The Effect And Mechanism Of Metformin On Cholangiocarcinoma RBE Cell Line

Objective:To investigate the effect of metformin and metformin combined with the AMPK inhibitor Compound C on human cholangiocarcinoma cell line RBE about proliferation, apoptosis and cell cycle. To investigate the activity change of AMPK and mTOR in the RBE cells.Methods:1. MTT detection of the effect of metformin on the cholangiocarcinoma RBE cell line:cholangiocarcinoma RBE cell was cultured in vitro, MTT was used to study the effect of metformin on cell growth with concentration0,5,10,20,30mmol/L metformin and groups (control,20mmol/L metformin,20umol/L Compound C,20umol/L Compound C+20mmol/L metformin) after24h,48h and72h.2. Flow cytometry detection of cell cycle distribution and apoptosis after metformin on the cholangiocarcinoma RBE cell line: cholangiocarcinoma RBE cell was cultured in vitro, flow cytometry was used to study the effect of metformin on cell cycle and apoptosis with concentration0,5,10,20,30mmol/L metformin and groups (control,20mmol/L metformin,20umol/L Compound C,20umol/L Compound C+20mmol/L metformin)48h. 3. Detection of the change of AMPK, p-AMPK, mTOR, p-mTOR after metformin on the cholangiocarcinoma RBE cell line: cholangiocarcinoma RBE cell was cultured in vitro, Western blot was used to study the effect of metformin on the change of AMPK, p-AMPK(Thr172), mTOR, p-mTOR(Ser2448) with concentration0,5,10,20,30mmol/L metformin and groups (control,20mmol/L metformin,20umol/L Compound C,20umol/L Compound C+20mmol/L metformin)48h.Results:1. MTT detection:As the concentration of metformin increased, cell growth was significantly decreased. At the concentration of10mmol/L,20mmol/L and30mmol/L, cell growth was decreased more in48h group than24h group. Group metformin combined with Compound C was not significantly different from the control.2. Flow cytometry detection:As the concentration of metformin increased, the number of G0/G1phase increased and the number of S phase decreased, and early apoptosis increased. Group metformin combined with Compound C was not significantly different from the control.3. Western Blot detection:As the concentration of metformin increased, AMPK and mTOR didn't change significantly, p-AMPK increased and p-mTOR decreased. Amount of p-AMPK and p-mTOR in group metformin combined with Compound C was between the control and group metformin.Conclusion:1. Metformin can inhibit the cell growth of cholangiocarcinoma RBE cells, and induce apoptosis and cell cycle arrest at G0/G1phase.2. Compound C can antagonize the effect of metformin on cholangiocarcinoma RBE cells.3. Metformin might inhibit cell growth of cholangiocarcinoma RBE cells though AMPK/mTOR signaling pathway.