6 months so I'm new to it if I knew this was the best ato I would have gotten it earlier , iHaTE float switches thy are ugly and too much wiring for me especially for a smaller tank with no sump a know Awesome Product By lwood on 5/24/2014 lwood would recommend this product to a friend Very efficient and easy to put together, not at all complicated like most ATO's easy of use is 10/10. Sleek design for smaller systems such as mine. I will give it a 7/10 on price but it more than makes up for it in efficiency. Wow. Works as advertised. By HappyRC on 5/19/2014 HappyRC would recommend this product to a friend Keeps my salinity in check. More importantly, when I am away from home for more than 1 week, my water level would get so low that I'd risk my overflow running dry. This ato device assures that my overflow will never run dry. My acrylic aquarium is a little thicker than the 1/2 inch that the product says it can do so I taped some Velcro below it to keep it from sliding down. NOTE: the device must be attached to a CLEAR glass/acrylic. awesome!!! By amerykno on 5/6/2014 amerykno would recommend this product to a friend this is an amazingly simple and excellent working product. Great ATO! By ronstopbl on 5/1/2014 ronstopbl would recommend this product to a friend Looked for quite some time for a good quality ATO system for my sump tank. With very limited room to work with, I finally narrowed it down to the AutoAqua ATO-Glad I did! This ATO is small and effective!. Very easy to set up and operate. Had one small alarm glitch which was quickly remedied by a call to tech dept. Quick reset and problem solved. Definitely a great purchase and addition to my set up! Thanks AUTOAQUA and BRS! Perfect By Steve on 4/22/2014 Steve would recommend this product to a friend It works flawlessly. Simple as that. Awesome ATO! By paul87 on 4/17/2014 paul87 would recommend this product to a friend This is my first ATO so I really can't say if its worse or better than any of the others. However I can say it works exactly as advertised and id super small and includes a pump! Installation is a breeze and its compact size and no moving parts sealed the deal for me. Great product good job AquaAuto! Minimal Footprint and Quick Setup By Brittany on 4/9/2014 Brittany would recommend this product to a friend I just received this ATO from BRS today. I had it up and running in less than 10 minutes. I have very limited space in my sump at the moment and I was still able to squeeze this baby in. The pump is so tiny I was skeptical but it sure does push some water! Time will tell how reliable it will be, but so far I am more than pleased. Great product at a great price. Thanks Coralvue and BRS! Write Your Own Review You're reviewing: AutoAqua Smart ATO - Auto Top Off System *How do you rate this product? *Would you recommend this product to a friend? Yes No *Nickname *Summary of Your Review *Review Submit Review


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I had a requirement to implement dynamic hierarchy in a project. One possible solution was to use a Graph database NoSQL solution like Neo4j. What I designed in a graph implementation in RDBMS. In this solution I designed a table has a Many To Many relationship with itself. An example of this is given here. I have a table (TABLE_A) that defines different entities and another table TABLE_B that defines type of the entities, and TABLE_B has a many to many relation with it self that defines relation between different entity types. This is store in a JoinTable, TABLE_C. Similarly TABLE_A has a many to many relationship with itself to define instances of different relations defined in TABLE_C. In Java I have used only two classes to implement this structure.
Now this many to many relationship in my case is not a always a many to many relationship. Sometimes it's a one to many and at time a one to one.
Firstly, is a No SQL Graph DB more suited to my requirement than a Relational DB since the data that I'm planning to test will be in the order of Hundred Thousand records.
Secondly, If RDBMS solution is better then is there a way to include a property in the join table TABLE_C that defines the multiplicity of the relation i.e. whether it's a one to many or a many to many or a one to one relation?


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Wanderlei vs Rmapge II and Fedor vs Cro Cop are down in History, in more recent years it has to be Shogun vs Hendo and 2013 fight of the year ( in my opinion) Wanderlei vs Stann
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POLL: What Is Your Favorite Fight Ever?
There's not many MMA fights that can legitimately be called the best ever. Today, some are calling last night's epic Gilbert Melendez vs. Diego Sanchez fight the best the Octgon has ever featured. Do you agree? If not, what is your favorite mixed martial arts war of all time?
26 Comments
October 21, 2013 at 8:34 am
Voted for my personal favorite Garcia vs Jung I' didn't see Don Fry vs Takiyama he craziest fight in history
October 21, 2013 at 8:55 am
My bad its the 4th option
October 20, 2013 at 7:24 pm
How is the fight that sparked the debate not even it's own option? I wouldn't pick it, but from the sound of some fans (and announcers) it could have been a viable option
October 20, 2013 at 7:28 pm
Yeah it should be. Not my vote, but it deserves a spot.
October 20, 2013 at 7:33 pm
Diego Sanchez vs. Clay Guida
October 20, 2013 at 7:33 pm
Nick Diaz vs Gomi… He pulled off a Gogoplata while superstoned. 50 metabolites is a fail.. He came in at 157..inspirational lol
October 21, 2013 at 6:49 am
Yeh, when I talk about it to people..they do not realize how good Gomi was at that time..he was the man.
October 20, 2013 at 8:11 pm
Ranking the best will always been subjective but I thought that fight last night was easily better than Griffin and Bonnar.
October 20, 2013 at 9:44 pm
Funny that Diego Sanchez's name shows up on the list three times.
It's testimony to how great he is and how he always shows up for a fight. Put him in the ring with someone of a like mind, as last night for example, and it's nothing but fireworks and thunder.
Again, I think if that had been a five round fight, Diego might have won it. He gets tougher as the bout goes on.
What to do with this guy, man? I would love to see Diego in a title fight. Even in loss he was amazing. He is just tough as nails and doesn't quit.
Even though un-ranked, I hope the UFC continues to give him top fighters. If he's not in the top ten, then surely he is #11.
That was just an awesome fight. Damn, I wish it had been 5 rounds.
I'm going to say, yeah. I love that fight. Maybe the list should be best 3 round fights and best 5 round fights. Jones / Gustafsson and Silva / Sonnen I, for those.
October 20, 2013 at 9:47 pm
I think if it went 5 rounds he would have lost from doctor stoppage early in the 4th. His ENTIRE eyebrow was sunk into that gash lol
October 20, 2013 at 11:54 pm
I personally don't have a favorite, but some underrated fights (ones that didn't make the list) are as follows:
Karo Parisyan vs. Nick Diaz
Karo Parisyan vs. Diego Sanchez
Ben Henderson vs. Donald Cerrone 1
Ben Henderson vs. Anthony Pettis 1
Rampage vs. Henderson
Eddie Alvarez vs. Mike Chandler
and maybe the most underrated… Frankie Edgar vs. Gray Maynard 2
* The fight you guys are probably thinking of for Matt Hughes/Frank Trigg is the second. The first was good, but the second is one of the most iconic moments for the start of the TUF era.
October 21, 2013 at 11:55 am
Yes they could all be added to this list.
October 21, 2013 at 6:31 am
Personally I don't get much out of the all heart not technique fights and as such Diego is one of my least liked fighters. It's just sad that fighters like Guida, Diego and Garcia get so many wins despite losing the fight. Getting your *** kicked while pretending to win isn't winning.
It's also really silly to have this poll so soon after the fight, to think that all the goldfish out there wouldn't vote for whatever brainless brawl happened most recently is just silly.
October 21, 2013 at 12:02 pm
Heart is one the very greatest aspects of MMA. It is a skill and a technique within itself to be able to overcome certain physical limitations with all out ferocity. If heart wins you a fight then what good was the superior skill set of the opponent? Diego makes a very tough fight as we have seen for any superior striker so that makes his heart worth virtually just as much. Pretending to win is GSP or Askren pointing out a decision win with no risk. Nobody really wins unless there is a finish. I used to dislike Diego because he acted like nobody stood a chance against him like when he was in TUF. He has changed though and has kept his aggression but let go of all the hate and stupidity.
October 21, 2013 at 12:05 pm
I started watching since UFC 1 and I actually think that may have been the ballsiest fight I have ever seen in the octagon. I think Don Frye was the only guy to compare to Diego that I can think of. Not pretty but definitely real fighters.
October 21, 2013 at 7:41 am
Sanchez/Melendez would`nt have made my list, good fight but not an epic one.
Sapp/big nog was fun to watch. Stann/Silva was another good one.
Three personal favs: Gomi/ Diaz, Fedor/Cro cop and Sonnen/Silva 1.
Can`t forget Jones/Gustaf most recently.
October 21, 2013 at 10:56 am
It's always tough answering this question only because there are so many factors. Some of these fights I saw as they happened which makes it a bit more exciting because I was watching it unfold at the same time as everyone else was. Some of these are more technically exciting while others are just barn burners and the pace and energy was unreal. Some of them have great grappling while some are slugfests. It's too tough to be honest but if I had to pick one on the list I'll be cliche and go with Bonnar vs. Griffin. Only because I like to think that at any time any fighter regardless of ranking can put on an amazing fight. It just takes the right mix of styles.
October 21, 2013 at 12:06 pm
True enough.
October 21, 2013 at 2:00 pm
anything with wanderlei, diego, nick diaz or shogun on it, even losing they will go forward zombie mode for the crowds.
Voted hendo-shogun, but wand-stann was another recent classic
October 21, 2013 at 3:15 pm
Yes actually Wand/Stann was a great one too just not as long.
October 21, 2013 at 9:12 pm
evangelista santos vs melvin manhoef
October 21, 2013 at 9:29 pm
watch it. its the best fight ever.
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