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 Bci-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.
Coffey
R N
RN
Department of Surgery, University College Dublin, Mater Misericordiae Hospital, Ireland.
Watson
R W
RW
Hegarty
N J
NJ
O'Neill
A
A
Gibbons
N
N
Brady
H R
HR
Fitzpatrick
J M
JM
D017304:Annexin A5 / Q000187:drug effects; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001865:Bongkrekic Acid / Q000494:pharmacology; D002277:Carcinoma / Q000473:pathology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000032:analysis; D004396:Coloring Agents; D004273:DNA, Neoplasm / Q000032:analysis; D003958:Diamide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism; D004792:Enzyme Precursors / Q000032:analysis; D005978:Glutathione / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008297:Male; D008298:Maleates / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects; D010084:Oxidation-Reduction; D011419:Propidium; D011471:Prostatic Neoplasms / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D011944:Receptors, Androgen / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein
D017304:Annexin A5; D000900:Anti-Bacterial Agents; C494811:BCL2L1 protein, human; D004396:Coloring Agents; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D008298:Maleates; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011944:Receptors, Androgen; D013439:Sulfhydryl Reagents; D051020:bcl-X Protein; D003958:Diamide; D001865:Bongkrekic Acid; D011419:Propidium; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C014476:diethyl maleate; D005978:Glutathione