Dataset Information

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages.

ABSTRACT: Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.

SUBMITTER: Kobayashi S

PROVIDER: S-EPMC7905073 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages.

Kobayashi Sho S Homma Takujiro T Fujii Junichi J

Biochemistry and biophysics reports 20210220

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-an ...[more]

PMID: 33665378

Similar Datasets

Project description:Initiation of nitric oxide (NO.)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese superoxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zinc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protein levels. To evaluate the decreased CuZnSOD expression and the initiation of apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expression of a functional and thus protective CuZnSOD was verified by attenuated superoxide (O2(.)-)-mediated apoptotic as well as necrotic cell death. In this study we showed that SOD-overexpressing macrophages (R-SOD1-12) were also protected against NO.-initiated programmed cell death. Protection was substantial towards NO. derived from exogenously added NO donors or when NO. was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimulation of parent and SOD-overexpressing cells with a combination of lipopolysaccharide and murine interferon gamma produced equivalent amounts of nitrite/nitrate, which ruled out attenuated inducible NO. synthase activity during protection. Because protection by a O2(.)--scavenging system during NO. -intoxication implies a role of NO. and O2(.)- in the progression of cell damage, we used uric acid to delineate the role of peroxynitrite during NO.-elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglutathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morpholinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO. -mediated apoptosis. Therefore protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO. cytotoxicity, whereas CuZnSOD overexpression protects macrophages from apoptosis.

Project description:Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1? for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Dataset Information

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages.

Publications

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets