NO-mediated chemoresistance in C6 glioma cells.

Expression of inducible nitric oxide synthase (iNOS) in malignant glioma and other tumors has been extensively documented. Massive production of NO by iNOS has been shown to exert tumoricidal effects. However, NO may enhance vasodilation and promote neovascularization, thereby facilitating tumor growth. Compared to the effects of NO on tumor cell death and survival, correlation between NO and cytotoxicity of chemotherapeutic reagents in glioma have been less well characterized. Another gene product often linked to tumor malignancy is hypoxia-inducible factor-1 (HIF-1). HIF-1 is a transcription factor that renders malignant tumors adaptive to hypoxic stress during massive vascularization and tumor invasion. Interestingly, HIF-1 also contributes to iNOS induction under hypoxia. We have characterized the interrelationship between iNOS, HIF-1 and chemoresistance. We note that increased NO synthesis by cytokine exposure or iNOS overexpression neutralized the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), but not cisplatin, in rat C6 glioma cells. Both BCNU and CCNU are chloroethylnitrosoureas that kill tumor cells via carbamoylating and alkylating actions. Further studies indicated that iNOS only neutralized carbamoylating action of chloroethylnitrosoureas. Expression of iNOS may inhibit HIF-1 activity under hypoxia in C6 glioma cells transfected with a VEGF promoter-driven luciferase gene. Pretreatment of C6 cells with N-acetyl-l-cysteine (NAC), an antioxidant, nullified the inhibitory effect of iNOS on HIF-1 binding. That NO generated by iNOS expression inhibits HIF-1 activity in hypoxic C6 cells reveals a negative feedback loop in the HIF-1 --> iNOS cascade. Together these results suggest a complicated role of NO in malignant tumor growth, survival and invasion.