OXIDATIVE STRESS, APOPTOSIS AND DRUG RESISTANCE

肿瘤细胞耐药性的产生是导致肿瘤化疗失败的主要原因。虽然药物的作用机制相当不同，但这些药物通常通过凋亡来杀死肿瘤细胞。对一对几乎等基因的药物敏感和耐药卵巢癌细胞株的比较研究发现，不同的细胞反应可能与防止耐药细胞的凋亡有关。许多抗癌药物和辐射可能通过破坏线粒体来诱导细胞中的活性氧物种(ROS)。我们观察到亚毒水平的ROS与药物协同作用于诱导耐药细胞的凋亡，其机制是：(1)抑制过表达的抗凋亡蛋白，(2)谷胱甘肽-S-转移酶，(3)促进双链断裂的修复，(4)诱导p53wt的核积聚。该项目的中心假设是，细胞凋亡是由慢性氧化应激以两种不同的方式触发的：(I)通过对线粒体的持续损伤，导致谷胱甘肽(GSH)外流，对线粒体膜和DNA的损伤，(Ii)通过阻止DNA交联剂和DNA拓扑异构酶抑制剂依托泊苷诱导的线粒体和核DNA双链断裂的修复。为了验证我们的假设，具体目的是：(1)检测线粒体GSH的耗竭是否在维持慢性氧化应激中起中心作用；(2)检测慢性ROS是否影响药物治疗诱导的线粒体DNA修复的效率和保真度；(3)确定慢性ROS如何导致P53的核积累并抑制核DNA修复；(4)测试慢性ROS是否通过DNA蛋白激酶途径影响DNA双链断裂修复；以及(5)确定氧化应激诱导耐药细胞凋亡的信号传递过程。这些研究将利用各种细胞和分子方法来确定慢性氧化应激使耐药细胞容易受到传统药物治疗引发的细胞凋亡的机制。这一转化研究项目的长期目标是全面了解ROS在耐药方面的多重和相互关联的分子效应，这将有助于开发新的策略来提高抗肿瘤药物的治疗指数，并将对癌症患者的治疗产生重大影响。

Development of drug resistance in tumor cells is the major cause for failure of cancer chemotherapy. Although the mechanisms of drug action are quite diverse, the drugs generally kill tumor cells via apoptosis. A comparative study of a nearly isogenic pair of drug sensitive and resistant ovarian carcinoma lines led to identification of distinct cellular responses that may be causally linked to prevention of apoptosis in resistant cells. Many anticancer drugs and radiation induce reactive oxygen species (ROS) in the cells, presumably by damaging mitochondria. We observed that sub-toxic levels of ROS acted synergistically with drugs in inducing apoptosis in the resistant cells by (i) overriding the effects of overexpressing anti-apoptotic proteins, (ii) glutathione-S-transferase, (iii) increased repair of double strand breaks and (iv) induced nuclear accumulation of p53wt. The central hypothesis of this project is that apoptosis is triggered by chronic oxidative stress in two distinct ways: (i) by continuous damage to the mitochondria, resulting in glutathione (GSH) efflux and damage to mitochondrial membrane and DNA, (ii) by prevention of repair of DNA double strand breaks in the mitochondrial and nuclear DNA induced by DNA cross-linking agents and DNA topoisomerase inhibitor, etoposide. To test our hypothesis the specific aims are: (1) to examine whether depletion of GSH from the mitochondria is central to maintenance of chronic oxidative stress, (2) to test if chronic ROS affect efficiency and fidelity of mitochondria DNA repair induced by drug-treatment, (3) to determine how chronic ROS result in nuclear accumulation of p53 and inhibit nuclear DNA repair, (4) to test if chronic ROS affects DNA double strand break repair via DNA protein kinase pathway, and (5) to identify the signaling process by oxidative stress which induce apoptosis in resistant cells. These studies will utilize a variety of cellular and molecular approaches to determine the mechanism by which chronic oxidative stress renders drug resistant cells susceptible to apoptosis triggered by conventional drug treatment. The long-term objective of this translational research project is a comprehensive understanding of the multiple and interlocking molecular effects of ROS on drug resistance that should help develop new strategies to improve the therapeutic index of antitumor drugs, and which will have a significant impact on the treatment of cancer patients.