Fellowship for Zhang: The Role of Mrp1 in Protecting against Doxorubicin - induced Cardiotoxicity

Normal tissue injury is a significant clinical problem that limits the use of otherwise effective cancer chemotherapeutic drugs. Doxorubicin (DOX) is a commonly used chemotherapeutics in the United States for a variety of malignant tumors, including breast cancer, leukemia, lymphoma, and sarcomas. However, it induces severe and dose-limiting cardiac toxicity. Although the mechanism of DOX cardiac toxicity has not been completely elucidated, it is believed that the superoxide anion and other reactive oxygen species (ROS) generated by DOX contributes significantly to cardiac pathology. Therefore, to understand the self-regulation and defense mechanisms of heart tissue under the oxidative stress may provide us new approaches to alleviate the clinical cardiotoxicity of chemotherapy. MRP1 (multidrug resistance associated protein 1) is a member of the C subfamily of the ATP binding cassette (ABC) transport proteins. It is ubiquitously expressed, especially in heart, skin, lung, brain capillary endothelial cells, and the small intestine, mediating the efflux of glutathione (GSH), glutathione disulfide (GSSG), multiple drugs and their conjugates, etc.. The long-term goal of the present application is to investigate the role of Mrp1 in protecting against DOX-induced cardiotoxicity. Here, we will test the hypothesis that Mrp1 is responsible for exporting the toxic products of oxidative stress following DOX treatment, thus alleviating the oxidative damage to the heart tissue. Three specific aims will be undertaken. Specific Aim 1: Characterize the role of Mrp1 in protecting the heart from DOX-induced injury in vivo. We will use the C57BL/6J (WT) and Mrp1-/- (KO) mouse as the animal model. Cardiac injury will be evaluated through ultrastructural examination, echocardiogram and cardiac injury serum markers. Specific Aim 2: Characterize the expression, subcellular localization and transport activity of Mrp1 in cultured neonatal mouse cardiomyocytes. Specific Aim 3: Characterize the mechanism of Mrp1 mediated cardioprotection using in vitro cultured cardiomyocytes. Specifically, cytotoxicity and apoptosis will be measured to determine the effect of Mrp1 on cell sensitivity of DOX. While oxidative stress in cultured WT and KO cardiomyocytes will be evaluated through the measurement of GSH/GSSG ratio, oxidative toxic products including GSSG and GS-HNE level and protein oxidation. We will also study the mitochondrial damage induced by DOX and the role of Mrp1 during this process. Overall, the present proposal will directly determine whether Mrp1 confers protection against DOX cardiotoxicity. It may provide novel insight into the role of Mrp1 in oxidative stress regulation, and thereby give us critical information regarding the potential adverse sequelae of introduction of MRP1 inhibitors as adjuncts to clinical chemotherapy of multidrug resistant tumors.