Metformin Promotes Sensitivity Of Gemcitabine Through Inhibiting PKM2 And Inducing Mitochondrial Apoptosis In Cholangiocarcinoma

Background Cholangiocarcinoma(CCA)is a malignant tumor with high mortality,no obvious early symptoms and rapid progression.The aggressiveness of tumor cell growth and insensitivity to therapeutic drugs are the main factors leading to the poor prognosis of CCA.Improving the sensitivity of tumor cells to therapeutic drugs is the main focus of cancer therapy.pyruvate kinase M2(PKM2)is an important rate-limiting enzyme in glycolysis pathway.PKM2 plays an important role in the development of drug resistance in tumor cells,and targeting PKM2 can effectively improve the sensitivity of tumor cells to chemotherapy.Metformin is a commonly used oral hypoglycemic agent,which has been found to have certain anticancer effects in vivo and in vitro studies,and can down-regulate the expression of PKM2 and inhibit the proliferation of tumor cells.Mitochondria are the energy source of cells and play an important role in the proliferation and differentiation of tumor cells.However,whether metformin can regulate the sensitivity of bile duct cancer cells to chemotherapy drugs and promote apoptosis through PKM2 remains unclear.Objective This study was to investigate the role of metformin in regulating mitochondrial apoptosis and the sensitivity of CCA chemotherapy,and the mechanism by which PKM2 is involved in the regulation of apoptosis of CCA.Methods CCA cell lines HCC9810 and RBE were treated with metformin in combination with PKM2-IN-1 or agonist ML265.Through MTT,transwell,qRT-PCR protein Western blot,cellular immunofluorescence,mitochondrial membrane potential detection,TUNEL,lactic acid and LDH release detection,in vivo animal experiments and other experimental techniques,To explore the role and mechanism of metformin in promoting gemcitabine sensitivity by regulating PKM2 in drug sensitivity,metastasis and migration of CCA.ResultsIn PKM2-IN-1 treated HCC9810 cells,the IC50 of metformin decreased from 75.6mM to 62.5mM.The IC50 of metformin in RBE cells treated with ML265 changed from 56.8mM to 78.3mM.Metformin combined with gemcitabine significantly inhibited the activity of bile duct cancer cells(P<0.05).Metformin combined with citabine and PKM2-in-1 or ML265 had synergistic effects on migration,invasion and apoptosis of bile duct cancer cells HCC9810 and RBE with different PKM2 background expression levels.In addition,metformin and gemcitabine inhibit the expression of PKM2 and PDHB in HCC9810 and RBE,activate mitochondrial apoptosis,and ultimately play a synergistic role in increasing the susceptibility of bile duct cancer cells to gemcitabine.The results of subcutaneous tumor experiment in nude mice in vivo showed that metformin combined with gemcitabine inhibited the growth of bile duct cancerous tumor.Conclusion These results suggest that metformin and gemcitabine have synergistic effects in inhibiting proliferation of bile duct cancer cells and promoting mitochondrial apoptosis of bile duct cancer cells.PKM2 may mediate the apoptotic effect of metformin in coordination with gemcitabine.