Metformin Induces Oxidative Stress-Mediated Apoptosis without the Blockade of Glycolysis in H4IIE Hepatocellular Carcinoma Cells

Metformin, a widely prescribed anti-diabetic drug, also exerts anti-cancer effects in different types of cancers. Although a number of molecular mechanisms have been suggested, the metabolic features underlying metformin's anti-cancer activity is not fully understood enough. Because cancer cells have been known to prefer inefficient aerobic glycolysis to support their proliferation, it is important to clarify by which metformin affects metabolism to suppress the proliferation of cancer cells. Here, we report the metabolic changes induced by metformin and its relevance to the induction of apoptosis in H4II rat hepatocellular carcinoma cells. H4IIE cells were treated with metformin and other reagents in culture media with various nutritional compositions. Glutamine as well as pyruvate enhanced the viability of H4IIE cells in glucose-deprived conditions. Protective effects of glucose and pyruvate were comparable at same concentrations (5 mM). Metformin induced apoptosis irrespective of any nutritional conditions. Glucose consumption and lactate production were stimulated by metformin. Inhibition of glycolysis by 2-deoxyglucose suppressed the metformin-induced lactate production but additively enhanced metformin's pro-apoptotic effect. These results indicate that metformin does not interfere but accelerate glycolysis. Unexpectedly, the production of reactive oxygen species (ROS) was markedly stimulated by metformin. A potent antioxidant, N-acetylcysteine (NAC) suppressed all pro-apoptotic changes as well as ROS generation induced by metformin. Taken together, metformin does not interfere with glycolysis but promotes apoptosis by enhancing oxidative stress.