Hypoxia inhibits human bladder smooth muscle cell proliferation: a potential mechanism of bladder dysfunction.

AIMS: Recent animal studies have suggested that bladder outflow obstruction causes bladder wall hypoxia during both the filling and the voiding phases of the micturition cycle. We have previously demonstrated that mechanical deformation of human detrusor leads to smooth muscle (SM) cell hypertrophy and hyperplasia, which may then contribute to hypoxia in the dysfunctional bladder. We hypothesise that the detrusor's response to a hypoxic environment contributes to bladder dysfunction. The aim of this study was to evaluate the effect of hypoxia on detrusor cell survival and growth. METHODS: Normal human detrusor muscle was obtained at radical cystectomy and primary cultures were established. Cells were then cultured in the presence of 1% oxygen in a hypoxic chamber for different times. Apoptosis was assessed by propidium iodide DNA staining and flow cytometry. Proliferation was assessed by radiolabelled thymidine incorporation. Cell supernatants were retained for growth factor estimation by enzyme linked immuno-sorbent assay (ELISA), and total cell and nuclear extracts were isolated for Western blotting. RESULTS: SM cells responded to the presence of hypoxia through significant upregulation of survival factors hypoxia inducible factor (HIF 1alpha) and vascular endothelial growth factor (VEGF) in a time-dependent manner. Hypoxia did not induce cell death, but significantly reduced the rate of proliferation over time, associated with an increase in the cell cycle inhibitor p27kip1. CONCLUSIONS: In an in vitro human detrusor cell culture model, cells demonstrate a resistance to hypoxia-induced apoptosis but proliferation is inhibited. We suggest that the anti-proliferative effects of hypoxia may limit the ability of detrusor cells to respond to, and compensate for, alterations in their environment contributing to bladder dysfunction.

Priming prostate carcinoma cells for increased apoptosis is associated with up-regulation of the caspases.

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.

Caspase 3 expression in benign prostatic hyperplasia and prostate carcinoma.

BACKGROUND: Apoptotic resistance to androgen ablation represents a significant problem in the treatment of prostate cancer. Over expression of antiapoptotic proteins such as Bcl-2 and mutations in p53 contribute to this resistance. The caspase family of proteases are central executioners of the cell death pathway. They are expressed in normal prostate secretory epithelial cells. Altered expression may represent an additional component leading to cell resistance. The aim of this study was to determine by immunohistochemistry caspase 3 expression in benign prostatic hyperplasia and prostate cancers. METHODS: Twenty-two patients with histologically determined prostate cancer and benign prostatic hyperplasia (BPH) were investigated. All specimens were obtained from patients undergoing surgical resection of the prostate. Immunohistochemical analysis was performed on formalin fixed paraffin embedded sections to assess caspase 3 expression. RESULTS: Caspase 3 was expressed in 18/22 (81.1%) samples, with high expression in BPH which demonstrated staining in both basal and secretory epithelial cells. Increasing grades of prostatic cancer showed a significant loss of expression in secretory epithelial layers and little staining in epithelial cells in high-grade prostatic carcinoma. CONCLUSIONS: Altered caspase 3 expression may represent an additional mechanism of apoptotic resistance to androgen ablation. Prostate 47:183-188, 2001.

Signaling for the caspases: their role in prostate cell apoptosis.

PURPOSE: The caspases are an evolutionary conserved family of cell death proteases. Their activation during apoptosis is an important underlying theme in prostate cancer therapy. We summarize the signaling pathways leading to the recruitment of the caspases and address the importance of recent therapeutic strategies aimed at specifically targeting these proteases in relation to prostate cancer. MATERIALS AND METHODS: We present a background introduction into the role of the caspases in apoptosis and how failure to signal effectively their activation may contribute to prostate cancer progression. Key studies aimed at specifically targeting the caspases as cancer therapy are discussed. RESULTS: Prostate carcinogenesis and apoptosis are related. The deregulation of apoptosis contributes to tumor initiation, metastasis and progression to the androgen insensitive state. Conversely the effectiveness of therapy often depends on its ability to induce apoptosis in prostate cancer cells. Identifying abnormalities in the apoptotic signaling pathway has greatly contributed to understanding the biology of prostate cancer. Elucidating caspase regulation has contributed to the design of novel therapies for prostate cancer. CONCLUSIONS: We summarize the physiological and pathological pathways leading to caspase activation in the prostate and describe novel approaches that target these proteases.

Glutathione depletion-induced neutrophil apoptosis is caspase 3 dependent.

Resolving inflammation is a vital step in preventing the persistence of inflammatory disorders. Neutrophils play a major role in tissue damage associated with an inflammatory response. Their death by apoptosis is central to the final resolution of this response. Thiol depletion with diethylmaleate (DEM) or diamide represent important triggers for neutrophil apoptosis. The mechanism by which this process occurs remains unknown. The apoptotic cascade is associated with a number of cellular changes, including caspase activation and mitochondrial permeability. The aims of this study were to determine the role of mitochondrial permeability and the caspase cascade in thiol depletion-induced neutrophil apoptosis. Total cellular glutathione was reduced by DEM and diamide. This reduction was associated with neutrophil apoptosis and an increase in caspase 3 activity. The effects of DEM were blocked by the caspase 3 inhibitor, Z-DEVD-FMK. Mitochondrial permeability that occurred was also increased during this induction of apoptosis. Bongkrekic acid, a mitochondrial membrane stabilizer, inhibited DEM-induced apoptosis. The inhibitors' effects of LPS or GM-CSF on spontaneous neutrophil apoptosis was reversed by DEM, which was mediated by an increase in caspase 3 activity and independent of mitochondrial disruption. Caspase activation is an important step in glutathione depletion-induced apoptosis in resting and inflammatory neutrophils. Regulation of caspase activity may represent a possible target to trigger apoptosis and resolve inflammatory disorders.

Heat-shock proteins inhibit induction of prostate cancer cell apoptosis.

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

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.