title: Nitrosylcobalamin promotes cell death via S nitrosylation of Apo2L/TRAIL receptor DR4.
abstract: We have previously demonstrated that nitrosylcobalamin (NO-Cbl), an analogue of vitamin B12 that delivers nitric oxide (NO), had potent antiproliferative activity against several human cancer cell lines. NO-Cbl induced apoptosis via a death receptor/caspase-8 pathway. In this study, we demonstrate that a functional Apo2L/TRAIL receptor was necessary for the induction of cell death by NO-Cbl. Furthermore, the Apo2L/TRAIL death receptor DR4 (TRAIL R1) was S nitrosylated following NO-Cbl treatment. Human melanoma (A375), renal carcinoma (ACHN), and ovarian carcinoma (NIH-OVCAR-3) cells were treated with NO-Cbl and subjected to the biotin switch assay; S-nitrosylated DR4 was detected in all three cell lines. NO-Cbl treatment did not cause S nitrosylation of DR5. The seven cysteine residues located in the cytoplasmic domain of DR4 were individually point mutated to alanines. NIH-OVCAR-3 cells expressing the DR4 C336A mutation lacked S nitrosylation following NO-Cbl treatment. Overexpression of wild-type DR4 sensitized cells to growth inhibition by NO-Cbl. Cells expressing the DR4 C336A mutant were more resistant to NO-Cbl and Apo2L/TRAIL than were the other six C-A mutations or wild-type cells. The C336A mutant also displayed blunted caspase-8 enzymatic activity following NO-Cbl treatment compared to the other mutants. Thus, DR4 residue C336 becomes S nitrosylated and promotes apoptosis following NO-Cbl treatment.
authors.affiliation: Center for Hematology and Oncology Molecular Therapeutics, Taussig Cancer Center, Cleveland, Ohio 44195, USA.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Tang
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bauer
authors.forename: Bei
authors.initials: B
authors.lastname: Morrison
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lindner
mesh_terms: D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D020287:In Situ Nick-End Labeling; D009603:Nitroso Compounds / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D009603:Nitroso Compounds; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C113298:nitrosylcobalamin; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D014805:Vitamin B 12