Mechanisms of Dicumarol-Induced Cytotoxicity

描述(申请人提供)：双库马罗尔是一种天然的抗凝血剂，从甜三叶草中提取。虽然像双库马罗尔这样的化合物已经用于癌症治疗，但人们对这些药物的作用机制知之甚少。在双黄酚治疗过程中，线粒体被认为是促氧化剂的产生部位，因为双黄酚被认为影响了苯二酚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。Dicumarol诱导的氧化应激可能代表了肿瘤细胞和正常细胞线粒体代谢的差异，这些细胞线粒体代谢适用于旨在改善癌症治疗的操作。为了从机制上了解双黄酚诱导的胰腺癌细胞的氧化应激，目前的提议将检验一种假设，即相对于正常人类细胞，线粒体产生的活性氧物种(ROS)介导了胰腺癌细胞对双黄酚诱导的代谢性氧化应激的易感性增加。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是否介导了双库马罗尔治疗胰腺癌细胞的细胞毒性和代谢氧化应激。用腺病毒载体瞬时转导肿瘤细胞表达抗氧化酶将被用来确定特定的ROS在双酚A诱导的细胞毒性和氧化应激中的作用。具体目标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相对于含有完全功能线粒体电子传递链的亲代Rho(+)细胞对双卡马罗尔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体I、II和/或III产生ROS的变化，这种变化可能有助于增加对双库马罗尔诱导的代谢性氧化应激的敏感性。如果能够严格地证明双黄素对人胰腺癌细胞的优先细胞毒性和氧化应激依赖于线粒体代谢产生的ROS的增加，那么本实验可以为基于正常细胞和胰腺癌细胞氧化代谢生化的根本差异而设计合理的联合治疗方案提供证据。

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.