title: 3,4-Dihydroxy-benzohydroxamic acid (Didox) suppresses pro-inflammatory profiles and oxidative stress in TLR4-activated RAW264.7 murine macrophages.
abstract: Didox (3,4-dihydroxy-benzohydroxamic acid), is a synthetic ribonucleotide reductase (RR) inhibitor derived from polyhydroxy-substituted benzohydroxamic acid, and originally developed as an anti-cancer agent. Some studies indicate that didox may have anti-oxidative stress-like properties, while other studies hint that didox may have anti-inflammatory properties. Using nitric oxide production in response to LPS treatment as a sensitive screening assay for anti-inflammatory compounds, we show that didox is very potent at levels as low as 6.25 μM, with maximal inhibition at 100 μM. A qRT-PCR array was then employed to screen didox for other potential anti-inflammatory and anti-oxidative stress-related properties. Didox was very potent in suppressing the expression of these arrayed mRNA in response to LPS, and in some cases didox alone suppressed expression. Using qRT-PCR as a follow up to the array, we demonstrated that didox suppresses LPS-induced mRNA levels of iNOS, IL-6, IL-1, TNF-α, NF-κβ (p65), and p38-α, after 24h of treatment. Treatment with didox also suppresses the secretion of nitric oxide, IL-6, and IL-10. Furthermore, oxidative stress, as quantified by intracellular ROS levels in response to macrophage activators LPS and phorbol ester (PMA), and the glutathione depleting agent BSO, is reduced by treatment with didox. Moreover, we demonstrate that nuclear translocation of NF-κβ (p65) in response to LPS is inhibited by didox. These findings were supported by qRT-PCR for oxidative stress genes SOD1 and catalase. Overall, this study supports the conclusion that didox may have a future role in managing acute and chronic inflammatory diseases and oxidative stress due to high production of ROS.
authors.affiliation: Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, South Africa.
authors.forename: Thabe M
authors.initials: TM
authors.lastname: Matsebatlela
authors.affiliation: Department of Biological Sciences, Clemson University, SC, USA.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Anderson
authors.affiliation: Department of Biological Sciences, Clemson University, SC, USA.
authors.forename: Vincent S
authors.initials: VS
authors.lastname: Gallicchio
authors.affiliation: Molecules for Health, Richmond, VA, USA.
authors.forename: Howard
authors.initials: H
authors.lastname: Elford
authors.affiliation: Department of Biological Sciences, Clemson University, SC, USA. Electronic address: cdrice@clemson.edu.
authors.forename: Charles D
authors.initials: CD
authors.lastname: Rice
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002374:Catalase / Q000235:genetics; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006877:Hydroxamic Acids / Q000494:pharmacology*; D015850:Interleukin-6 / Q000235:genetics; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000276:immunology; D013482:Superoxide Dismutase / Q000235:genetics; D000072105:Superoxide Dismutase-1; D051197:Toll-Like Receptor 4 / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000893:Anti-Inflammatory Agents; D016166:Free Radical Scavengers; D006877:Hydroxamic Acids; D015850:Interleukin-6; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D002374:Catalase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C035419:3,4-dihydroxybenzohydroxamic acid
keywords: Didox; Inflammation; Oxidative stress; RAW264.7 cells; TLR-4; iNOS
references.citation: BMC Immunol. 2007 Jan 12;8:1
references.pmid: 17222336
references.citation: J Biol Chem. 2005 Nov 25;280(47):38969-75
references.pmid: 16176925
references.citation: Cancer Res. 1979 Mar;39(3):844-51
references.pmid: 427774
references.citation: Toxicol Appl Pharmacol. 2001 Aug 15;175(1):1-9
references.pmid: 11509021
references.citation: Nat Rev Drug Discov. 2004 Jan;3(1):17-26
references.pmid: 14708018
references.citation: Nature. 2008 Jul 24;454(7203):436-44
references.pmid: 18650914
references.citation: Oncogene. 2004 May 27;23(25):4400-12
references.pmid: 15077192
references.citation: J Allergy Clin Immunol. 2009 Sep;124(3):514-21
references.pmid: 19541349
references.citation: J Immunol. 2008 Sep 15;181(6):4347-53
references.pmid: 18768893
references.citation: J Pathol. 2008 Jan;214(2):161-78
references.pmid: 18161744
references.citation: Epilepsia. 2014 Jan;55(1):184-92
references.pmid: 24299259
references.citation: Cardiovasc Toxicol. 2003;3(2):141-51
references.pmid: 14501032
references.citation: Neurology. 2003 Jan 28;60(2):307-14
references.pmid: 12552050
references.citation: J Immunol. 2000 Jul 15;165(2):1013-21
references.pmid: 10878378
references.citation: J Inflamm (Lond). 2010 Aug 18;7:43
references.pmid: 20718971
references.citation: FEBS Lett. 2001 Mar 2;491(3):227-32
references.pmid: 11240132
references.citation: Pharmacol Ther. 2002 Oct;96(1):23-43
references.pmid: 12441176
references.citation: Eur J Cancer. 2006 Apr;42(6):717-27
references.pmid: 16520032
references.citation: Biochemistry. 2009 Jun 9;48(22):4980-7
references.pmid: 19374446
references.citation: Toxicol Appl Pharmacol. 2013 Jan 1;266(1):157-66
references.pmid: 23107598
references.citation: Cold Spring Harb Perspect Biol. 2009 Nov;1(5):a000141
references.pmid: 20066113
references.citation: Semin Immunopathol. 2008 Jul;30(3):279-89
references.pmid: 18536919
references.citation: Int J Mol Sci. 2011;12(5):3117-32
references.pmid: 21686173
references.citation: Br J Cancer. 1990 Mar;61(3):447-50
references.pmid: 2183873
references.citation: J Biol Chem. 1994 Feb 18;269(7):4705-8
references.pmid: 7508926
references.citation: J Leukoc Biol. 1994 Feb;55(2):253-8
references.pmid: 8301222
references.citation: BMC Gastroenterol. 2007 Jul 04;7:25
references.pmid: 17610716
references.citation: Br J Dermatol. 2012 Nov;167 Suppl 3:3-11
references.pmid: 23082810
references.citation: J Immunol Methods. 1983 Dec 16;65(1-2):55-63
references.pmid: 6606682
references.citation: Cytokine. 2008 May;42(2):145-151
references.pmid: 18304834
references.citation: Nature. 2008 Jul 24;454(7203):428-35
references.pmid: 18650913
references.citation: Nat Rev Immunol. 2005 Oct;5(10):749-59
references.pmid: 16175180
references.citation: Immunopharmacol Immunotoxicol. 2013 Feb;35(1):34-42
references.pmid: 23131135
references.citation: Adv Enzyme Regul. 1980;19:151-68
references.pmid: 6175187
references.citation: N Engl J Med. 2005 Jul 7;353(1):33-45
references.pmid: 16000354
references.citation: PLoS One. 2010 Feb 11;5(2):e9165
references.pmid: 20161797
references.citation: Biochem Pharmacol. 2010 May 1;79(9):1272-80
references.pmid: 20067776
references.citation: PLoS One. 2014 Nov 17;9(11):e112619
references.pmid: 25402485
references.citation: Eur J Immunol. 2011 Sep;41(9):2498-502
references.pmid: 21952805
references.citation: Int Immunol. 2009 Apr;21(4):317-37
references.pmid: 19246554
references.citation: Br J Cancer. 1988 Jul;58(1):70-2
references.pmid: 3048353
references.citation: J Leukoc Biol. 1990 May;47(5):440-8
references.pmid: 2159514
references.citation: Immunobiology. 2013 Nov;218(11):1402-10
references.pmid: 23891329
references.citation: Toxicol Sci. 2010 Nov;118(1):7-18
references.pmid: 20538741
references.citation: J Biol Chem. 1996 Jan 5;271(1):337-42
references.pmid: 8550583
references.citation: Circulation. 2005 Mar 22;111(11):1448-54
references.pmid: 15781756
references.citation: Science. 2010 Jan 15;327(5963):296-300
references.pmid: 20075245
references.citation: J Inflamm (Lond). 2010 Jan 08;7:1
references.pmid: 20148136
references.citation: Curr Opin Immunol. 2007 Dec;19(6):615-22
references.pmid: 17977705
references.citation: J Clin Invest. 2012 Mar;122(3):787-95
references.pmid: 22378047
references.citation: Scand J Immunol. 2009 Jun;69(6):479-91
references.pmid: 19439008
references.citation: Curr Pain Headache Rep. 2007 Dec;11(6):423-7
references.pmid: 18173976
references.citation: Nature. 1994 Dec 22-29;372(6508):739-46
references.pmid: 7997261
references.citation: Circulation. 2002 Mar 5;105(9):1135-43
references.pmid: 11877368
references.citation: J Biol Chem. 1970 Oct 25;245(20):5228-33
references.pmid: 4319235
references.citation: Ther Clin Risk Manag. 2009;5:889-96
references.pmid: 19956553
references.citation: Nat Immunol. 2010 Oct;11(10):889-96
references.pmid: 20856220
references.citation: Ann Intern Med. 2008 Jun 17;148(12):939-55
references.pmid: 18458272
references.citation: J Immunol. 1977 Sep;119(3):950-54
references.pmid: 894031
references.citation: Pharmacol Ther. 1985;29(2):239-54
references.pmid: 3939037
references.citation: Health Technol Assess. 2004 Jul;8(28):iii, 1-120
references.pmid: 15245690