Le Son B, Hailer M Katie, Buhrow Sarah, Wang Qi, Flatten Karen, Pediaditakis Peter, Bible Keith C, Lewis Lionel D, Sausville Edward A, Pang Yuan-Ping, Ames Matthew M, Lemasters John J, Holmuhamedov Ekhson L, Kaufmann Scott H
Department of Oncology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
J Biol Chem. 2007 Mar 23;282(12):8860-72. doi: 10.1074/jbc.M611777200. Epub 2007 Jan 9.
Adaphostin is a dihydroquinone derivative that is undergoing extensive preclinical testing as a potential anticancer drug. Previous studies have suggested that the generation of reactive oxygen species (ROS) plays a critical role in the cytotoxicity of this agent. In this study, we investigated the source of these ROS. Consistent with the known chemical properties of dihydroquinones, adaphostin simultaneously underwent oxidation to the corresponding quinone and generated ROS under aqueous conditions. Interestingly, however, this quinone was not detected in intact cells. Instead, high performance liquid chromatography demonstrated that adaphostin was concentrated by up to 300-fold in cells relative to the extracellular medium and that the highest concentration of adaphostin (3000-fold over extracellular concentrations) was detected in mitochondria. Consistent with a mitochondrial site for adaphostin action, adaphostin-induced ROS production was diminished by >75% in MOLT-4 rho(0) cells, which lack mitochondrial electron transport, relative to parental MOLT-4 cells. In addition, inhibition of oxygen consumption was observed when intact cells were treated with adaphostin. Loading of isolated mitochondria to equivalent adaphostin concentrations caused inhibition of uncoupled oxygen consumption in mitochondria incubated with the complex I substrates pyruvate and malate or the complex II substrate succinate. Further analysis demonstrated that adaphostin had no effect on pyruvate or succinate dehydrogenase activity. Instead, adaphostin inhibited reduced decylubiquinone-induced cytochrome c reduction, identifying complex III as the site of inhibition by this agent. Moreover, adaphostin enhanced the production of ROS by succinate-charged mitochondria. Collectively, these observations demonstrate that mitochondrial respiration rather than direct redox cycling of the hydroquinone moiety is a source of adaphostin-induced ROS and identify complex III as a potential target for antineoplastic agents.
阿达福司汀是一种二氢醌衍生物，作为一种潜在的抗癌药物正在进行广泛的临床前测试。先前的研究表明，活性氧（ROS）的产生在该药物的细胞毒性中起关键作用。在本研究中，我们调查了这些ROS的来源。与二氢醌的已知化学性质一致，阿达福司汀在水性条件下同时被氧化为相应的醌并产生活性氧。然而，有趣的是，在完整细胞中未检测到这种醌。相反，高效液相色谱表明，相对于细胞外培养基，阿达福司汀在细胞中的浓度可高达300倍，并且在线粒体中检测到阿达福司汀的最高浓度（比细胞外浓度高3000倍）。与阿达福司汀作用的线粒体部位一致，相对于亲本MOLT-4细胞，在缺乏线粒体电子传递的MOLT-4 rho(0)细胞中，阿达福司汀诱导的ROS产生减少了>75%。此外，当用阿达福司汀处理完整细胞时，观察到氧消耗受到抑制。将分离的线粒体加载到等效的阿达福司汀浓度下，会导致在用复合物I底物丙酮酸和苹果酸或复合物II底物琥珀酸孵育的线粒体中，非偶联氧消耗受到抑制。进一步分析表明，阿达福司汀对丙酮酸或琥珀酸脱氢酶活性没有影响。相反，阿达福司汀抑制了还原型癸基泛醌诱导的细胞色素c还原，确定复合物III是该药物的抑制位点。此外，阿达福司汀增强了琥珀酸负载的线粒体产生活性氧的能力。总的来说，这些观察结果表明，线粒体呼吸而非对苯二酚部分的直接氧化还原循环是阿达福司汀诱导的ROS的来源，并确定复合物III是抗肿瘤药物的潜在靶点。
