Abstract
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.
Highlights
The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation
COV434 cells are responsive to follicle stimulating hormone (FSH) stimulation in our hands, as indicated by the increase in estradiol synthesis when treated with 10lM androstenedione and 200 ng/ml FSH (Fig. 1)
FSH treatment appeared to modestly increase intracellular GSH levels at 24 h, but not at later time points (Fig. 2A). This contrasts with the pronounced stimulatory effect of FSH we have observed on GSH synthesis in cultured rat preovulatory follicles (Tsai-Turton and Luderer, 2006)
Summary
The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation. 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. We have shown that the destruction of granulosa cells by cyclophosphamide in rats treated in vivo occurs by the induction of apoptosis (Lopez and Luderer, 2004) Both the death receptor and the mitochondrial apoptotic pathways have been implicated in the induction of apoptosis by cyclophosphamide in developing embryos (Little and Mirkes, 2002; Mirkes and Little, 2000) and hair follicles (Lindner et al, 1997). We describe experiments conducted using these cells to test the hypotheses that (1) GSH depletion and cyclophosphamide treatment induce apoptosis in human granulosa cells, (2) GSH depletion sensitizes human granulosa cells to the induction of apoptosis by cyclophosphamide, and (3) cyclophosphamide treatment causes GSH depletion and oxidative stress in granulosa cells, which mediate the induction of apoptosis
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