Oxidative Pentose Cycle in Hypoxic Cancer Cell Response

描述(申请人提供)：在体外和体内，低氧在癌细胞对DNA损伤剂的抗性中的作用已经被清楚地证明。一些临床研究也证明了低氧肿瘤的治疗效果不佳。DNA修复、修复蛋白KF和细胞内GSH解毒在细胞对DNA损伤拓扑异构酶II抑制剂和伽马辐射的反应中起主要作用。虽然这些因子的功能可以被功能性硫醇的氧化抑制，但细胞通过氧化戊糖磷酸循环(OPPC)产生NADPH来抵抗硫醇氧化还原变化。例如，羟乙基二硫化物(HEDS)是一种独特的无毒硫醇氧化剂，在用OPPC修饰正常CHO细胞中的硫醇氧化还原过程中并不有效。我们的初步研究首次表明，OPPC活性受损的CHO突变体是HEDS介导的DNA损伤剂增敏的理想候选者。OPPC的这一独特性质消除了HEDS介导的硫醇氧化还原修饰和DNA损伤敏化，将被用于在不影响葡萄糖熟练的正常细胞的情况下，使缺乏葡萄糖的缺氧癌细胞增敏。葡萄糖缺乏在几种实体瘤的低氧细胞中很常见，这是因为血管生成不良和代谢活性较高。 我们假设，葡萄糖/OPPC耗竭的低氧癌细胞对HEDS介导的氧化还原[蛋白质硫醇(PSH)和谷胱甘肽(GSH)]修饰敏感，并通过抑制DNA修复、DNA修复蛋白功能、GSH解毒和抗细胞凋亡因子等多种机制增敏“和肿瘤和DNA损伤”Topo II抑制剂和伽马辐射。 我们将使用生化和分子方法在缺氧的人类癌细胞中检验我们的假设。首先，我们将确定硫醇氧化还原修饰在葡萄糖缺乏性缺氧癌细胞对Topo II抑制剂和伽马辐射增敏中的应用。还将进行其他研究，以测试HEDS在体内低糖缺氧性肿瘤的疗效。其次，利用siRNA和抗体技术，我们将确定OPPC的特定抑制剂是否也可以诱导HEDS介导的氧化还原修饰，以及随后缺氧癌细胞对DNA损伤剂的敏化。最后，我们将通过检测氧化还原修饰对DNA双链断裂、DNA修复蛋白KF、GSH解毒和促凋亡因子的影响来确定氧化还原修饰介导的低氧敏化的机制。

DESCRIPTION (provided by applicant): The role of hypoxia in the resistance of cancer cells to DNA damaging agents has been clearly demonstrated in in vitro and in vivo. Several clinical studies have also demonstrated poor therapeutic outcome for hypoxic tumors. DNA repair, repair protein Kf, and intracellular GSH detoxification play a major role in cellular response to DNA damaging topoisomerase II inhibitors and gamma radiation. Although the functions of these factors can be inhibited by oxidation of functional thiols, cells resist thiol redox changes by producing NADPH through oxidative pentose phosphate cycle (OPPC). For example hydroxyethyldisulfide (HEDS), a unique non-toxic thiol oxidant, is not effective in modifying the cellular thiol redox in normal CHO cells with OPPC. Our preliminary studies indicated, for the first time, that CHO mutants impaired with OPPC activity are the ideal candidates for HEDS-mediated sensitization to DNA damaging agents. This unique property of OPPC, which eliminates HEDS-mediated thiol redox modification and DNA damage sensitization, will be exploited to sensitize hypoxic cancer cells deprived of glucose, a substrate for OPPC, without affecting glucose proficient normal cells. Glucose deprivation is common in hypoxic cells of several solid tumors because of poor vascularization and higher metabolic activity. We hypothesize that glucose/OPPC depleted hypoxic cancer cells are susceptible to HEDS-mediated redox [Protein thiol (PSH) & glutathione (GSH)] modification and sensitization to "and tumor and DNA damaging" Topo II inhibitors and gamma radiation through multiple mechanisms that include inhibition of DNA repair, DNA repair protein function, GSH detoxification, and anti-apoptotic factors. We will test our hypothesis in hypoxic human cancer cells using biochemical and molecular approaches. First, we will determine the application of thiol redox modification in the sensitization of glucose deprived hypoxic cancer cells to topo II inhibitors and gamma radiation. Additional studies will also be conducted to test the efficacy of HEDS in low glucose hypoxic tumor in in vivo. Second, using siRNA and antibody technologies, we will determine whether specific inhibitors of OPPC can also induce HEDS-mediated redox modification, and subsequent sensitization of hypoxic cancer cells to DNA damaging agents. Finally, we will determine the mechanisms of redox modification- mediated hypoxic sensitization by determining its effect on DNA double strand breaks, DNA repair protein Kf, GSH detoxification and pro-apoptotic factors.