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

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, 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.

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  • Authors+Show Affiliations

    ,

    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.

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    Source

    MeSH

    Animals
    Antibiotics, Antineoplastic
    Antioxidants
    Apoptosis
    Cell Line
    Dose-Response Relationship, Drug
    Doxorubicin
    Enzyme Inhibitors
    Free Radical Scavengers
    Heart Diseases
    Male
    Matrix Metalloproteinase 2
    Matrix Metalloproteinase 9
    Mice
    Mice, Inbred C57BL
    Mice, Knockout
    Mitochondria, Heart
    Myocardial Contraction
    Myocytes, Cardiac
    Necrosis
    Nitric Oxide
    Nitric Oxide Donors
    Nitric Oxide Synthase Type II
    Peroxynitrous Acid
    Poly(ADP-ribose) Polymerases
    Superoxides
    Tyrosine
    Ventricular Function, Left
    Ventricular Pressure

    Pub Type(s)

    Journal Article
    Research Support, N.I.H., Extramural
    Research Support, N.I.H., Intramural
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    19286953

    Citation

    Mukhopadhyay, Partha, et al. "Role of Superoxide, Nitric Oxide, and Peroxynitrite in Doxorubicin-induced Cell Death in Vivo and in Vitro." American Journal of Physiology. Heart and Circulatory Physiology, vol. 296, no. 5, 2009, pp. H1466-83.
    Mukhopadhyay P, Rajesh M, Bátkai S, et al. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol. 2009;296(5):H1466-83.
    Mukhopadhyay, P., Rajesh, M., Bátkai, S., Kashiwaya, Y., Haskó, G., Liaudet, L., ... Pacher, P. (2009). Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. American Journal of Physiology. Heart and Circulatory Physiology, 296(5), pp. H1466-83. doi:10.1152/ajpheart.00795.2008.
    Mukhopadhyay P, et al. Role of Superoxide, Nitric Oxide, and Peroxynitrite in Doxorubicin-induced Cell Death in Vivo and in Vitro. Am J Physiol Heart Circ Physiol. 2009;296(5):H1466-83. PubMed PMID: 19286953.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. AU - Mukhopadhyay,Partha, AU - Rajesh,Mohanraj, AU - Bátkai,Sándor, AU - Kashiwaya,Yoshihiro, AU - Haskó,György, AU - Liaudet,Lucas, AU - Szabó,Csaba, AU - Pacher,Pál, Y1 - 2009/03/13/ PY - 2009/3/17/entrez PY - 2009/3/17/pubmed PY - 2009/6/20/medline SP - H1466 EP - 83 JF - American journal of physiology. Heart and circulatory physiology JO - Am. J. Physiol. Heart Circ. Physiol. VL - 296 IS - 5 N2 - 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. SN - 0363-6135 UR - https://www.unboundmedicine.com/medline/citation/19286953/Role_of_superoxide_nitric_oxide_and_peroxynitrite_in_doxorubicin_induced_cell_death_in_vivo_and_in_vitro_ L2 - http://www.physiology.org/doi/full/10.1152/ajpheart.00795.2008?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -