Inhibition of glutathione reductase and ovarian cancer drug resistance reversal

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DESCRIPTION (provided by applicant): Ovarian cancer is the leading cause of death among gynecological malignancies in the United States. Drug resistance is a major cause of failure in ovarian cancer treatment. Therefore, developing novel approaches to combat ovarian cancer drug resistance has been an extensive and ongoing research effort. An increase in intracellular glutathione (GSH) has been demonstrated to be the major factor in ovarian cancer drug resistance to the first line and second line ovarian cancer chemotherapeutic agents such as cisplatin and doxorubicin. We have obtained preliminary data with a GSH-mediated drug resistant human ovarian cancer cell line (OVCAR-3) showing that inhibition of glutathione reductase (GR) and a combined inhibition of GR and gamma-glutamylcysteine synthetase (GCS) that catalyzes the first and rate-determining step in GSH biosynthesis significantly increased the sensitivity of the cell line to melphalan, a chemotherapeutic agent. GR is an enzyme catalyzing the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). Inhibition of this enzyme caused both reduction of intracellular GSH and accumulation of GSSG resulting in an increased cellular oxidative stress. This application is aimed to investigate whether inhibition of GR or a combined inhibition of GR and GCS could be effective approaches in combating GSH-mediated ovarian cancer drug resistance to the first line and second line chemotherapeutic agents. In addition, biochemical parameters that might be affected by the inhibition and might contribute to drug resistance reversal will be determined to help understand drug resistance reversal mechanism. These biochemical parameters include intracellular activities of GR, GCS, glutathione synthetase, and glutathione S-transferase (GST), intracellular levels of GSH, GSSG, total thiols and total disulfides. Further, potential toxicity GR inhibition and a combined inhibition of GR and GCS will be investigated. The project includes in vitro (with a cell line) and in vivo (with mice) studies. OVCAR-3 cells will be used as a drug resistance model cell line in the investigation. G0026, a potent irreversible GR inhibitor recently developed in our laboratory, and buthionine sulfoximine (BSO), an inhibitor of GCS, will be used to test the hypothesis. Cisplatin and doxorubicin will be employed respectively as representative first line and second line ovarian cancer chemotherapeutic agents. Completion of this project will enable us to determine whether GR inhibition and/or a combined inhibition of GR and GSH biosynthesis would have a promising potential as a novel approach in combating ovarian cancer resistance to chemotherapy. The long term objective of this project is to extend these approaches to other GSH-mediated drug resistant cancers.