Inhibition of glutathione reductase and ovarian cancer drug resistance reversal

描述(申请人提供)：卵巢癌是美国妇科恶性肿瘤的主要死因。耐药性是卵巢癌治疗失败的主要原因。因此，开发新的方法来对抗卵巢癌的耐药性一直是一项广泛而持续的研究努力。细胞内谷胱甘肽(GSH)的增加是卵巢癌对顺铂和阿霉素等一线和二线化疗药物耐药的主要因素。我们用GSH介导的耐药人卵巢癌细胞株(OVCAR-3)获得的初步数据表明，抑制谷胱甘肽还原酶(GR)和联合抑制GR和催化GSH生物合成的第一步和速率决定步骤的谷氨酰半胱氨酸合成酶(GCS)显著增加了细胞对化疗药物马法兰的敏感性。GR是一种催化氧化谷胱甘肽(GSSG)还原为还原型谷胱甘肽(GSH)的酶。抑制这种酶会导致细胞内GSH的减少和GSSG的积累，从而导致细胞氧化应激的增加。本研究旨在探讨抑制GR或联合抑制GR和GCS是否是对抗GSH介导的卵巢癌对一线和二线化疗药物耐药性的有效途径。此外，还将确定可能受抑制影响并可能有助于耐药逆转的生化参数，以帮助了解耐药逆转机制。这些生化参数包括细胞内GR、GCS、谷胱甘肽合成酶和谷胱甘肽S转移酶的活性，细胞内GSH、GSSG、总硫醇和总二硫的水平。此外，潜在的毒性GR抑制以及GR和GCS的联合抑制将被研究。该项目包括体外(用细胞系)和体内(用小鼠)研究。本研究将以OVCAR-3细胞作为耐药模型细胞株。本实验室最近开发的一种有效的不可逆GR抑制剂G0026和GCS的抑制剂丁硫氨酸亚磺胺(BSO)将用于验证这一假说。顺铂和阿霉素将分别作为卵巢癌一线和二线的代表性化疗药物。这个项目的完成将使我们能够确定GR抑制和/或联合抑制GR和GSH生物合成是否有希望成为对抗卵巢癌化疗耐药的新方法。该项目的长期目标是将这些方法扩展到其他GSH介导的耐药癌症。

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.