Wang G W, Klein J B, Kang Y J
Department of Medicine, University of Louisville, Louisville, Kentucky 40202, USA.
J Pharmacol Exp Ther. 2001 Aug;298(2):461-8.
Previous studies using transgenic mice in which metallothionein (MT) was overexpressed only in the heart have demonstrated that MT protects from oxidative cardiac injury induced by doxorubicin (DOX), an important anticancer agent. MT cardioprotection is associated with its antiapoptotic effect. The present study was undertaken to test the hypothesis that MT suppresses DOX-induced apoptosis through inhibition of mitochondrial cytochrome c release and caspase-3 activation. Primary cultures of cardiomyocytes isolated from the hearts of transgenic neonatal mice and nontransgenic controls were treated with DOX at a clinically relevant concentration (1.0 microM) for varying time periods. Apoptosis was detected in nontransgenic cardiomyocyte cultures by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Annexin V-fluorescein isothiocyanate binding. This apoptotic effect was significantly suppressed in the MT-overexpressing transgenic cardiomyocytes. Western blot analysis revealed that DOX caused mitochondrial cytochrome c release. Furthermore, caspase-3 activation was observed. The activation of this apoptotic pathway by DOX was dramatically inhibited in the MT-overexpressing cardiomyocytes. To elucidate the role of reactive oxygen species (ROS) in the activation of the cytochrome c-mediated caspase-3 activation pathway, the intracellular levels of ROS and their localization were detected by fluorescent confocal microscopy. Mitochondrial ROS concentrations were dramatically elevated by DOX in nontransgenic cardiomyocytes. This elevation was completely inhibited almost in the MT-overexpressing cardiomyocytes. Thus, these results demonstrate that MT suppresses DOX-induced apoptosis in cardiomyocytes through, at least in part, inhibition of the cytochrome c-mediated apoptotic pathway.
以往使用仅在心脏中过表达金属硫蛋白（MT）的转基因小鼠进行的研究表明，MT可保护心脏免受阿霉素（DOX，一种重要的抗癌药物）诱导的氧化损伤。MT的心脏保护作用与其抗凋亡作用相关。本研究旨在验证MT通过抑制线粒体细胞色素c释放和半胱天冬酶-3激活来抑制DOX诱导的细胞凋亡这一假说。从转基因新生小鼠和非转基因对照小鼠心脏分离的原代心肌细胞培养物，用临床相关浓度（1.0微摩尔）的DOX处理不同时间段。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记和膜联蛋白V-异硫氰酸荧光素结合检测非转基因心肌细胞培养物中的细胞凋亡。在MT过表达的转基因心肌细胞中，这种凋亡效应得到显著抑制。蛋白质印迹分析显示，DOX导致线粒体细胞色素c释放。此外，观察到半胱天冬酶-3激活。在MT过表达的心肌细胞中，DOX对这一凋亡途径的激活受到显著抑制。为了阐明活性氧（ROS）在细胞色素c介导的半胱天冬酶-3激活途径激活中的作用，通过荧光共聚焦显微镜检测细胞内ROS水平及其定位。在非转基因心肌细胞中，DOX使线粒体ROS浓度显著升高。在MT过表达的心肌细胞中，这种升高几乎完全受到抑制。因此，这些结果表明，MT至少部分地通过抑制细胞色素c介导的凋亡途径来抑制DOX诱导的心肌细胞凋亡。
