INVESTIGATIONS OF SHETA2 MEDIATED MITOCHONDRIA CHANGES IN HUMAN CANCER CULTURES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The novel anticancer drug SHetA2 directly interacts with the mitochondria resulting in mitochondrial membrane changes, mitochondrial-generated superoxide anion and ultimately apoptosis. While recent experiments in our laboratory indicate that superoxide formation is a relatively late event in the apoptotic process, the order of these events is uncertain. We will investigate mitochondrial density and membrane polarization changes, as well as superoxide generation in isolated mitochondria treated with SHets and attempt to order these events. To test if mitochondrial derived ROS occurs prior to mitochondrial membrane depolarization we will treat mitochondria with SHets in the absence and presence of mitochondrial targeted antioxidants. If the model is correct, targeted antioxidants will prevent SHet induced superoxide generation but not mitochondrial swelling or depolarization. Another test to determine the order of ROS generation and mitochondrial membrane changes will be to treat isolated mitochondria with SHetA2 in the absence and presence of Cyclosporin A (CsA), a potent blockers of the mtPTP. If our model is correct, CsA will prevent mtPTP as well as ROS generation. We will determine the site of superoxide generation in human Ovarian Cancer cells. Tissue cultures will be treated with SHets and the mitochondria selective fluorophore MitoSOXTM Red a novel, fluorogenic, membrane permeant that is rapidly targeted to the mitochondria and selectively oxidized to a red dye by superoxide. If the model is correct the addition of targeted antioxidants will prevent SHet induced superoxide generation but not mitochondrial swelling or mtPTP.