Qanungo Suparna, Wang Mi, Nieminen Anna-Liisa
Department of Anatomy and Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2004 Nov 26;279(48):50455-64. doi: 10.1074/jbc.M406749200. Epub 2004 Sep 16.
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.
在本研究中，我们调查了还原型谷胱甘肽（GSH）和核因子-κB（NFκB）在缺氧诱导的细胞凋亡中的作用。缺氧在转染了Ras和E1A的小鼠胚胎成纤维细胞中引起p53依赖性细胞凋亡。N-乙酰-L-半胱氨酸（NAC）而非其他抗氧化剂，如维生素E类似物曲克芦丁和表没食子儿茶素-3-没食子酸酯，增强了缺氧诱导的半胱天冬酶-3激活和细胞凋亡。NAC还增强了缺氧在两种人类癌细胞系，即MIA PaCa-2胰腺癌细胞和A549肺癌细胞中诱导的细胞凋亡。在小鼠胚胎成纤维细胞中，所有三种抗氧化剂均阻断了缺氧诱导的活性氧生成。NAC并未增强缺氧诱导的细胞色素c释放，但增强了聚（ADP核糖）聚合酶的切割，表明NAC作用于线粒体后水平。用GSH单酯孵育细胞可模拟NAC介导的细胞凋亡增强作用，GSH单酯增加细胞内GSH的方式与NAC类似。缺氧促进了κB抑制蛋白（IκBα）的降解、NFκB-p65易位至细胞核、NFκB与DNA结合以及随后NFκB的反式激活，这增加了X染色体连锁凋亡抑制蛋白水平。NAC未能阻断IκBα的降解以及NFκB的p65亚基隔离至细胞核。然而，NAC确实消除了缺氧诱导的NFκB与DNA结合、NFκB依赖性基因表达以及X染色体连锁凋亡抑制蛋白的诱导。总之，NAC通过一种明显涉及GSH依赖性抑制NFκB反式激活的机制增强了缺氧诱导的细胞凋亡。
