ABSTRACT
Chronic hypoxia up-regulated the mRNA and protein expression of inducible nitric oxide synthase (iNOS) in EMT-6 tumour cells exposed to interferon (IFN)-gamma and interleukin (IL)-I beta. Low concentrations of cytokines (1 unit ml(-1)) in 1% but not in 21% oxygen induced a remarkable increase in NO production and a 1.8-fold hypoxic cell radiosensitization. Therefore, chronic hypoxia may potentially be exploited to increase tumour cell radioresponse through the cytokine-inducible iNOS pathway.
Subject(s)
Cytokines/pharmacology , Nitric Oxide Synthase/drug effects , Oxygen/pharmacology , Animals , Blotting, Northern , Blotting, Western , Cell Survival/drug effects , Cell Survival/radiation effects , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Gene Expression Regulation, Enzymologic/drug effects , Interferon-gamma/pharmacology , Interleukin-1/pharmacology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitrites/metabolism , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/radiation effectsABSTRACT
The radiosensitizing activity of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was assessed in a model of non-metabolic hypoxia achieved in an atmosphere of 95% nitrogen-5% carbon dioxide. A 10 min preincubation of hypoxic EMT-6 cells (10 x 10(6) ml(-1)) with 0.1 and 1 mM SNAP before radiation resulted in an enhancement ratio of 1.6 and 1.7 respectively. The level of spontaneous NO release, measured by a NO specific microsensor, correlated directly with the concentration of SNAP and was enhanced 50 times in the presence of cells. Dilution of the cell suspension from 10 to 0.1 x 10(6) ml(-1) resulted in a 16-fold decline in NO release, but only a twofold decrease in radiosensitization was observed. Preincubation of hypoxic cells with SNAP for 3 min up to 30 min caused an increasing radiosensitizing effect. Extended preincubation of 100 min led to the loss of radiosensitization although the half-life of SNAP is known to be 4-5 h. Taken together, these observations suggest that SNAP generates NO predominantly by a bioreductive mechanism and that its biological half-life is unlikely to exceed 30 min. The lack of correlation between free NO radical and radiosensitizing activity may reflect a role of intracellular NO adducts which could contribute to radiosensitization as well.
Subject(s)
Cell Hypoxia/radiation effects , Nitric Oxide/biosynthesis , Penicillamine/analogs & derivatives , Radiation-Sensitizing Agents/pharmacology , Animals , Cell Count , Cell Hypoxia/drug effects , Cell Survival , Dose-Response Relationship, Radiation , Half-Life , Mice , Penicillamine/pharmacology , Time Factors , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/radiation effectsABSTRACT
EMT-6 cells treated for 16 h with 1-10 units/ml IFN-gamma 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 N(G)-monomethyl-L-arginine. Collectively, these data demonstrate that IFN-gamma can radiosensitize EMT-6 cells through iNOS induction and that NO is the effector molecule 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.
