Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Abstract

Mukhopadhyay P, Rajesh M, Bátkai S, Yoshihiro K, Haskó G, Liaudet L, Szabó C, Pacher P. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol 296: H1466 -H1483, 2009. First published March 13, 2009 doi:10.1152/ajpheart.00795.2008.-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, p22phox , 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. heart failure; apoptosis; inducible nitric oxide synthase; hemodynamics BECAUSE OF ITS unmatched efficacy and broad-spectrum effect, doxorubicin (DOX; Adriamycin), an anthracycline antibiotic, continues to be a widely used chemotherapeutic agent to treat a variety of cancers (55) despite its potential to elicit serious dose-dependent cardiotoxicity often leading to degenerative cardiomyopathy...

[11C]Acetate rest–stress protocol to assess myocardial perfusion and oxygen consumption reserve in a model of congestive heart failure in rats

“…Overtime, doxorubicin induces a cardiomyopathy resulting from myocytes apoptosis [12,39]. The damage is linked to oxidative stress at the level of the mitochondria that will interfere with oxidative phosphorylation.…”

The Possible Protective Role of Melatonin on Doxorubicin Induced Cardiomyopathy of Adult Male Albino Rats

“…Different mechanisms may play a role in the effects induced by DOX, including suppression of protein synthesis and nucleic acid formation, lysosomal deformities, changes in adrenergic function, mitochondrial abnormalities, changed sarcolemmal Ca2+ transport, calcium overload and energy metabolism disorders may lead to lipid peroxidation in myocardial fibers; and an imbalance of myocardial electrolytes [2] . Effects of DOX may be attributed to Oxidative/nitrosative stress [3] . production of reactive oxygen species (ROS) may be the cause of effects of DOX or through induction of nitric oxide synthases (NOS), leading to nitric oxide (NO) formation.…”

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

“…The mechanism of DOX's cardiotoxicity is complex and may involve oxidative (2,3), nitrosative and nitrative stress (4,5), mitochondrial dysfunction/ toxicity (1,(6)(7)(8), dysregulation of various metabolic (9) and lipid signaling pathways (10)(11)(12), activation of various stress kinases and cell death mechanisms (both apoptotic and necrotic) (13), triggering of secondary inflammation and remodeling (14), eventually culminating in cardiac dysfunction and heart failure (1,15).…”

“…In this study, we aimed to explore the effects of CBD in a well-established, clinically relevant mouse model of DOX-induced cardiomyopathy (4,5,23), particularly focusing on oxidative and nitrative stress and mitochondrial dysfunction/ biogenesis. Our results may also provide a novel mechanistic insight on the protective effects of CBD in various models of tissue injury and a promising tool for the prevention of devastating cardiovascular complications of DOX chemotherapy.…”