Ineffective GSH regeneration enhances G6PD-knockdown Hep G2 cell sensitivity to diamide-induced oxidative damage

Glucose-6-phosphate dehydrogenase (G6PD) has been recently found to play growth-regulatory roles in nucleated cells. To identify any other physiologic roles of G6PD, we generated G6PD-knockdown Hep G2 cells and investigated their susceptibility to oxidants. Hep G2 cells expressing shRNA against G6PD (Gi) were more susceptible to diamide-induced cytotoxicity than control cells expressing scrambled control shRNA (Sc). The level of reactive oxygen species in the Gi cells substantially exceeded that in Sc cells. This was accompanied by increased membrane peroxidation and the appearance of high-molecular-weight aggregates of membrane-associated cytoskeletal proteins in Gi cells. G6PD knockdown was associated with an impaired ability to regenerate glutathione. Diamide caused a considerable decrease in cellular glutathione level and a concomitant increase in glutathione disulfide in Gi cells. Consistent with this finding, N-acetylcysteine mitigated diamide-induced oxidative stress and cell death. Our findings suggest that G6PD confers protection against oxidant-induced cytotoxicity through effective glutathione regeneration.