Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of NFκB in pancreatic cancer cells Free

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Aggressive human malignancies characteristically outgrow their blood supply and become hypoxic. Strategies to sensitize tumor cells to hypoxic cell death could therefore be useful in cancer treatment. Previously, we reported that N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis in murine embryonic fibroblasts by blocking the NFκB survival pathway (Qanungo et al., J. Biol. Chem. 279, 50455-50464, 2004). The goal of the present study was to investigate the mechanisms responsible for inactivation of NFκB and subsequent apoptotic death by GSH supplementation in hypoxic MIA PaCa-2 human pancreatic cancer cells. Hypoxia alone for 22 h in 0.6%-2.1% O₂ induced very little apoptosis in MIA PaCa-2 cells (1.9% during normoxia vs. 3.1%-4.7% in hypoxia). In contrast, NAC (10 mM) significantly enhanced hypoxic apoptosis in O₂-dependent manner (2.4% during normoxia vs. 10.3%-22.6% in hypoxia). Hypoxia increased p65-NFκB DNA binding assessed by ELISA and NFκB transactivation assessed by luciferase assay by 2.6- and 2.8-fold, respectively, compared to normoxia. NAC blocked these events without having an effect on p65-NFκB total protein levels or p65-NFκB nuclear translocation during hypoxia. Pharmacological inhibition of the NFκB pathway with JSH-23 (10 μM) also induced hypoxic apoptosis, indicating that the NFκB signaling pathway is a major protective mechanism against hypoxic apoptosis in pancreatic cancer cells. Since transcription factors have critical cysteine residues that are crucial for their DNA binding activity, we determined p65-NFκB sulfhydryl modification. In cell lysates after hypoxia and subsequent N-ethylmaleimide treatment to block free thiols, dithiothreitol (DTT, a reducing agent) was not able to increase binding of p65-NFκB to DNA, suggesting that most sulfhydryls in the p65-NFκB protein were in the reduced and activated form after hypoxia. With hypoxic cells that were also treated with NAC, DTT increased p65-NFκB DNA binding indicating that disulfides were inhibiting NFκB. To further determine the nature of NFκB inactivation, cells were treated with NEM followed by glutaredoxin, which selectively reduces protein-glutathione-mixed disulfides and causes protein-SSG deglutathionylation. Glutaredoxin reversed inhibition of p65-NFκB DNA binding in cell extracts treated with hypoxia plus NAC and restored NFκB activity. In conclusion, the results indicate that S-glutathionylation of p65-NFκB is most likely responsible for NAC-mediated NFκB inactivation and enhanced apoptosis in hypoxic cancer cells. Thus, manipulations to increase GSH in solid tumors could potentially be used therapeutically to sensitize tumors to hypoxic apoptosis.