ABSTRACT
Hypoxic EMT-6 tumour cells displayed a high level of inducible nitric oxide synthase (iNOS) and an increased radiosensitivity after a 16 h exposure to lipopolysaccharide, a known activator of nuclear factor-kappaB (NF-kappaB). Both iNOS activation and radioresponse were impaired by the NF-kappaB inhibitors phenylarsine oxide and lactacystin. Contrasting to other studies, our data show that inhibition of NF-kappaB may impair the radioresponse of tumour cells through downregulation of iNOS.
Subject(s)
Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , NF-kappa B/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Radiation Tolerance/drug effects , Animals , Anti-Bacterial Agents/pharmacology , Arsenicals/pharmacology , Cell Hypoxia , Cysteine Endopeptidases/metabolism , Down-Regulation , Lactams , Mice , Multienzyme Complexes/metabolism , NF-kappa B/physiology , Nitric Oxide Synthase Type II , Oxygen/metabolism , Proteasome Endopeptidase Complex , Radiation Tolerance/physiology , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Mammary Neoplasms, Experimental/metabolism , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/radiation effects , Nitric Oxide/metabolism , Radiation Tolerance/drug effects , Animals , Cell Hypoxia , Enzyme Activation , Hydrazines/pharmacology , Interferon-gamma/pharmacology , Mice , Nitric Oxide/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase Type II , Penicillamine/analogs & derivatives , Penicillamine/pharmacology , Tumor Cells, CulturedABSTRACT
A high-performance liquid chromatographic method is described for the determination of vinblastine in various normal mouse tissues, such as lung, heart, liver, kidney and muscles, and in implanted MO4 tumours. Vincristine was used as the internal standard. Freshly obtained mouse tissue or tumour tissue was frozen at -20 degrees C and then lyophilized. After lyophilisation, the dry tissues were pulverized and homogeneously mixed, and an aliquot was suspended in 0.1 M hydrochloric acid. The drugs of interest were then isolated from this suspension using ion-pair extraction at pH 3 with octylsulphate as counter-ion. The obtained extracts were analysed on a reversed-phase system with a cyanopropyl stationary phase. The detection limit was 1 ng/l in plasma and 10 ng/g in tissue. The extraction recoveries of vincristine and vinblastine were between 45 and 67%, and there were no interferences from blank components. Preliminary pharmacokinetic data for different mouse tissues and tumour implanted in muscle tissue are presented.
Subject(s)
Vinblastine/pharmacokinetics , Animals , Chromatography, High Pressure Liquid , Fibrosarcoma/chemistry , Kidney/chemistry , Lung/chemistry , Male , Mice , Muscles/chemistry , Myocardium/chemistry , Neoplasm Transplantation , Spectrophotometry, Ultraviolet , Tissue Distribution , Tumor Cells, CulturedABSTRACT
A high-performance liquid chromatographic method is described for separating and determining navelbine and possible metabolites in plasma, cell culture medium and MO4 cells. Navelbine is extracted from these fluids by ion-pair extraction with sodium octylsulphate as the counter-ion at pH 3. The system uses a cyano column as the stationary phase and a mobile phase of acetonitrile-0.12 M phosphate buffer (pH 3) (60:40, v/v). Application of the method to a study of the pharmacokinetic behaviour of navelbine in MO4 mouse fibrosarcoma cells is reported.
