Identification of glucose-independent metabolic pathways associated with anti-proliferative effect of metformin, their coordinate derangement with cMyc downregulation and reversibility in liver cancer cells.

Several studies indicated anti-cancer effects of metformin in liver cancer. This was attributed to the activation of LKB-AMPK axis, which is associated with anti-hyperglycaemic effect and cytotoxicity. However, despite lack of evidence on cytotoxic effect of physiological metformin concentrations and ability of cancer cells to survive under glucose-deprivation, no study has examined the glucose-independent effect of non-cytotoxic metformin or metabolic reprogramming associated with it. In addition, no study has ever been conducted on reversibility of anti-cancer effects of metformin. Here, the dose-dependent effects of metformin on HepG2 cells were examined in presence and absence of glucose. The longitudinal evolution of metabolome was analyzed along with gene and protein expression as well as their correlations with and reversibility of cellular phenotype and metabolic signatures. Metformin concentrations up to 2.5mM were found to be non-cytotoxic but anti-proliferative irrespective of presence of glucose. Apart from mitochondrial impairment, derangement of fatty acid desaturation, one-carbon, glutathione and polyamine metabolism were associated with non-cytotoxic metformin treatment irrespective of glucose supplementation. Depletion of pantothenic acid, downregulation of essential amino acid uptake, metabolism and purine salvage were identified as novel glucose-independent effects of metformin. These were significantly correlated with cMyc expression and reduction in proliferation. Rescue experiments established reversibility upon metformin withdrawal and tight association between proliferation, metabotype and cMyc expression. Taken together, derangement of novel glucose-independent metabolic pathways and concomitant cMyc downregulation co-ordinately contribute to anti-proliferative effect of metformin even at non-cytotoxic concentrations, which is reversible and may influence its therapeutic utility.