The effect of metformin on human clear cell renal cell carcinoma energy metabolism. Identification of adenine nucleotide translocator as a novel target of metformin

Although metformin (MET), the well-known antidiabetic drug, exhibits clear antineoplastic effects and is reported to target mitochondria, several issues are still open in this regard, thus limiting its utilization as an anticancer drug alone or in combination with other molecules. Here a functional investigation was carried out to reveal how MET impacted on mitochondrial functions and cell energy metabolism in human cultured clear cell renal carcinoma cells (ccRCCs), in which the anticancer effect of MET is already known. The in vitro effect of increasing MET concentrations on cell viability, necrosis and apoptosis of ccRCCs was checked and compared to normal immortalized HK2 cells. At the same time, the effect of MET on mitochondrial functions, ATP synthesis via oxidative phosphorylation, cellular ATP level, L-lactate production and export, glucose consumption and key mitochondrial and cytosolic enzyme activities was also investigated in cancer cells. MET affected ccRCC proliferation and impaired mitochondrial respiration, membrane potential generation and ATP production by targeting complex I (CI), III and IV of the respiratory chain and by inhibiting the activity of the adenine nucleotide translocator (ANT), the latter first identified here as a target of MET. Notably, the sensitivity of ANT and CI activity to increasing MET concentrations differed markedly, the former being considerably inhibited already at a low, near-clinically relevant concentrations, while the latter only at concentrations > 1 mM. The drug also induced a glycolytic shift in ccRCCs and increased the activity of the mitochondrial flavoenzymes succinate dehydrogenase and D-lactate dehydrogenase, and of the key enzymes of the pay-off phase of glycolysis, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase and pyruvate kinase. Nevertheless, cellular ATP level dropped markedly, and the intracellular L-LAC amount was almost doubled in the presence of MET. Interestingly, MET-induced glycolytic shift showed a drug concentration dependence similar to that seen for CI inhibition, suggesting not ANT but rather CI inhibition may be the trigger for metabolic rewiring. These findings give new insightsinto MET mechanisms of action which may potentially improve its application and outcome in cancer as well as in other pathologies.