Introduction

Treatments for organ-confined prostate cancer include surgery in the form of radical prostatectomy or radiotherapy. Unfortunately, despite increased awareness, many patients still present with metastatic disease. Hormonal treatment in the form of combination therapy (luteinizing hormone-releasing hormone analogues and antiandrogens) or surgical castration (orchidectomy) remains the main treatment option for this subset of patients. Androgen ablation is, however, finite as the emergence of the hormone-refractory state occurs within 12–18 months. Currently, no curative treatment exists for hormone-refractory disease, although significant advances have been made in the palliation of advanced disease.¹

Bicalutamide (Casodex; Astra Zeneca, London, UK) is a non-steroidal antiandrogen used in the treatment of locally advanced and metastatic prostate cancer. Owing to its tolerable side effect profile, ease of administration and long half-life, it offers an attractive alternative to the steroidal antiandrogens.² Clinically, it may be used as a single agent³ or in combination with luteinizing hormone-releasing hormone analogues. Ostensibly, it functions as a receptor antagonist, preventing the binding of dihydrotestosterone. This inhibits the androgen-dependent gene expression culminating in apoptosis of the prostate cancer cell. However, the exact mechanism by which bicalutamide induces prostate cell apoptosis remains ill-defined.

There is also emerging evidence that bicalutamide may have effects independent of its antiandrogenic ability. LNCaP cells, when cultured in charcoal-stripped medium alone (which depletes androgen), show markedly reduced growth patterns, yet bicalutamide specifically induces cell death. It remains unclear how these indirect effects of bicalutamide-induced apoptosis are mediated, but a number of theories have been proposed. Firstly, in animal models, bicalutamide has been shown to decrease prostatic blood flow⁴ and may mediate apoptosis via a hypoxic pathway. Nickerson and Pollak⁵ have shown that bicalutamide can increase mRNA levels of insulin-like growth factor binding proteins 2–4, which sequester active insulin-like growth factor-1, in turn preventing the cytoprotective and pro-proliferatory effects of insulin-like growth factor-1.

Conversely, in advanced disease, bicalutamide may function as an androgen receptor agonist. Other authors have reported that bicalutamide works as an agonist in cells derived from prolonged periods of androgen ablation, leading to an androgen-independent (LNCaP-abl) cell line.⁶ In LNCaP cells exposed to bicalutamide therapy, raised levels of Bcl-2 and decreased androgen receptor levels reflect the changes that occur in early hormone-refractory disease.⁷ There is also evidence to suggest that bicalutamide may actually promote the development of metastatic disease by inducing extracellular matrix proteases facilitating the development of the invasive state in a small number of cells (0.2–0.4%).⁸

Prostate cancer can be thought of as a disease continuum with differing degrees of response to hormonal treatment. Benign prostate cells are hormone responsive and contain androgen receptors. On initiation of hormonal blockade, these cells respond accordingly and undergo apoptosis. Malignant prostate cells become androgen independent despite hormonal manoeuvres and proliferate. In advanced disease, some of these cells are thought to contain androgen receptors, which remain active to therapeutic measures. In this study, we chose two cell lines representing the hormonal therapeutic spectrum of prostate cell physiology: (1) PWR-1E cells, which are benign, androgen sensitive and receptor positive, and (2) PC-3 cells, which are derived from malignant prostate tissue and are androgen independent. Comparing these two cell lines mirrors the clinical dilemma of hormonal treatment. Androgen-dependent disease responds to hormonal ablation, whereas androgen-independent cells, found in the hormone-refractory state, exhibit a diminished therapeutic response.

Apoptosis is a complex, tightly controlled method of cellular auto-regulation⁹ mediated by caspase-dependent¹⁰ and caspase-independent¹¹ pathways. Defining the precise cellular mechanism by which bicalutamide induces apoptosis would enhance our understanding of its role in prostate cancer. Promoting apoptosis in advanced disease with bicalutamide, either as a single agent or as an adjunct to multimodality therapy, would represent a major therapeutic breakthrough for prostate cancer research.

Materials and methods

Cell culture

PC-3 cells were maintained in RPMI-1640, which was supplemented with 0.5% glucose, 10% heat-inactivated fetal bovine serum, 50 U ml^-1 penicillin, 50 g ml^-1 streptomycin and 2 mM L-glutamine. DU-145 cells were cultured and stored identically. PWR-1E cells were grown in keratinocyte-free medium supplemented with 50 U ml^-1 penicillin and 50 g ml^-1 streptomycin, 150 l of epidermal growth factor (from a stock concentration of 2.0 ng ml^-1) and 25 mg ml^-1 of bovine pituitary extract. All three cell lines were grown routinely in a humidified atmosphere of 5% CO₂ in T-75 cm² vented tissue culture flasks. All cell lines were purchased from the American Type Tissue Collection (LGC Promochem, Teddington, UK).

Reagents

Bicalutamide ((2-R,S)-4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)-propionanilide) was donated to the laboratory by Professor A Von-Angerer. zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) was supplied by R&D Systems, Oxford, United Kingdom. DMSO (dimethylsulphoxide) was supplied by Fluka Chemica, Dublin, Ireland (product no. 41460). All other reagents were obtained from Sigma-Aldrich Laboratories, Dublin, Ireland: calpain 2 inhibitor (product no. A6060), TLCK (N-tosyl-L-lysyl-chloromethylketone; product no. T7524) and 1% crystal violet solution (product no. 88H0752).

Assessment of cell death

Assessment of apoptosis was carried out using the classical method of DNA fragmentation. Cells (1 10⁵) were trypsinized for 5 min and centrifuged at 1100 r.p.m. for 5 min. Following this, they were solubilized in 400 l of hypotonic fluorochrome solution containing 50 g ml^-1 propidium iodide (PI), 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA and 0.1% Triton X-100. Samples were then stored on ice in the dark for 10–15 min before flow cytometric analysis. Triton X-100 treatment permits PI to enter the cell and intercalate with DNA. Increased DNA fragmentation (apoptosis) is shown graphically by decreased PI uptake in the sub-G₀ region. A minimum of 5000 events were analysed. Mean percentage apoptosis was calculated by expressing the number of sub-G₀ events (gated area A) (Figure 1) as a fraction of total viable counts and multiplying this figure by 100%, where gated areas B and C represent G₁ and G₂/M cell events. Apoptotic nuclei were differentiated from normal nuclei by the presence of hypodiploid DNA. The forward threshold was raised to exclude debris from the sub-G₀ population.

Figure 1.

Graphic illustration of fluorescence-activated cell source data obtained by flow cytometry. Mean percentage apoptosis was calculated by expressing the number of sub-G₀ events as a fraction of total viable counts. On the histograms listed, the gated area denoted by A represents sub-G₀ events (equating to apoptosis) and areas B and C represent all G₁ and G₂/M events.

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Necrotic cells are characterized by loss of cell membrane integrity and they allow PI to enter the cell. Unlike apoptotic cells, DNA cleavage does not occur, but owing to membrane disruption, necrotic cells show higher PI staining. In this work, necrosis was quantified as the percentage of cells with increased PI uptake. Cells (1 10⁵) were trypsinized for 5 min and centrifuged at 1100 r.p.m. for 5 min. Following this, they were resuspended in 400 l of hypotonic fluorochrome solution (minus 0.1% Triton X-100) and stored on ice in the dark for 10–15 min before flow cytometric analysis. A minimum of 5000 events were analysed. Necrotic cells appeared as a population shifted to the right in histograms relative to normal non-permeable cells. All measurements were performed under the same settings using an Epics XL-MCL Coulter Elite Flow Cytometer.

Viability

To determine cell viability, crystal violet staining was used. Briefly, following treatment with different concentrations of bicalutamide, cells were fixed with 500 l of 2% glutaraldehyde per well and left at room temperature for 30 min. Following the removal of glutaraldehyde, 1% crystal violet solution was added to each well. The plates were then left on an orbital shaker at 4000 r.p.m. for 1 h. The crystal violet was then washed off and solubilized using 400 l of 1% Triton X-100 (per well) and again left on an orbital shaker at 4000 r.p.m. for 1 h. Finally, 100 l of lysate from each well was transferred to a 96-well plate and absorbance read using a spectrophotometer (absorbance 590 nm; Tecan UK Ltd, Reading, UK).

Statistical methods

Statistical analysis was carried out using the Student's t-test (independent, two-tailed, type 3 (two-sample unequal variance)), using the Excel package on Microsoft Office XP edition. Significance was assumed for values of P<0.05. All experiments were performed on a minimum of four occasions unless stated otherwise (n=number of independent experiments).

Results

As hypothesized, bicalutamide induced apoptosis at 24 h in the androgen-dependent PWR-1E cells in a dose-dependent manner independent of growth conditions. However, bicalutamide also induced apoptosis at 24 h in the androgen-independent PC-3 cells, but the effects were more pronounced in the PWR-1E cell line at all doses (Figure 2a). Cell membrane integrity (viability) remained unaffected after 24 h of bicalutamide treatment in both cell lines (data not shown).

Figure 2.

(a) Effects of bicalutamide on PWR-1E and PC-3 cell apoptosis. PWR-1E and PC-3 cells (1 10⁵) were cultured under normal growth conditions for 24 h before bicalutamide treatment (0, 10, 50 and 100 M). Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. ^*P<0.05 vs untreated controls (n=5). (b) Effects of bicalutamide on PWR-1E cell apoptosis under both conditions. PWR-1E cells (1 10⁵) were cultured with growth factor (BPE+) or without (BPE-) for 24 h before bicalutamide treatment (0, 10, 50 and 100 M). Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. ^*P<0.05 vs untreated controls (n=5). (c) Effects of bicalutamide on PC-3 cell apoptosis under both conditions. PC-3 cells (1 10⁵) were cultured in 10% FCS or 1% FCS for 24 h before bicalutamide treatment (0, 10 and 50  M). Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. ^*P<0.05 vs untreated controls (n=5). FCS, fetal calf serum; PI, propidium iodide.

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Growth factor withdrawal for 24 h before bicalutamide treatment was undertaken to determine if cells in a G₀ phase of cell growth were more or less susceptible to the apoptotic effects of bicalutamide. No significant alteration in apoptotic potential was demonstrated (Figure2b; PWR-1E with growth factor bovine pituitary extract (BPE)+; 10 M 11.564.42%, 50 M 15.286.0% and 100 M 20.433.89%) vs PWR-1E without growth factor (BPE-; 10 M 9.024.13%, 50 M 12.622.7% and 100 M 21.384.25%)).

In the androgen-independent cell line again, no significant alteration in apoptotic potential was demonstrated (Figure 2c; PC-3 with growth factor (10% fetal calf serum (FCS); 10 M 5.863.8%, 50 M 9.383.79% and 100 M 11.615.06%) vs PC-3 without growth factor (1% FCS; 10 M 2.370.5%, 50 M 5.341.9% and 100 M 11.186.3%)). To determine if prolonged exposure to bicalutamide had an effect on apoptotic potential, treatment time was extended to 48 h up to a maximum dose of 50 M bicalutamide only. These experiments were performed only under full growth conditions. In the androgen-dependent PWR-1E cell line at 48 h, there was no appreciable increase in the amount of apoptosis induced at the higher dose of 50 M bicalutamide (Figure 3a) (50 M 15.286.0% apoptosis at 24 h vs 50 M 14.23.85% at 48 h). However, in the androgen-independent PC-3 cell at 48 h, an increase in the rate amount of bicalutamide-induced apoptosis was demonstrated (Figure 3b) (50 M 9.393.79% apoptosis at 24 h vs 50 M 21.756.01% apoptosis at 48 h). Having identified an increase in apoptosis at 48 h in an androgen-independent cell line, we sought to verify this finding using a different metastatic line (DU-145). Interestingly, bicalutamide did induce apoptosis in DU-145 cells at 48 h (Figure 3c; DU-145, 50 M 20.434.73%). At 48 h, no differences in viability were detected in PWR-1E, PC-3 or DU-145 cell lines (data not shown).

Figure 3.

(a) Effects of bicalutamide on PWR-1E cell apoptosis at 48 h under normal growth conditions. PWR-1E cells (1 10⁵) were cultured with pituitary extract (BPE) for 24 h before bicalutamide treatment (0, 10 and 50 M) for 24 and 48 h. Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. ^*P<0.05 vs untreated controls (n=3). (b) Effects of bicalutamide on PC-3 cell apoptosis at 48 h under normal growth conditions. PC-3 cells (1 10⁵) were cultured in 10% FCS for 24 h before bicalutamide treatment (0, 10 and 50 M) for 24 and 48 h. Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. ^*P<0.05 vs untreated controls (n=3). (c) Effects of bicalutamide on DU-145 cell apoptosis at 48 h. DU-145 cells (1 10⁵) were cultured in 10% FCS for 24 h before bicalutamide treatment (0, 10 and 50 M) for 48 h only. Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. ^*P<0.05 vs untreated controls (n=3). FCS, fetal calf serum; PI, propidium iodide.

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To specifically determine if bicalutamide-induced apoptosis was caspase dependent, the pan-caspase inhibitor zVAD-FMK was used. Bicalutamide-induced apoptosis was successfully inhibited by pan-caspase inhibition in the androgen-dependent PWR-1E cells (Figure 4a). However, in the androgen-independent PC-3 cells (Figure 4b), no significant inhibition was noted using the higher dose of 50 M bicalutamide (P=0.06).

Figure 4.

Effects of the pan-caspase inhibition on bicalutamide-induced apoptosis in the PWR-1E (a) and PC-3 (b) cells. PWR-1E (a) and PC-3 (b) cells (1 10⁵) were grown to confluence and preincubated with 100 M of zVAD-FMK for 1 h and then treated with bicalutamide (0, 10 and 50 M) for 48 h under normal growth conditions. Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry (n=4). (a) ^**P<0.005 vs 10 Mbicalutamide, ^*P<0.05 vs 50 M bicalutamide (n=4). (b) ^†P>0.05 vs 50 Mbicalutamide (n=4). PI, propidium iodide; zVAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.

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Thus, having demonstrated disparities in caspase dependency between androgen-dependent and androgen-independent cells, our next step was to determine the role of calpains in bicalutamide function. To this effect we used a calpain 2 inhibitor. In the PWR-1E cells, bicalutamide-induced apoptosis was not inhibited by calpain 2 blockade (Figure 5a). The PC-3 cell line showed a partial yet significant (P<0.05) inhibition of apoptosis by calpain 2 blockade (Figure 5b). Viability remained unaffected in both cell lines following calpain blockade (data not shown).

Figure 5.

Effects of the calpain inhibition on bicalutamide-induced apoptosis in PWR-1E and PC-3 cells. PWR-1E (a) and PC-3 (b) cells (1 10⁵) were grown to confluence and preincubated with 5 M of the calpain 2 inhibitor for 1 h and then treated with bicalutamide (0, 10 and 50 M) for 48 h under normal growth conditions. Following trypsinization, cells were stained with PI and Triton X solution to assess apoptosis and then analysed by flow cytometry. Statistical analysis carried out using Student's t-test. (a) ^*P<0.05 vs 50 M bicalutamide (n=4). (b) ^*P<0.05 vs 50 Mbicalutamide (n=4). PI, propidium iodide.

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Discussion

How bicalutamide induces prostate cell apoptosis definitively remains the focus of much research. It is known that bicalutamide does not prevent nuclear translocation and as the bicalutamide/receptor complex is in the nucleus, it can bind androgen-responsive elements. The complex, however, remains transcriptionally inactive and prevents androgen receptor N- and C-terminal association.¹² Bicalutamide has also been shown to increase mRNA levels of insulin-like growth factor binding proteins, helping to sequester active insulin-like growth factor-1, which has a cumulative effect of preventing insulin-like growth factor-1 binding to its receptor, and thus elicits an antiapoptotic effect.⁵ There is emerging evidence to show that bicalutamide exerts its proapoptotic effects independent of mitochondrial membrane changes and that a by-product of caspase 8 is produced as a result of caspase 3 action.¹³

Paradoxically, there is evidence to suggest that bicalutamide in advanced disease may be agonistic in nature. Some authors have reported that bicalutamide works as an agonist in cells derived from prolonged periods of androgen ablation, leading to an androgen-independent (LNCaP-abl) cell line.⁶ Specific to this work, we observed that low-dose bicalutamide (10 m) consistently exhibited lower rates of induced apoptosis when compared to control (Figures 2a, c, 3b, c and 5b). This was shown to be not significant but may reflect the potential of low-dose bicalutamide to render the cells quiescent. In LNCaP cells exposed to bicalutamide therapy, raised levels of Bcl-2 are found, reflecting the changes that occur in early hormone-refractory disease.⁷ Bicalutamide, via promoting extracellular matrix protease formation may facilitate cellular invasiveness and aid the metastatic process.⁸ In addition, in the presence of increased androgen receptor levels (which has been shown to be the most consistent change in the development of prostate cancer), bicalutamide switches from an antagonist to an agonist and serves to assemble coactivators to act on androgen-responsive genes.¹⁴ Prostate cancer is a slow growing disease, which in the advanced stages is androgen independent. In comparing the amount of apoptosis induced by bicalutamide, in two different prostate cell lines the clinical scenario was mirrored. Following androgen ablation, androgen-dependent cells activate a cell death pathway (apoptosis) ensuring that daily cell death exceeds proliferation.¹⁵

It is important, briefly, to comment on the dosing regimen of bicalutamide and the clinical correlation (where a 150-mg dose can be used as monotherapy and where 50 mg is used in conjunction with luteinizing hormone-releasing hormone analogues). The active antiandrogenic component of bicalutamide (the R enantiomer) has a plasma half-life of approximately 7 days. In clinical practice, doses up to 150 mg of bicalutamide exhibit a linear dose-related curve, and correspondingly maximum prostate specific antigen (PSA) reduction is seen at these doses.¹⁶ In this study, we did not perform intracellular assays of bicalutamide but we did employ doses of 50 and 100 g to mirror the clinically relevant doses, although in vivo the exact intraprostatic concentrations of bicalutamide remain undetermined.

In our initial experiments, at 24 h, the androgen-independent PC-3 cells showed a less marked apoptotic response to bicalutamide as compared to the androgen-dependent PWR-1E cell line (Figure 2). Growth factor withdrawal (simulating cellular stress) had no significant effect on either cell line in terms of the amount of apoptosis induced by bicalutamide. However, after 48 h, the apoptotic rate of the PC-3 cell was increased (Figure 3b). Thus, the malignant cancer cell, represented here by the PC-3 cell, may require extra time to create the necessary apoptotic machinery, and therefore it is not as sensitive to apoptosis as the receptor-bearing cell, represented here by the PWR-1E cell. This may account for the more pronounced rate of apoptosis at 48 h in the PC-3 cell. However, the PC-3 cell line does not express an androgen receptor and we demonstrated that bicalutamide, a receptor antagonist, induced apoptosis in this cell line.

This represented an interesting finding in that a drug (bicalutamide) that ostensibly acts as a receptor antagonist induced cell death in a receptor-negative cell line. At 48 h, we also showed that bicalutamide induced appreciable apoptosis in a different malignant androgen-independent cell line (DU-145). Vicentini et al.¹⁷ also demonstrated that high-dose bicalutamide inhibited growth curves in androgen receptor-negative PC-3 cells, although the method of cell death was thought to be cytotoxic and also receptor independent. The same authors also showed similar effects in DU-145 cells.

Furthermore, it was effectively demonstrated that altering the cellular environment to slow proliferation of the cell had minimal effect on apoptotic susceptibility and did not differ significantly in terms of an apoptotic response when compared to cells grown in normal media.

As we had shown that bicalutamide induces apoptosis in cells with no apparent androgen receptor, the mechanism that is responsible for causing cell death is not yet clear. Our next step was to determine the role of the caspases in bicalutamide function and to examine specifically if there were disparities in caspase dependency between the two cell lines. To this effect, we assessed the role of pan-caspase inhibition (using zVAD-FMK) on bicalutamide-induced apoptosis in vitro.

Differences in caspase dependency between androgen-dependent (PWR-1E) cells and androgen-independent cells (PC-3) with respect to bicalutamide-induced apoptosis were noted. In the androgen-dependent PWR-1E cell, bicalutamide function is caspase dependent. In the androgen-independent PC-3 cell line, pan-caspase inhibition had minimal effect (P=0.06) on bicalutamide-induced apoptosis at the higher dose 50 M. Thus, bicalutamide-induced apoptosis was a predominantly caspase-independent event with respect to the PC-3 cell. This gave rise to two fundamental questions: (a) why is there a disparity between the cell lines (PWR-1E and PC-3) using the same drug? and (b) if it is only partially caspase dependent, what cell death pathway does bicalutamide use to induce PC-3 cell apoptosis? From our results, it is evident that bicalutamide can induce apoptosis of androgen receptor blockade. Expression of antiapoptotic Bcl-2 members such as Bcl-xl (preventing cytochrome c release, mitochondrial incapacitation and subsequent caspase activation) has been implicated in the resistance of PC-3 cells to apoptosis.¹⁸

As we had determined that bicalutamide-induced apoptosis may be a partially caspase-independent event in PC-3 cells, we next determined the role of calpains in bicalutamide-induced apoptosis.

Calpains are calcium-dependent cysteine proteases found in all eukaryotic species. Structurally two forms exist, namely and m calpain, and they are produced with their endogenous cytosolic inhibitor, calpastatin,¹⁹ which regulates calpain activity in vivo. They have a broader range of targets than the caspases, with regards to substrate cleavage, and are involved in functions such as cytoskeleton remodelling of proteins (such as fodrin), steroid receptor removal, protein kinase degradation and processing of calcium ATPase.¹⁹ In HL-60 cells, pan-caspase inhibition affects calpain cleavage of Bax, whereas use of calpeptin, a calpain inhibitor, does not affect caspase activity, highlighting a possible temporal role between caspase and subsequent calpain activation.²⁰ Inhibition of calpain activity, which can affect growth factor signalling, has been demonstrated to reduce prostate tumour cell invasiveness in vitro.²¹

Inhibition of calpain function in prostate cancer cells yields conflicting results. Calpain inhibition did not affect the benign PWR-1E cell line. In androgen-independent PC-3 cells, bicalutamide acts in a partially calpain-dependent manner. In CPTX prostate epithelial cells, thapsigargin induces apoptosis with a concomitant reduction in insulin receptor substrate-1 protein levels. Use of a calpain 2 inhibitor prevents this, highlighting a role for calpains in thapsigargin-mediated prostate cell apoptosis.²² This supports our finding with the PC-3 cell line, as CPTX cells are derived from a primary prostate carcinoma and are thought not to express androgen receptors. Specific to PC-3 cells, calpain 2 inhibition alone has been shown not to be cytotoxic, but in malignant lymphoid cells, caspase inhibition inhibits calpain 2, again highlighting a possible sequential, dependent relationship between the caspase and calpain families.²³

Lu and Mellgren²⁴ demonstrated that caspase inhibition prevented apoptosis induced by serine protease inhibition, pointing towards an involvement of the serine proteases in apoptosis with caspases being the ultimate cell death pathway. Although we did not directly assess the impact of serine protease inhibition in this work, we did demonstrate that pan-caspase inhibition did affect bicalutamide action in androgen-dependent cells.

Recent data have demonstrated a differential effect of bicalutamide on different cell lines. Telomerase activity is altered after 3 days of bicalutamide therapy in androgen-sensitive cells (accompanied by decreased levels of androgen receptors and chaperone proteins), and yet it remains unaffected in insensitive cells.²⁵ This same study also illustrated a caspase-independent apoptosis by bicalutamide in androgen-insensitive cells.

In conclusion, these results demonstrate the proapoptotic effect of bicalutamide on benign androgen-dependent and malignant androgen-independent prostatic epithelial cells. In keeping with the work by Vicentini et al.,¹⁷ we demonstrated that bicalutamide can induce cell death in receptor-negative cells.

This apoptotic event is partially reliant on calpain activity in androgen-independent cells. In addition, given our specific aim at apoptotic pathway delineation, we demonstrated that bicalutamide, in androgen-independent cells, acts in a predominantly caspase-independent manner and yet is also partially reliant on calpains to induce apoptosis.

Thus, this study provides evidence that bicalutamide persists in providing a proapoptotic effect on prostate cancer cells in vitro, even in androgen-independent cells. Further work at defining specific apoptotic pathways will help to determine the exact role of how bicalutamide induces apoptosis in androgen-independent cells. Extending the use of antiandrogens in hormone-refractory disease will broaden the spectrum of prostate cancer treatment available.

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Acknowledgements

Michael St John Floyd Jr was a British Urological Foundation Scholar during the course of this work.