Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes
Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes
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DOI：
10.1007/s00441-012-1475-8
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发表时间：
2012-11-01
影响因子：
3.6
通讯作者：
Zuppinger, Christian
中科院分区：
文献类型：
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作者：
Dimitrakis, Polychronis;Romay-Ogando, Maria-Iris;Zuppinger, Christian
蒽环类药物在癌症治疗中的临床应用受到涉及心肌细胞损伤和死亡的剂量依赖性心脏毒性的限制。我们已经验证了蒽环类药物影响成年心肌细胞蛋白质降解途径的假设。为此，我们评估了阿霉素（Doxo）对长期培养的成年大鼠心肌细胞凋亡，自噬和蛋白酶体/泛素系统的影响。多聚泛素化蛋白的积累，组织蛋白酶D阳性溶酶体的增加和肌原纤维降解观察到在Doxo处理的心肌细胞。蛋白酶体的胰凝乳蛋白酶样活性最初增加，然后被Doxo在48 h的时间过程中抑制。蛋白酶体20 S蛋白被更高剂量的Doxo下调。MURF-1，一种特异性靶向肌原纤维蛋白的泛素连接酶，在所有测量浓度下都被Doxo抑制。微管相关蛋白1轻链3B（LC 3）阳性斑点和LC 3-I和-II蛋白均由Doxo以剂量依赖性方式诱导，通过使用与LC 3结合的绿色荧光蛋白的慢病毒表达和实时成像证实。亲溶酶体药物氯喹导致自噬体积累，其随着伴随的Doxo处理而增加，表明增强的自噬通量。我们的结论是，Doxo导致下调的蛋白质降解机制的心肌细胞与聚泛素化蛋白和自噬体的积累。虽然自噬最初被刺激作为对细胞毒性应激的补偿反应，但随后在较高剂量和较长暴露时间下发生凋亡和坏死。这一机制可能通过加速有丝分裂后的成年心肌细胞的衰老和衰老心脏对蒽环类药物癌症治疗的敏感性而导致蒽环类药物的晚期心脏毒性。
The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein 1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy.