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

Partha Mukhopadhyay, Mohanraj Rajesh, Sándor Bátkai, Yoshihiro Kashiwaya, György Haskó, Lucas Liaudet, Csaba Szabo, Pál Pacher

Research output: Contribution to journalArticle

223 Citations (Scopus)

Abstract

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, p40phox, p47phox, 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume296
Issue number5
DOIs
StatePublished - May 2009

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Peroxynitrous Acid
Superoxides
Doxorubicin
Nitric Oxide
Cell Death
NADPH Oxidase
Apoptosis
Necrosis
Xanthine Oxidase
Nitric Oxide Synthase
Catalase
In Vitro Techniques
Protein Isoforms
Allopurinol
Nitric Oxide Donors
Poly(ADP-ribose) Polymerases
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
In Situ Nick-End Labeling
Nitric Oxide Synthase Type II

Keywords

  • Apoptosis
  • Heart failure
  • Hemodynamics
  • Inducible nitric oxide synthase

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Cite this

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. / Mukhopadhyay, Partha; Rajesh, Mohanraj; Bátkai, Sándor; Kashiwaya, Yoshihiro; Haskó, György; Liaudet, Lucas; Szabo, Csaba; Pacher, Pál.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 296, No. 5, 05.2009.

Research output: Contribution to journalArticle

Mukhopadhyay, Partha ; Rajesh, Mohanraj ; Bátkai, Sándor ; Kashiwaya, Yoshihiro ; Haskó, György ; Liaudet, Lucas ; Szabo, Csaba ; Pacher, Pál. / Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. In: American Journal of Physiology - Heart and Circulatory Physiology. 2009 ; Vol. 296, No. 5.
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