title: Protein nitration and nitrosylation by NO-donating aspirin in colon cancer cells: Relevance to its mechanism of action.
abstract: Nitric oxide-donating aspirin (NO-ASA) is a promising agent for cancer prevention. Although studied extensively, its molecular targets and mechanism of action are still unclear. S-nitrosylation of signaling proteins is emerging as an important regulatory mechanism by NO. Here, we examined whether S-nitrosylation of the NF-κB, p53, and Wnt signaling proteins by NO-ASA might explain, in part, its mechanism of action in colon cancer. NO-ASA releases significant amounts of NO detected intracellularly in HCT116 and HT-29 colon cells. Using a modified biotin switch assay we demonstrated that NO-ASA S-nitrosylates the signaling proteins p53, β-catenin, and NF-κB, in colon cancer cells in a time- and concentration-dependent manner. NO-ASA suppresses NF-κB binding to its cognate DNA oligonucleotide, which occurs without changes in the nuclear levels of the NF-κB subunits p65 and p50 and is reversed by dithiothreitol that reduces -S-NO to -SH. In addition to S-nitrosylation, we documented both in vitro and in vivo widespread nitration of tyrosine residues of cellular proteins in response to NO-ASA. Our results suggest that the increased intracellular NO levels following treatment with NO-ASA modulate cell signaling by chemically modifying key protein members of signaling cascades. We speculate that S-nitrosylation and tyrosine nitration are responsible, at least in part, for the inhibitory growth effect of NO-ASA on cancer cell growth and that this may represent a general mechanism of action of NO-releasing agents.
authors.affiliation: Division of Cancer Prevention, Stony Brook University, HSC, T17-080, Stony Brook, NY 11794-8173, USA.
authors.forename: Jennie L
authors.initials: JL
authors.lastname: Williams
authors.forename: Ping
authors.initials: P
authors.lastname: Ji
authors.forename: Nengtai
authors.initials: N
authors.lastname: Ouyang
authors.forename: Levy
authors.initials: L
authors.lastname: Kopelovich
authors.forename: Basil
authors.initials: B
authors.lastname: Rigas
mesh_terms: D000818:Animals; D001241:Aspirin / Q000031:analogs & derivatives* / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D051176:beta Catenin / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D020030:Nitric Oxide Donors; D051176:beta Catenin; C098312:nitroxy-butyl-acetylsalicylic acid; D009569:Nitric Oxide; D014443:Tyrosine; D001241:Aspirin