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Cyclooxygenase-2 (COX-2) and the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), have been implicated in the progression of hormone-refractory prostate cancer; however, a mechanistic link between the bioactive peptide and COX-2 expression in prostate cells has not been made.
We report that BBS stimulates COX-2 mRNA and protein expression, and the release of prostaglandin E2 from the GRP receptor (GRPR)-positive, androgen-insensitive prostate cancer cell line, PC-3. BBS-stimulated COX-2 expression is mediated, in part, by p38MAPK and PI3 kinase (PI3K)/Akt pathways, and blocked by a GRPR antagonist. The PI3K/Akt pathway couples GRPR to the transcription factor, activator protein-1 (AP-1), and enhanced COX-2 promoter activity. Although BBS stimulates nuclear factor-kappaB (NF-κB) in PC-3, NF-κB does not regulate GRPR-mediated COX-2 expression. The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA. Expression of recombinant GRPR in the androgen-sensitive cell line LNCaP is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways.
Our study establishes a mechanistic link between GRPR activation and enhanced COX-2 expression in prostate cancer cell lines, and suggests that inhibiting GRPR may, in the future, provide an effective therapeutic alternative to non-steroidal anti-inflammatory drugs for inhibiting COX-2 in patients with recurrent prostate cancer.
Keywords:gastrin-releasing peptide receptor; signal transduction; prostate cancer; neuroendocrine differentiation; hormone-refractory
Prostate cancer is the most commonly diagnosed form of cancer among men in the United States and second only to lung cancer as a cause of cancer-related death. In 2010, the American Cancer Society estimates that over 217,000 new cases of prostate cancer will be diagnosed and more than 32,000 men will die, most from metastatic, androgen (hormone)-refractory disease (American Cancer Society. Cancer Facts & Figures 2010, Atlanta: American Cancer Society; 2010; http://www.cancer.org webcite).
Hormone-refractory prostate cancer is characterized, in part, by focal expansion of a malignant cell subpopulation with neuroendocrine (NE) features. NE cells lack expression of androgen receptors, express NE markers, such as neuron-specific enolase and chromogranin A, and contain numerous secretory granules rich in neuropeptides including calcitonin, calcitonin gene-related peptide [1], parathyroid hormone-related protein [2], and the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP). Although the impact of NE differentiation on poor prognosis and androgen independence has been extensively studied [3], the molecular mechanisms linking NE tumor cells and their bioactive neuropeptides to disease progression are still unclear.
Increased expression of cyclooxygenase-2 (COX-2), an enzyme that catalyzes the synthesis of prostanoids such as prostaglandin E2 (PGE2) from arachidonic acid [4-6], was identified as an independent predictor of prostate cancer progression [7]. Clinical trials using COX-2 inhibitors in patients with biochemical recurrence of prostate cancer have suggested that COX-2 inhibition may improve survival [8,9], and pre-clinical studies with cell lines and animal models have established a functional link between COX-2 expression and an aggressive cancer phenotype. Specifically, Dandekar and coworkers [10] have demonstrated that overexpression of COX-2 in human prostate cancer cell lines induced chemotherapeutic resistance, decreased apoptosis, and increased tumor angiogenesis and growth. In a transgenic mouse model of prostate carcinogenesis, pharmacological inhibitors of COX-2 suppressed tumor growth and decreased metastatic spread [11,12]. Together, these studies implicate COX-2 in prostate cancer progression; however, the molecular mechanisms leading to its increased expression and the relationship between enhanced expression and NE differentiation requires further investigation.
COX-2 expression can be induced by multiple factors including growth factors, proinflammatory cytokines, and peptide hormones [13-15]. BBS is a 14-amino acid peptide originally isolated from the skin of the frog, Bombina bombina, and is a functional homologue to GRP. In humans, GRP binds with high affinity to the GRP receptor (GRPR), a member of the G protein-coupled receptor superfamily [16]. Clinical, histological, and experimental observations have implicated GRP and GRPR in the pathophysiology of prostate cancer progression. Logothetis and Hoosein [17] reported that 40% of patients with hormone-refractory prostate cancer had significantly elevated levels of GRP in their serum. GRP and GRPR are expressed by NE cells in prostate cancer tissue and by prostate cancer-derived cell lines [18,19]; BBS stimulates the growth of both orthotopic and ectopic prostate cancer cell xenografts in athymic nude mice through GRPR-mediated mechanisms [20,21]. BBS also promotes expression of metalloproteinases [22] and increases prostate cancer cell migration and invasion [23-25]. Previously, we reported that BBS stimulates the expression of the proangiogenic genes IL-8 and vascular endothelial growth factor (VEGF) in human prostate cancer cell lines [26].
Since COX-2 and GRPR both regulate cellular processes that contribute to the progression and metastatic spread of prostate cancers and, because BBS has been shown to regulate COX-2 expression in cells from other tissues [27-29], we reasoned that GRPR activation and COX-2 expression may be mechanistically linked in prostate cancer cells. Here, we report that BBS stimulates an increase in COX-2 mRNA, protein expression, and the release of PGE2 from the GRPR-positive, androgen-insensitive prostate cancer cell line, PC-3. The stimulatory effects of BBS on COX-2 expression and PGE2 production are mediated by p38MAPK and PI3 kinase (PI3K)/Akt pathways and blocked by the selective GRPR antagonist BIM26226. The PI3K/Akt pathway couples GRPR to the activation of the transcription factor, activator protein-1 (AP-1), and enhances COX-2 promoter activity. BBS also stimulates nuclear factor-kappaB (NF-κB) activation in PC-3; however, NF-κB does not regulate GRPR-mediated COX-2 expression. The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA. Expression of recombinant GRPR in the GRPR-negative, androgen-sensitive cell line LNCaP, is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways. Together, these results define a molecular mechanism for enhanced COX-2 expression in prostate cancer cells, and suggest a means by which NE-differentiated tumor cells and their bioactive neuropeptides may contribute to disease progression.
BBS stimulates COX-2 mRNA and protein expression
To determine whether BBS stimulates COX-2 expression, we treated the androgen-insensitive prostate cancer cell line PC-3 with BBS and measured steady-state levels of COX-2 mRNA and protein at various time points. Compared to vehicle-treated [phosphate buffered saline (PBS)] control cultures, COX-2 mRNA was increased at 1 h following addition of BBS (10 nM) and peaked between 2 and 6 h (Figure 1A). Increased COX-2 protein was also detected at 1 h following BBS treatment, peaked between 4 and 8 h, and returned to baseline levels by 24 h (Figure 1B). Consistent with the lack of change in basal COX-2 mRNA levels over the time course (Figure 1A); we did not observe a change in the basal expression of COX-2 protein in non-treated cells (data not shown). Induction of both COX-2 mRNA and protein expression was dependent on the concentration of BBS. Increased COX-2 mRNA and protein levels were detected in cells treated with as little as 0.1 nM BBS for 4 h and maximal induction was observed in cells treated with 1 to 10 nM BBS (Figure 1C and 1D, respectively).
Figure 1. BBS treatment induces a time- and dose-dependent increase in COX-2 mRNA and protein expression. A) Autoradiography of Northern blots demonstrating time-dependent increase in COX-2 mRNA steady-state levels in PC-3 cells treated with 10 nM of BBS. Blots were reprobed for 18S ribosomal RNA to ensure equivalent loading and transfer of total RNA in each lane. B) Immunoblot showing time-dependent increase in COX-2 protein levels following treatment with BBS (10 nM). Blots were reprobed for β-actin to ensure the equivalent loading and transfer of protein samples. C, D) Effects of a 4-h treatment with different concentrations of BBS on COX-2 mRNA (C) and protein (D) expression.
GRPR mediates BBS-stimulated COX-2 protein expression and PGE2 synthesis
COX-2 converts arachidonic acid, released from phospholipids by the action of phospholipase A2, to prostaglandin H2 - the common precursor of all prostaglandins, including PGE2. To assess whether BBS stimulation of COX-2 expression was associated with an increased prostaglandin synthesis, the levels of PGE2 released from PC-3 cells were measured using an enzyme-linked immunosorbent assay. Compared to vehicle-treated control cultures, BBS induced a time-dependent increase in the PGE2 levels that peaked between 4 and 16 h (Figure 2A, open bar vs. black bar, † p ≤ 0.01). Pretreatment with the GRPR-selective antagonist, BIM26226 ([D-F5-Phe6, D-Ala11]Bombesin(6-13)Ome] [30], (1 μM) for 30 min blocked the BBS-induced increase in PGE2 (Figure 2A, black bar vs. cross-hatched bar), *p ≤ 0.01). Consistent with this observation, BIM26226 also inhibited BBS-stimulated increases in COX-2 protein expression (Figure 2B).
Figure 2. GRP receptor mediates BBS-stimulated PGE2 release and COX-2 expression. A) PC-3 cells were pre-treated with BIM26226 (1 μM) for 30 min and then incubated with or without 10 nM of BBS over a time course. The amount of PGE2 released into the culture media was assessed by enzyme-linked immunoassay (Methods). The data was normalized to total cellular protein per well. [† BBS vs. vehicle (no treatment), p < 0.01; * BBS+BIM26226 vs. BBS alone, p < 0.01]. B) Immunoblot showing the effects of BIM26226 on BBS-stimulated COX-2 protein expression. PC-3 cells were pretreated with BIM26226 for 30 min and then treated with BBS for 4 h. Blots were reprobed for β-actin to ensure the equivalent loading and transfer of protein samples.
BBS activates multiple intracellular signaling pathways in PC-3 cells
COX-2 expression is regulated by multiple intracellular signaling pathways including the MAPK pathways, MEK/ERK and p38MAPK, and the PI3K/Akt pathway [31]. To determine if these pathways were activated by BBS, PC-3 cells were treated with peptides over a time course and the activation state of each pathway assessed by immunoblotting. BBS treatment (10 nM) induced a time-dependent increase in the levels of activated (phosphorylated) ERK1 (pERK1) and ERK2 (pERK2) (Figure 3A). The levels increased rapidly with a peak phosphorylation occurring between 1 and 15 min and remained elevated above baseline 60 min after treatment with BBS (Figure 3A). BBS stimulated a transient activation of p38MAPK. The levels of phospho-p38MAPK (pp38MAPK) peaked between 5 and 30 min and, in contrast to the activation of either ERK1 and 2 or Akt, returned to baseline levels by 60 min (Figure 3A). The levels of phospho-Akt (pAkt) increased at 5 min, reached a plateau by 15 min, and remained elevated for the duration of the time course (Figure 3A). Over the same time course, we did not observe any change in the activation state of these pathways in non-stimulated cells (data not shown).
Figure 3. The PI3K/Akt and p38MAPK pathways mediate BBS-stimulated COX-2 expression and PGE2 synthesis. A) PC-3 cells were treated with 10 nM BBS over a time course, and the levels of phosphorylation (activated) of ERK1 and ERK2 (pERK1 and pERK2), p38MAPK (pp38MAPK) and Akt (pAkt) were detected by immunoblotting with phospho-specific antibodies. The level of total Akt was assessed to insure the equivalent loading of protein samples in each lane. B, C) Effects of the p38MAPK inhibitor SB203580, the PI3K/Akt inhibitor, LY294002, and the ERK inhibitor, PD98059 on BBS-stimulated COX-2 mRNA (B) and protein (C) expression. Blots were reprobed for 18S ribosomal or β-actin to ensure the equivalent loading and transfer of RNA and protein samples, respectively. D) Effects of the SB203580, LY294002, PD98059 on BBS-stimulated (6 h) PGE2 elaboration from PC-3 cells [† BBS vs. vehicle, p ≤ 0.01; * BBS+SB203580 or LY294002 vs. BBS alone, p ≤ 0.01, n = 3].
BBS-stimulated COX-2 expression is regulated by the p38MAPK and PI3K/Akt pathways, but not the MEK/ERK signaling axis
To assess the roles of the p38MAPK, PI3K/AKt, and MEK/ERK pathways in BBS-stimulated COX-2 expression, PC-3 cells were pretreated (30 min) with either the p38MAPK inhibitor (SB203580, 10 μM), the PI3K inhibitor (LY294002, 25 μM) or the MEK1,2 inhibitor (PD98059, 20 μM) and then stimulated with BBS (10 nM) for 4 h. Agonist treatment failed to increase either COX-2 mRNA or protein levels when the cells were pretreated with either SB203580 or LY294002 (Figure 3B and 3C). In contrast, pretreatment with PD98059 did not inhibit BBS-stimulated increases in COX-2 mRNA nor protein expression. Consistent with these observations, SB203580 or LY294002 inhibited BBS-stimulated PGE2 release from PC-3 cells, whereas PD98059 had no effect (Figure 3D).
Inhibition of PI3K/Akt pathway reduces BBS-stimulated COX-2 promoter activity
The cellular levels of COX-2 mRNA can be regulated both by enhanced gene transcription and inhibition of message degradation [31,32]. To determine whether BBS treatment enhanced COX-2 gene transcription, PC-3 cells were first transiently transfected with a transcription reporter construct consisting of 1.4 kb of the human COX-2 promoter coupled to a luciferase gene and then stimulated with BBS over a time course. BBS induced a time-dependent increase (1.4- to 2.3-fold) in COX-2 promoter activity when compared to vehicle-treated control cell cultures (Figure 4A). To determine whether the p38MAPK or PI3K/Akt pathways were involved in BBS-stimulated COX-2 transcription, cells were pretreated with SB203580 (10 μM) or LY294002 (25 μM) for 30 min followed by a 6-h treatment with BBS (10 nM). Compared to BBS treatment alone, LY294002 inhibited approximately 50% of the increase in BBS-stimulated luciferase activity (Figure 4B). In contrast, SB203580 had no effect on BBS-stimulated COX-2 promoter activity (Figure 4B), suggesting that the PI3K/Akt pathway, not the p38MAPK pathway, is involved in BBS-induced COX-2 gene transcription in PC-3 cells.
Figure 4. The PI3K/Akt pathway regulates BBS-stimulated COX-2 promoter activity and AP-1 activation. PC-3 cells were co-transfected with plasmids containing the 1.4 kb human COX-2 promoter 5' of a luciferase reporter gene and β-galactosidase. A) Twenty-four hours after transfection, cells were treated either with vehicle or 10 nM BBS for 2, 4, and 6 h. Changes in luciferase activity (mean ± S.D, n = 3) are expressed relative to vehicle treated at each time point after normalizing for transfection efficiency using β-galactosidase activity [* BBS vs. time-point matched vehicle control, p < 0.05]. B) Pre-treatment with LY294002 (25 μM) partially inhibited BBS-stimulated COX-2 promoter activity [* LY294002 vs. BBS alone, p < 0.05, n = 3], whereas SB203580 (10 μM) had no effect. C) Autoradiogram showing binding of nuclear proteins to 32P-labeled oligonucleotide containing the AP-1 consensus sequence [lane 1, radiolabeled probe only; lane 2, nuclear proteins + radio-labeled probe and excess unlabeled probe; lane 3, nuclear protein from vehicle-treated cells; lane 4 nuclear proteins from cells treated with BBS (10 nM) for 30 min; lanes 5 and 6, nuclear proteins from cells pretreated with LY294002 (25 μM) for 30 min followed by vehicle or BBS for 30 min]. D) PC-3 cells were treated with BBS (10 nM) or TNF-α (10 ng/ml) alone or in combination with inhibitors for 30 min, fixed, and immunostained with an antibody to the p65 subunit of NF-κB. Quantification of the effects of curcumin (Cur) (20 μM) and LY294002 (LY) (25 μM) on BBS-stimulated NF-κB nuclear translocation. Data are expressed as the ratio of NF-κB-positive stained nuclei divided by the total number of cells per high power field (200X). Four fields were counted for each condition from 3 independent experiment [† BBS or BBS + LY294002 vs. vehicle or LY294002 alone, p ≤ 0.001; * BBS + curcumin vs. BBS alone or TNFα + curcumin vs. TNFα alone, p ≤ 0.001].
LY294002 inhibits BBS-stimulated AP-1 binding activity but not NF-κB nuclear translocation
The human COX-2 promoter contains multiple regulatory sites that bind transcription factors including nuclear factor-κB (NF-κB) [33,34] and AP-1 [35]. Electrophoresis mobility shift assays showed that BBS treatment of PC-3 cells induced an increase in AP-1 binding activity (Figure 4C) that was inhibited by pretreatment with LY294002 (25 μM) for 30 min (Figure 4C), suggesting that PI3K/Akt-mediated AP-1 activation is involved in BBS regulation of the COX-2 promoter activity. In addition to activation of AP-1, we previously reported that BBS also induced NF-κB activation in PC-3 cells [26]. NF-κB is an inducible dimeric transcription factor that belongs to the Rel/NF-κB family of proteins [36]. Activation of NF-κB involves its dissociation from the inhibitor protein, IκB, followed by its translocation to the nucleus where it binds to specific DNA sequences in the promoter regions of multiple genes including COX-2 [37]. To confirm that BBS activated NF-κB, PC-3 cells were treated with peptide for 30 min, fixed, and immunostained with an antibody to the p65 subunit of NF-κB. Treated cells demonstrated increased nuclear NF-κB immunoreactivity when compared with vehicle-treated cultures (Figure 4D). Pretreating cells with the NF-κB inhibitor, curcumin (diferuloylmethane, 20 μM), inhibited both BBS- and TNF-α-induced NF-κB translocation to the nucleus (Figure 4D). To assess the role of the PI3K/Akt pathway in BBS-induced NF-κB activation, cells were pretreated with LY294002 for 30 min, followed by 30 min treatment with agonist. LY294002 had no effect on the BBS-induced NF-κB translocation (Figure 4D). Since LY294002 inhibits COX-2 mRNA expression (Figure 3A), COX-2 promoter activity (Figure 4B) and AP-1 binding (Figure 4C), these data suggest that AP-1 and not NF-κB regulates BBS-stimulated COX-2 promoter, in part, through activation of the PI3K/Akt pathway.
p38MAPK activity enhances the stability of COX-2 mRNA
Since inhibition of the p38MAPK pathway did not block BBS-stimulated COX-2 promoter activity (Figure 4B), we assessed its potential role in regulating COX-2 mRNA stability. PC-3 cells were stimulated for 4 h with BBS (10 μM) and then treated with actinomycin D (5 μM) alone or in combination with either SB203580 or LY294002. RNA was isolated from the cells over a time course and the level of COX-2 mRNA at each time point assessed by Northern blotting (Figure 5A). COX-2 mRNA degraded slowly in BBS-stimulated cells treated with actinomycin D alone or in combination with LY294002; less than 20% of the COX-2 message was lost over the 90-min time course (Figure 5A and 5B). In contrast, the levels of COX-2 mRNA decreased rapidly in cells treated with a combination of actinomycin D and SB203580; approximately 50% of the COX-2 message was degraded in cells treated for 30 min (Figure 5B), suggesting that p38MAPK activity stabilizes BBS-induced increases in COX-2 mRNA levels.
Figure 5. p38MAPK activity enhances COX-2 mRNA stability. A) Autoradiogram of Northern blot showing the effects of p38MAPK and PI3K/Akt inhibition on COX-2 mRNA degradation. Cells were stimulated with BBS for 4 h, and attenuated 4 μM actinomycin D was added (t = 0) alone or with SB203580 or LY294002 and total RNA isolated at 15, 30, 60, and 90 min. Blots were reprobed for 18S ribosomal RNA to ensure equivalent loading and transfer of total RNA in each lane. B) Graph of densitometric analysis of blot in panel A.
Expression of GRPR is sufficient to confer BBS-inducible COX-2 expression in LNCaP cells
LNCaP prostate cancer cells, in contrast to the PC-3 cells, are androgen-responsive, poorly invasive [23] and do not have specific GRPR binding sites, as assessed by radioligand binding to 125I[Tyr4]-Bombesin [38] and [Ca2+]i mobilization in response to BBS [39]. To determine whether the acquisition of the GRPR was sufficient to confer BBS-inducible COX-2 expression, LNCaP cells were stably transfected with an expression plasmid containing GRPR cDNA downstream of the constitutively-active CMV promoter [40]. GRPR mRNA expression was confirmed by Northern blot (Figure 6A), and the functional status of the receptor was demonstrated by intracellular Ca2+ imaging using Fura-2 (Figure 6B). Immunoblots revealed that BBS stimulated COX-2 protein expression in GRPR-transfected cells, but had no effect on LNCaP cells expressing control vector (Figure 6C). Also, similar to PC-3 cells, pretreatment of GRPR-transfected LNCaP cells with either SB203580 (10 μM) or LY294002 (25 μM) completely inhibited BBS-stimulated PGE2 release, suggesting that GRPR regulates COX-2 activity via the p38MAPK and PI3K/Akt pathways (Figure 6D).
Figure 6. Expression of GRPR in LNCaP cells confers BBS-stimulated COX-2 expression and PGE2 synthesis. A) Northern blot confirming the expression of GRPR in transfected LNCaP cells (n = 1). B) Time-dependent change in intracellular Ca2+ concentration measured with Fura-2; comparison of GRPR transfected (LNCaP/GRPR) to control vector-transfected LNCaP cells (LNCaP/Vector) (tracing represents the average change in [Ca2+]i from 40 cells). C) Immunoblot showing effects of BBS treatment on COX-2 protein expression in LNCaP/Vector and LNCaP/GRPR cells. PC-3 cells were used as a positive control. Blots were reprobed for β-actin to ensure the equivalent loading and transfer of protein samples. D) Effects of SB203580 and LY294002 on BBS-stimulated PGE2 elaboration form LNCaP/GRPR cells. Cells were pretreated for 30 min with the inhibitors and then stimulated with BBS for 6 h. PGE2 levels in the culture media were determined as described (Material and Methods) [† BBS vs. vehicle, p < 0.01; * BBS+SB203580 or LY294002 vs. BBS alone, p < 0.01].
Although the aberrant overexpression of COX-2, the BBS-like peptide, GRP, and GRPR has been documented for prostate cancers with NE features, particularly in the setting of recurrent disease [3,7,41], a mechanistic link between GRPR activation and COX-2 expression in prostate cancer cells has not been made. The studies presented here establish that BBS can stimulate COX-2 expression and begins to define the molecular signal transduction pathways linking GRPR to COX-2.
Expression of COX-2 can be regulated by multiple signaling pathways that affect both gene transcription and post-transcriptional mRNA processing [42-45]. We found that BBS-stimulated COX-2 expression requires the activation of both the p38MAPK and PI3K/Akt pathways in PC-3 and LNCaP cells expressing recombinant GRPR. LY294002 partially inhibited BBS-stimulated COX-2 promoter activity, decreased COX-2 mRNA and protein levels, and reduced PGE2 secretion, whereas, inhibition of p38MAPK destabilized BBS-stimulated COX-2 mRNA, resulting in a decrease of both COX-2 protein expression and PGE2 production. The MEK inhibitor, PD98059, had no effect on either BBS-stimulated COX-2 expression or PGE2 secretion, in contrast to previously published data from our laboratory showing that, in intestinal epithelial cells, BBS stimulated COX-2 expression through a MEK/ERK-dependent pathway [40].
Sequence analysis of the 5'-flanking region of the human COX-2 gene has identified multiple potential transcriptional regulatory elements including two NF-κB sites located at 213 to 222 and 438 to 447 [46,47] base pairs 5' of the transcriptional start site, and an AP-1 site located ~60 base pairs upstream from the start site [31]. Although we have previously reported NF-κB-dependent regulation of VEGF and IL-8 expression [26] in PC-3 cells, in the present study NF-κB does not appear to be involved in GRPR-regulation of the endogenous COX-2 promoter. This conclusion is based on the observation that LY294002 can inhibit COX-2 mRNA expression and partially reduced promoter activity, but does not block BBS-stimulated NF-κB activation. In contrast, LY294002 does inhibit BBS-induced AP-1 binding, suggesting that AP-1 may be the primary transcription factor involved in BBS-stimulated COX-2 mRNA expression in PC-3 cells. These findings are consistent with data from intestinal epithelial cells showing that BBS-stimulated COX-2 expression via an AP-1-dependent pathway [40].
Despite the fact that more than 80% of patients with late stage prostate cancer initially respond to androgen-ablation therapy [48], more than half will progress to a hormone-refractory disease within 16 to 18 months after treatment [49]. Elucidation the molecular mechanisms mediating the conversion of androgen-sensitive prostate cancers to hormone-refractory disease is one of the most critical tasks in improving current treatment strategies and patient survival. A recent study evaluating archival prostate cancer specimens by immunohistochemical methods identified COX-2 levels as an independent predictor of prostate cancer recurrence [7]. At 62-months follow-up, COX-2 immunostaining predicted cancer progression with 82% sensitivity and 81% specificity. Additionally, two clinical trials have generated early, but promising, data that inhibition of COX-2 can benefit patients with biochemical recurrence of prostate cancer (i.e., the same transcription factor can regulate increasing PSA levels, but no clinical evidence of disease following androgen-ablation therapy). In the first study, Pruthi and associates [8] studied 12 patients who were given the selective COX-2 inhibitor, celecoxib, following a diagnosis of biochemical recurrence. Five of the 12 patients had decreased PSA levels, and three of the 12 had stabilization of their PSA levels at 3, 6, and 12 months following the initiation of therapy. In a second larger randomized control trial comparing placebo (n = 40) to celecoxib (n = 38), the group receiving celecoxib for 6 months showed a significantly decreased rate of rise in PSA levels when compared to the placebo control patients [9]. Together, these trials suggest that targeting COX-2 can be beneficial. Unfortunately, the studies were terminated by the United States Food and Drug Administration because of the potential risk of cardiovascular complications with the current cadre of COX-2 inhibitors.
Novel strategies for inhibiting COX-2 could once again make it a viable therapeutic target in the future. Inhibiting GRPR may provide an effective therapeutic alternative for decreasing COX-2 expression and activity in patients with recurrent prostate cancer. Proof of this concept is provided by a recent pre-clinical study that evaluated the effects of the GRPR antagonist, RC-3940-II, in an orthotopic non-small-cell lung carcinoma model [29]. Similar to prostate cancers, lung cancers express GRP and GRPR [50] where they promote tumor progression and metastatic spread through autocrine and paracrine mechanisms. Hohla and colleagues [29] showed that daily treatment of NSCLC tumor-bearing mice with RC-3940-II reduced the mean lung tumor weight by up to 53%. Importantly, the decreased tumor growth was associated with antagonist-induced decreases in p-Akt levels and COX-2 expression suggesting, together with the data presented herein, that GRPR blockage may be an effective means of decreasing COX-2 expression within receptor-positive tumor tissue.
Our study establishes a mechanistic link between GRPR activation and enhanced COX-2 expression in prostate cancer cell lines, and suggests that inhibiting GRPR may provide an alternative to non-steroidal anti-inflammatory drugs for inhibiting COX-2 in patients with recurrent prostate cancer.
BBS was purchased from Bachem (Torrance, CA). Inhibitors of p38MAPK (SB203580), MEK-1 and -2 (PD98059), and PI3-kinase (PI3K) (LY294002) were purchased from CalBioChem (San Diego, CA). The antibody to phospho-p38MAPK was purchased from Promega (Madison, WI). Antibodies to ERK-1 and -2, phospho-ERK1 and ERK2, p38MAPK, and human COX-2 antibody were obtained from Santa Cruz (Santa Cruz, CA). Antibodies to Akt and phosphor-Akt were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The β-actin antibody was purchased from SIGMA (St. Louis, MO). Arachidonic acid was purchased from Cayman Chemical (Ann Arbor, MI). GRPR expression vector was a gift from Dr. James F. Battey (National Institutes of Health, Bethesda, MD).
Cell culture
The PC-3 and LNCaP cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured at 37°C in RPMI 1640 supplement with L-glutamine, 10% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories Inc., Logan, UT) and 1 mM sodium pyruvate (Sigma-Aldrich Corp., St. Louis, MO). For all experiments, the cells were cultured in serum-free media for 18-24 h prior to treatments.
RNA isolation and Northern blot analysis
Cells (1.5 × 106) were plated in 100 mm tissue culture dishes. After 24 h, the cells were serum starved for an additional 20 h and then treated as described in the figure legends. RNA was isolated using ULTRASPEC™RNA Isolation System (Biotecx, Houston, TX), resolved on 1% agarose/formaldehyde gels, and transferred onto Hybond-N+ membrane (Amersham Biosciences Corp, Piscataway, NJ). The membrane was hybridized with human COX-2 cDNA probe labeled with [α-32P]dATP (Perkin Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX). The specific hybridization was visualized by autoradiography. The membrane was re-hybridized with a probe for 18S ribosomal RNA to confirm RNA integrity and equivalent loading of each sample. With the exception of the Figure 6A, all RNA isolations and Northern blots were repeated a minimum of three times.
Western blot analysis
Cells were incubated in serum-free media for the indicated period of time, treated with peptide hormone and/or drug as described in the figure legends and lysed using a solution containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 5 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM NaF, 1 mM PMSF, and 1 Protease Inhibitor Cocktail Tablet/50 ml (Roche Applied Science, Indianapolis, IN). The detergent-insoluble cellular material was removed by centrifugation at 14000 rpm for 15 min, and the protein concentration of the supernatant was determined using BioRad Dye Reagent Concentrate (BioRad Laboratories, Inc., Hercules, CA). Proteins (40-100 μg/lane) were resolved on 4-12% continuous gradient Bis-Tris-HCl buffered polyacrylamide gels (Invitrogen, Carlsbad, CA), transferred to a PVDF membrane (Millipore Corporation, Bedford, MA), and blocked with 5% non-fat milk. Membranes were probed with the primary antibodies at dilutions indicated in the figure legends. Specific immunoreactive bands were detected using enhanced chemiluminescence (ECL)™ Western Blotting Detection Reagent (Amersham Biosciences Corp, Piscataway, NJ) and Kodak X-Omat film. All protein isolations and Western blots were repeated a minimum of three times.
PGE2 assay
Cells (1.2 × 105/well) were seeded in 12-well plates for 24 h. After culturing for 20 h in serum-free media, the cells were treated with BBS for the period of 4, 8, 10, 16, and 24 h. Arachidonic acid (15 μM) was added 30 min prior to the collection of media. Culture media (100 μl) from each well were analyzed for PGE2 by using Biotrak Enzyme immunoassay (EIA) system (Amersham Biosciences, Piscataway, NJ). All assays where repeat at least three times.
Luciferase reporter gene assay
Cells (1.2 × 105/well) were seeded into 12-well plates and co-transfected with 250 ng of plasmid DNA containing the human COX-2 promoter coupled to a luciferase reporter gene and 30 ng of plasmid containing β-galactosidase using LipofectAMINE™ Reagent (Invitrogen, Carlsbad, CA). Cells were treated as described in the figure legends. Luciferase and β-galactosidase activity were assayed using Enhanced Luciferase Assay Kit (BD Biosciences, San Diego, CA) and Galacto-Light Plus™ Systems (Applied Biosystems, Bedford, MA), respectively. The transfections and luciferase assays were repeated three times.
Immunofluorescence microscopy
PC-3 cells were cultured on glass coverslips. Before immunostaining for NF-κB p65 subunit, the cells were treated either with vehicle, curcumin (20 μM), BBS (10 nM), TNF-α (10 ng/ml), or a combination of curcumin and BBS or TNF-α for 30 min at 37°C, fixed with 4% paraformaldehyde (15 min), permeabilized with 0.3% Triton X-100 (10 min), and incubated in blocking solution (1% BSA in PBS; 20 min). After incubating the cells with anti-NF-κB antiserum (1:100 dilution; San Cruz Biotechnology Inc., San Cruz, CA) for 1 h at room temperature, the cells were washed three times with PBS and incubated with a goat anti-rabbit IgG antibody labeled with Alexa 488 (Molecular Probes, Inc., Eugene, OR; 1:2000; 30 min). The immunostaining procedure was repeated at least three times. Specific immunostaining was visualized with a Nikon Eclipse fluorescence microscope.
Electrophoretic mobility shift assay
Nuclear extracts were prepared as previously described [26]. Oligonucleotides (Promega, Madison, WI) of which the sequence corresponding to the AP-1 binding site consensus sequence (5'- CGC TTG ATG AGT CAG CCG GAA -3') were end-labeled with [γ-32P]ATP and T4 polynucleotide kinase respectively. Electrophoretic mobility shift assay (EMSA) reaction mixtures contained 50,000 cpm of 32P-end-labeled oligonucleotide, 20 μg of nuclear protein extract, and gel shift binding buffer (Promega) in a final volume of 20 μl. Reaction mixtures were resolved on 4% nondenaturing polyacrylamide gel electrophoresis at 200 V for 2 h. Gels were dried and visualized by autoradiography. Preparation of nuclear extracts and EMSA was performed a minimum of three times.
Intracellular Ca2+ measurements
Cells, grown on 25-mm glass coverslips, were washed with a physiological medium (KRH) containing NaCl (125 mM), KCl (5 mM), KH2PO4 (1.2 mM), MgSO4 (1.2 mM), CaCl2 (2 mM), glucose (6 mM), HEPES (25 mM; pH 7.4), and loaded with 2 μM Fura-2 AM (Molecular Probes, Inc.) for 50 min at 25°C. The cells were treated with BBS (10 nM), and single cell changes in the concentration of free intracellular Ca2+ ([Ca2+]i) were recorded using a Nikon Diaphot inverted microscope (Garden City, NY) and a CCD camera (Dage-MTI, Inc., Michigan City, IN). Data points were collected every 1-8S from ~35 cells/coverslip and processed using ImageMaster software. Data are presented as the mean change in [Ca2+]i.
Statistical analysis
Statistical analysis was performed using GraphPad InStat 3.0 (GraphPad Software, Inc.). Statistical significance was assumed if P ≤ 0.05.
List of abbreviations
COX-2: Cyclooxygenase-2; BBS: bombesin; GRP: gastrin-releasing peptide; PI3K: PI3 kinase; AP-1: activator protein-1; NF-κB: nuclear factor-kappaB; NE: neuroendocrine; GRPR: GRP receptor; EIA: Enzyme immunoassay
Authors' contributions
XDW performed the majority of the cellular, molecular, and protein assays. KI performed the calcium and prostaglandin assays. CC participated in the study's design and coordination, statistical analyses, and helped draft the manuscript. MRH conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.
We thank Eileen Figueroa and Steve Schuenke for their assistance in the preparation of this manuscript. This work is supported by grants from the National Institutes of Health (5P01 DK035608 and 5R01 DK048345, and 5K08 CA125209-02).
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Guo YS, Hellmich MR, Wen XD, Townsend CM Jr: Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells.
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Dixon DA, Kaplan CD, McIntyre TM, Zimmerman GA, Prescott SM: Post-transcriptional control of cyclooxygenase-2 gene expression. The role of the 3'-untranslated region.
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The Leuprolide Study Group: Leuprolide versus diethylstilbestrol for metastatic prostate cancer.
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Nature 1985, 316(6031):823-826. PubMed Abstract | Publisher Full Text


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A Watchdog team blog
Lawmakers demand accountabilty for child-care spending
Lawmakers today called on the Department of Children and Families to provide a monthly report on activities to curb fraud and collect misspent money within the beleaguered Wisconsin Shares program.
The two co-chairs of the Joint Committee on Finance have requested that Reggie Bicha, secretary of the department, submit reports detailing expenditures on fraud prevention and investigations and well as specifics on how much the department collects every month from providers who were overpaid.
The move comes in the wake of recent reports by the Journal Sentinel exposing lax oversight of the program and how parents and providers continue to scam the $350 million taxpayer- financed system.
To read the newspaper's entire "Cashing in on Kids" series, click here.
To read today's letter from lawmakers to Bicha,click here.
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subset: commoncrawl


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Why is Firefly's Cancellation So Unforgiveable?
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September 19 2009, 10:27 AM
Vice Admiral
Location: Fifth Circle of Hell, IE: Pennsylvania
Re: Why is Firefly's Cancellation So Unforgiveable?
For many people, it was just the last straw that broke the camels back. You know?
We had suffered oh so many prior cancellations of good shows that only died because they where on FOX, a network notorious for perhaps the most cancellations per-year in the entire industry. Space: Above and Beyond, Dark Angel, so on and so forth.
Firefly was just the one that happened to be, simultaneously, people's breaking point.
Now I myself didn't like the show when it was on TV. Because it was aired out of order, because it was spotty on times, I couldn't get into it. I saw the first few episodes, I watched the premiere, but without the pilot it failed to click with me. I, like many, where thrown in the deep end without a life vest and gave up quickly. It was only later when I, too, got tired of the browncoats lamenting it's loss, that I was challenged -- and provided with the episodes -- to watch it in order, in full.
I did so. I was intrigued by the end of the pilot. I was hooked by the end of the second regular episode, which I'd seen on broadcast but been lost during.
FOX is... all together, bad.
They're a bad news network. They're a bad TV network. They're a bad business network. They're all run badly.
Network FOX seems to be run by impatient 8 year olds. They got spoiled very early on, which is the root of things. Many FOX station carried TNG in it's first run syndication, when they had little of their own programming. It introduced them to sci-fi and very good ratings. Then they did X-Files, which again, sci-fi and very good ratings. Those two shows basically convinced them, sci-fi was super-ratings. So when they put a sci-fi show on that failed to achieve such heights? It got the axe quickly. Because they couldn't give anything a fair shake.
Hell, the only sci-fi show not to be cancelled on FOX in recent years, prior to Fringe, was Dark Angel. And it only got a second season because it was Jim Cameron's, and he had enough pull to keep it around that long. Otherwise it would have died in season 1 too.
The sad fact is, FOX is the only network to regularly give true sci-fi shows a shake; none of the other networks want to touch it. After LOST in 2004, they all tried here and there, but no one made a complete effort.
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Five Signs He is About to Propose or Thinking About Marriage
Hi ladies, do you wonder if your current beau is thinking about marriage or is about to propose? Here are five common signs that I discovered among my circle of girlfriends and myself
These are the 5 signs and the first tip was the most obvious one for me -
1) He stops investing in expensive "guy toys" or "guy hobbies"
If he was constantly spending money modifying his car, spending lots of money on himself in the earlier part of the relationship and then now stops to save money or get into financial planning that will enable an increase in income, wedding bells could be ringing soon. He understands that he will need to be able to finance a home and support a wife and family.
2) He asks you for your opinion on having kids or jokes constantly about how your children might be like or look like.
3) He glances at jewellery shop windows displaying rings whilst out on a date with you and attempts to find out the size of your ring finger or asks a friend of yours to find out.
4) He tells you he has plans for the future and actively includes you in those plans. He might ask you what your plans are for the future, your career plans any further schooling or plans on moving to another city/country
5) He gets a close friend to hint about his intentions. An arrangement for a surprise engagement party might be on the way.
There you go ladies, the 5 signs, if you experience more than 3 of the above, i say, whip out the bridal catalogue!
Love and Blessings!
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|Photo by Johan Persson
The critically acclaimed revival of The Glass Menagerie received seven nominations, including Best Actress (Cherry Jones), Best Featured Actress (Celia Keenan-Bolger) and Best Featured Actor (Brian J. Smith). Zachary Quinto, making his Broadway debut as the aspiring writer Tom, was the only member of the cast to not be recognized.
After stripping naked in Equus and dancing up a storm in How to Succeed in Business Without Really Trying, Daniel Radcliffe proved his stage talents yet again in the title role of the dark comedy The Cripple of Inishmaan, yet the nominators overlooked his work for a third time.
Denzel Washington took home a Tony Award for his last Broadway turn in August Wilson's Fences, but his performance as Walter Lee Younger in Kenny Leon's revival of A Raisin in the Sun did not earn him a nomination.
Sally Bowles' dreams of stardom are still beyond her reach; though many thought Michelle Williams gave a "perfectly marvelous" performance while making her Broadway debut as the ill-fated nightclub singer in Cabaret, she was also overlooked by Tony nominators. Not to mention the fact that the Tony-winning revival was passed up for another Tony win, leaving the Best Musical Revival category with one less that its allowed four nominees.
The competition was notably intense for the role of Helen Sinclair in the musical adaptation of Woody Allen's movie "Bullets Over Broadway," and the role went to Marin Mazzie. Mazzie, who has three Tony nominations under her belt already, did not receive one for her performance as the narcissistic, alcoholic diva.
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Tuesday Jun 01 2010
Miniscapes put gardens on small scale
By: Gloria Young Home & Garden
When the economy slowed business for landscape designer Katrina Leonidov Fairchild, she took a creative path to downsizing. The Meadow Vista resident set her sights on miniature gardens. Created in 14-to-16- inch diameter containers, they're smaller versions of landscapes Fairchild would actually create on a life-size scale. "I'm interested in the elements that make up the design, from the same perspective that I tackle a site for someone who wants a design," she said. Most are built around structures — gazebos, pergolas, patios. For her first miniature design, she started with a cabin. "I adorned it with rock on the fireplace and painted it to look like a cabin you'd find in Tahoe," she said. "One thing led to another and I found some plants that mimic redwoods or pines and then added rock and stones and basically it started to look like places I've visited." That got her started thinking about other places she's seen. "I created one on La Paz, Mexico," she said. Another of the gardens had a Harley theme. "I bought a miniature Harley and included it in one of the landscapes, calling it 'Ride into Tahoe,'" she said. She drew upon her Texas heritage for another garden scene. Planted with cactus, it has a motorcycle riding through the dessert. Then there's her mini-take on the California coast. "I made it look like it was on top of a cliff overlooking the water," Fairchild said. For the water, Fairchild used recycled blue glass. One of the scenes reflects time she spent in Africa. "It was called 'Remember Africa,' and I made it in a little tin bucket and incorporated some grasses and gravel and two lions. The lions were plastic but very realistic. I usually don't use plastic but this was an exception." Fairchild keeps the miniature gardens as true-to-life as possible. "I strive to use real materials — real stone, gravel, real wood furniture," she said. "I refrain from plastic or anything fake looking. I even use real driftwood. When I take walks, I'm always looking down searching for things I can use in the landscapes." When she started displaying her designs at home shows and fairs, they struck a chord. "People actually stopped to analyze them and look at them," Fairchild said. "They'd begin smiling. I asked them, 'Do you see yourself in that landscape?' and they'd say yes." A design with an arbor caught the eye of Nevada City resident Natasha Shubin. "It was an emotional thing," she said Monday. "When I saw it, it was like a little yard and a little garden and it was just like coming back to my childhood. I just wanted to go in there and be in there — like Alice in Wonderland." Shubin said she keeps the miniscape on her deck, within easy view from a nearby window. When she creates the designs, Fairchild plans them around where they'll fit into a home landscape. "Just like doing a regular planting plan, I evaluate what kind of plants go together," she said. "I group them into ones that like full sun, part shade or full shade; and that need the same amount of water. Others I create for indoors." Maintaining these small gardens takes some special care. "Because these are plants that grow slowly, you have to back off on fertilizing. That's different than something that's growing life size," she explained. "You don't want them to be fast-growing. And you must trim them back in small amounts from time to time. You can even use scissors." Depending on location indoors or in the garden, most of the miniscapes will need only a little water once a week or every two weeks, she said. At the same time, she stresses that they're not bonsai designs. "I don't tinker with the roots," she said. "I don't control the plants. It's a totally different thing." When Fairchild began selling the designs, she noticed that customers would ask a lot of questions about how to put them together. So she decided to offer a class in making the miniature gardens and also to sell the tiny items needed to create them. "I thought, I'm always about helping people put their gardens together, so why not share this expertise in landscape design and apply it to miniatures," she said. Her first class on miniature gardens will be June 19. Cost of Fairchild's ready-made miniature gardens ranges from $50 to $100, depending on the amount of materials used. She also will do special orders reflecting hobbies or special interests.


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Roslyn Schwartz
Author, Illustrator
Contact Information
wolfest [at] cooptel [dot] qc [dot] ca
2051 Rue Wolfe
Montreal QC
p. 514-523-6771
Selected Bibliography
Tales from Parc la Fontaine
(Annick Press, 2006)
Complete Adventures of the Mole Sisters
(Annick Press, 2004)
The Mole Sisters and the Fairy Ring
(Annick Press, 2003)
The Mole Sisters and the Way Home
(Annick Press, 2003)
The Mole Sisters and the Cool Breeze
(Annick Press, 2002)
The Mole Sisters and the Question
(Annick Press, 2002)
The Mole Sisters and the Moonlit Night
(Annick Press, 2001)
The Mole Sisters and the Blue Egg
(Annick Press, 2001)
The Mole Sisters and the Busy Bees
(Annick Press, 2000)
The Mole Sisters and the Wavy Wheat
(Annick Press, 2000)
The Mole Sisters and the Piece of Moss
(Annick Press, 1999)
The Mole Sisters and the Rainy Day
(Annick Press, 1999)
Yo Baby!
(Annick Press, 2002)
Rose and Dorothy
(Kids Can Press, 1990)
|Location: Montreal, QC
|Availability: national
|Available: Any time
Reading Location:
libraries, schools
SK to 2
Audience Size:
10 to 50 (1-2 classes)
$500 plus GST and travel expenses for two 45-minute presentations; question time and booksigning.
English, French
Author/illustrator Rosyln Schwartz of Montreal brings a life long love of children's literature to her fresh, charming and intriguing books for very young readers and listeners. She shows students from kingergarten to grade two the ten small books in her Mole Sisters series and tells them how they came to be, from the beginning idea made into a story, then pictures, then nine more stories and many more appealing, amusing illustrations.
Using the Mole Sister finger puppets, she first tells a story (or two or three), perhaps "The Mole Sisters and the Moonlit Night" or "The Mole Sisters and the Busy Bee," all of them gently adventurous, optimistic and funny.
As reviewers have said, "In every adventure, the sisters seek out new bearings based solely on their own curiosity, which leads them to some delightful situations.... After their escapades, they rest ... ending on a very sweet, mellow note sure to leave young ones smiling."
After being read to, the children together develop a brand new story of their own, with Roslyn doing the storyboard sketches they suggest, on the spot. (An easel or blackboard is necessary.)
Older children are given sheets of paper and shown how to make them into a dummy book, in which they can rough out their own words and pictures.
Roslyn also brings with her a number of rough sketches, preliminary drawings and originals of the final coloured-pencil artwork to show the students how illustrations are created. As well, she shows them some of the many messy notebooks and scraps of paper that an author keeps, writing down any ideas that come, to assemble into a story, poem or picture. The kids are also interested in her Vole Brothers tales set up as comic strips for Chirp magazine--there are many ways, more than ever nowadays, to present a story. Questions are welcomed.
Special Equipment:
an easel and/or blackboard and chalk
Book Sales:
Copies of books can be brought to be sold and autographed depending on numbers and advance notice.
Roslyn Schwartz is the author and illustrator of more than a dozen books for children. In 2003, she was the winner of the Vicky Metcalf Award awarded by the Canadian Authors Association for a body of work. Born in Montreal, she grew up in England and returned to Canada in her 20s. In Britain she attended one of the experimental, artistic Rudolph Steiner schools, so was encouraged to spend much of her classroom time drawing.
Roslyn is best known for the unique and optimistic Mole sister series for very young children, with rights sold in 11 countries and a TV spin-off. Roslyn learned the art of storyboarding that she shares with students by directing two animated films for the National Film Board of Canada.
At heart, she feels the Mole sisters are the two parts of any one of us, planning, preparing, enjoying and even fretting together.


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well im new to this and i just where my Fila cross trainers with my custom fit orthotics that i had made last year by my podiatrist after surgery for planters fasciitis on both feet.should i be wearing something else?
well im new to this and i just where my Fila cross trainers with my custom fit orthotics that i had made last year by my podiatrist after surgery for planters fasciitis on both feet.should i be wearing something else?
I use oldstyle toe clips and straps, and I wear indoor soccer shoes.
"I had this baby hand made in Tuscany, from titanium blessed by the pope. It weighs less than a fart, and costs more than a divorce..."
http://www.bicyclehero.com/us/shimano-sh-mp66w-bmx-mountain-bike-shoes-white-44.html) with 2-hole cleats (currently Bebop), although you could just pass on removing the plug in the sole and ride them on platform pedals.
Another vote for 2-sided SPD pedals. I also like to have mountain bike shoes that have rubber in the arch. Most have a slick nylon or fiberglass arch, that is really easy to slip off of at a traffic intersection. Slipping off is dangerous. I've seen people wreck that way numerous times.
You can push through the intersection with the rubber arches without being clipped in. Saves me lots of worry at busy intersections.
I also have an old stiff front-fork mountain bike that I use as a commuter with old fashioned toe clips. That works fine for carrying groceries and such, but I can't pull up on the toe clips as hard on hills as I can with SPD pedals.
My Bike Blog
1987 Trek 1000 Aluminum
1993 Cannondale M300
2008 Specialized Allez Elite Compact
I wear Sidi mountian bike shoes and use Shimano M324 SPD pedals.
Take life one day at a time and live well each day..
2009 Trek 7500 (mine)
2009 Trek 7300 (hers)
I brought my bike in for the derraileurs to be adjusted yesterday. While I was waiting I found these Keen bike sandles, ordinarily in the $100-$115 range:
They were on sale for $89, one pair, my size. I waffled and didn't buy and regretted later because they were a perfect fit. I went online and found the exact same shoes on Amazon for $69. I ordinarily would have supported my LBS but I couldn't pass up this deal.
I just wear regular ole Nike tennis shoes. No clips on the pedals or anything.
I ride two different department store bikes of doom. Neither have exploded or caused small children to cry yet.
198? Free Spirit Sovereign 12 speed road bike (Sears) - the smooth and comfy one. The commuter/hauler/touring bike.
2011 GMC Denali 14 speed road bike (Wal-Mart) - the fast frankenbike. The racer.
Goldfinch- 69 bucks is the cheapest I've ever seen Keen's. Thanks for the link, I ordered a pair. Stouts is a site to keep an eye on that carries Keens too. I have found that the even the biggest sites don't have every color and size variations at all times. Do you think the better the deal the less likely you are to find exacctly what you want?
Last edited by DownTheRoad; 06-15-11 at 10:09 PM.
I like these, sole is stiff and they don't slip, plus, easy to stop and get the
rocks out.
Keen Sandals, rain or shine, stiff and dry out well.
Shimano mountain bike shoes with SPD cleats.
@ Bike Journal
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Bought them this year and since they are an older model I only paid $100 for them at my LBS. I also use SPD-SL pedals with them.
Next to switching to bibs these are the most comfortable addition to my cycling gear.
Brahma low cut boots from Wal-mart. A couple of the styles are very light, and have stiff soles; and less than $30-
My commute is 12 miles each way, the Brahmas are comfortable for the ride and all day at work (office).
I am a fan of Sidi shoes but understand they are expensive. I recently bought a pair of Pearl Izumi MTB shoes at REI. They were on sale - I think around $70. Although I've added a cleat, I can see where these shoes would work great without one (I use the same pedal system - Time - on my MTB and road bikes - just different style platforms). The shoes are stiff enough to be supportive but comfortable for walking around. I highly recommend them (as I see others have as well).
One word of Sidi's... yes I know they cost just south of $300 but most comfortable shoes ever and they last. I beat my MTB shoes up terribly and am still using a pair I bought a number of seasons ago. I can't say the Sidi's are the most comfortable walking around shoes (even though I buy the Dominators with an MTB sole) which is why I bought the PI for longer,more difficult rides where i know i have to walk.
I am a firm believer in using the right equipment for the task. For instance, cycling shorts... nothing beats them for comfort and function. Cycling shoes same thing. The investment in good stuff is worth it.
Private docent led mountain bike rides through Limestone Canyon. Go to letsgooutside.org and register today! Also available: hikes, equestrian rides and family events as well as trail maintenance and science study.
Adidas road shoes (closeout, cheap at local bike shop) + Speedplay pedals (new for 2011, formerly Time pedals)
For my commute (20-34 miles round trip, depending on how I feel), plain old sneakers with wide bmx style pedals work fine for me.
For longer rides, I'm currently using shimano "multi-use touring shoes" and dual sided spd mountain pedals.
I mostly wear Speedo's version of the aquasock which has almost zero padding for better foot/pedal transfer and keeps my feet dry if I have to cross a stream. In the summer I sometimes ride wearing my rope sandals: I have a pair from Nomadic State of Mind, but there are several similar manufacturers. Again, little padding for less squishiness.
I wear these everyday with slacks for work. I get home and change into my baggies and t-shirt and then put these back on. I am crazy about them Very stiff, look good and extremely comfortable.
That said, the more serious mtn bikers I have been around swear by these. I am surprised no one has mentioned these yet.
This summer I am wearing my regular Keen sandals. They're not bike sandals but they seem rigid enougn. In the winter I wear my Shimano mountain bike shoes.
1975 Peugeot UO-18
Just wanting to have some dang fun!
"What she loved was simply life." Mrs. Dalloway
"Free the cheese!"
Diesel Dan: HTFU!
I've been happiest with the Chrome shoes I bought last fall.
Read Simply Cycle
"I can still do everything I used to, but now I'm mature enough to take a nap without being told." - Me
"You don't deteriorate from age,you age from deterioration" --Joe Weider


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"The cards came printed on heavy paper and the colors were bright and fun. I ordered them for my daughter's birthday. Everyone that saw them loved them. The picture on the card made them even better. I would buy from tiny prints again."
"Beautiful card and I'm always impressed with the turn around time of Tiny Prints. So wonderful for a busy mother who is running behind on birthday planning"
"Again, these were perfect for a nine year old's "Lego Birthday Party"....I used a photo of him in his Lego costume from last Halloween!"
"I loved the simplicity of it. I got many compliments. My only concerned was the writing in pink on the back could've been a bit darker. Besides that the paper quality was great and my photo came out beautifully."
"I really liked everything about the card. My only wish is that I was able to write a message on the back - especially since it's a pool party and wanted to add additional info about rain location etc."


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Whoa! Never Saw That Coming!
it's a postoperative bandage on her nose, she wears it so her nose job doesn't screw. lol why do i even know that?
What the hell is wrong with her nose? It looks like its disintegrating!
Is that sand on her nose?
dude atleast she looks more like the REAL heidi except for the fake boobs.i really liked her then
- Photo Credit:
- PacificCoastNewsOnline.com
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- Heidi Back to Her Roots
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- Heidi Montag
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Reviewed By Larry R (Shippensburg, PA) Reviewed for a 2015 GMC Canyon —4/16/2015 5:19 AM
This is my second pair of Putco bed rails. I installed the first pair on a 2003 pick-up and they still looked great after twelve years. I did not hesitate to purchase a set for my new 2015 pick-up and looking forward to many years of use. The quality is outstanding.


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Naureen S. Malik
American Electric Power Co.'s (AEP) fourth-quarter profit dropped 26% amid higher taxes and a required refund to customers, but revenue rose on a continued rebound in demand from industrial customers.
The Columbus, Ohio-based utility is one of the largest in the U.S., serving 5.2 million customers across 11 states stretching from Texas to Ohio.
The power generator's core results improved in recent quarters amid higher demand, driven by increased manufacturing, mining and other industrial activity in parts of the Midwest and South Central regions. Favorable weather conditions also bolstered demand for power during the quarter and throughout 2010. But the company continues to face significant hurdles in Ohio where its regulated power rates are higher than the going market rate in this partly deregulated market, resulting in persistent customer switching.
American Electric responded to recession-related struggles by cutting costs and will create sustainable savings, company executives said. "We clearly have demonstrated and will continue to demonstrate our ability not only to manage [operating and maintenance expenses], but also to manage the capital spending on our system," Chairman and Chief Executive Michael Morris said during a conference call Friday morning.
The company achieved "tremendous rate success" last year and set the stage for favorable rate activities in 2011 and beyond, he said. The Ohio rate case "will take center stage" as the year unfolds, he added.
In the most recent quarter, though, benefits from weather and cost-cutting initiatives were largely offset by a refund to Ohio utility customers required by regulators and higher health-care costs.
The company posted earnings of $176 million, or 37 cents a share, down from $238 million, or 50 cents, a year earlier. Restructuring charges cut the latest result by a penny. Revenue increased 3% to $3.4 billion.
Analysts polled by Thomson Reuters most recently forecast earnings of 40 cents on $3.56 billion in revenue.
The utility operations' income tax expenses soared to $111 million from $29 million and an order from the Public Utility Commission of Ohio to return $43 million to customers also reduced results by 6 cents a share. Virginia regulators also declined the company's request to recover $54 million in costs for a carbon capture.
AEP executives expect industrial sales to continue to improve in 2011. Last year's results also benefited from higher rates approved by regulators. Across AEP, the average return on equity "was extremely strong" at 10.75%, CEO Morris said.
Retail deliveries to industrial customers jumped 7%, while output to residential and commercial users was up 1.4% and 0.7%, respectively. Wholesale deliveries dropped 9.5%. On a weather-adjusted basis, total power demand across AEP's system rose 1.1% in 2010 and is expected to grow 1.7% this year.
The company also announced plans to repurchase $400 million to $700 million in shares this year, funded by tax savings.
Shares of AEP, which affirmed its forecast for the year, fell 1.4% to $36.16. The stock is up 3.1% over the past 12 months.
--Matt Jarzemsky contributed to this article.
Copyright © 2011 Dow Jones Newswires


text: Crump Dancing At Your Son's Funeral #FAIL By DJ Supreme September 19, 2011 5:10 PM Share on Facebook Share on Twitter I guess everyone mourns in their own way, but this has to be the most ridiculous thing I have ever seen. What do you think ? Filed Under: Fail Category: Weird News Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! Best of WBLK Recommended For You Best of the Web


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Used Trucks Calgary
(19) for Used Trucks in Calgary
Eastside FiatAlberta, 3501 - 8Th Ave. North EastT2A 5H6 Calgary Downtown (403) 273-4313Closed
Eastside FIAT is your Calgary FIAT dealer. It sells and services FIAT vehicles in greater Calgary, Alberta. New FIAT cars and used cars, SUVs, trucks available.
DRIVING FORCE Vehicle Rentals, Sales & LeasingAB, 2332 - 23 Street NeT2E 8N3 Calgary Vista Heights 403-768-2692Closed
trucks to five tons, plus a full line of Fleet Management Services. If you need a specialty vehicle, Driving Force is your number one choice. Wheelchair accessible vans, 7- and 15-passenger vans,...
Request for Quotation
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Calgary Peterbilt LtdAlberta, 11550 44 St SeT2Z 4A2 Calgary Alyth - Bonnybrook - Manchester (403) 248-7383Closed
..., RETAIL, SERVICES, TRUCK PARTS, TRUCK SERVICE, NEW, Truck Equipment, Part, TRUCK DEALER, USED
Ryder CanadaNE, 2140 23 St NeT2E 8J8 Calgary Vista Heights 403 569 8805
... and logistics solutions worldwide since 1933. Whether you need to rent a truck or trucks, looking to purchase a used truck or lease a fleet, Ryder has the right vehicle to fit your needs. Maximize...
Maxim Used Truck SalesAB, 819 46 Avenue SeT2G 2A5 Calgary Alyth - Bonnybrook - Manchester (403) 571-1275
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Danco Equipment can equip your truck with cranes, power take offs, winches, service bodies, product pumps, heavy, light duty snow and ice removal equipment, dump bodies decks, hydraulic pumps, motors ... AND ICE CONTROL, SNOW, ICE, USED EQUIPMENT, PUMP, HOIST, USED, DECKS, VALVES, BLOWERS, DRIVE...
Bottom Line Auto BrokersAlberta, 7015 Macleod Trail Sw, Suite 400T2H 2K6 Calgary (403) 984-9191Closed
... and paperwork. Let us take care of your entire negotiation for you. From new car negotiations to used car locating and even US auto importing, we are sure to find the vehicle of your dreams.
The Car Store SalesAlberta, 121 42 Av SeT1X 1E1 CalgaryClosed
Calgary Used cars and trucks The Car Store Used Calgary Cars and Trucks in Calgary. Online consignment, new or pre-owned cars, vans, trucks and more.
Commercial Automotive Services (Calgary) IncorporatedAB, 2815 Bonnybrook Road SeT2G 4N1 Calgary Alyth - Bonnybrook - Manchester (403) 261-4800
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Metro-Ford Sales LtdAB, StreetT1X 1E1 Calgary (403) 233-8195
... Automobile Dealers, Truck Dealers, Car, Dealer, used cars dealer, Dealer New Cars
Hino CentralAlberta, 3660 - 50 Avenue SeT2B 3N9 Calgary 403-768-2982
... Calgary's Hino Central is the only place to go. We carry new and used Hino trucks, and other used trucks, to suit every need. From class-4 COE models to conventional cab class-7 trucks, including...
Glover IdealeaseAlberta, 5425 90 Ave SeT2C 4Z6 Calgary Acadia (587) 324-8279
... Used Dealers, Truck Rental & Leasing, Automobile, Automotive, Location de camions, Concessionaires...
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MILLENIUM MOTORSAlberta, 603 36 Ave NeT2E 2L7 Calgary Sunridge (403) 735-6050Closed
... used car, truck
DLH Enterprises IncAlberta, 209 – 279 Midpark Way South EastT2X 1M2 Calgary Midnapore (403) 775-7240
... Used Truck Dealers
Hyatt InfinitiAlberta, 46 Heritage Meadows Rd S.ET2H 3C1 Calgary Haysboro
... Used Truck Dealer, Used Car Dealer, Auto Body Parts Supplier, Auto Repair Shop, Car Dealer,...
Fraserway r v Centre LtdAB, 2475 Pegasus Rd NeT2E 8G8 Calgary (403) 291-5080
... Adventurer Camper, world's first, best, lightweight camper, built for, made for half ton trucks, 1/2 ton, original, new, Toyota, Tundra, Titan, Nissan, Dakota, Dodge Ram, Chevy Silverado, F150 ...
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... used concrete pump trailer, entrepreneurs, truck-mounted telescopic conveyors, concrete boom pump,...
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This afternoon's meeting at Warwick has been abandoned.
Clerk of the course Sulekha Varma had planned to inspect the track at 8am after reporting the course to be only just raceable on Wednesday evening. However, further rain overnight has resulted in false patches of ground, forcing Varma to make an earlier call.
"We had another 1.5 millimetres of rain overnight and that just left some areas of false ground, particularly at the end of the back straight. It's not safe to race, so we have called off the meeting," she said.
"We don't race again until August 21 now, so hopefully the weather will have improved by then as that's our fourth abandonment this year which is less then helpful."


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If you're a fan of understated French chic, unisex accessories or three-letter brands, you're in luck. Awesome Gallic brand A.P.C. has teamed up with the eyewear experts at L.G.R. on a pair of classic sunglasses you'll want to wear all summer long.
Available in either black or rich tortoise for $350, the handmade-in-Italy shades look great on both guys or gals, meaning you can share your pair with your brother/husband/boyfriend—that is, if you're willing to part with them at all. Plus, the lenses in the chestnut-hued pair are photochromatic, meaning that they darken naturally in the sunlight. Pick up yours at APC.fr today—or swing by your nearest A.P.C. boutique, where they're also in stock.
More on Luckymag.com:
- A.P.C. Lands in NYC's West Village
- 50 Sunglasses Under $50
- Vanessa Seward's Capsule for A.P.C. Is All You Need This Spring
- RIP Donna Summer: Remembering the Disco Queen's Awesome Style
- Peep Emma Stone's New Revlon Ad
Keep up with the Lucky team on Twitter: Follow @LuckyMagazine


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Using High Performance Computing to Examine Fluid Flow and Chemistry in Geologic Systems
This project uses MSI HPC resources to run fully coupled, multi-phase, multi-component, non-isothermal simulations to examine fluid flow and chemistry in a variety of geologic systems. One of our main focuses is geologic Carbon Capture, Utilization, and Storage (CCUS), which is a promising technology for mitigating the detrimental effects of global climate change by creating carbon-negative electricity. Another of our main focuses is fluid flow in low-permeability igneous rocks, which could potentially sustain life in extreme environments. MSI resources are utilized to run massively parallel simulations in both of these research themes using the simulators TOUGH2, PFLOTRAN, and LBHydra.
Return to this PI's main page.


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The Great Masters of Baroque (1600-1750) - Featuring Seasonal Choral Works - Welcome to the highest quality classical stream on the net! - Visit baroque247.com for additional streams & downloads.
A relaxing, therapeutic, and stress reducing form of music and sounds that is often referred to as ambient. We call it 'BRAVE NEW WORLD' music because it utilizes a whole new concept regarding form. Enjoy, relax, star gaze, meditate, or sleep to it.
Peaceful and Relaxing, Quiet and Soothing... The Best Mix of NewAge and Ambient Music. ♫ Listen to NewAge music the way it should be heard.. now broadcasting at CD quality 128k. ►TheCosmicIsland.com◄
Sounds of the yogic path... 10 hours of music to inspire your practice, on and off the mat.
We play all types of tango music... from early tango, through Golden Age, to contemporary tango. Also, Please visit my Tango Blog at http://www.tangology101.com
Visit us at www.sacredsoundsradio.com- 24/7 commercial free-- the very finest Yoga for your Ears music --join the thousands who listen FREE via our HD sound technology today!
Selection of Early Music, ranging from Hildegard von Bingen to the height of the English Renaissance
Take a relaxing trip to the sun-drenched beaches of Brazil with Live365's Original Bossa Nova Station! The Bossa Nova Breakfast features Classic & Contemporary Bossa Nova, Samba & Brazilian Jazz.
The Best in WUMB's Celtic Music, with Gail Gilmore - from Boston
New Age 2
Where's Walt the Wizard?
Music and teachings on the path of Kundalini Maha Yoga
Samba, Axe, Brazilian Jazz, Nu Bossa and more: the full panorama of Brazilian muisc! Brazilian Blend at www.BrazilClubUSA.com.
Sahaja Yoga Meditation


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Forget password?
Remember Me
Ice cold PBR, bottles, cans+ Draft at both bars!! Always ice coooold!
Be sure to check out some of the daily specials - they're usually VERY good. The Highlander was featured on Diners Drive-ins and Dives from the Food Network.
A hard-hitting menu but the burgers can't be beat! Brought to you by THE EXPENDABLES opening August 13!
The pot stickers are ridiculously good, the music selection is awesome and the drinks are well priced. Go in the Spring and spend a night on the patio or hit up the back bar for tunes and pool.
fun fun fun!
Best Places to Check out in United States Pt 1
"Diners, Drive-Ins & Dives" (Part 1, AL - KS)


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Simpson Bay Hotels
9 Hotels Found
Sonesta Maho Beach Resort & Casino 3.5Simpson Bay, St. Martin/St. MaartenBy far the biggest resort in St. Maarten, this 534-room behemoth is a veritable village with two towers, two pools, three restaurants, the island's largest casino, direct beach access, and a 40-boutique shopping center. Most of the rooms have fresh, modern decor and balconies, though the expensive Wi-Fi…
Royal Palm Beach Resort 3.0Simpson Bay, St. Martin/St. MaartenA central location on a quieter portion of Simpson Bay places this 140-room resort close to virtually every attraction on the island's Dutch side. Solid (though not quite spectacular) amenities include a beachfront restaurant, a pool with swim-up bar, a fitness center, a cocktail lounge, and family-oriented…
The Atrium Beach Resort & Spa, a Festiva Resort 3.0Simpson Bay, St. Martin/St. MaartenFor a medium-sized hotel, this 87-room beachfront property has a lot to offer: a beautiful beachfront with water sports and beach bars, a small pool, a lovely spa and salon, and a fitness center. Updated rooms are bright and colorful, and all have balconies and full kitchens. There is no on-site restaurant…
The Azure Hotel 3.0Simpson Bay, St. Martin/St. MaartenLocated on Simpson Bay in a residential neighborhood just south of the airport, the Azure Hotel is a small, intimate property with only eight rooms and plenty of natural beauty. The property is located directly on beautiful and scenic Simpson Bay with white sand and turquoise water. The small size of…
Mary's Boon Beach Resort 3.0Simpson Bay, St. Martin/St. MaartenThis 37-room beach hotel along Simpson Bay is certainly no luxury resort -- rooms show wear and tear, the pool is untended, and extra features are few. But an amazing beachfront location, small spa, and lush grounds full of character make it almost impossible not to appreciate Mary's Boon. Some people…
Simpson Bay Resort & Marina 3.5Simpson Bay, St. Martin/St. MaartenThis upper-middle-range resort in popular and beautiful Simpson Bay on the Dutch side is one of the best values on the island. It's enormous, especially for St. Maarten, with 342 rooms, six pools, four restaurants, and a long beach. Most of the rooms are stylish and modern (all rooms will be updated…
The Villas at Simpson Bay Resort & Marina 3.5Simpson Bay, St. Martin/St. MaartenThe Villas at Simpson Bay Resort & Marina, located in popular and beautiful Simpson Bay on the south (Dutch) side of the island, has access to adjacent sister property Simpson Bay Resort's many amenities. The 83 Two-Bedroom Villas (each can be split up into a One-Bedroom Suite and Junior Suite) are housed…
Simpson Bay Suites 3.5Simpson Bay, St. Martin/St. MaartenThis 18-unit condo hotel in Simpson Bay offers some of St. Maarten's most affordable and spacious rooms. And on that strength alone it's a great choice for families who want plenty of space to stretch out and a full kitchen for homemade meals. Still, this place is cheap for a reason: minimal service…
Most Romantic Hotels in St. Martin/St. Maarten
4 Hotels
Best Luxury Hotels in St. Martin/St. Maarten
2 Hotels
Best Boutique Hotels in St. Martin/St. Maarten
5 Hotels


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MK Early Releases Lead to Some Pre-Release Info Leaks
By Burn1n9m4n 10 Comments
Well it figures that some of the retailers for the new Mortal Kombat have already broken the street date. For those of you that already know this you can probably skip this part of this blog post, but for those of you that don't know some of the Mom and Pop retailers have already started selling the game as of Friday. The reason I found out about this is because of my perusal of the forums at PS3Trophies.org. Apparently some details have come to light about the Krypt, Unlocking Cyber Sub-Zero and Quan Chi, and the alternate fatalities and costumes within the game. I'm going to create a spoiler break here, so if you don't want to have the surprise ruined please don't click the reveal link and navigate away from here right now.
Well that's all that I've got for now. But considering the game comes out tomorrow I'm sure that those of you that have waited this long will be able to wait a bit longer. However, in closing I would like to ask you all to watch closely:
By the way, if anyone is interested or has already begun to make a guide for this game I would love to contribute to it. If not then I may make my own at somepoint.


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Both Sides of the COIN
Abandoning counterinsurgency doctrine after Afghanistan would doom the U.S. military to irrelevance and impotence, writes Christopher Sims and Fernando Luján. Not so, says Bing West; like it or not, the United States will be much less ambitious in future wars.
See more in Afghanistan; Defense and Security


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Set up your remote control
Use this tool to find the codes of your devices and to get specific instructions on programming the codes into your Comcast remote.
New to the Community? Start here.
I cannot connect to the internet with my own comcast service. I have a Mac OS X 10.5.8. My roommate is able to connect with her PC but I cannot. I have to use someone else's wireless but sometimes the service fluctuates and that's why I got my own service. I have ordered a wireless router but for now my service is just by Ethernet.
When I click on Ethernet it says Ethernet: Self-assigned IP Address. It also says: Ethernet has a self-assigned IP address and may not be able to connect. The IP address is listed as:169.254.177.218
I would like to figure this out since I am paying for it after all. The borrowed wireless has worked great since I bought service so I didn't even notice the Ethernet wasn't working for a while. I know the technician took for ever trying to get my computer set up and now I suspect he never actually got it to work...
Solved! Go to solution
When you switch the device connected to the modem, you have to reset the modem. If it's an ordinary modem, unplug it for a minute; if it's a telephony modem, press the Reset button.
Getting a router will save you this hassle, and allow you to use the Internet at the same time as your roommate.
Thank you, I am now connected with the airport turned off. I did what you said and unplugged the modem for a few minuets then plugged it back in while my computer was also connected. It did not work at first until I clicked advanced in Network Preferences and hit Renew DHCP Lease. It was then that the DNS Server and Search Domains section that had been blank was filled in. So I take it I will have to do this every time I need to use the Ethernet cable to connect? That is going to be a pain.
I'm not sure why you needed to Renew DHCP Lease. Normally the Mac detects the Ethernet being disconnected and reconnected, and renews automatically.
Do yourself a favor and get a router to avoid the pain. New ones cost around $50; if cost is an issue, you should be able to find cheap ones on eBay.
Pulled out into its own thread from this solved one.
Use this tool to find the codes of your devices and to get specific instructions on programming the codes into your Comcast remote.
Alerts are an easy, quick way to manage your account and get information - like payment confirmations and your current balance.
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