Mechanisms of Dicumarol-Induced Cytotoxicity

描述（由申请方提供）：双香豆素是一种天然存在的抗凝剂，从甜三叶草中获得。虽然双香豆素等化合物已用于癌症治疗，但对这些药物的作用机制知之甚少。线粒体被假设为双香豆素处理期间促氧化剂产生的位点，因为双香豆素被认为影响醌介导的电子转移反应，导致超氧化物（O2-）和过氧化氢（H2 O2）的产生。双香豆素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，这些差异适用于旨在改善癌症治疗的操作。为了获得对胰腺癌细胞中二香豆素诱导的氧化应激的机制性理解，目前的提议将测试以下假设：相对于正常人类细胞，活性氧（ROS）的线粒体产生介导胰腺癌细胞对二香豆素诱导的代谢氧化应激的易感性增加。我们计划通过追求以下三个具体目标来测试我们的假设：具体目标1：确定线粒体O2-和/或H2 O2是否介导胰腺癌细胞中双香豆素治疗所见的细胞毒性和代谢氧化应激。使用腺病毒载体进行瞬时转导以增加肿瘤细胞抗氧化酶的表达，将用于确定特定活性氧在双香豆素诱导的细胞毒性和氧化应激中的作用。具体目标二：确定相对于含有完全功能性线粒体电子传递链的亲代rho（+）细胞，功能性线粒体电子传递链缺陷的rho（0）人癌细胞是否表现出对双香豆素诱导的细胞毒性和氧化应激的敏感性改变。具体目标3：使用电子传递链阻断剂确定癌细胞（相对于正常人细胞）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，这可能导致对双香豆素诱导的代谢氧化应激的敏感性增加。如果可以严格证明，与正常人细胞类型相比，双香豆素诱导的人胰腺癌细胞优先细胞毒性和氧化应激依赖于线粒体代谢产生的ROS增加，所提出的实验可以提供证据，支持基于正常与胰腺癌细胞中氧化代谢的生物化学之间的根本差异的联合模式癌症治疗的合理设计。

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.