Inhibition of glutathione reductase and ovarian cancer drug resistance reversal

描述（由申请人提供）：卵巢癌是美国妇科恶性肿瘤中的主要死亡原因。耐药性是卵巢癌治疗失败的主要原因。因此，开发对抗卵巢癌耐药性的新方法一直是一项广泛和持续的研究工作。细胞内谷胱甘肽（GSH）的增加已被证明是卵巢癌对一线和二线卵巢癌化疗药物（如顺铂和多柔比星）耐药的主要因素。我们已经获得了GSH介导的耐药人卵巢癌细胞系（OVCAR-3）的初步数据，显示谷胱甘肽还原酶（GR）的抑制和GR和γ-谷氨酰半胱氨酸合成酶（GCS）的联合抑制（催化GSH生物合成的第一步和速率决定步骤）显著增加了细胞系对化疗药物美法仑的敏感性。GR是一种催化氧化型谷胱甘肽（GSSG）还原为还原型谷胱甘肽（GSH）的酶。这种酶的抑制引起细胞内GSH的减少和GSSG的积累，导致细胞氧化应激增加。本申请旨在研究GR的抑制或GR和GCS的联合抑制是否可以是对抗GSH介导的卵巢癌对一线和二线化疗药物的耐药性的有效方法。此外，将确定可能受抑制影响并可能有助于耐药性逆转的生化参数，以帮助了解耐药性逆转机制。这些生化参数包括GR、GCS、谷胱甘肽合成酶和谷胱甘肽S-转移酶（GST）的细胞内活性，GSH、GSSG、总巯基和总二硫化物的细胞内水平。此外，将研究潜在毒性GR抑制以及GR和GCS的联合抑制。该项目包括体外（细胞系）和体内（小鼠）研究。OVCAR-3细胞将用作研究中的耐药模型细胞系。G 0026，一个强大的不可逆的GR抑制剂，在我们的实验室最近开发的，和丁噻呋亚砜亚胺（BSO），一个抑制剂的GCS，将被用来测试的假设。顺铂和阿霉素将分别用作代表性的一线和二线卵巢癌化疗药物。该项目的完成将使我们能够确定GR抑制和/或GR和GSH生物合成的联合抑制是否具有作为对抗卵巢癌化疗耐药性的新方法的有前途的潜力。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.