N-Acetyl-l-cysteine Enhances Apoptosis through Inhibition of Nuclear Factor-κB in Hypoxic Murine Embryonic Fibroblasts

In this study, we investigated the role of reduced glutathione (GSH) and nuclear factor-κB (NFκB) in hypoxia-induced apoptosis. Hypoxia caused p53-dependent apoptosis in murine embryonic fibroblasts transfected with Ras and E1A. N-Acetyl-l-cysteine (NAC) but not other antioxidants, such as the vitamin E analog trolox and epigallocatechin-3-gallate, enhanced hypoxia-induced caspase-3 activation and apoptosis. NAC also enhanced hypoxia-induced apoptosis in two human cancer cell lines, MIA PaCa-2 pancreatic cancer cells and A549 lung carcinoma cells. In murine embryonic fibroblasts, all three antioxidants blocked hypoxia-induced reactive oxygen species formation. NAC did not enhance hypoxia-induced cytochrome c release but did enhance poly-(ADP ribose) polymerase cleavage, indicating that NAC acted at a post-mitochondrial level. NAC-mediated enhancement of apoptosis was mimicked by incubating cells with GSH monoester, which increased intracellular GSH similarly to NAC. Hypoxia promoted degradation of an inhibitor of κB(IκBα), NFκB-p65 translocation into the nucleus, NFκB binding to DNA, and subsequent transactivation of NFκB, which increased X chromosome-linked inhibitor of apoptosis protein levels. NAC failed to block degradation by IκBα and sequestration of the p65 subunit of NFκB to the nucleus. However, NAC did abrogate hypoxia-induced NFκB binding to DNA, NFκB-dependent gene expression, and induction of X chromosome-linked inhibitor of apoptosis protein. In conclusion, NAC enhanced hypoxic apoptosis by a mechanism apparently involving GSH-dependent suppression of NFκB transactivation.