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 redistribution of this thiol antioxidant is associated with the onset of apoptosis. The aim of this study was to determine the effects of a thiol‐depleting agent, diethylmaleate (DEM), on androgen sensitive and insensitive prostate 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 iodide incorporation using flow cytometry and was confirmed by DNA gel electrophoresis. Intracellular GSH was quantified using a thiol quantitation kit and the generation of reactive oxygen intermediates was measured using dihydrorhodamine 123. Western blot assessed caspase‐3, caspase‐8, Bcl‐2, and Bcl‐XL protein expression. Mitochondrial permeability was measured using DiOC6 and stabilized using bongkrekic acid. RESULTS DEM and diamide induced apoptosis in both androgen sensitive and insensitive 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 DEM on mitochondrial permeability but was unable to prevent the induction of apoptosis. Decreased Bcl‐2 and Bcl‐XL 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 insensitive prostate carcinoma cells. Direct activation of this effector caspase may serve as a useful strategy for inducing apoptosis in prostate carcinoma cells. Cancer 2000;88:2092–104. © 2000 American Cancer Society. Thiol depletion of glutathione through diethylmaleate leads to the direct activation of caspase‐3 and apoptotic induction in both androgen sensitive and insensitive prostate carcinoma cells. Direct activation of this downstream effector caspase is independent of the overexpression of Bcl‐2 and may prove an effective strategy for inducing apoptosis in prostate carcinoma cells.