Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.
Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.
Mukhopadhyay
Partha
P
Section on Oxidative Stress and Tissue Injury, Laboratory of Physiological Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, MSC-9413, Bethesda, MD 20892-9413, USA.
Rajesh
Mohanraj
M
Bátkai
Sándor
S
Kashiwaya
Yoshihiro
Y
Haskó
György
G
Liaudet
Lucas
L
Szabó
Csaba
C
Pacher
Pál
P
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D006331:Heart Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009336:Necrosis; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D016277:Ventricular Function, Left / Q000187:drug effects; D017725:Ventricular Pressure / Q000187:drug effects
D000903:Antibiotics, Antineoplastic; D000975:Antioxidants; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D020030:Nitric Oxide Donors; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D004317:Doxorubicin; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011065:Poly(ADP-ribose) Polymerases; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse