Cellular inactivation of nitric oxide induces p53-dependent apoptosis in human melanoma cells

Purpose: To examine the role of endogenous nitric oxide (NO^•) and influence of p53 status during apoptosis induced by a selective iNOS inhibitor, N-[(3-aminomethyl) benzyl] acetamidine (1400W), and/or an NO^• scavenger carboxy-PTIO (c-PTIO) in two isogenic human melanoma cell lines, wild-type p53 (A375) and p53 mutant (SK mel-28) cells.
Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Annexin V/propidium iodide assay were used to test for antiproliferation and apoptosis, respectively. Griess and reverse transcription-polymerase chain reaction (RT-PCR) reactions were carried out to assay NO^• production and the mRNA levels of inhibitors of apoptosis (IAP).
Results: c-PTIO and 1400W, alone or in combination, inhibited cell growth and promoted apoptosis via sub-G1 cell cycle arrest mediated by decrease in NO^•. Apoptosis was delayed and greatly reduced in magnitude in SK mel-28 cells, underscoring the importance of p53 modulation of the response. In both cell types, apoptosis induced by iNOS inhibition and/or NO^• depletion was blocked by an exogenous NO^• donor, sodium nitroprusside. It was also found that inhibitors of apoptosis family (survivin, XIAP and cIAP1) were significantly depressed, which appear to play an important role in the regulation of p53- mediated apoptotic response under these conditions.
Conclusion: The data obtained provide insight into the mechanism of cell proliferation action of endogenous NO^•, based on p53 status, and indicate manipulation of iNOS may offer exciting opportunities to improve the effectiveness of melanoma treatment.