Abstract 576: Hydrogen peroxide and iron released from nitroprusside cooperate to induce melanoma cell death: role of the p53 genotype

Abstract

Abstract BACKGROUND. We recently reported that sodium nitroprusside or nitroferricyanide (III) (SNP), an iron nitrosyl complex clinically used as a powerful vasodilator, promotes melanoma cell apoptosis independently of the status of the p53 tumor suppressor gene. This occurred with intact SNP but was delayed when using its light-exhausted form (lex-SNP) devoid of nitric oxide (NO) (1). OBJECTIVES. Since extracellular NO and hydrogen peroxide (H2O2) are increased after SNP treatment (2), to define antitumor effects of H2O2 plus NO or iron, we used sub-toxic exogenous H2O2 singly or together with either: a) SNP; b) lex-SNP; c) potassium ferricyanide or d) ferric ammonium sulphate, against genetically-matched human C8161 melanoma harboring wild type (wt) or mutant p53, since the latter contributes to drug resistance. METHODS. Cell survival was assayed by crystal violet staining or by determining percentage of calcein AM retention and lack of propidium iodide uptake. DNA damage was assessed by laser scanning cytometry and PARP fragmentation was measured by immune blotting. RESULTS. While each treatment alone showed low efficacy, a synergistic cytotoxicity was demonstrated only between either intact or lex-SNP + H2O2. Shorter treatments of these combinations were preferentially cytotoxic against wt p53 melanoma compared to their mutant p53 matched counterparts, in crystal violet survival assays. However, longer treatments with [SNP + H2O2] induced apoptosis-associated PARP cleavage irrespective of cellular p53 genotype. All these changes were antagonized by anti-oxidant N-acetyl cysteine (NAC) or metal chelator o-phenanthroline, suggesting that metal-dependent redox changes are important in this anti-tumor activity. CONCLUSIONS. When using alternative iron sources like those provided by potassium ferricyanide, ferric ammonium sulphate or sodium nitroferricyanide (III) (SNP), we show for the first time that in vitro anti-tumor combination therapy with sub-toxic H2O2 is achieved only with iron made available by SNP, since H2O2 did not synergize with iron provided by potassium ferricyanide or ferric ammonium sulphate. Lex-SNP devoid of NO (1) also potentiated the effect of H2O2, implying that the cooperative effect of [SNP + H2O2] is independent of its NO-donating ability. REFERENCES (1) Gomez-Sarosi LA, Strasberg-Rieber M, Rieber M. ERK activation increases nitroprusside induced apoptosis in human melanoma cells irrespective of p53 status: role of superoxide dismutases. Cancer Biol Ther 2009; 8:1173-82. (2) Rabkin SW and Kong JY. Nitroprusside induces cardiomyocyte death: interaction with hydrogen peroxide. Am J Physiol Heart Circ Physiol 2000; 279:H3089-H3100. 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 576.