The Role of nitric oxide in apoptotic susceptibility of human lung cancer and normal lung cells

Abstract

Interfering apoptotic susceptibility is important in progression of many diseases, in particular cancers. Cancer cell-acquired resistance to cisplatin-induced apoptosis is a major limitation for efficient therapy, as frequently observed in human lung cancer. Moreover, escaping from Fas ligand (FasL)-induced apoptosis is a potential mechanism of cancer progression. Nitric oxide (NO) is a key regulator of apoptosis, but its role in cisplatin- and FasL-induced cell death and the underlying mechanism are largely unknown. Evidences indicated that increased NO production in lung injury, lung inflammation, and lung carcinomas, correlated with the incidence of chemotherapeutic resistance. The present study showed that NO impaired the apoptotic function of cells and increased their resistance to cisplatin- and FasL-induced apoptosis in both human lung carcinoma H460 cells and human lung epithelial BEAS cells. The NO donors sodium nitroprusside (SNP) and dipropylenetriamine (DPTA) NONOate were able to inhibit cisplatin- and FasL-induced cell death, whereas the NO inhibitors Aminoguanidine (AG) and 2-(4-carboxyphenyl)-4,4,5,5-tetra-methylimidazoline-1-oxy1-3oxide (PTIO) had opposite effect. Cisplatin resistance in H460 cells was mediated by Bcl-2, and NO upregulated its expression by preventing the degradation of Bcl-2 via ubiquitin-proteasome pathway. Cisplatin-induced generation of reactive oxygen species (ROS) caused dephosphorylation and degradation of Bcl-2. In contrast, elevation of NO had no effect on Bcl-2 phosphorylation but induced S-nitrosylation of the protein, which inhibited its ubiquitination and subsequent proteasomal degradation, Also, this study revealed the mechanism of NO in regulating FasL-induced apoptosis in BEAS cells. FasL-induced downregulation of FLICE inhibitory protein (FLIP) was mediated by a ubiquitin-proteasome pathway that was negatively regulated by NO. S-nitrosylation of FLIP was an important mechanism rendering FLIP resistant toubiquitination and proteasomal degradation. Deletion analysis showed that the caspase-like domain of FLIP was a key target for S-nitrosylation by NO, and mutation of its cysteine 254 and cysteine 259 residues completely inhibited S-nitrosylation, leading to increase ubiquitination and proteasomal degradation of FLIP. These findings indicated a novel pathway for NO regulation of Bcl-2 which provided a key mechanism for cisplatin resistance and its potential modulation for improved cancer chemotherapy. Also, this study revealed a novel pathway for NO regulation of FLIP that provided a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.