Abstract 204: Disruption of S-nitrosothiols decreases chemoresistance in melanoma cells

Exposure to nitric oxide (NO • ) during inflammation has been implicated in the development of various forms of cancer, including melanoma. Clinical and epidemiologic studies indicate that expression of inducible nitric oxide synthase (NOS2), as well as NO • production by tumor cells, correlate with patient mortality. NO • favors resistance to drug-induced apoptosis in melanoma cells and may do so by caspases inactivation through S-nitrosation, which could represent a mechanism responsible for NO • inhibition of apoptosis. Here we further investigate how S-nitrosation impacts apoptotic and survival pathways in human melanoma. We demonstrate that A375, SK-Mel 100 and SK-Mel 28 cells constitutively express NOS2 and that an inhibitor of NO • synthesis decreases cell proliferation. Moreover, this same treatment, or the pre-incubation with the NO • scavenger carboxy-PTIO, leads to enhanced toxicity of cisplatin, suggesting that endogenous NO • regulates growth and enhances anti-apoptotic mechanisms in melanoma cells. This anti-apoptotic effect may be due to protein S-nitrosation promoted by endogenous NO • , because disruption of nitrosothiols by dithiothreitol promotes a remarkable increase in cisplatin-induced cell death in comparison with cells treated with cisplatin alone (80% vs. 20% cell death, respectively). On the other hand, when accumulation of cellular nitrosothiols is favored via inhibition of thioredoxin reductase by 1-chloro-2,4-dinitrobenzene, increased resistance to cisplatin is seen in A375 cells. Using the biotin switch technique (BST), a low, yet detectable level of endogenous protein S-nitrosation is seen in A375 cells. The properties conferred by exogenous NO • to melanoma cells were studied in a controlled-delivery system to simulate NO • concentrations known to occur in inflamed tissues. Using this system, the toxicity of NO • was shown to be lower in A375, SK-Mel 100 and SK-Mel 28 cells when compared to other non-melanoma cell lines. The possible protective effect of a chronic exposure to a non-toxic dose of exogenous NO • was investigated. A375 cells were treated with 0.64 µM NO • or argon (control) for 24 hours and then cisplatin was added to the system. Cell viability was assessed 24 hours later with trypan blue staining. While nearly 50% cell death was seen in argon+cisplatin-treated cells, no significant cell death occurred under NO • +cisplatin treatment, which points to an anti-apoptotic effect of low, sustained doses of NO • in this system. Finally, using the BST, the level of global protein S-nitrosation in the cells exposed to 0.64 µM NO • was shown to be considerably increased. Collectively, these results show that NO • favors survival and proliferation of melanoma cells in vitro, possibly by S-nitrosation of components of the signaling cascades. Identification of the proteins susceptible to this NO • -mediated modification will help to devise rational strategies for disease intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 204.