Download (4-HC) and Glutathione (GSH)

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4-Hydroxycyclophosphamide (4-HC) and Glutathione (GSH) Depletion Induces Apoptosis
in Human COV434 Granulosa Cells
Brian Luong
Mentor: Ulrike Luderer
Glutathione depletion induces apoptosis in many cells. We have shown that GSH depletion induces
granulosa cell apoptosis in rat antral follicles. We have also shown that the anti-cancer drug
cyclophosphamide induces apoptosis in granulosa cells of rat follicles. However, the effects of
cyclophosphamide and GSH depletion on human granulosa cells have not been elucidated. Using the
COV434 human granulosa cell line, we tested the following hypotheses: 1) 4-HC, a preactivated form
of cyclophosphamide, induces apoptosis; 2) GSH depletion induces apoptosis; and 3) GSH depletion
enhances the apoptotic effects of 4-HC. Cells treated with 100 µM buthionine sulfoximine (BSO), a
specific inhibitor of GSH synthesis, were assessed for apoptosis by terminal
deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) and Hoechst 33342
staining. Additionally, WST-1 assay was performed in each experiment to assess cell viability. GSH
depletion induced apoptosis within 24h. Next, cells were treated with 1-50 µM 4-HC. 4-HC
treatment, which resulted in dose-dependent induction of apoptosis within 12h. Combined treatment
with BSO and 4-HC resulted in greater levels of apoptosis than with either treatment alone. In all
experiments, a decrease in cell viability was observed simultaneously with apoptosis. We then tested
if 4-HC induced apoptosis via the mitochondrial pathway by looking at the presence of specific
molecular markers such as Bax expression and caspase-3 activation. There were increased levels of
Bax and cleaved caspase-3 proteins in cells treated for 12h with 4-HC relative to control. These
findings suggest that GSH plays a role in protecting human granulosa cells against endogenous and
4-HC induced apoptosis.