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Molecular and Cellular Biology, August 2006, p. 5588-5594, Vol. 26, No. 15
0270-7306/06/$08.00+0     doi:10.1128/MCB.00199-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Nitrosylcobalamin Promotes Cell Death via S Nitrosylation of Apo2L/TRAIL Receptor DR4

Center for Hematology and Oncology Molecular Therapeutics, Taussig Cancer Center,¹ Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195²

Received 3 February 2006/ Returned for modification 7 March 2006/ Accepted 9 May 2006

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.

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