Analysis of glucose-independent metabolic pathways associated with anti-proliferative effect of metformin and their 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, glucose- and central carbon-independent effects of metformin and their reversibility remain unexplored. 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 ≤2.5mM were found to be non-cytotoxic but anti-proliferative irrespective of presence of glucose. Mitochondrial impairment along with derangement of one-carbon, glutathione and polyamine metabolism was associated with non-cytotoxic metformin treatment irrespective of glucose supplementation. Depletion of pantothenic acid, upregulation of fatty acid desaturation and 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 pathways and concomitant cMyc downregulation coordinately contribute to anti-proliferative effect, which is reversible and may influence therapeutic utility of metformin.