Deoxycholic Acid Inhibits Proliferation And Induces Apoptosis Via NFκB Pathway In Human Cholangiocarcinoma Cell Lines QBC939

Deoxycholic acid inhibits proliferation and induces apoptosis via NF k B pathway in human cholangiocarcinoma cell lines QBC939Miao Zhu,Hanlin Zhao,Yujie Sun, Xiao Han Objective:Cholangiocarcinoma is an uncommon malignancy arising from the epithelial cells of the biliary tract. These tumors may arise anywhere along the intrahepatic or extrahepatic biliary tree. Patients with cholangiocarcinoma typically present at advanced stages, and cure rates are low, even with aggressive therapy.Bile acids are steroid molecules that are synthesized from cholesterol in the liver. They function as important regulators of cholesterol metabolism in the hepatocyte by modulating the expression of genes encoding cholesterol-degrading enzymes as well as cholesterol and phospholipid transport proteins. However, under certain pathophysiological conditions such as cholestasis, bile acids can promote cellular apoptosis and necrosis. Especially,it was reported that hydrophobic bile acids such as deoxycholic acid (DCA) could inhibit proliferation of pancreatic cancer and colon cancer.Whether deoxycholic acid can influence cholangiocarcinoma remains a subject of controversy. In order to investigate the direct effects of deoxycholicacid on cholangiocarcinoma, human cholangiocarcinoma cell lines QBC939 were selected and used.In the present study, we demonstrated deoxycholic acid can inhibit QBC939 proliferation and promote its apoptosis by suppressing the activity of NF- k B in the dose-dependent way. Materials And Methods:Reagent An IL6-NF- k B luciferase construct was generous gift from Dr.j-j Li, I k B was obtained from B&D Inc. Lipofectmine2000 was purchased from Invitrigen Inc,Luciferase assay kit was purchased from Promega Inc. Cell culture QBC939 cells were grown in DMEM medium supplemented with 10% heat-inactivated fetal calf serum, (100 units/ml penicillin, 100 ug/ml streptomycin) at 37°C with 5% CC^.The cultures were fed with fresh medium two times/week. Cell Viability assay:1. Proliferation of QBC939 cells in response to DCA was investigated using MTT assay method. For each experiment, cells were seeded in 96-well plates at 2 * 104 cells/well and Medium was then replaced with media containing DCA in different concentration incubated for an additional 24h, Cells then were incubated with MTT for 4h before adding formazon solution.Two hours later, absorbance at a wavelength of 570 nm was measured using a microplate reader(Bio-Rad Inc).2. Proliferation of QBC939 cells in response to I k B was investigated usingMTT assay method.cells were seeded in 96-well plates at 2 * 104 cells/well,then transfected with I k B in different concentration, 12 hours later ,cells were then incubated with MTT for 4 hours, and read absorbance at A570.Cell Survival Was Calculated: (%) = (Atreatment-Acontrol/Anegative-Acontrol) X100%Transfection and Luciferase Activity Assays The ability of DCA to activate NF-kB was investigated using a luciferase reporter system in which the luciferase gene was under the control of a NF-kB dependent promoter. A plasmid containing the β -galactosidase gene driven by the cytomegalovirus promoter was obtained from Clontech Laboratories. QBC393 cells were cotransfected with two plasmid (An IL6-NF- k B luciferase construct/AP-1 vector plus 0 -gal) using the LIPOFECTAMINE 2000 methods(Life Technologies) according to the manufacturer's instructions. Tranfected cells were cultured in serum-free, DMEM medium for 24 h before treatment with DCA in different concentions. Luciferase activity was measured with a luminometer(TD-20/20;Turner Designs Inc.) using Luciferase Assay System(Promega) according to the instruction. β -galactosidase activity was detected to normalize the luciferase activity.Flow cytometry Apoptosis assays were performed as follows. QBC939 cells were seeded in 6-well plates at 1 x 106cells/well.24 hours later, medium was replaced with media containing DCA incubated for an additional 24h ortransfected with I k -B plasmid for 12 hours.The cells were sent to theAnalytical Cytometry Core at Gulou Hospit...