title: Nitric oxide-releasing prodrug triggers cancer cell death through deregulation of cellular redox balance.
abstract: JS-K is a nitric oxide (NO)-releasing prodrug of the O (2)-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action.
authors.affiliation: Basic Science Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Maciag
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Holland
authors.forename: Y-S
authors.initials: YS
authors.lastname: Robert Cheng
authors.forename: Luis G
authors.initials: LG
authors.lastname: Rodriguez
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Anderson
authors.forename: Larry K
authors.initials: LK
authors.lastname: Keefer
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D001391:Azo Compounds / Q000138:chemical synthesis / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000378:metabolism* / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000138:chemical synthesis / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010879:Piperazines / Q000138:chemical synthesis / Q000494:pharmacology*; D011355:Prodrugs / Q000138:chemical synthesis* / Q000494:pharmacology; D026361:Reactive Nitrogen Species / Q000378:metabolism
chemical_list: D001391:Azo Compounds; D020030:Nitric Oxide Donors; C474958:O(2)-(2,4-dinitrophenyl) 1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate; D010879:Piperazines; D011355:Prodrugs; D026361:Reactive Nitrogen Species; D009569:Nitric Oxide; D005978:Glutathione; D000111:Acetylcysteine
keywords: ATF, activating transcription factor; Arylated diazeniumdiolate; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; DCF-DA, 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSH, glutathione; GSSG, glutathione disulfide (oxidized GSH); Glutathione; HBSS, Hank's balanced salt solution; IPA, Ingenuity Pathway Analysis; JS-K, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate; LC/MS, liquid chromatography/mass spectrometry; Leukemia; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NO, nitric oxide; NSCLC, non-small cell lung cancer; Nitric oxide; PARP, poly (ADP-ribose) polymerase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPK/JNK, stress activated protein kinase/c-jun N-terminal kinase.
references.citation: Methods Enzymol. 1985;113:548-55
references.pmid: 4088074
references.citation: Science. 1983 Oct 28;222(4622):390-3
references.pmid: 6414084
references.citation: Br J Pharmacol. 2004 May;142(2):231-55
references.pmid: 15155533
references.citation: J Pharmacol Exp Ther. 2011 Feb;336(2):313-20
references.pmid: 20962031
references.citation: Blood. 2003 May 15;101(10):4098-104
references.pmid: 12531810
references.citation: Neurosurgery. 2012 Feb;70(2):497-510; discussion 510
references.pmid: 21849924
references.citation: J Org Chem. 2001 May 4;66(9):3090-8
references.pmid: 11325274
references.citation: Nature. 2004 Oct 28;431(7012):1112-7
references.pmid: 15475948
references.citation: Biochem J. 2007 Jan 1;401(1):1-11
references.pmid: 17150040
references.citation: Bioorg Med Chem. 2008 Nov 15;16(22):9764-71
references.pmid: 18930407
references.citation: Cancer Cell. 2008 Dec 9;14(6):458-70
references.pmid: 19061837
references.citation: Free Radic Biol Med. 2012 Jan 1;52(1):1-6
references.pmid: 22027063
references.citation: Blood. 2007 Jul 15;110(2):709-18
references.pmid: 17384201
references.citation: Breast Cancer Res. 2008;10(3):R44
references.pmid: 18474097
references.citation: Science. 2000 May 5;288(5467):870-4
references.pmid: 10797012
references.citation: Proc Natl Acad Sci U S A. 1982 Dec;79(24):7824-7
references.pmid: 6961453
references.citation: Exp Cell Res. 1996 Jan 10;222(1):117-24
references.pmid: 8549653
references.citation: J Med Chem. 2011 Nov 24;54(22):7751-8
references.pmid: 22003962
references.citation: Mol Aspects Med. 2009 Feb-Apr;30(1-2):13-28
references.pmid: 18786560
references.citation: Int J Cancer. 1976 May 15;17(5):565-77
references.pmid: 178611
references.citation: J Exp Med. 1995 May 1;181(5):1661-72
references.pmid: 7722446
references.citation: Bioorg Med Chem. 2012 May 1;20(9):3094-9
references.pmid: 22480849
references.citation: Int J Oncol. 2011 Apr;38(4):963-71
references.pmid: 21271218
references.citation: J Cell Physiol. 2003 Dec;197(3):426-34
references.pmid: 14566972
references.citation: Blood. 2000 Sep 15;96(6):2140-8
references.pmid: 10979959
references.citation: Free Radic Biol Med. 2001 Jan 15;30(2):213-21
references.pmid: 11163539
references.citation: Anal Biochem. 1980 Jul 15;106(1):207-12
references.pmid: 7416462
references.citation: Mol Cancer Ther. 2003 Apr;2(4):409-17
references.pmid: 12700285
references.citation: J Immunol. 2005 Dec 15;175(12):8424-30
references.pmid: 16339585
references.citation: Proc Natl Acad Sci U S A. 1981 Dec;78(12):7492-6
references.pmid: 6950392
references.citation: Am J Physiol. 1989 Oct;257(4 Pt 1):L163-73
references.pmid: 2572174
references.citation: Methods Enzymol. 2002;348:93-112
references.pmid: 11885298
references.citation: Nat Med. 2005 Dec;11(12):1306-13
references.pmid: 16286925
references.citation: J Med Chem. 2006 Jul 13;49(14):4356-66
references.pmid: 16821795