The Mechanisms Of Compound C Induces Autophagy In Human Cholangiocarcinoma Cells

objective: To investigate the molecular mechanisms of the induction of autophagy by the AMPK inhibitor Compound C in human cholangiocarcinoma cells.Methods: In this experiment,Human cholangiocarcinoma QBC939 and RBE cells were treated with Compound C.The autophagy inhibitor 3-MA and Bafilomycin A1(BAF)alone or in combination with Compound C treated cells.The Akt/mTOR inhibitor LY294002(LY)/Rapamycin(Rap)alone or in combination with Compound C treated cells.The JNK inhibitor SP600125(SP)and the p53 inhibitor Pifithrin-α(PFT-α)or transfection of siRNA interfered with JNK and transfection of siRNA interfered with p53 and the p38 MAPK inhibitor SB203580(SB)alone or in combination with Compound C treated cells.Cell immunofluorescence staining tests cell autophagy.Cytotoxicity was detected by LDH cytotoxicity Assay Kit.Western blot was used to analyze the protein expression levels of autophagy and apoptosis related molecules LC3A/B,PARP and p53.Real-time PCR was used to detect the mRNA expression level of p53 and MDM2.Result:(1)Compound C induces cell death in human CCA cells: Lactate dehydrogenase(LDH)release results indicate that Compound C effectively induced the death of QBC939 and RBE cells.Western blot results showed that Compound C treatment induced the activation of cleaved PARP in QBC939 and RBE cells,which was consiscent with the results of cell death.These results suggest that Compound C induces Cell death in human CCA cells in a dose and time dependent manner.(2)Compound C induces autophagy in human CCA cells.Cell immunofluorescence results showed that Compound C induces autophagy in human CCA cells in a dose and time-dependent manner.In addition,Western blot results showed that Compound C induces autophagy-related protein LC3A/B expression.The results suggest Compound C effectively induces autophagy in human CCA cells.(3)Inhibition of autophagy to increases Compound C-induced cell death of human CCA cells.LDH release results showed that autophagy inhibitors,3-MA and BAF,could significantly increased compound C-induced cell death of QBC939 and RBE cells after blocking autophagy.In addition,Western blot results also showed that the blockade of autophagy with 3-MA and BAF significantly increased Compound C-induced apoptosis of QBC939 and RBE cells,consistent with the results of LDH.The results suggest that inhibition of autophagy increased Compound C-induces killing effect in human CCA cell.(4)Akt/mTOR pathway is not involved in Compound C-induced autophagy in human CCA cells: Western blot results showed that compound C treatment increased the levels of phosphorylated Akt in QBC939 and RBE cells,and had no obvious influence on the levels of phosphorylated p70S6 K.In addition,Compound C-inducedautophagy of QBC939 and RBE are not affected after LY/Rap blockade of Akt/mTOR pathway.The results suggest that Akt/mTOR pathway is not involved in the regulation of Compound C-induced autophagy in human CCA cells.(5)Blocking JNK inhibits Compound C-induced autophagy in human CCA cells: Western blot results showed that SP blocked the activity of JNK and significantly attenuated autophagy induced by Compound C in QBC939 and RBE cells.Transfection of siRNA interfered with the expression of JNK also significantly attenuated Compound C-induced autophagy in QBC939 and RBE cells.These results suggest that JNK is involved in the regulation of Compound C-induced autophagy in human CCA cells.(6)CCA cells: Western blot results showed that,although Compound C activated the p38 MAPK activity in QBC939 and RBE cells,SB increased the autophagy induced by Compound C after SB blocked the activity of p38 MAPK.The results suggest that p38 MAPK is involved in the regulation of autophagy in Compound C-induced in human CCA cells.(7)p38 MAPK inhibition promotes compound C-induced autophagy via JNK activation in human CCA cells: Western blot results showed that SB blocked the p38 MAPK activity of cells and promoted the up-regulation of JNK activity in QBC939 and RBE cells.In addition,blockade of JNK activity by SP attenuated the inhibition of p38 MAPK and promotes Compound C-induced autophagy in QBC939 and RBE cells.These results suggest that p38 MAPK inhibition promotes compound C-induced autophagy via JNK activation in human CCA cells.(8)Blocking p53 inhibits Compound C-induced autophagy in human CCA cells: Real-time quantitative PCR and Western blot showed that Compound C induces p53 expression in QBC939 and RBE cells.In addition,Western blot results showed that PFT-α blocked the activity of p53,significantly weakened Compound C-induced autophagy in QBC939 and RBE cells.Transfection of si RNA to interfere with the expression of p53 significantly attenuated Compound C-induced autophagy in QBC939 and RBE cells.These results suggest that p53 is involved in the process of Compound C induced autophagy in human CCA cells.(9)Akt/mTOR pathway is not involved in Compound C-induced apoptosis in human CCA cells: Western blot results showed that the apoptosis induced by Compound C in QBC939 and RBE cells was not affected by LY/Rap blocking Akt/mTOR pathway.The results suggest that the Akt/mTOR pathway is not involved in Compound C-induced apoptosis in human CCA cells.Conclusion: Compound C has the effect of cell death on human CCA cells.Autophagy could be induced and protect human CCA cells from the killing effect by Compound C.It is JNK and p53 pathway rather than Akt/mTOR pathway involved in Compound C-induced autophagy in human CCA cells.