Cisplatin-induced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alpha-ketoglutarate dehydrogenase complex in LLC-PK1 cells.
Cisplatin-induced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alpha-ketoglutarate dehydrogenase complex in LLC-PK1 cells.
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顺铂诱导的毒性与体内小鼠肾线粒体中的铂沉积以及 LLC-PK1 细胞中 α-酮戊二酸脱氢酶复合物的选择性失活有关。
DOI：
10.1021/bi060027g
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Hanigan,MarieH
中科院分区：
文献类型：
--
作者：
Zhang,Lei;Cooper,ArthurJL;Krasnikov,BorisF;Xu,Hui;Bubber,Parvesh;Pinto,JohnT;Gibson,GaryE;Hanigan,MarieH
抗癌药物顺铂具有肾毒性和神经毒性。先前的数据支持顺铂被生物活化为肾毒性物质的假设。所提出的生物活化的最后一步是形成铂-半胱氨酸S-缀合物，然后进行吡哆醛5 '-磷酸（PLP）依赖性半胱氨酸S-缀合物β-裂解酶反应。该反应会在体内产生丙酮酸盐、铵和高活性的铂（Pt）-硫醇化合物，这些化合物会与蛋白质结合。在这项工作中，PLP依赖的半胱氨酸S-共轭β-裂解酶的细胞定位和身份进行了研究。研究表明，铂与接受顺铂治疗的小鼠肾脏中的蛋白质结合。Pt结合蛋白的浓度在线粒体组分中比在胞质组分中更高。用氨基氧乙酸（AOAA，PLP酶抑制剂）治疗小鼠，这以前已被证明可以阻断顺铂的肾毒性，减少了Pt与线粒体蛋白的结合，但对Pt与胞质组分中蛋白结合的量没有影响。这些数据表明线粒体酶催化PLP依赖性半胱氨酸S-缀合物β-裂解酶反应。PLP依赖性线粒体天冬氨酸转氨酶（mitAspAT）是一种线粒体酶，可催化与卤代烯烃的半胱氨酸S-共轭物的β-消除反应。我们推断，这种酶也可能催化β-裂解酶与顺铂-半胱氨酸S-缀合物的反应。在本研究中，mitAspAT在LLC-PK 1细胞中稳定过表达。顺铂在过表达mitAspAT的LLC-PK 1细胞的融合单层中的毒性显著高于仅含载体的对照细胞。AOAA完全阻断了融合的mitAspAT转染细胞中的顺铂毒性。Pt−硫醇化合物可以快速结合蛋白质和与PLP依赖性半胱氨酸S-共轭β-裂解酶紧密相关的β-内酰胺酶。用50或100 μM顺铂处理3 h，然后从培养基中去除顺铂24 h，导致mitAspAT转染细胞和对照细胞中α-酮戊二酸脱氢酶复合物（KGDHC）活性明显丧失。暴露于100 μM顺铂导致过表达mitAspAT的细胞中KGDHC活性的损失显著大于对照细胞。在两种细胞类型中，三磷酸腺苷酶活性均降低，但仅在较高水平的顺铂暴露下。AspAT活性也显著降低顺铂治疗。相比之下，其他几种参与能量/氨基酸代谢的酶（胞质和线粒体）在LLC-PK 1细胞中不受顺铂处理的显著影响，无论mitAspAT是否过表达。KGDHC和乌头酸酶在暴露于顺铂代谢物的肾细胞中对失活的敏感性可能是由于mitAspAT与线粒体中的KGDHC和乌头酸酶接近。这些发现支持线粒体半胱氨酸S-缀合物β-裂解酶将顺铂-半胱氨酸S-缀合物转化为毒物的假设，并且数据与mitAspAT在顺铂的生物活化中起作用的假设一致。
The anticancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum−cysteineS-conjugate followed by a pyridoxal 5‘-phosphate (PLP)-dependent cysteineS-conjugate β-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)−thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteineS-conjugate β-lyase were investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteineS-conjugate β-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes β-elimination reactions with cysteineS-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a β-lyase reaction with the cisplatin−cysteineS-conjugate. In this study, mitAspAT was stably overexpressed in LLC-PK1cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK1cells that overexpressed mitAspAT than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt−thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteineS-conjugate β-lyase. Treatment with 50 or 100 μM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of α-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 μM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK1cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteineS-conjugate β-lyase converts the cisplatin−cysteineS-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.