| Background:PGE2is one of the predominant metabolic products of arachidonic acid catalyted by cyclooxy gcnase-2(COX-2). It has been found that PGE2mediates its biological effects through binding and activating4different G protein-coupled receptors (EP1, EP2, EP3, EP4) and relates to tumor closely, including tumor proliferation, invasion and migration, tumor angiogenesis.CAMP response element binding protein(CREB) is a transcriptional enhancer factor.It has a high affinity with cAMP response element(CRE) and can enhance cAMP induced gene transcription. CREB plays transcriptional regulatory function by its own phosphorylation, and activates a series of downstream signal transduction pathways.It has been shown that CREB can promote metastasis and invasion in different types of tumorsOur previous studies have shown that PGE2could significantly enhance HuCCT1cell invasion. However,it is still unclear about the relationship between PGE2and CREB,the mechanism of the action of CREB regulated by PGE2in the growth and invasiveness of HuCCTl cells.In this study, HuCCTl cells have been treated with PGE2,intracellular calcium chelator, PI3K inhibitor to detect the level of Phospho-CREB,we aim to clarify the role of CREB regulated by PGE2in the growth of HuCCTl cells and the probable signal transduction pathway.Objectives:To investigate the effect of PGE2on the activation of CREB and its related mechanism in HuCCTl cells.Methods:1.41cases of intrahepatic cholangiocarcinoma tissue and20cases of normal bile duct tissue from the First Affiliated Hospital of Nanjing Medical University were collected to detect the expression of Phospho-CREB by immunohistochemistry. The relationship between the expression of Phospho-CREB and clinicopathological parameters was analyzed statistically.2. The cholangiocarcinoma HuCCT1cells were cultured in conventional approach.3. HuCCT1cells were treated with exogenous PGE2, cells migration and invasion was examined by Transwell.4. HuCCT1cells were treated with exogenous PGE2for Omin、5min、15min、30min、45min、60min respectively, Western blot test was conducted to observe the alteration of Phospho-CREB.5. HuCCTl cells were treated with exogenous PGE2, the concentration of Ca2+in the cell was examined by laser scanning confocal microscope.6. Western blot test was performed to investigate the expression level of Phospho-CREB in HuCCTl cells after the treatment of PGE2, PGE2+BAPTA-AM.7. Western blot test was performed to investigate the expression level of Phospho-CREB in HuCCT1cells after the treatment of PGE2, PGE2+LY294002. Results:1. Immunohistochemistry was used to detect the expression of Phospho-CREB in41cases of intrahepatic cholangiocarcinoma tissue and20cases of normal bile duct tissue. The positive expression rates of Phospho-CREB in41cases of intrahepatic cholangiocarcinoma and20cases of normal bile duct tissue were73.2%and35.0%,respectively (P<0.05). The expression of Phospho-CREB was not statistically related to sex,age,tumor size,TNM staging,lymphnode metastasis and perineural invasion. The positive expression rates of Phospho-CREB in high-moderately differentiated tumors and poorly differentiated tumors were59.1%and89.5%,respectively (P<0.05)2. Cell invasion was found to increase by3.2times when the cells were treated with5μM PGE2for24h.3. HuCCT1cells were treated with5μmol/L PGE2for5min、15min、30min、45min、60min, the Western blot test showed that the level of Phospho-CREB was increased by64.5%、135.5%、141.9%、98.4%、59.7%, represtively, compared with the control group.The maximum was reached in30min.4. HuCCTl cells were treated with5μmol/L PGE2for120s,the concentration of Ca2+in cells detected by laser scanning confocal microscope was increased averagely by30%.5. HuCCTl cells were treated with5μmol/L PGE2for30min, the Western blot test showed that the level of Phospho-CREB and Phospho-AKT were increased by77.3%and30.4%. However, intracellular calcium chelator BAPTA-AM (5μmol/L) suppressed PGE2-mediated expression of Phospho-CREB and Phospho-AKT by58.9%and26.0%(P<0.05) when HuCCTl cells were treated with5μmol/L PGE2for30min after being pretreated with BAPTA-AM for30min.6. HuCCT1cells were treated with5μmol/L PGE2for30min, the Western blot test showed that the level of Phospho-CREB was increased by90.3%. However, PI3K inhibitor LY294002(5μmol/L) suppressed PGE2-mediated expression of Phospho-CREB by59.3%(P<0.05) when HuCCT1cells were treated with5μmol/L PGE2for30min after being pretreated with LY294002for30min.Conclusion:PGE2might up-regulate the expression level of Phospho-CREB to promote the growth and invasiveness in HuCCTl cells, which was partly related to the Ca2+/PI3K/AKT/CREB signaling pathway. |
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