Resistance to caspase-dependent, hypoxia-induced apoptosis is not hypoxia-inducible factor-1 alpha mediated in prostate carcinoma cells.

BACKGROUND: Hypoxia occurs in association with cancer development, the result being a more aggressive and metastatic cancer phenotype. Hypoxia, which activates hypoxia-inducible factor-1 alpha (HIF-1alpha), is associated with a number of cellular changes including increased apoptotic resistance. The authors hypothesized that HIF-1alpha is central to the cell's ability to resist apoptosis induced during the hypoxia selection process. METHODS: PWR-1E, LNCaP, LNCaP-HOF, PC-3, and DU-145 cells were cultured in normoxic and hypoxic conditions. Apoptosis was assessed by propidium iodide DNA staining. Cleavage of specific substrates was used to assess caspase activity and Western blotting was used to assess mitochondrial release of cytochrome c and second mitochondria-derived activator caspase (SMAC)/Diablo. A dominant negative HIF-1alpha construct was transfected into the PC-3 and LNCaP cells to block HIF-1alpha activity. RESULTS: PC-3 and DU-145 were resistant to apoptosis induced by exposure to hypoxia, but the PWR-1E and LNCaP cells were susceptible. This induction of apoptosis in the LNCaP cells was caspase dependent but independent of cytochrome c release. Blocking the activity of HIF-1alpha had no effect on increased apoptotic susceptibility in the PC-3 cells. LNCaP-HOF cells, which were resistant to hypoxia-induced apoptosis, showed no increase in HIF-1alpha expression or activity. CONCLUSIONS: Apoptotic resistance is already established in cells that survive a hypoxic insult and whereas increased HIF-1alpha activity may be essential for the development of a more aggressive cancer phenotype, it may not be responsible for the initial selection of an apoptotic resistance phenotype.

Caffeic acid phenethyl ester-induced PC-3 cell apoptosis is caspase-dependent and mediated through the loss of inhibitors of apoptosis proteins.

OBJECTIVE: To investigate the effects of a novel agent, caffeic acid phethyl ester (CAPE) on nuclear factor (NF)-kappaB activation and apoptosis in the androgen-independent PC3 prostate cancer cell line. MATERIALS AND METHODS: PC-3 cells were assessed for NF-kappaB activation induced by paclitaxel and tumour necrosis factor-alpha (TNF-alpha), using a p65 enzyme-linked immunosorbent assay, with or without CAPE treatment. The corresponding apoptosis was assessed with propidium iodide DNA staining using flow cytometry. The pan-caspase inhibitor Z-VAD-FMK was used to investigate the mechanism of apoptosis. Alterations in the expression of inhibitor of apoptosis proteins (IAP), cIAP-1, cIAP-2 and XIAP, were detected using western blot analysis. RESULTS: CAPE prevented paclitaxel and TNFalpha-mediated NF-kappaB activation. Its ability to induce apoptosis in a dose-dependent manner was associated with the loss of cIAP-1, cIAP-2 and XIAP expression. Pretreatment with Z-VAD-FMK prevented CAPE-induced apoptosis and the loss of the IAPs. CONCLUSIONS: CAPE is an effective inhibitor of NF-kappaB activation in PC-3 cells, but the mechanism of apoptosis, and the corresponding loss of IAP expression, is caspase-dependent.

An antisense oligonucleotide to cIAP-1 sensitizes prostate cancer cells to fas and TNFalpha mediated apoptosis.

BACKGROUND: The inhibitors of apoptosis (IAP) proteins are a family of structurally homologous caspase inhibitors. We synthesized an antisense oligonucleotide (AO) to target a region within the BIR domain of cIAP-1 and examined its ability to facilitate apoptosis in prostate cancer cells. METHODS: We transfected the IAP AO into PC3 and DU145 cells and determined alterations in IAP expression using Western blotting. Apoptosis and viability were assessed using propidium iodide (PI) DNA incorporation with flow cytometry. Pacitaxel, caffeic acid phenethyl ester (CAPE), Fas antibody, and TNFalpha were used as 'second hit' agents in association with the AO. RESULTS: Western blotting showed a down-regulation in cIAP-1 expression and higher levels of spontaneous apoptosis in both cell types with no alteration in overall cell viability. AO sensitized PC3 cells, to Fas antibody and TNFalpha-mediated apoptosis, but not to apoptosis mediated by paclitaxel or CAPE. CONCLUSIONS: cIAP-1 down-regulation increased spontaneous apoptosis in prostate cancer cells and sensitized PC3 cells to receptor-mediated apoptosis.

Inhibitors of apoptosis proteins in prostate cancer cell lines.

BACKGROUND: The caspases are the central executioners of apoptosis. The inhibitors of apoptosis proteins (IAPs) are a family of recently described caspase inhibitors. We hypothesised that tumor resistance to apoptosis could be due in part to IAP expression. METHODS: The expression of NAIP, cIAP-1, cIAP-2, XIAP, and survivin was investigated in the prostate cancer cell lines LNCaP, PC3, and DU145. RNase protection assays and Western blotting were used to assess RNA and protein expression. Apoptotic susceptibility was determined using etoposide and assessed by propidium iodide (PI) DNA incorporation using flow cytometry. RESULTS: DU145 and PC3 cells were more resistant to apoptosis than LNCaP cells. All the IAPs were identified in the cell lines with variation in IAP expression between different cell types. Immunohistochemistry demonstrated cIAP-1 expression in PC3 cells was nuclear, while the expression of cIAP-2 and XIAP was perinuclear. Growing LNCaP cells in charcoal-stripped or androgen-supplemented medium resulted in no alteration in IAP expression. CONCLUSIONS: This study characterises the expression of IAP in three of the most commonly used prostate cancer cells. IAP may make an important contribution to apoptotic resistance in patients with prostate cancer.

Effects of cyclic stretch on prostatic cells in culture.

PURPOSE: The fundamental process in the development of benign prostatic hyperplasia (BPH) is a loss of homeostasis between cell proliferation and apoptosis. Prostatic smooth muscle cells contract under adrenergic control. The response of a cell to stretch may have a role in the pathogenesis of BPH. MATERIALS AND METHODS: Monolayer cultures of human prostatic stromal and epithelial cell lines were exposed to cyclic stretch for 48 hours. RESULTS: Cyclic stretch conferred resistance to etoposide induced apoptosis. Underlying this apoptotic resistance was increased expression of the anti-apoptotic Bcl-2 family of proteins. As measured by thymidine incorporation, the rate of proliferation also increased in benign epithelial cells under cyclic stretch conditions. Furthermore, an increase in the production of platelet-derived growth factor by stromal cells and transforming growth factor-beta by epithelial cells occurred under such conditions. CONCLUSIONS: The observed changes in proliferation and apoptosis may contribute to the understanding of BPH, ultimately leading to therapeutic and preventive applications.

Androgen-mediated resistance to apoptosis.

BACKGROUND: Previous studies demonstrate that androgen is capable of exerting a protective effect in the androgen-sensitive human prostate cancer cell line LNCaP. Limited studies, however, have addressed the underlying mechanisms involved, in particular the effects of androgen on both pro- and anti-apoptotic gene expression. METHODS: We investigated the effects of androgen on apoptotic sensitivity and the expression of the caspases and specific members of the Bcl-2 family in the LNCaP cell line. The effects of androgen on NF-kappaB activation were also investigated by using a gel mobility shift assay. RESULTS: 5alpha-Dihydrotestosterone (5-alphaDHT) conferred resistance to radiation (5 Gy) and etoposide-induced apoptosis in the LNCaP cell line. This finding was associated with a time-dependent decrease in the expression of the caspases and pro-apoptotic Bcl-2 family members. 5-alphaDHT did not confer protection against apoptosis in the LNCaP line transfected with the IkappaB super repressor of NF-kappaB, nor in the androgen insensitive PC-3 and DU-145 cell lines. CONCLUSION: The ability of 5-alphaDHT to raise the apoptotic threshold in the LNCaP cell line by altering specific pro-apoptotic gene expression suggests that androgen may serve as a general survival signal against diverse pathways that ultimately signal for apoptosis. We hypothesize that NF-kappaB serves as an important mediator in androgen survival signaling.