ABSTRACT
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
