Mechanisms of Dicumarol-Induced Cytotoxicity

描述（由申请人提供）：双umarol是从甜三叶草中提取的天然抗凝血剂。虽然双酚醇等化合物已被用于癌症治疗，但对这些药物的作用机制知之甚少。线粒体被假设是在双豆醇治疗过程中产生促氧化剂的部位，因为双豆醇被认为会影响醌介导的电子转移反应，导致超氧化物（O2-）和过氧化氢（H2O2）的产生。双异丙醇诱导的氧化应激可能代表肿瘤细胞和正常细胞线粒体代谢之间的差异，这种差异适用于旨在改善癌症治疗的操作。为了进一步了解双酚醇诱导胰腺癌细胞氧化应激的机制，本研究将验证线粒体产生活性氧（ROS）介导胰腺癌细胞相对于正常人类细胞对双酚醇诱导的代谢氧化应激的易感性增加的假设。我们计划通过追求以下三个特定目标来验证我们的假设：特定目标1：确定线粒体O2-和/或H2O2是否介导胰腺癌细胞的细胞毒性和代谢氧化应激。利用腺病毒载体瞬时转导增加肿瘤细胞中抗氧化酶的表达，将用于确定特异性ROS在双酚醇诱导的细胞毒性和氧化应激中的作用。特异性目标2：确定缺乏功能性线粒体电子传递链的rho(0)人癌细胞是否表现出对双酚醇诱导的细胞毒性和氧化应激的易感性改变，相对于具有完全功能线粒体电子传递链的亲本rho（+）细胞。特异性目标3：使用电子传递链阻滞剂确定癌细胞（相对于正常人类细胞）是否表现出线粒体电子传递链复合物I、II和/或III产生ROS的改变，这可能有助于增加双酚醇诱导的代谢氧化应激的易感性。如果能够严格证明双糖醇诱导的人类胰腺癌细胞相对于正常人类细胞类型的优先细胞毒性和氧化应激依赖于线粒体代谢产生ROS的增加，那么所提出的实验可以提供证据，支持基于正常细胞与胰腺癌细胞氧化代谢生物化学的根本差异，合理设计联合模式的癌症治疗。

DESCRIPTION (provided by applicant): Dicumarol is a naturally occurring anticoagulant obtained from sweet clover. Though such compounds as dicumarol have been utilized in cancer therapy, little is known about the mechanism of action of these drugs. Mitochondria have been hypothesized to be the site of prooxidant production during dicumarol treatment since dicumarol is thought to affect quinone-mediated electron transfer reactions leading to the production of superoxide (O2-), and hydrogen peroxide (H2O2). Dicumarol-induced oxidative stress could represent a difference between tumor cell and normal cell mitochondrial metabolism amenable to manipulations designed to improve cancer therapy. To gain a mechanistic understanding of dicumarol-induced oxidative stress in pancreatic cancer cells, the current proposal will test the hypothesis that mitochondrial production of reactive oxygen species (ROS) mediates the increased susceptibility of pancreatic cancer cells to dicumarol-induced metabolic oxidative stress, relative to normal human cells. We plan to test our hypothesis by pursuing the following three specific aims: Specific Aim 1: Determine if mitochondrial O2- and/or H2O2 mediate the cytotoxicity and metabolic oxidative stress seen with dicumarol treatment in pancreatic cancer cells. Transient transduction using adenoviral vectors to increase tumor cell expression of antioxidant enzymes will be used to determine the role of specific ROS in dicumarol-induced cytotoxicity and oxidative stress. Specific Aim 2: Determine if rho (0) human cancer cells, deficient in functional mitochondrial electron transport chains demonstrate altered susceptibility to dicumarol-induced cytotoxicity and oxidative stress, relative to parental rho (+) cells containing fully functional mitochondrial electron transport chains. Specific Aim 3: Determine using electron transport chain blockers if cancer cells (relative to normal human cells) demonstrate alterations in production of ROS by mitochondrial electron transport chain Complexes I, II, and/or III that could contribute to increased susceptibility to dicumarol-induced metabolic oxidative stress. If it could be rigorously demonstrated that dicumarol-induced preferential cytotoxicity and oxidative stress in human pancreatic cancer cells vs. normal human cell types was dependent on increased production of ROS by mitochondrial metabolism, the proposed experiments could provide evidence supporting the rational design of combined modality cancer therapy based on a fundamental difference between the biochemistry of oxidative metabolism in normal vs. pancreatic cancer cells.