diethyl-maleate and Carcinoma

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

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Topics: Animals; Breast Neoplasms; Bridged Bicyclo Compounds; Buthionine Sulfoximine; Carcinoma; Carcinosarcoma; CHO Cells; Colonic Neoplasms; Cricetinae; Ethylmaleimide; Eukaryotic Cells; Evaluation Studies as Topic; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Glutathione; Humans; Maleates; Mammary Neoplasms, Experimental; Methionine Sulfoximine; Mice; o-Phthalaldehyde; Phenylmercury Compounds; Pyrazoles; Species Specificity; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Topics: Animals; Anticarcinogenic Agents; Antioxidants; Benzodioxoles; Butylated Hydroxyanisole; Caffeic Acids; Carcinogens; Carcinoma; Catechols; Cell Division; Hydroquinones; Kidney Neoplasms; Male; Maleates; Methylnitronitrosoguanidine; Organ Specificity; Papilloma; Phenols; Rats; Rats, Inbred F344; Stomach; Stomach Neoplasms

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.

Topics: Animals; Buthionine Sulfoximine; Carcinoma; Cell Line; Cells, Cultured; Cricetinae; Dose-Response Relationship, Radiation; Glutathione; Humans; Hypoxia; Lung; Lung Neoplasms; Maleates; Methionine Sulfoximine; Oxygen Consumption; Radiation-Sensitizing Agents; Time Factors