INVESTIGATIONS OF SHETA2 MEDIATED MITOCHONDRIA CHANGES IN HUMAN CANCER CULTURES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。新型抗癌药物SHetA 2直接与线粒体相互作用，导致线粒体膜变化、线粒体产生的超氧阴离子和最终凋亡。虽然我们实验室最近的实验表明，超氧化物的形成是一个相对较晚的事件在凋亡过程中，这些事件的顺序是不确定的。我们将研究线粒体密度和膜极化的变化，以及超氧化物的产生与SHets处理孤立的线粒体，并试图订购这些事件。为了测试线粒体衍生的ROS是否在线粒体膜去极化之前发生，我们将在不存在和存在线粒体靶向抗氧化剂的情况下用SHet处理线粒体。如果该模型是正确的，靶向抗氧化剂将防止SHet诱导的超氧化物生成，但不会防止线粒体肿胀或去极化。确定ROS产生和线粒体膜变化的顺序的另一个测试将是在不存在和存在环孢菌素A（CsA）（一种有效的mtPTP阻断剂）的情况下用SHetA 2处理分离的线粒体。如果我们的模型是正确的，CsA将阻止mtPTP以及ROS的产生。我们将确定超氧化物在人卵巢癌细胞中产生的位点。将用SHets和线粒体选择性荧光团MitoSOXTM Red处理组织培养物，MitoSOXTM Red是一种新型的荧光膜渗透剂，可快速靶向线粒体并通过超氧化物选择性氧化为红色染料。如果该模型是正确的，则添加靶向抗氧化剂将防止SHet诱导的超氧化物生成，但不会防止线粒体肿胀或mtPTP。

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.