EMT-6 cells treated for 16 h with 1–10 units/ml IFN-γ showed a gradual activation of inducible nitric oxide synthase (iNOS) in Western and Northern blots, a simultaneous raise in NO output, and an increase in hypoxic cell radiosensitivity almost to the level of aerobic cells. Both the NO signal and radiosensitization were counteracted by the NO scavenger oxyhemoglobin, by the specific iNOS inhibitor aminoguanidine, and by the l-arginine analogue NG-monomethyl-l-arginine. Collectively, these data demonstrate that IFN-γ can radiosensitize EMT-6 cells through iNOS induction and that NO is the effector molecular responsible for radiosensitization. Compared with the spontaneous NO releaser (2)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino)diazen-1-ium-1,2-diolate], the iNOS-generated NO signal appeared to be 10 times lower yet resulting in the same enhancement ratio of 2.4. Direct stimulation of NO synthesis in tumor cells through the l-arginine/iNOS pathway represents a novel approach to exploit the radiosensitizing properties of NO.
This research was funded by Grants G.0055.96 and G.0195.98 from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen and Sportvereniging tegen Kanker and Vlaamse Kankerliga.