Glutathionylation of p65NF-κB correlates with proliferating/ apoptotic hepatoma cells exposed to pro- and anti-oxidants

Oxidative stress influences a variety of regulatory proteins, including nuclear factor-κB (NF-κB). NF-κB is critical for maintaining the proliferation/apoptosis balance in hepatocytes. In this study we investigated the causal links between glutathione, NF-κB and hepatocyte damage. HepG2 and 3B cells were exposed to different doses of H₂O₂ or N-acetylcysteine (NAC) and the proliferation/apoptosis rate, glutathione forms, and p65NF-κB glutathionylation and activity were analysed. Our results demonstrate that H₂O₂ stopped proliferative response at low doses, but induced apoptosis only at high doses. In contrast, NAC exerted, proportionally to its concentration, a dual role simultaneously increasing both proliferation and apoptosis. Interestingly, the levels of protein-bound glutathione were increased by H₂O₂ and decreased by NAC. Moreover, the antibody recognizing the glutathionylated proteins co-precipitated and -localized with the cytoplasmic inactive form of p65NF-κB in H₂O₂- and NAC-treated cells, even when, in 1 mM NAC-treated cells, a part of p65 was glutathione-free and localized into the nucleus. Apoptotic cells were characterised principally by a cytoskeletal staining of glutathionylation and retention of NF-κB in the cytoplasmic region; whereas in proliferating cells, glutathionylated proteins were concentrated into the perinuclear region and p65NF-κB was traslocated into the nucleus. While cytoplasmic NF-κB retention correlated well with an increased apoptotic rate, a greater expression of this protein was observed in association with the NAC-dependent. In conclusion, our findings suggest that glutathionylation inhibits NF-κB activity causing reduced hepatocyte survival, which is common in several liver diseases.