Metabolic Oxidative Stress in Human Cancer Cells

描述（由申请人提供）：自20世纪20年代以来，已经观察到癌细胞（相对于正常细胞）表现出糖酵解和戊糖循环活性的增加速率以及呼吸速率的轻微降低，但是这对癌症治疗的意义尚不清楚。最近的研究表明，相对于正常细胞，葡萄糖剥夺优先诱导人类癌细胞的细胞毒性和氧化应激[附录1]。线粒体已被假设为在葡萄糖剥夺过程中产生促氧化剂的位点。如果这是普遍正确的，葡萄糖剥夺诱导的氧化应激可能代表了肿瘤细胞线粒体代谢的缺陷，可用于改善癌症治疗。当前的提案将测试线粒体产生活性氧簇（ROS）的假设;即，超氧化物和过氧化氢）介导人癌细胞相对于正常细胞对葡萄糖剥夺诱导的代谢氧化应激的增加的易感性。具体目标1将使用电子传递链阻断剂（即，抗霉素A、粘噻唑和鱼藤酮），如果完整的人癌细胞（或分离的线粒体）显示线粒体电子传递链复合物I、II和/或III的ROS产生的改变，其有助于相对于正常细胞增加对葡萄糖剥夺诱导的氧化应激的易感性。具体目标2将确定相对于含有完全功能性电子传递链的亲代rho（+）细胞，功能性线粒体电子传递链缺陷的rho（0）癌细胞是否表现出对葡萄糖剥夺诱导的细胞毒性和氧化应激的敏感性改变。特异性目的3将使用腺病毒载体以及稳定转染的细胞系来确定是否过度表达抑制超氧化物和过氧化氢的抗氧化酶（即，过氧化氢酶、超氧化物歧化酶）能够改变癌细胞中葡萄糖剥夺的生物效应。具体目标4将确定2-脱氧-d-葡萄糖是否能够模拟目标1-3中观察到的葡萄糖剥夺效应。长期目标是提供癌细胞对葡萄糖剥夺诱导的氧化应激的差异易感性的严格机制理解，以基于正常细胞与癌细胞中氧代谢之间的差异开发联合模式癌症治疗。

DESCRIPTION (provided by applicant): Since the 1920's it has been observed that cancer cells (relative to normal cells) demonstrate increased rates of glycolysis and pentose cycle activity as well as slightly decreased rates of respiration but the significance of this to cancer therapy is unclear. Recent studies have shown that glucose deprivation preferentially induces cytotoxicity and oxidative stress in human cancer cells, relative to normal cells [Appendix 1]. Mitochondria have been hypothesized to be the site of prooxidant production during glucose deprivation. If this were generally true, glucose deprivation-induced oxidative stress could represent a defect in tumor cell mitochondrial metabolism amenable to manipulations designed to improve cancer therapy. The current proposal will test the hypothesis that mitochondrial production of reactive oxygen species (ROS; i.e., superoxide and hydrogen peroxide) mediates the increased susceptibility of human cancer cells to glucose deprivation-induced metabolic oxidative stress, relative to normal cells. Specific Aim 1 will determine using electron transport chain blockers (i.e., antimycin A, myxothiazol, and rotenone), if intact human cancer cells (or isolated mitochondria) demonstrate alterations in ROS production by mitochondrial electron transport chain Complexes I, II, and/or III that contribute to increased susceptibility to glucose deprivation-induced oxidative stress, relative to normal cells. Specific Aim 2 will determine if rho(0) cancer cells, deficient in functional mitochondrial electron transport chains demonstrate altered susceptibility to glucose deprivation-induced cytotoxicity and oxidative stress, relative to parental rho(+) cells containing fully functional electron transport chains. Specific Aim 3 will determine using adenoviral vectors as well as stably transfected cell lines, if over expession of antioxidant enzymes that scavenge superoxide and hydrogen peroxide (i.e., catalase, superoxide dismutases) are capable of altering the biological effects of glucose deprivation in cancer cells. Specific Aim 4 will determine if 2-deoxy-d-glucose is capable of mimicking the effects of glucose deprivation seen in Aims 1-3. The long-term goal is to provide a rigorous mechanistic understanding of the differential susceptibility of cancer cells to glucose deprivation-induced oxidative stress for the purpose of developing combined modality cancer therapy based on differences between oxygen metabolism in normal vs. cancer cells.