Tsai-Turton Miyun, Luong Brian T, Tan Youming, Luderer Ulrike
Department of Community and Environmental Medicine, University of California, Irvine, California 92617, USA.
Toxicol Sci. 2007 Jul;98(1):216-30. doi: 10.1093/toxsci/kfm087. Epub 2007 Apr 13.
The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation. We previously showed that both cyclophosphamide treatment and GSH depletion induced granulosa cell apoptosis in rats, but the role of GSH in apoptosis in human ovarian cells has not been studied. Using the COV434 human granulosa cell line, we tested the hypotheses that (1) GSH depletion or treatment with 4-hydroperoxycyclophosphamide (4HC), a preactivated form of cyclophosphamide, induces apoptosis, (2) GSH depletion potentiates 4HC-induced apoptosis, and (3) 4HC-induced apoptosis is mediated by GSH depletion and oxidative stress. Cells were treated with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, with or without follicle stimulating hormone (FSH) or serum. A significant increase in the number of apoptotic cells, assessed by terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling (TUNEL) and Hoechst 33342 staining, occurred with BSO treatment. Treatment with 4HC dose-dependently induced apoptosis by TUNEL, Hoechst staining, and caspase 3 activation. Treatment with 4HC caused an increase in reactive oxygen species generation, measured by dichlorofluorescein fluorescence, oxidative DNA damage, measured by 8-hydroxyguanosine immunostaining, and an oxidation of the redox potential for the oxidized glutathione/reduced glutathione couple. Total intracellular GSH declined after 4HC treatment, preceding the onset of cell death. Treatment with antioxidants inhibited 4HC-induced apoptosis. Combined treatment with BSO and 4HC caused greater induction of apoptosis than either treatment alone. These findings are consistent with roles for oxidative stress and GSH depletion in mediating the induction of apoptosis in COV434 cells by cyclophosphamide.
抗癌药物环磷酰胺可诱导颗粒细胞凋亡，并通过谷胱甘肽（GSH）结合作用进行解毒。我们之前的研究表明，环磷酰胺处理和GSH耗竭均可诱导大鼠颗粒细胞凋亡，但GSH在人卵巢细胞凋亡中的作用尚未得到研究。利用COV434人颗粒细胞系，我们检验了以下假设：（1）GSH耗竭或用环磷酰胺的预活化形式4-氢过氧环磷酰胺（4HC）处理可诱导凋亡；（2）GSH耗竭会增强4HC诱导的凋亡；（3）4HC诱导的凋亡是由GSH耗竭和氧化应激介导的。用谷胱甘肽合成的特异性抑制剂丁硫氨酸亚砜胺（BSO）处理细胞，同时或不添加促卵泡激素（FSH）或血清。通过末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记（TUNEL）和Hoechst 33342染色评估，BSO处理后凋亡细胞数量显著增加。用4HC处理可通过TUNEL、Hoechst染色和半胱天冬酶3激活剂量依赖性地诱导凋亡。用4HC处理会导致通过二氯荧光素荧光测量的活性氧生成增加、通过8-羟基鸟苷免疫染色测量的氧化性DNA损伤增加以及氧化型谷胱甘肽/还原型谷胱甘肽对的氧化还原电位氧化。4HC处理后细胞内总GSH下降，早于细胞死亡的发生。用抗氧化剂处理可抑制4HC诱导的凋亡。BSO和4HC联合处理比单独任何一种处理诱导的凋亡程度更大。这些发现与氧化应激和GSH耗竭在介导环磷酰胺诱导COV434细胞凋亡中的作用一致。
