S-Nitrosation regulates the activation of endogenous procaspase-9 in HT-29 human colon carcinoma cells

Nitric oxide-mediated signals have been suggested to regulate the activity of caspases negatively, yet literature has provided little direct evidence. We show in this paper that cytokines and nitric-oxide synthase (NOS) inhibitors regulate S-nitrosation of an initiator caspase, procaspase-9, in a human colon adenocarcinoma cell line, HT-29. A NOS inhibitor, N(G)-methyl-l-arginine, enhanced the tumor necrosis factor-alpha (TNF-alpha)-induced cleavage of procaspase-9, procaspase-3, and poly-(ADP-ribose) polymerase, as well as the level of apoptosis. N(G)-Methyl-l-arginine, however, did not affect the cleavage of procaspase-8. These results suggest that nitric oxide regulates the cleavage of procaspase-9 and its downstream proteins and, subsequently, apoptosis in HT-29 cells. Labeling S-nitrosated cysteines with a biotin tag enabled us to reveal S-nitrosation of endogenous procaspase-9 that was immunoprecipitated from the HT-29 cell extracts. Furthermore, the treatment with TNF-alpha, as well as NOS inhibitors, decreased interferon-gamma-induced S-nitrosation in procaspase-9. Our results show that S-nitrosation of endogenous procaspase-9 occurs in the HT-29 cells under normal conditions and that denitrosation of procaspase-9 enhances its cleavage and consequent apoptosis. We, therefore, suggest that S-nitrosation regulates activation of endogenous procaspase-9 in HT-29 cells.