OXIDATIVE STRESS, APOPTOSIS AND DRUG RESISTANCE

肿瘤细胞耐药性的产生是肿瘤化疗失败的主要原因。 虽然药物作用的机制是相当多样的，但药物通常通过凋亡杀死肿瘤细胞。 一项对药物敏感和耐药卵巢癌细胞系的近等基因对的比较研究，鉴定了不同的细胞反应，这些反应可能与耐药细胞中细胞凋亡的预防有因果关系。许多抗癌药物和辐射诱导细胞中的活性氧（ROS），可能是通过破坏线粒体。我们观察到，亚毒性水平的ROS与药物协同作用，诱导耐药细胞的凋亡，（i）推翻过表达抗凋亡蛋白的影响，（ii）谷胱甘肽-S-转移酶，（iii）增加双链断裂的修复和（iv）诱导p53 wt的核积累。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）流出并损伤线粒体膜和DNA，（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中DNA双链断裂的修复。 为了检验我们的假设，具体目标是：（1）检查GSH从线粒体的消耗是否是维持慢性氧化应激的中心，（2）测试慢性ROS是否影响由药物处理诱导的线粒体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）检测慢性ROS是否通过DNA蛋白激酶途径影响DNA双链断裂修复;（5）鉴定氧化应激诱导耐药细胞凋亡的信号传导过程。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对常规药物治疗引发的凋亡敏感的机制。 该转化研究项目的长期目标是全面了解ROS对耐药性的多重和连锁分子效应，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.