Metformin sensitizes cholangiocarcinoma cell to cisplatin-induced cytotoxicity through oxidative stress mediated mitochondrial pathway.

Metformin (Met), an essential antidiabetic agent, shows antitumor activity in some cancers. A previous study showed that Met enhanced cytotoxic activity of cisplatin (Cis) in cholangiocarcinoma (CCA) in association with the activation of AMP-activated protein kinase and suppression of Akt-mTOR. However, these effects do not entirely explain the observed chemosensitizing effect. The present study investigated the interaction of Met and Cis over the enhanced antitumor effect. KKU-100 and KKU-M156 cells were used in the study. Cytotoxicity was assessed by acridine orange-ethidium bromide staining. Reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψm) were measured by dihydroethidium and JC-1 fluorescent methods. Cellular glutathione (GSH) and redox ratio were analyzed by enzymatic coupling assay. Proteins associated with antioxidant system and cell death were evaluated by western immunoblot. Cytotoxicity of Cis was enhanced by Met in association with ROS formation and GSH redox stress. The antioxidants, N-acetylcysteine and TEMPOL, and MPTP inhibitor, cyclosporine, attenuated cytotoxicity in association with suppression of ROS formation and the losses of Δψm. Met in combination with Cis suppressed expression of Nrf2 and altered the expression of Bcl2 family proteins. The chemosensitizing effect of Met in combination with Cis is causally associated with increased oxidative stress-mediated mitochondrial cell death pathway. Met may improve the efficacy of Cis in the treatment of cancer.