The present study aimed to assess the effect of NO• on melanoma A375 cell growth and apoptotic cell death. Trypan blue exclusion assay was employed to detect the cytotoxicity induced by controlled steady-state concentrations (given in µM • min) of NO•. The characteristics of the cellular cell cycle and apoptosis in NO•-treated A375 cells were also analyzed by Annexin V/PI and DNA fragmentation assays. Western blotting was applied to detect the expression of apoptosis-related proteins (p53, Bax, Fas, DR5, caspase-3 and -9, and PARP). When exposed to preformed 100% NO• for 8 h reactor system, a cumulative dose of 3360 μM • min reduced the viability by 22% 24 h after treatment and promoted apoptosis, 2.9- and 12.2-folds 24 and 48 h after treatment higher than the argon control, respectively. Cell cycle analysis 48 h after treatment revealed S-phase arrest in cells treated with 3360 μM • min NO•. It was also observed that the expression of p53, DR5, caspase 9 and PARP increased significantly upon NO• treatment. In addition, the present study assessed the inhibitory effects of endogenous NO• on the proliferation of human melanoma cells by employing specific (AMG, 1400W and/or SMTC) and nonspecific (NMA) NO• synthase (NOS) inhibitors resulting in melanoma cell growth inhibition; the highest cytotoxic effect was seen when inducible NOS inhibition by 1 mM 1400W treatment. Collectively, the present data suggest that NO• is involved in a key mechanism limiting melanoma proliferation and apoptosis, which may play in improving the efficacy of melanoma treatment.
Apoptosis, Cell proliferation, Human melanoma cells, Nitric oxide
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