N-Acetyl-L-cysteine enhances apoptosis through inhibition of nuclear factor-kappaB in hypoxic murine embryonic fibroblasts

In this study, we investigated the role of reduced glutathione (GSH) and nuclear factor-kappaB (NFkappaB) 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 kappaB(IkappaBalpha), NFkappaB-p65 translocation into the nucleus, NFkappaB binding to DNA, and subsequent transactivation of NFkappaB, which increased X chromosome-linked inhibitor of Apoptosis protein levels. NAC failed to block degradation by IkappaBalpha and sequestration of the p65 subunit of NFkappaB to the nucleus. However, NAC did abrogate hypoxia-induced NFkappaB binding to DNA, NFkappaB-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 NFkappaB transactivation.