BACKGROUND
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  <dig>  the american cancer society estimates that over  <dig>  new cases of prostate cancer will be diagnosed and more than  <dig>  men will die, most from metastatic, androgen -refractory disease .
hormone-refractory prostate cancer is characterized, in part, by focal expansion of a malignant cell subpopulation with neuroendocrine  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  <cit> , parathyroid hormone-related protein  <cit> , and the bombesin -like peptide, gastrin-releasing peptide .
although the impact of ne differentiation on poor prognosis and androgen independence has been extensively studied  <cit> , the molecular mechanisms linking ne tumor cells and their bioactive neuropeptides to disease progression are still unclear.
increased expression of cyclooxygenase- <dig> , an enzyme that catalyzes the synthesis of prostanoids such as prostaglandin e <dig>  from arachidonic acid  <cit> , was identified as an independent predictor of prostate cancer progression  <cit> .
clinical trials using cox- <dig> inhibitors in patients with biochemical recurrence of prostate cancer have suggested that cox- <dig> inhibition may improve survival  <cit> , and pre-clinical studies with cell lines and animal models have established a functional link between cox- <dig> expression and an aggressive cancer phenotype.
specifically, dandekar and coworkers  <cit>  have demonstrated that overexpression of cox- <dig> 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- <dig> suppressed tumor growth and decreased metastatic spread  <cit> .
together, these studies implicate cox- <dig> 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- <dig> expression can be induced by multiple factors including growth factors, proinflammatory cytokines, and peptide hormones  <cit> .
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 , a member of the g protein-coupled receptor superfamily  <cit> .
clinical, histological, and experimental observations have implicated grp and grpr in the pathophysiology of prostate cancer progression.
logothetis and hoosein  <cit>  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  <cit> ; bbs stimulates the growth of both orthotopic and ectopic prostate cancer cell xenografts in athymic nude mice through grpr-mediated mechanisms  <cit> .
bbs also promotes expression of metalloproteinases  <cit>  and increases prostate cancer cell migration and invasion  <cit> .
previously, we reported that bbs stimulates the expression of the proangiogenic genes il- <dig> and vascular endothelial growth factor  in human prostate cancer cell lines  <cit> .
since cox- <dig> 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- <dig> expression in cells from other tissues  <cit> , we reasoned that grpr activation and cox- <dig> expression may be mechanistically linked in prostate cancer cells.
here, we report that bbs stimulates an increase in cox- <dig> mrna, protein expression, and the release of pge <dig> from the grpr-positive, androgen-insensitive prostate cancer cell line, pc- <dig>  the stimulatory effects of bbs on cox- <dig> expression and pge <dig> production are mediated by p38mapk and pi <dig> kinase /akt pathways and blocked by the selective grpr antagonist bim <dig>  the pi3k/akt pathway couples grpr to the activation of the transcription factor, activator protein- <dig> , and enhances cox- <dig> promoter activity.
bbs also stimulates nuclear factor-kappab  activation in pc-3; however, nf-κb does not regulate grpr-mediated cox- <dig> expression.
the p38mapk pathway increases bbs-stimulated cox- <dig> expression by slowing the degradation of cox- <dig> mrna.
expression of recombinant grpr in the grpr-negative, androgen-sensitive cell line lncap, is sufficient to confer bbs-stimulated cox- <dig> expression via the p38mapk and pi3k/akt pathways.
together, these results define a molecular mechanism for enhanced cox- <dig> expression in prostate cancer cells, and suggest a means by which ne-differentiated tumor cells and their bioactive neuropeptides may contribute to disease progression.
RESULTS
bbs stimulates cox- <dig> mrna and protein expression
to determine whether bbs stimulates cox- <dig> expression, we treated the androgen-insensitive prostate cancer cell line pc- <dig> with bbs and measured steady-state levels of cox- <dig> mrna and protein at various time points.
compared to vehicle-treated  control cultures, cox- <dig> mrna was increased at  <dig> h following addition of bbs  and peaked between  <dig> and  <dig> h .
increased cox- <dig> protein was also detected at  <dig> h following bbs treatment, peaked between  <dig> and  <dig> h, and returned to baseline levels by  <dig> h .
consistent with the lack of change in basal cox- <dig> mrna levels over the time course ; we did not observe a change in the basal expression of cox- <dig> protein in non-treated cells .
induction of both cox- <dig> mrna and protein expression was dependent on the concentration of bbs.
increased cox- <dig> mrna and protein levels were detected in cells treated with as little as  <dig>  nm bbs for  <dig> h and maximal induction was observed in cells treated with  <dig> to  <dig> nm bbs .
grpr mediates bbs-stimulated cox- <dig> protein expression and pge <dig> synthesis
cox- <dig> converts arachidonic acid, released from phospholipids by the action of phospholipase a <dig>  to prostaglandin h <dig> - the common precursor of all prostaglandins, including pge <dig>  to assess whether bbs stimulation of cox- <dig> expression was associated with an increased prostaglandin synthesis, the levels of pge <dig> released from pc- <dig> cells were measured using an enzyme-linked immunosorbent assay.
compared to vehicle-treated control cultures, bbs induced a time-dependent increase in the pge <dig> levels that peaked between  <dig> and  <dig> h .
pretreatment with the grpr-selective antagonist, bim <dig> ome]  <cit> ,  for  <dig> min blocked the bbs-induced increase in pge <dig> , *p ≤  <dig> ).
consistent with this observation, bim <dig> also inhibited bbs-stimulated increases in cox- <dig> protein expression .
bbs activates multiple intracellular signaling pathways in pc- <dig> cells
cox- <dig> expression is regulated by multiple intracellular signaling pathways including the mapk pathways, mek/erk and p38mapk, and the pi3k/akt pathway  <cit> .
to determine if these pathways were activated by bbs, pc- <dig> cells were treated with peptides over a time course and the activation state of each pathway assessed by immunoblotting.
bbs treatment  induced a time-dependent increase in the levels of activated  erk <dig>  and erk <dig>  .
the levels increased rapidly with a peak phosphorylation occurring between  <dig> and  <dig> min and remained elevated above baseline  <dig> min after treatment with bbs .
bbs stimulated a transient activation of p38mapk.
the levels of phospho-p38mapk  peaked between  <dig> and  <dig> min and, in contrast to the activation of either erk <dig> and  <dig> or akt, returned to baseline levels by  <dig> min .
the levels of phospho-akt  increased at  <dig> min, reached a plateau by  <dig> min, and remained elevated for the duration of the time course .
over the same time course, we did not observe any change in the activation state of these pathways in non-stimulated cells .
bbs-stimulated cox- <dig> 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- <dig> expression, pc- <dig> cells were pretreated  with either the p38mapk inhibitor , the pi3k inhibitor  or the mek <dig>  inhibitor  and then stimulated with bbs  for  <dig> h. agonist treatment failed to increase either cox- <dig> mrna or protein levels when the cells were pretreated with either sb <dig> or ly <dig> .
in contrast, pretreatment with pd <dig> did not inhibit bbs-stimulated increases in cox- <dig> mrna nor protein expression.
consistent with these observations, sb <dig> or ly <dig> inhibited bbs-stimulated pge <dig> release from pc- <dig> cells, whereas pd <dig> had no effect .
inhibition of pi3k/akt pathway reduces bbs-stimulated cox- <dig> promoter activity
the cellular levels of cox- <dig> mrna can be regulated both by enhanced gene transcription and inhibition of message degradation  <cit> .
to determine whether bbs treatment enhanced cox- <dig> gene transcription, pc- <dig> cells were first transiently transfected with a transcription reporter construct consisting of  <dig>  kb of the human cox- <dig> promoter coupled to a luciferase gene and then stimulated with bbs over a time course.
bbs induced a time-dependent increase  in cox- <dig> promoter activity when compared to vehicle-treated control cell cultures .
to determine whether the p38mapk or pi3k/akt pathways were involved in bbs-stimulated cox- <dig> transcription, cells were pretreated with sb <dig>  or ly <dig>  for  <dig> min followed by a 6-h treatment with bbs .
compared to bbs treatment alone, ly <dig> inhibited approximately 50% of the increase in bbs-stimulated luciferase activity .
in contrast, sb <dig> had no effect on bbs-stimulated cox- <dig> promoter activity , suggesting that the pi3k/akt pathway, not the p38mapk pathway, is involved in bbs-induced cox- <dig> gene transcription in pc- <dig> cells.
ly <dig> inhibits bbs-stimulated ap- <dig> binding activity but not nf-κb nuclear translocation
the human cox- <dig> promoter contains multiple regulatory sites that bind transcription factors including nuclear factor-κb   <cit>  and ap- <dig>  <cit> .
electrophoresis mobility shift assays showed that bbs treatment of pc- <dig> cells induced an increase in ap- <dig> binding activity  that was inhibited by pretreatment with ly <dig>  for  <dig> min , suggesting that pi3k/akt-mediated ap- <dig> activation is involved in bbs regulation of the cox- <dig> promoter activity.
in addition to activation of ap- <dig>  we previously reported that bbs also induced nf-κb activation in pc- <dig> cells  <cit> .
nf-κb is an inducible dimeric transcription factor that belongs to the rel/nf-κb family of proteins  <cit> .
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- <dig>  <cit> .
to confirm that bbs activated nf-κb, pc- <dig> cells were treated with peptide for  <dig> min, fixed, and immunostained with an antibody to the p <dig> subunit of nf-κb.
treated cells demonstrated increased nuclear nf-κb immunoreactivity when compared with vehicle-treated cultures .
pretreating cells with the nf-κb inhibitor, curcumin , inhibited both bbs- and tnf-α-induced nf-κb translocation to the nucleus .
to assess the role of the pi3k/akt pathway in bbs-induced nf-κb activation, cells were pretreated with ly <dig> for  <dig> min, followed by  <dig> min treatment with agonist.
ly <dig> had no effect on the bbs-induced nf-κb translocation .
since ly <dig> inhibits cox- <dig> mrna expression , cox- <dig> promoter activity  and ap- <dig> binding , these data suggest that ap- <dig> and not nf-κb regulates bbs-stimulated cox- <dig> promoter, in part, through activation of the pi3k/akt pathway.
p38mapk activity enhances the stability of cox- <dig> mrna
since inhibition of the p38mapk pathway did not block bbs-stimulated cox- <dig> promoter activity , we assessed its potential role in regulating cox- <dig> mrna stability.
pc- <dig> cells were stimulated for  <dig> h with bbs  and then treated with actinomycin d  alone or in combination with either sb <dig> or ly <dig>  rna was isolated from the cells over a time course and the level of cox- <dig> mrna at each time point assessed by northern blotting .
cox- <dig> mrna degraded slowly in bbs-stimulated cells treated with actinomycin d alone or in combination with ly294002; less than 20% of the cox- <dig> message was lost over the 90-min time course .
in contrast, the levels of cox- <dig> mrna decreased rapidly in cells treated with a combination of actinomycin d and sb203580; approximately 50% of the cox- <dig> message was degraded in cells treated for  <dig> min , suggesting that p38mapk activity stabilizes bbs-induced increases in cox- <dig> mrna levels.
expression of grpr is sufficient to confer bbs-inducible cox- <dig> expression in lncap cells
lncap prostate cancer cells, in contrast to the pc- <dig> cells, are androgen-responsive, poorly invasive  <cit>  and do not have specific grpr binding sites, as assessed by radioligand binding to 125i-bombesin  <cit>  and i mobilization in response to bbs  <cit> .
to determine whether the acquisition of the grpr was sufficient to confer bbs-inducible cox- <dig> expression, lncap cells were stably transfected with an expression plasmid containing grpr cdna downstream of the constitutively-active cmv promoter  <cit> .
grpr mrna expression was confirmed by northern blot , and the functional status of the receptor was demonstrated by intracellular ca2+ imaging using fura- <dig> .
immunoblots revealed that bbs stimulated cox- <dig> protein expression in grpr-transfected cells, but had no effect on lncap cells expressing control vector .
also, similar to pc- <dig> cells, pretreatment of grpr-transfected lncap cells with either sb <dig>  or ly <dig>  completely inhibited bbs-stimulated pge <dig> release, suggesting that grpr regulates cox- <dig> activity via the p38mapk and pi3k/akt pathways .
discussion
although the aberrant overexpression of cox- <dig>  the bbs-like peptide, grp, and grpr has been documented for prostate cancers with ne features, particularly in the setting of recurrent disease  <cit> , a mechanistic link between grpr activation and cox- <dig> expression in prostate cancer cells has not been made.
the studies presented here establish that bbs can stimulate cox- <dig> expression and begins to define the molecular signal transduction pathways linking grpr to cox- <dig>
expression of cox- <dig> can be regulated by multiple signaling pathways that affect both gene transcription and post-transcriptional mrna processing  <cit> .
we found that bbs-stimulated cox- <dig> expression requires the activation of both the p38mapk and pi3k/akt pathways in pc- <dig> and lncap cells expressing recombinant grpr.
ly <dig> partially inhibited bbs-stimulated cox- <dig> promoter activity, decreased cox- <dig> mrna and protein levels, and reduced pge <dig> secretion, whereas, inhibition of p38mapk destabilized bbs-stimulated cox- <dig> mrna, resulting in a decrease of both cox- <dig> protein expression and pge <dig> production.
the mek inhibitor, pd <dig>  had no effect on either bbs-stimulated cox- <dig> expression or pge <dig> secretion, in contrast to previously published data from our laboratory showing that, in intestinal epithelial cells, bbs stimulated cox- <dig> expression through a mek/erk-dependent pathway  <cit> .
sequence analysis of the 5'-flanking region of the human cox- <dig> gene has identified multiple potential transcriptional regulatory elements including two nf-κb sites located at  <dig> to  <dig> and  <dig> to  <dig>  <cit>  base pairs 5' of the transcriptional start site, and an ap- <dig> site located ~ <dig> base pairs upstream from the start site  <cit> .
although we have previously reported nf-κb-dependent regulation of vegf and il- <dig> expression  <cit>  in pc- <dig> cells, in the present study nf-κb does not appear to be involved in grpr-regulation of the endogenous cox- <dig> promoter.
this conclusion is based on the observation that ly <dig> can inhibit cox- <dig> mrna expression and partially reduced promoter activity, but does not block bbs-stimulated nf-κb activation.
in contrast, ly <dig> does inhibit bbs-induced ap- <dig> binding, suggesting that ap- <dig> may be the primary transcription factor involved in bbs-stimulated cox- <dig> mrna expression in pc- <dig> cells.
these findings are consistent with data from intestinal epithelial cells showing that bbs-stimulated cox- <dig> expression via an ap-1-dependent pathway  <cit> .
despite the fact that more than 80% of patients with late stage prostate cancer initially respond to androgen-ablation therapy  <cit> , more than half will progress to a hormone-refractory disease within  <dig> to  <dig> months after treatment  <cit> .
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- <dig> levels as an independent predictor of prostate cancer recurrence  <cit> .
at 62-months follow-up, cox- <dig> immunostaining predicted cancer progression with 82% sensitivity and 81% specificity.
additionally, two clinical trials have generated early, but promising, data that inhibition of cox- <dig> can benefit patients with biochemical recurrence of prostate cancer .
in the first study, pruthi and associates  <cit>  studied  <dig> patients who were given the selective cox- <dig> inhibitor, celecoxib, following a diagnosis of biochemical recurrence.
five of the  <dig> patients had decreased psa levels, and three of the  <dig> had stabilization of their psa levels at  <dig>   <dig>  and  <dig> months following the initiation of therapy.
in a second larger randomized control trial comparing placebo  to celecoxib , the group receiving celecoxib for  <dig> months showed a significantly decreased rate of rise in psa levels when compared to the placebo control patients  <cit> .
together, these trials suggest that targeting cox- <dig> 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- <dig> inhibitors.
novel strategies for inhibiting cox- <dig> could once again make it a viable therapeutic target in the future.
inhibiting grpr may provide an effective therapeutic alternative for decreasing cox- <dig> 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  <cit> .
similar to prostate cancers, lung cancers express grp and grpr  <cit>  where they promote tumor progression and metastatic spread through autocrine and paracrine mechanisms.
hohla and colleagues  <cit>  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- <dig> expression suggesting, together with the data presented herein, that grpr blockage may be an effective means of decreasing cox- <dig> expression within receptor-positive tumor tissue.
CONCLUSIONS
our study establishes a mechanistic link between grpr activation and enhanced cox- <dig> expression in prostate cancer cell lines, and suggests that inhibiting grpr may provide an alternative to non-steroidal anti-inflammatory drugs for inhibiting cox- <dig> in patients with recurrent prostate cancer.

 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 the american cancer society estimates that over new cases of prostate cancer will be diagnosed and more than men will die, most from metastatic, androgen -refractory disease. hormone-refractory prostate cancer is characterized, in part, by focal expansion of a malignant cell subpopulation with neuroendocrine 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, parathyroid hormone-related protein, and the bombesin -like peptide, gastrin-releasing peptide. although the impact of ne differentiation on poor prognosis and androgen independence has been extensively studied, the molecular mechanisms linking ne tumor cells and their bioactive neuropeptides to disease progression are still unclear. increased expression of cyclooxygenase-, an enzyme that catalyzes the synthesis of prostanoids such as prostaglandin e from arachidonic acid, was identified as an independent predictor of prostate cancer progression. clinical trials using cox- inhibitors in patients with biochemical recurrence of prostate cancer have suggested that cox- inhibition may improve survival, and pre-clinical studies with cell lines and animal models have established a functional link between cox- expression and an aggressive cancer phenotype. specifically, dandekar and coworkers have demonstrated that overexpression of cox- 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- suppressed tumor growth and decreased metastatic spread. together, these studies implicate cox- 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- expression can be induced by multiple factors including growth factors, proinflammatory cytokines, and peptide hormones. 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, a member of the g protein-coupled receptor superfamily. clinical, histological, and experimental observations have implicated grp and grpr in the pathophysiology of prostate cancer progression. logothetis and hoosein 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; bbs stimulates the growth of both orthotopic and ectopic prostate cancer cell xenografts in athymic nude mice through grpr-mediated mechanisms. bbs also promotes expression of metalloproteinases and increases prostate cancer cell migration and invasion. previously, we reported that bbs stimulates the expression of the proangiogenic genes il- and vascular endothelial growth factor in human prostate cancer cell lines. since cox- 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- expression in cells from other tissues, we reasoned that grpr activation and cox- expression may be mechanistically linked in prostate cancer cells. here, we report that bbs stimulates an increase in cox- mrna, protein expression, and the release of pge from the grpr-positive, androgen-insensitive prostate cancer cell line, pc- the stimulatory effects of bbs on cox- expression and pge production are mediated by p38mapk and pi kinase /akt pathways and blocked by the selective grpr antagonist bim the pi3k/akt pathway couples grpr to the activation of the transcription factor, activator protein-, and enhances cox- promoter activity. bbs also stimulates nuclear factor-kappab activation in pc-3; however, nf-κb does not regulate grpr-mediated cox- expression. the p38mapk pathway increases bbs-stimulated cox- expression by slowing the degradation of cox- mrna. expression of recombinant grpr in the grpr-negative, androgen-sensitive cell line lncap, is sufficient to confer bbs-stimulated cox- expression via the p38mapk and pi3k/akt pathways. together, these results define a molecular mechanism for enhanced cox- 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- mrna and protein expression to determine whether bbs stimulates cox- expression, we treated the androgen-insensitive prostate cancer cell line pc- with bbs and measured steady-state levels of cox- mrna and protein at various time points. compared to vehicle-treated control cultures, cox- mrna was increased at h following addition of bbs and peaked between and h. increased cox- protein was also detected at h following bbs treatment, peaked between and h, and returned to baseline levels by h. consistent with the lack of change in basal cox- mrna levels over the time course; we did not observe a change in the basal expression of cox- protein in non-treated cells. induction of both cox- mrna and protein expression was dependent on the concentration of bbs. increased cox- mrna and protein levels were detected in cells treated with as little as nm bbs for h and maximal induction was observed in cells treated with to nm bbs. grpr mediates bbs-stimulated cox- protein expression and pge synthesis cox- converts arachidonic acid, released from phospholipids by the action of phospholipase a to prostaglandin h - the common precursor of all prostaglandins, including pge to assess whether bbs stimulation of cox- expression was associated with an increased prostaglandin synthesis, the levels of pge released from pc- cells were measured using an enzyme-linked immunosorbent assay. compared to vehicle-treated control cultures, bbs induced a time-dependent increase in the pge levels that peaked between and h. pretreatment with the grpr-selective antagonist, bim ome], for min blocked the bbs-induced increase in pge, *p ≤ ). consistent with this observation, bim also inhibited bbs-stimulated increases in cox- protein expression. bbs activates multiple intracellular signaling pathways in pc- cells cox- expression is regulated by multiple intracellular signaling pathways including the mapk pathways, mek/erk and p38mapk, and the pi3k/akt pathway. to determine if these pathways were activated by bbs, pc- cells were treated with peptides over a time course and the activation state of each pathway assessed by immunoblotting. bbs treatment induced a time-dependent increase in the levels of activated erk and erk. the levels increased rapidly with a peak phosphorylation occurring between and min and remained elevated above baseline min after treatment with bbs. bbs stimulated a transient activation of p38mapk. the levels of phospho-p38mapk peaked between and min and, in contrast to the activation of either erk and or akt, returned to baseline levels by min. the levels of phospho-akt increased at min, reached a plateau by min, and remained elevated for the duration of the time course. over the same time course, we did not observe any change in the activation state of these pathways in non-stimulated cells. bbs-stimulated cox- 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- expression, pc- cells were pretreated with either the p38mapk inhibitor, the pi3k inhibitor or the mek inhibitor and then stimulated with bbs for h. agonist treatment failed to increase either cox- mrna or protein levels when the cells were pretreated with either sb or ly. in contrast, pretreatment with pd did not inhibit bbs-stimulated increases in cox- mrna nor protein expression. consistent with these observations, sb or ly inhibited bbs-stimulated pge release from pc- cells, whereas pd had no effect. inhibition of pi3k/akt pathway reduces bbs-stimulated cox- promoter activity the cellular levels of cox- mrna can be regulated both by enhanced gene transcription and inhibition of message degradation. to determine whether bbs treatment enhanced cox- gene transcription, pc- cells were first transiently transfected with a transcription reporter construct consisting of kb of the human cox- promoter coupled to a luciferase gene and then stimulated with bbs over a time course. bbs induced a time-dependent increase in cox- promoter activity when compared to vehicle-treated control cell cultures. to determine whether the p38mapk or pi3k/akt pathways were involved in bbs-stimulated cox- transcription, cells were pretreated with sb or ly for min followed by a 6-h treatment with bbs. compared to bbs treatment alone, ly inhibited approximately 50% of the increase in bbs-stimulated luciferase activity. in contrast, sb had no effect on bbs-stimulated cox- promoter activity, suggesting that the pi3k/akt pathway, not the p38mapk pathway, is involved in bbs-induced cox- gene transcription in pc- cells. ly inhibits bbs-stimulated ap- binding activity but not nf-κb nuclear translocation the human cox- promoter contains multiple regulatory sites that bind transcription factors including nuclear factor-κb and ap-. electrophoresis mobility shift assays showed that bbs treatment of pc- cells induced an increase in ap- binding activity that was inhibited by pretreatment with ly for min, suggesting that pi3k/akt-mediated ap- activation is involved in bbs regulation of the cox- promoter activity. in addition to activation of ap- we previously reported that bbs also induced nf-κb activation in pc- cells. nf-κb is an inducible dimeric transcription factor that belongs to the rel/nf-κb family of proteins. 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-. to confirm that bbs activated nf-κb, pc- cells were treated with peptide for min, fixed, and immunostained with an antibody to the p subunit of nf-κb. treated cells demonstrated increased nuclear nf-κb immunoreactivity when compared with vehicle-treated cultures. pretreating cells with the nf-κb inhibitor, curcumin, inhibited both bbs- and tnf-α-induced nf-κb translocation to the nucleus. to assess the role of the pi3k/akt pathway in bbs-induced nf-κb activation, cells were pretreated with ly for min, followed by min treatment with agonist. ly had no effect on the bbs-induced nf-κb translocation. since ly inhibits cox- mrna expression, cox- promoter activity and ap- binding, these data suggest that ap- and not nf-κb regulates bbs-stimulated cox- promoter, in part, through activation of the pi3k/akt pathway. p38mapk activity enhances the stability of cox- mrna since inhibition of the p38mapk pathway did not block bbs-stimulated cox- promoter activity, we assessed its potential role in regulating cox- mrna stability. pc- cells were stimulated for h with bbs and then treated with actinomycin d alone or in combination with either sb or ly rna was isolated from the cells over a time course and the level of cox- mrna at each time point assessed by northern blotting. cox- mrna degraded slowly in bbs-stimulated cells treated with actinomycin d alone or in combination with ly294002; less than 20% of the cox- message was lost over the 90-min time course. in contrast, the levels of cox- mrna decreased rapidly in cells treated with a combination of actinomycin d and sb203580; approximately 50% of the cox- message was degraded in cells treated for min, suggesting that p38mapk activity stabilizes bbs-induced increases in cox- mrna levels. expression of grpr is sufficient to confer bbs-inducible cox- expression in lncap cells lncap prostate cancer cells, in contrast to the pc- cells, are androgen-responsive, poorly invasive and do not have specific grpr binding sites, as assessed by radioligand binding to 125i-bombesin and i mobilization in response to bbs. to determine whether the acquisition of the grpr was sufficient to confer bbs-inducible cox- expression, lncap cells were stably transfected with an expression plasmid containing grpr cdna downstream of the constitutively-active cmv promoter. grpr mrna expression was confirmed by northern blot, and the functional status of the receptor was demonstrated by intracellular ca2+ imaging using fura-. immunoblots revealed that bbs stimulated cox- protein expression in grpr-transfected cells, but had no effect on lncap cells expressing control vector. also, similar to pc- cells, pretreatment of grpr-transfected lncap cells with either sb or ly completely inhibited bbs-stimulated pge release, suggesting that grpr regulates cox- activity via the p38mapk and pi3k/akt pathways. discussion although the aberrant overexpression of cox- the bbs-like peptide, grp, and grpr has been documented for prostate cancers with ne features, particularly in the setting of recurrent disease, a mechanistic link between grpr activation and cox- expression in prostate cancer cells has not been made. the studies presented here establish that bbs can stimulate cox- expression and begins to define the molecular signal transduction pathways linking grpr to cox- expression of cox- can be regulated by multiple signaling pathways that affect both gene transcription and post-transcriptional mrna processing. we found that bbs-stimulated cox- expression requires the activation of both the p38mapk and pi3k/akt pathways in pc- and lncap cells expressing recombinant grpr. ly partially inhibited bbs-stimulated cox- promoter activity, decreased cox- mrna and protein levels, and reduced pge secretion, whereas, inhibition of p38mapk destabilized bbs-stimulated cox- mrna, resulting in a decrease of both cox- protein expression and pge production. the mek inhibitor, pd had no effect on either bbs-stimulated cox- expression or pge secretion, in contrast to previously published data from our laboratory showing that, in intestinal epithelial cells, bbs stimulated cox- expression through a mek/erk-dependent pathway. sequence analysis of the 5'-flanking region of the human cox- gene has identified multiple potential transcriptional regulatory elements including two nf-κb sites located at to and to base pairs 5' of the transcriptional start site, and an ap- site located ~ base pairs upstream from the start site. although we have previously reported nf-κb-dependent regulation of vegf and il- expression in pc- cells, in the present study nf-κb does not appear to be involved in grpr-regulation of the endogenous cox- promoter. this conclusion is based on the observation that ly can inhibit cox- mrna expression and partially reduced promoter activity, but does not block bbs-stimulated nf-κb activation. in contrast, ly does inhibit bbs-induced ap- binding, suggesting that ap- may be the primary transcription factor involved in bbs-stimulated cox- mrna expression in pc- cells. these findings are consistent with data from intestinal epithelial cells showing that bbs-stimulated cox- expression via an ap-1-dependent pathway. despite the fact that more than 80% of patients with late stage prostate cancer initially respond to androgen-ablation therapy, more than half will progress to a hormone-refractory disease within to months after treatment. 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- levels as an independent predictor of prostate cancer recurrence. at 62-months follow-up, cox- immunostaining predicted cancer progression with 82% sensitivity and 81% specificity. additionally, two clinical trials have generated early, but promising, data that inhibition of cox- can benefit patients with biochemical recurrence of prostate cancer. in the first study, pruthi and associates studied patients who were given the selective cox- inhibitor, celecoxib, following a diagnosis of biochemical recurrence. five of the patients had decreased psa levels, and three of the had stabilization of their psa levels at and months following the initiation of therapy. in a second larger randomized control trial comparing placebo to celecoxib, the group receiving celecoxib for months showed a significantly decreased rate of rise in psa levels when compared to the placebo control patients. together, these trials suggest that targeting cox- 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- inhibitors. novel strategies for inhibiting cox- could once again make it a viable therapeutic target in the future. inhibiting grpr may provide an effective therapeutic alternative for decreasing cox- 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. similar to prostate cancers, lung cancers express grp and grpr where they promote tumor progression and metastatic spread through autocrine and paracrine mechanisms. hohla and colleagues 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- expression suggesting, together with the data presented herein, that grpr blockage may be an effective means of decreasing cox- expression within receptor-positive tumor tissue.  our study establishes a mechanistic link between grpr activation and enhanced cox- expression in prostate cancer cell lines, and suggests that inhibiting grpr may provide an alternative to non-steroidal anti-inflammatory drugs for inhibiting cox- in patients with recurrent prostate cancer. 
we report that bbs stimulates cox- mrna and protein expression, and the release of prostaglandin e from the grp receptor -positive, androgen-insensitive prostate cancer cell line, pc- bbs-stimulated cox- expression is mediated, in part, by p38mapk and pi kinase /akt pathways, and blocked by a grpr antagonist. cyclooxygenase- and the bombesin -like peptide, gastrin-releasing peptide, have been implicated in the progression of hormone-refractory prostate cancer; however, a mechanistic link between the bioactive peptide and cox- expression in prostate cells has not been made. our study establishes a mechanistic link between grpr activation and enhanced cox- 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- in patients with recurrent prostate cancer. expression of recombinant grpr in the androgen-sensitive cell line lncap is sufficient to confer bbs-stimulated cox- expression via the p38mapk and pi3k/akt pathways. the p38mapk pathway increases bbs-stimulated cox- expression by slowing the degradation of cox- mrna. although bbs stimulates nuclear factor-kappab in pc- nf-κb does not regulate grpr-mediated cox- expression. the pi3k/akt pathway couples grpr to the transcription factor, activator protein-, and enhanced cox- promoter activity.
 cyclooxygenase- and the bombesin -like peptide, gastrin-releasing peptide, have been implicated in the progression of hormone-refractory prostate cancer; however, a mechanistic link between the bioactive peptide and cox- expression in prostate cells has not been made.  we report that bbs stimulates cox- mrna and protein expression, and the release of prostaglandin e from the grp receptor -positive, androgen-insensitive prostate cancer cell line, pc- bbs-stimulated cox- expression is mediated, in part, by p38mapk and pi kinase /akt pathways, and blocked by a grpr antagonist. the pi3k/akt pathway couples grpr to the transcription factor, activator protein-, and enhanced cox- promoter activity. although bbs stimulates nuclear factor-kappab in pc- nf-κb does not regulate grpr-mediated cox- expression. the p38mapk pathway increases bbs-stimulated cox- expression by slowing the degradation of cox- mrna. expression of recombinant grpr in the androgen-sensitive cell line lncap is sufficient to confer bbs-stimulated cox- expression via the p38mapk and pi3k/akt pathways.  our study establishes a mechanistic link between grpr activation and enhanced cox- 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- in patients with recurrent prostate cancer. gastrin-releasing peptide receptorsignal transductionprostate cancerneuroendocrine differentiationhormone-refractory 