Endogenously produced NO reduces sensitivity to cis-platin and stabilization of HIF-1a in human melanoma and colon carcinoma cells: role of S-nitrosation  Melanoma patients experience inferior survival after bio-chemotherapy when their tumors contain numerous cells that express iNOS, and contain elevated levels of nitrotyrosine derived from NO.  Evidence suggests that NO promotes tumor growth and increases resistance to chemotherapy, but responsible mechanisms are unclear. We have shown that NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to cisplatin in human melanoma cells in vitro. Cells express iNOS constitutively, generating sustained nanomolar levels of intracellular NO, which is further increased in cells surviving cisplatin treatment.  Reduction of NO levels by scavenging or inhibition of synthesis enhances cisplatin-induced apoptosis, as do agents disrupting S-nitrosation; in contrast, stabilization of S-nitrosothiols (SNOs) decreases its cytotoxic potency.  Evidence indicates that these effects are attributable to inactivation of caspase-3 by S-nitrosation.  Hypoxia-inducible factor-1 alpha (HIF-1a) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, HIF-1a level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation and proteasomal degradation. During hypoxia, degradation decreases and its intracellular level is increased. We have shown that NO and ROS produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1a accumulation in hypoxia. The antioxidant NAC and NO synthase inhibitor L-NMMA reduces HIF-1a stabilization and decreases HIF-1a hydroxylation. Thiol reduction with dithiothreitol decreases HIF-1a stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favors its accumulation, suggesting that ROS- and NO-mediated HIF-1a stabilization involves S-nitrosation.  This is confirmed by the finding of increased PHD2 S-nitrosation during hypoxia, supporting a regulatory mechanism of HIF-1a during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity by its S-nitrosation.

Endogenously produced NO reduces sensitivity to cis-platin and stabilization of HIF-1a in human melanoma and colon carcinoma cells: Role of S-nitrosation

246th National Meeting (2013)

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