diethyl-maleate and Prostatic-Neoplasms

The potential to prime prostatic carcinoma cell lines for apoptosis represents an exciting strategy for the treatment of patients with this disease. The ability and the underlying molecular mechanisms involved in sensitizing both androgen-sensitive and androgen-insensitive cell types to a range of apoptotic-inducing agents are investigated by the authors.. Primary and secondary cell lines were pretreated with diethyl-maleate (DEM) prior to the induction of apoptosis by Fas antibody (1 microg/mL), cycloheximide (1 microg/mL), etoposide (62.5 microM), and radiation (5 grays). It was demonstrated previously that DEM (50 microM) increases the sensitivity to apoptosis induced by these agents. The effects of DEM on both protein and RNA expression was determined by Western blot analysis and a ribonuclease protection assay, respectively. The effects of DEM on intracellular glutathione (GSH) levels and its intracellular distribution also were assessed.. DEM did not affect the expression of the caspases at the transcriptional level but was associated with increased procaspase-3 and caspase-8 protein levels. DEM preincubation restored sensitivity to Fas antibody and radiation-induced apoptosis in cells from the LNCaP-bcl-2 transfectant cell line that, normally, are resistant to these apoptotic stimuli. It is that DEM chemically depletes intracellular thiol levels. Although no depletion in total intracellular thiol GSH was observed at these concentrations of DEM, trafficking of GSH from the nucleus to the cytosol was demonstrated.. Identification of the caspases as a potential target for chemical manipulation may serve as an effective, adjuvant-based approach in the treatment of patients with prostate carcinoma and, in particular, for immunotherapy and radiation-based strategies that rely on the activation of these death-effector proteases.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma; Caspases; Etoposide; Genes, bcl-2; Glutathione; Humans; Immunotherapy; Intracellular Fluid; Male; Maleates; Prostatic Neoplasms; Radiotherapy; Sulfhydryl Compounds; Tumor Cells, Cultured; Up-Regulation

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.. 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.. 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.. 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.

Topics: Annexin A5; Anti-Bacterial Agents; Apoptosis; bcl-X Protein; Bongkrekic Acid; Carcinoma; Caspase 3; Caspase 8; Caspase 9; Caspases; Coloring Agents; Diamide; DNA, Neoplasm; Enzyme Inhibitors; Enzyme Precursors; Glutathione; Humans; Male; Maleates; Mitochondria; Oxidation-Reduction; Propidium; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Androgen; Sulfhydryl Reagents; Tumor Cells, Cultured

Resistance to apoptosis remains a significant problem in the treatment of prostate cancer. Heat-shock proteins (HSP) have been correlated with tumor progression. The role of HSP in prostate cancer resistance to apoptosis is unknown.. PC-3 and LNCaP prostate cancer cells were heat-shocked and then treated with or without diethyl-maleate, etoposide, cycloheximide, or 3 Gray irradiation. Percent apoptosis was assessed by propidium iodide DNA incorporation. Protein was also extracted for analysis by SDS-PAGE Western blotting.. Western blotting confirmed an increase in HSP 27 and 72. These cells were resistant to both chemical- and radiation-induced apoptosis. Cycloheximide and specific oligonucleotides to HSP 72 blocked the increased expression of HSP 72 and the resistance to apoptosis. Mcl-1, Bcl-2, Bcl-X(L), and glutathione-S-transferase (GST) expression were increased in a time-dependent manner after heat shock.. This study demonstrates that HSP expression, specifically HSP 72, inhibits apoptosis in prostate tumor cell lines, which may be mediated by the production of survival factors.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Blotting, Western; Cycloheximide; Etoposide; Glutathione; Glutathione Transferase; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Kinetics; Male; Maleates; Molecular Chaperones; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Nucleic Acid Synthesis Inhibitors; Prostatic Neoplasms; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Tumor Cells, Cultured