Thiol-mediated apoptosis in prostate carcinoma cells

BACKGROUND. Glutathione (GSH) maintains an optimum cellular redox potential . Chemical depletion, physical efflux from the cell, or intracellular redis tribution of this thiol antioxidant is associated with the onset of apoptos is. The aim of this study was to determine the effects of a thiol-depleting agent, diethylmaleate (DEM), on androgen sensitive and insensitive prostat e carcinoma cells. METHODS. LNCaP and PC-3 cell lines were induced to undergo apoptosis by DEM and diamide. Apoptosis was quantified by annexin V binding and propidium i odide incorporation using flow cytometry and was confirmed by DNA gel elect rophoresis. Intracellular GSH was quantified using a thiol quantitation kit and the generation of reactive oxygen intermediates was measured using dih ydrorhodamine 123. Western blot assessed caspase-3, caspase-8, Bcl-2, and B cl-X-L protein expression. Mitochondrial permeability was measured using Di OC(6) and stabilized using bongkrekic acid. RESULTS. DEM and diamide induced apoptosis in both androgen sensitive and i nsensitive cells. Apoptosis was also induced in an LNCaP transfectant cell line overexpressing Bcl-2. Apoptosis was caspase-3 dependent and caspase-8 independent. Bongkrekic acid partially prevented the effects of DER I on mi tochondrial permeability but was unable to prevent the induction of apoptos is. Decreased Bcl-2 and Bcl-X-L protein expression was observed at the time of initial caspase-3 activation. CONCLUSIONS. This study demonstrates that thiol depletion can be used as an effective means of activating caspase-3 in both androgen sensitive and ins ensitive prostate carcinoma cells. Direct activation of this effector caspa se may serve as a useful strategy for inducing apoptosis in prostate carcin oma cells. (C) 2000 American Cancer Society.