The role of oxidative stress 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 cells of the colonic mucosa, and that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Secondary bile acids are known promoters of colon cancer, but the mechanism by which they promote cancer is still largely unknown. We have shown that high physiologic concentrations (0.5 mM) of NaDOC activates the redox-sensitive transcription factor, NF-κB, and also causes the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of cells. These observations indicate that this bile salt induces oxidative stress within the cells. Since peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, L-NAME (N-nitro-L-arginine methyl ester), an inhibitor of nitric oxide synthase, CuDIPSH, a superoxide dismutase mimetic compound, Trolox, a water-soluble analog of α-tocopherol, Melatonin, a fat and water soluble antioxidant, N-acetyl-cysteine, a GSH enhancer, U-74389G, a lazeroid that inhibits superoxide anion and free radical lipid peroxidation and U83836E, a lazeroid that is 100X more potent than trolox, alone, or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. We also investigated the effects of inhibitors of certain pathways known to generate ROS, mitochondrial complexes I and II of the electron transport chain and arachidonic acid metabolism, on bile salt-induced apoptosis. Both rotenone and TTFA, inhibitors of mitochondrial complex I and complex II respectively, protected HT-29 cells from NaDOC-induced apoptosis. The inhibitor of COX-1, Sulindac Sulfide, sensitized cells to NaDOC-induced apoptosis and so did the combination of the COX-2 and LOX inhibitors, NS-398 and Esculetin. These results suggest that nitric oxide and reactive oxygen species (ROS) may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that antioxidants may possess anti- cancer properties