Effect Of Prostaglandin E₂ On The Proliferation Of Human Cholangiocarcinoma Cell And The Mechanism

Background:Recently, a heightened interest in the role of cyclooxyenase-2 (COX-2)-derived prostaglandin E₂ (PGE₂) in neoplasia has emerged, and COX-2/PGE₂ show high expression in many tumor tissues. However, the biological role and molecular mechanism of PGE₂ in the tumorigenesis have not been established. COX-2 derived prostaglandin E₂ (PGE₂) can promote tumor growth by binding its receptors and activating signaling pathways which control cell proliferation, migration, apoptosis, and/or angiogenesis. Efforts to identify the molecular mechanisms by which PGE₂ promotes tumor growth may provide opportunities for cancer prevention and treatment. This study was designed to examine the effect of prostaglandin E₂ on the proliferation of human cholangiocarcinoma CCLP and HuCCT1 cells and the mechanism.Objectives:This study was designed to examine the effect ofprostaglandin E₂ on the proliferation of human cholangiocarcinoma CCLP and HuCCT1 cells and the mechanism.Methods:1. Cell culture: human cholangiocarcinoma cell lines (CCLP and HuCCT1) were cultured in DMEM and RPMI-1640.2. The cell growth rate of CCLP and HuCCT1 treated with PGE₂ was detected by viability and growth rate (WST-1) assay.3. Intracellular free calcium was measured after treated with PGE₂, by laser scanning confocal microscopy using fluo-3/AM as fluorescence probe.4. The level of cell cAMP was measured by immunoassay.5. Expression and intracellular location of p-CREB in CCLP and HuCCT1 cells were detected by Fluorescent Immunocytochemistry after treated with PGE₂ or forskolin or BAPTA-AM.6. Cell p-CREB level was detected using Western blot assay after treated with PGE₂ or forskolin or BAPTA-AM.7. Six cases of human cholangiocarcinoma samples from Jiangsu Province Hospital were collected for detecting of p-CREB by immunohistochemical assay.Results:1. Cell viability and growth rate (WST-1) assay showed 1,5,10, 20μmol/L PGE₂ promoted the growth of HuCCT1 cell, and cell growth rates were 105.36%, 112.45%, 120.73% and 128.21%. The result of CCLP cell was similarity to the HuCCT1 cell.2. 10μmol/L PGE₂ increased cell fluorescence intensity, and fluorescence intensity increased by 50%, which suggested PGE₂ increased intracellular free calcium in HuCCT1 cell. The result of CCLP cell was similarity to the HuCCT1 cell.3. PGE₂ increased intracellular cAMP in HuCCT1 cell, and intracellular cAMP were 183.94 pmol/L, 189.01 pmol/L and 202.12 pmol/L treated by 0.1μmol/L, 1μmol/L, 10μmol/L PGE₂. The result of CCLP cell was similarity to the HuCCT1 cell.4. Fluorescent Immunocytochemistry assay showed p-CREB located in cellular nucleus of CCLP and HuCCT1 cells. PGE2 and forskolin promoted the expression of p-CREB. However, BAPTA-AM inhibited the expression of p-CREB.5. Western blot showed the same results as the Fluorescent Immunocytochemistry assay.6. Immunohistochemical methods showed highly expression of p-CREB in cholangiocarcinoma tissues.Conclusion:PGE₂ can increase the intracellular free calcium and cAMP levels in cholangiocarcinoma CCLP and HuCCT1 cells and promote the expression of CREB and its phosphorylation while promotes CCLP and HuCCT1 cell growth. This suggests that PGE₂ stimulates cholangiocarcinoma CCLP and HuCCT1 cell growth though Ca²⁺,cAMP/p-CREB signaling pathway.