Regulation Of Proliferation Signal Transduction Pathway By PGE₂ In Human Cholangiocarcinoma Cells

Background:Cholangiocarcinoma is a common primary hepatobiliary malignancythat often arises from longstanding bile duct inflammation, injury, andreparative biliary epithelial cell proliferation. Prostaglandins, especiallyPGE₂, appear to participate in the development of inflammatory reactionsand in oncogenesis. Prostaglandin E₂ is an important short-lived lipidmediator involved in diverse activities, such as inflammation, survival, andwound healing. The rate-limiting step of prostaglandin synthesis is catalyzedby the cyclooxygenase enzyme. Two primary COX isoenzymes have beencharacterized: 1) constitutive COX-1, which can be detected in most tissuesunder normal physiologic conditions and 2) inducible COX-2, which can berapidly induced under a variety of pathophysiologic conditions.COX-2-derived PGE₂ is an important mediator of inflammation.Nonsteroidal anti-inflammatory drugs, like aspirin, are widely used in thetreatment of inflammation. Nonsteroidal anti-inflammatory drugs exert theireffect by inhibiting COX-2 activity and PGE₂ synthesis. Epidemiologic studies and clinical trials have shown that the long-termuse of aspirin and other NSAIDs decreases the incidence of severalmalignancies. These findings have stirred a heightened interest in the role ofCOX-2 and inflammation in neoplasia. The connection between chronicinflammation and cancer has been elucidated. Although a number of studieshave revealed that COX-2 and aberrant PGE₂-mediated signaling pathwaysplay a critical role in the proliferation and survival of cancer cells, thedefinitive mechanism by which COX-2 and PGE₂-signaling pathwaysmediate these effects remains unknown. Furthermore, the safety of selectiveCOX inhibitors is recently concerned, which has prompted a search foralternative means to target this pathway.The action of PGE₂ is mediated by four G protein-coupled receptors:EP1, EP2, EP3 and EP4. Previous studies have shown that GPCRs cancrosstalk with the epidermal growth factor receptor pathway. AberrantPGE₂-induced activation of the epidermal growth factor receptor and Akt areimplicated in human cholangiocarcinoma cell growth and survival. However,the effect of PGE₂ on the signaling pathway of proliferation has not beenclearly defined. In the present study, we investigated the mechanism ofPGE₂-induced signaling pathway of proliferation in humancholangiocarcinoma cells. Objective:1. To investigate the expression of EP receptor in HuCCT1,SG231 andCCLP1 cell lines.2. To detect the biological effects of PGE₂,EP receptor and Erk signalingpathway on the proliferation of human cholangiocarcinoma cells.3. To explore the molecular mechanism of PGE2-induced signaling pathwayof proliferation in human cholangiocarcinoma cells.Methods:1. Human cholangiocarcinoma cells were cultured as routine.2. The expression of EP receptor was determined by RT-PCR and westernblotting analyses.3. The cell viability was determined using the cell proliferation reagentWST-1.4. The intracellular concentration of calcium was measured using laserconfocal scanning microscope.5. The expression levels of protein kinase phosphorylation were determinedby western blotting analyses.6. The activation of EP1 receptors involved in PGE2-stimulated Erkactivation and the intracellular calcium concentration increase waselucidated using selective EP1 receptor subtype antagonists and agonist.7. The activation of Erk involved in increase of the intracellular calcium concentration and EGFR phosphorylation was elucidated using theintracellular calcium chelator BAPTA-AM and EGF receptor kinaseinhibitor AG1478.8. The activation of Akt involved in increase of the intracellular calciumconcentration and EGFR phosphorylation was elucidated using theintracellular calcium chelator BAPTA-AM and EGF receptor kinaseinhibitor AG1478.Results:1. We characterized the expression of all four EP isoforms at the protein levelby western blotting and mRNA level by PT-PCR. All four EP receptorsubtypes were detected in human cholangiocarcinoma cells.2. PGE₂ and EP1 receptor agonist 17-P-T-PGE₂ promote human cholan-giocarcinoma cell growth and EP1 receptor antagonist SC51322 inhibitedcell growth.3. The effect of PD98059 (inhibitor of Erk) on PGE₂-iduced cholangio-carcinoma cell growth was examined by WST-1 assay. Compared withcontrol, PGE₂ significantly increase cell growth. The increase wasabrogated by the Erk inhibitor PD98059 treatment with a statisticalsignificance. PD98059 treatment markedly reduced the cell growth.4. Treatment of cells with PGE₂ and EP1 receptor agonist 17-P-T-PGE₂increase intracellular calcium concentration. EP1 receptor antagonist SC51322 inhibited the increase of intracellular calcium concentrationstimulated by PGE₂.5. Selective EP1 receptor antagonist SC51322 inhibited the activation ofPGE₂ and EP1 receptor agonist 17-P-T-PGE₂-stimulated Erk activationand the intracellular calcium concentration increase.6. The intracellular calcium chelator, BAPTA-AM, was shown to blockPGE₂ and EP1 receptor agonist 17-P-T-PGE₂-induced EGFRphosphorylation.7. The intracellular calcium chelator, BAPTA-AM, was shown to blockPGE₂-induced Erk phosphorylation. PGE₂-induced Erk phosphorylationwas abrogated by pre-treatment with the EGF receptor kinase inhibitor,AG1478.8. The intracellular calcium chelator, BAPTA-AM, was shown to blockPGE₂-induced Akt phosphorylation. PGE₂-induced Akt phosphorylationwas abrogated by pre-treatment with the EGF receptor kinase inhibitor,AG1478.Conclusion:These findings suggest that in human cholangiocarcinoma cells, PGE₂stimulated cell growth involved Erk signaling pathway. PGE₂ inducedphosphorylation of Erk is, at least in part, mediated through EP1 receptorsand EGFR phosphorylation induced by the intracellular calciumconcentration increase. Furthermore, PGE₂ induced activation of Akt is mediated through EGFR phosphorylation induced by increase of intracellularcalcium concentration.