In murine embryonic fibroblasts,
N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2
pancreatic cancer cells,
hypoxia increased p65-NFkappaB
DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB
protein levels and p65-NFkappaB nuclear translocation during
hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after
hypoxia and treatment with
N-ethylmaleimide (
thiol alkylating agent),
dithiothreitol (
disulfide reducing agent) was not able to increase binding of p65-NFkappaB to
DNA, suggesting that most sulfhydryls in p65-NFkappaB
protein were in reduced and activated forms after
hypoxia, thereby being blocked by
N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC,
dithiothreitol increased p65-NFkappaB
DNA binding.
Glutaredoxin (GRx), which specifically catalyzes reduction of
protein-SSG mixed
disulfides, reversed inhibition of p65-NFkappaB
DNA binding in extracts from cells treated with
hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under
hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes
protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.