Metabolic Oxidative Stress in Human Cancer Cells

描述(申请人提供)：自1920年S以来，人们观察到癌细胞(相对于正常细胞)表现出糖酵解和戊糖循环活性增加，以及呼吸速率略有下降，但这对癌症治疗的意义尚不清楚。最近的研究表明，与正常细胞相比，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]。线粒体被认为是葡萄糖剥夺过程中产生促氧化剂的场所。如果这是普遍的事实，那么葡萄糖剥夺诱导的氧化应激可能代表着肿瘤细胞线粒体代谢的缺陷，可以通过旨在改善癌症治疗的操作来进行。目前的提议将检验以下假设：相对于正常细胞，线粒体产生的活性氧物种(ROS；即超氧化物和过氧化氢)介导了人类癌细胞对葡萄糖剥夺诱导的代谢性氧化应激的敏感性增加。如果完整的人类癌细胞(或分离的线粒体)表现出线粒体电子传递链复合体I、II和/或III的ROS产生的变化，与正常细胞相比，线粒体电子传递链复合体I、II和/或III有助于增加对葡萄糖剥夺诱导的氧化应激的敏感性，特定目标1将确定使用电子传递链阻滞剂(即抗霉素A、霉唑和鱼藤酮)是否表明完整的人类癌细胞(或分离的线粒体)产生ROS的变化。具体目标2将确定Rho(0)癌细胞，缺乏功能性线粒体电子传递链，相对于含有完整功能电子传递链的亲代Rho(+)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。具体目标3将确定使用腺病毒载体和稳定转染的细胞系，过度表达清除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)是否能够改变癌细胞中葡萄糖剥夺的生物效应。特定的目标4将确定2-脱氧-d-葡萄糖是否能够模拟目标1-3中看到的葡萄糖剥夺的影响。其长期目标是提供对肿瘤细胞对葡萄糖剥夺诱导的氧化应激的不同易感性的严格的机制理解，以开发基于正常细胞和癌细胞之间的氧代谢差异的联合治疗方法。

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.