Suppression by Metallothionein of Doxorubicin-induced Cardiomyocyte Apoptosis through Inhibition of p38 Mitogen-activated Protein Kinases*
- From the Departments of ‡Medicine and§Pharmacology and Toxicology, University of Louisville,¶Jewish Hospital Heart and Lung Institute, and **Veterans Affairs Medical Center, Louisville, Kentucky 40292
Abstract
Cardiomyopathy induced by doxorubicin (DOX) has long been a major impediment of clinical applications of this effective anticancer agent. Previous studies have shown that cardiac-specific metallothionein (MT)-overexpressing transgenic mice are highly resistant to DOX-induced cardiotoxicity. To investigate cellular and molecular mechanisms by which MT participates in this cytoprotection, transgenic mice containing high levels of cardiac MT and non-transgenic controls were treated intraperitoneally with DOX at a single dose of 15 mg/kg and sacrificed on the 4th day after treatment. Myocardial apoptosis was detected by a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and confirmed by electron microscopy of immunogold staining of apoptotic nuclei. Dual staining of cardiac α-sarcomeric actin using an immunohistochemical method further identified apoptotic myocytes. Apoptosis was significantly inhibited in the transgenic myocardium. The anti-apoptotic effect of MT was further revealed in primary cultures of neonatal mouse cardiomyocytes. Furthermore, DOX activated p38 mitogen-activated protein kinase (MAPK), which was critically involved in the apoptotic process, as demonstrated by inhibition of DOX-induced apoptosis by a p38-specific inhibitor, SB203580. Both DOX-induced p38 MAPK activation and apoptosis were dramatically inhibited in the transgenic cardiomyocytes. The results thus demonstrate that DOX induces apoptosis in cardiomyocytes both in vivo andin vitro and MT suppresses this effect through at least in part inhibition of p38 MAPK activation.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants CA68125 and HL59225, Established Investigator Award 9640091N from the American Heart Association National Center (to Y. J. K.), a research grant from the Department of Veterans Affairs (to J. B. K.), and research grants from the Jewish Hospital Foundation, Louisville, KY (to Y. J. K. and J. B. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ University Scholar of the University of Louisville. To whom correspondence should be addressed: Dept. of Medicine, University of Louisville School of Medicine, 511 S. Floyd St., MDR 530, Louisville, KY 40202. Tel.: 502-852-8677; Fax: 502-852-6904; E-mail: yjkang01@athena.louisville.edu.
- Abbreviations:
- DOX
-
doxorubicin
- MT
-
metallothionein
- MAPK
-
mitogen-activated protein kinase
- TUNEL
-
terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
- FITC
-
fluorescein isothiocyanate
- PS
-
phosphatidylserine
- TdT
-
terminal deoxynucleotidyltransferase
- PIPES
-
1,4-piperazinediethanesulfonic acid
- ROS
-
reactive oxygen species
- PBS
-
phosphate-buffered saline
-
- Received September 28, 1999.
- Revision received January 27, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
