Adriamycin-induced cardiomyocyte and endothelial cell apoptosis: in vitro and in vivo studies.
Adriamycin is a potent, broad-spectrum chemotherapeutic agent effective against solid tumors and malignant hematological disease. The major limiting factor for adriamycin is its cardiotoxicity. Thus, the objective of this study was to investigate the role of cardiomyocyte and endothelial cell apoptosis in adriamycin-induced cardiomyopathy, in vivo and in vitro. For in vivo study, intraperitoneal injections of adriamycin were administered to nine adult male Wistar rats and normal saline to six rats as control. Eight of the nine rats in the adriamycin group, but none in the control group, developed marked ascites and DNA ladders in agarose gel electrophoresis of genomic DNA extracted from the rat hearts (P<0.001). The ratio of apoptotic nuclei in the cardiomyocytes was significantly higher for the adriamycin-treated rats (162+/-149/10(6) cells) than for the controls (4.2+/-1.3/10(6) cells; P<0.01) by TUNEL assay. Increased endothelial cell apoptosis was detected in the small coronary vessels of the myocardium of the adriamycin-treated rats. Increased immuno-reactive Caspase-3 expression was also noted for both cardiomyocytes and endothelial cells of adriamycin-treated rats. In vitro adriamycin treatment for cultured neonatal rat cardiomyocytes and human umbilical vein endothelial cells, respectively, showed a dose-related increase in apoptosis as determined by flowcytometry, DNA ladder analysis, TUNEL assay and/or electron-microscope examination. A dose-related increase in the expression of Fas antigen, Bax and Caspase-3, as well as a decrease in the expression of Bcl-2, were determined for the adriamycin-treated cardiomyocytes using Northern blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and ribonuclease protection assay. RT-PCR also revealed increased Fas antigen expression, decreased Bcl-2 expression, and no change in Bax expression for the adriamycin-treated human umbilical vein cells. Further, pretreatment with broad caspase inhibitor, but not neutralizing FasL antibody, resulted in inhibition of adriamycin-induced endothelial cell apoptosis. In conclusion, these results indicate that both adriamycin-induced cardiomyocyte and endothelial cell death can occur via apoptosis which is dose-related, and can occur both in vitro and in vivo with changes in the expression of the apoptosis-related genes. Adriamycin-induced endothelial cell apoptosis is mediated by caspase activation but is Fas/FasL signal pathway independent. Our data provides evidence that both cardiomyocyte and endothelial cell apoptosis may play an important role in adriamycin-induced cardiomyopathy.
Wu
Semon
S
The First Cardiovascular Division, Department of Internal Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan, Republic of China.
Ko
Yu-Shien
YS
Teng
Ming-Sheng
MS
Ko
Yu-Lin
YL
Hsu
Lung-An
LA
Hsueh
Chuen
C
Chou
Yung-Yin
YY
Liew
Choong-Chin
CC
Lee
Ying-Shiung
YS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000235:genetics; D009202:Cardiomyopathies / Q000139:chemically induced* / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000302:isolation & purification; D002478:Cells, Cultured; D004317:Doxorubicin / Q000633:toxicity*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000473:pathology; D005434:Flow Cytometry; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar
D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004317:Doxorubicin; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases