Search

Loss of p53 function by inactivating mutations results in abrogation of NO·-induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO· in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO· was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO· also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO· treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO· exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.

Supplementary Table 1 from Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53

Supplementary Figure 1 from Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53

Melanoma patients experience inferior survival after bio-chemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS), and elevated levels of nitrotyrosine, a product derived from nitric oxide (NO). Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anti-cancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols decreased its cytotoxic potency. Activity of the pro-apoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/S-nitrisothiols, whereas lower activity resulted from cisplatin combined with stabilization of S-nitrisothiols. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2 (PHD2), the enzyme responsible for targeting the pro-survival transcription factor HIF-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy. Citation Format: Luiz C. Godoy, Chase C.T. Anderson, Rajdeep Chowdhury, Laura J. Trudel, Gerald N. Wogan. Melanoma fights cisplatin with NO: S-nitrosation as a mechanism supporting drug-resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2013-894

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.