Role of nitric oxide and peroxynitrite in bile salt-induced apoptosis: Relevance to colon carcinogenesis

Previous work from our laboratory indicated that the bile salt sodium deoxy cholate (NaDOC) induced apoptosis in cultured cells and in normal goblet ce lls of the colonic mucosa. We also reported that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Using immunofluorescence in conjunction with confocal microscopy, we now report t hat high physiological concentrations (0.5 mM) of NaDOC result in the forma tion of nitrotyrosine residues, a footprint for the formation of reactive n itrogen species, including peroxynitrite, in plasma membrane-associated pro teins of HT-29 cells. Because peroxynitrite is formed from the reaction bet ween nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatme nt of cells with the inhibitor/antioxidants, N-nitro-L-arginine methyl eate r; an inhibitor of nitric oxide synthase, copper (II) 3,5-diisopropyl salic ylate hydrate, a superoxide dismutase mimetic compound and Trolox, a water- soluble analog of alpha-tocopherol, alone or in combination, sensitized cel ls to apoptosis induced by 0.5 mM NaDOC. These results suggest that nitric oxide may be parr of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that nitric oxide does not appear to protect cells against NaDOC-induced apoptosis by scavenging superoxide anio n.