title: NG as a novel nitric oxide donor induces apoptosis by increasing reactive oxygen species and inhibiting mitochondrial function in MGC803 cells.
abstract: NG, O(2)-(2,4-dinitro-5-{[2-(12-en-28-β-D-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl] amino} phenyl) 1-(N-hydroxyethylmethylamino) diazen-1-ium-1,2-diolate, was identified in our laboratory as a novel nitric oxide-releasing prodrug with antitumor effects. A previous study showed that NG inhibited cell growth, and induced apoptosis in HepG2 cells. In this study, the inhibitory effects of NG on the viability of MGC803 cells were examined using methylthiazolyl tetrazolium biomide (MTT) assay, neutral red assay and trypan blue exclusion test. The results showed that NG had strong cytotoxicity to induce apoptosis, which was characterized by a significant externalization of phosphatidylserine, nuclear morphological changes and enhanced Bax-to-Bcl-2 ratio. Moreover, the release of cytochrome c (Cyt c) from mitochondria and the activation of caspase-9/3 were also detected, indicating that NG may induce apoptosis through a mitochondrial-mediated pathway. NG induced mitochondrial dysfunction in MGC803 cells by altering membrane potential (△Ψm), the inhibition of complexes I, II and IV consequently decreasing ATP level. Furthermore, the treatment of MGC803 cells with NG caused a marked rise in oxidative stress as characterized by accumulation of reactive oxygen species (ROS), excessive malondialdehyde (MDA) production and a reduction in glutathione hormone (GSH) level and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. In addition, pretreatment with N-acetylcysteine (NAC), a GSH synthesis precursor, was partially protective against the NG-induced ROS generation and cell apoptosis. In contrast, pretreatment of MGC803 cells with L-buthionine-S, R-sulfoximine (BSO), a GSH synthesis inhibitor, increased the ROS levels, and aggravated cell apoptosis by NG. These results suggest that NG-induced apoptosis in MGC803 cells is mediated, at least in part, by the increase in ROS production, oxidative stress and mitochondrial dysfunction.
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Department of Pharmacy, Medical College, Henan University of Science and Technology, Luoyang 471003, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Jiani
authors.initials: J
authors.lastname: Tan
authors.affiliation: Center of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Fu
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Electronic address: jihuicpu@163.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Ji
authors.affiliation: Center of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Electronic address: zyhtgd@sohu.com.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001391:Azo Compounds / Q000494:pharmacology*; D053453:Caspase 9 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D008315:Malondialdehyde / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D011355:Prodrugs / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology*; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology; D013482:Superoxide Dismutase / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001391:Azo Compounds; D020030:Nitric Oxide Donors; C576900:O2-(2,4-dinitro-5-((2-(12-en-28-galactopyranosyloleanolate-3-yl)oxy-2-oxoethyl)amino)phenyl) 1-(N-hydroxyethylmethylamino)diazen-1-ium-1,2-diolate; D011355:Prodrugs; D019253:Proto-Oncogene Proteins c-bcl-2; D012503:Saponins; D051028:bcl-2-Associated X Protein; C065566:buthionine sulfoximine ethyl ester; D008717:Methionine Sulfoximine; D009569:Nitric Oxide; D008315:Malondialdehyde; D000255:Adenosine Triphosphate; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D053453:Caspase 9; D005978:Glutathione; D000111:Acetylcysteine
keywords: Apoptosis; Glutathione; Mitochondria; Oxidative stress; Reactive oxygen species