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Differential Mechanisms of Bicalutamide-Induced Apoptosis in Prostate Cell Lines

Differential Mechanisms of Bicalutamide-Induced Apoptosis in Prostate Cell Lines

and Prostatic Diseases (2009) 12, 25–33 & 2009 Nature Publishing Group All rights reserved 1365-7852/09 $32.00 www.nature.com/pcan ORIGINAL ARTICLE

Differential mechanisms of -induced apoptosis in prostate cell lines

M St John Floyd Jr, SJ Teahan, JM Fitzpatrick and RWG Watson UCD School of Medicine and Medical Science, UCD Conway Institute of Biomolecular and Biomedical Research, Mater Misericordiae University Hospital, University College Dublin, Dublin, Ireland

Bicalutamide is a non-steroidal used in the treatment of . Although widely accepted as an , the mechanism by which it induces apoptosis remains unclear. Defining exact pathways by which bicalutamide induces its apoptotic effects would help to advance its clinical applications. We aimed to (a) examine the apoptotic effects of bicalutamide at 24 h and (b) comment on the role of the caspases and calpains in mediating bicalutamide-induced apoptosis in androgen-dependent and androgen-independent cells. PWR-1E, PC-3 and DU-145 cells were treated with bicalutamide and assessed for apoptosis by flow cytometry at 24 h. DU-145 cells were used to compare differences between two different metastatic receptor-negative cells and to verify apoptotic induction at 48 h. To delineate a specific pathway of action for bicalutamide, PC-3 and PWR-1E cells were pretreated with specific inhibitors of caspase- dependent (zVAD-FMK) and caspase-independent pathways (calpain 2 inhibitor). Bicalutamide induced apoptosis in androgen-dependent PWR-1E cells via a caspase-dependent and calpain- independent mechanism. In androgen-independent PC-3 cells, bicalutamide also induced apoptosis by mechanisms that were partially inhibited by pan-caspase inhibition but were partially calpain dependent. Understanding into how bicalutamide exerts its effects in androgen- independent cells will yield further insights into the treatment of -refractory disease. Prostate Cancer and Prostatic Diseases (2009) 12, 25–33; doi:10.1038/pcan.2008.23; published online 13 May 2008

Keywords: bicalutamide; apoptosis; caspase; androgen independent; calpain

Introduction to its tolerable side effect profile, ease of administration and long half-life, it offers an attractive alternative to the Treatments for organ-confined prostate cancer include steroidal .2 Clinically, it may be used as a surgery in the form of radical prostatectomy or radio- single agent3 or in combination with luteinizing hor- therapy. Unfortunately, despite increased awareness, mone-releasing hormone analogues. Ostensibly, it func- many patients still present with metastatic disease. tions as a receptor antagonist, preventing the binding of Hormonal treatment in the form of combination therapy . This inhibits the androgen-depen- (-releasing hormone analogues and dent gene expression culminating in apoptosis of the antiandrogens) or surgical castration (orchidectomy) prostate cancer cell. However, the exact mechanism by remains the main treatment option for this subset of which bicalutamide induces prostate cell apoptosis patients. Androgen ablation is, however, finite as the remains ill-defined. emergence of the hormone-refractory state occurs within There is also emerging evidence that bicalutamide may 12–18 months. Currently, no curative treatment exists for have effects independent of its antiandrogenic ability. hormone-refractory disease, although significant ad- LNCaP cells, when cultured in charcoal-stripped med- vances have been made in the palliation of advanced ium alone (which depletes androgen), show markedly disease.1 reduced growth patterns, yet bicalutamide specifically Bicalutamide (Casodex; Astra Zeneca, London, UK) is induces cell death. It remains unclear how these indirect a non- used in the treatment of effects of bicalutamide-induced apoptosis are mediated, locally advanced and metastatic prostate cancer. Owing 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 Correspondence: M St John Floyd Jr, UCD School of Medicine and hypoxic pathway. Nickerson and Pollak5 have shown Medical Science, UCD Conway Institute of Biomolecular and that bicalutamide can increase mRNA levels of - Biomedical Research, Mater Misericordiae University Hospital, like growth factor binding proteins 2–4, which sequester University College Dublin, Dublin 4, Ireland. E-mail: [email protected] active insulin-like growth factor-1, in turn preventing the Received 20 October 2007; revised 5 February 2008; accepted 8 March cytoprotective and pro-proliferatory effects of insulin- 2008; published online 13 May 2008 like growth factor-1. Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al

26 Conversely, in advanced disease, bicalutamide may Reagents function as an . Other authors Bicalutamide ((2-R,S)-40-cyano-3-(4-fluorophenylsulpho- have reported that bicalutamide works as an agonist in nyl)-2-hydroxy-2-methyl-30-(trifluoromethyl)-propiona- cells derived from prolonged periods of androgen nilide) was donated to the laboratory by Professor A ablation, leading to an androgen-independent (LNCaP- Von-Angerer. zVAD-FMK (benzyloxycarbonyl-Val-Ala- abl) cell line.6 In LNCaP cells exposed to bicalutamide Asp-fluoromethylketone) was supplied by R&D Systems, therapy, raised levels of Bcl-2 and decreased androgen Oxford, United Kingdom. DMSO (dimethylsulphoxide) receptor levels reflect the changes that occur in early was supplied by Fluka Chemica, Dublin, Ireland hormone-refractory disease.7 There is also evidence to (product no. 41460). All other reagents were obtained suggest that bicalutamide may actually promote from Sigma-Aldrich Laboratories, Dublin, Ireland: cal- the development of metastatic disease by inducing pain 2 inhibitor (product no. A6060), TLCK (N-tosyl-L- extracellular matrix proteases facilitating the develop- lysyl-chloromethylketone; product no. T7524) and 1% ment of the invasive state in a small number of cells crystal violet solution (product no. 88H0752). (0.2–0.4%).8 Prostate cancer can be thought of as a disease continuum with differing degrees of response to hormo- Assessment of cell death nal treatment. Benign prostate cells are hormone respon- Assessment of apoptosis was carried out using the sive and contain androgen receptors. On initiation of classical method of DNA fragmentation. Cells (1 Â105) hormonal blockade, these cells respond accordingly and were trypsinized for 5 min and centrifuged at 1100 r.p.m. undergo apoptosis. Malignant prostate cells become for 5 min. Following this, they were solubilized in 400 mlof androgen independent despite hormonal manoeuvres hypotonic fluorochrome solution containing 50 mgmlÀ1 and proliferate. In advanced disease, some of these cells propidium iodide (PI), 3.4 mM sodium citrate, 1 mM Tris, are thought to contain androgen receptors, which remain 0.1 mM EDTA and 0.1% Triton X-100. Samples were then active to therapeutic measures. In this study, we chose stored on ice in the dark for 10–15 min before flow two cell lines representing the hormonal therapeutic cytometric analysis. Triton X-100 treatment permits PI to spectrum of prostate cell physiology: (1) PWR-1E cells, enter the cell and intercalate with DNA. Increased DNA which are benign, androgen sensitive and receptor fragmentation (apoptosis) is shown graphically by de- positive, and (2) PC-3 cells, which are derived creased PI uptake in the sub-G0 region. A minimum of from malignant prostate tissue and are androgen 5000 events were analysed. Mean percentage apoptosis independent. Comparing these two cell lines mirrors was calculated by expressing the number of sub-G0 events the clinical dilemma of hormonal treatment. Androgen- (gated area A) (Figure 1) as a fraction of total viable counts dependent disease responds to hormonal ablation, and multiplying this figure by 100%, where gated areas B whereas androgen-independent cells, found in the and C represent G1 and G2/M cell events. Apoptotic hormone-refractory state, exhibit a diminished thera- nuclei were differentiated from normal nuclei by the peutic response. presence of hypodiploid DNA. The forward threshold Apoptosis is a complex, tightly controlled method of was raised to exclude debris from the sub-G0 population. cellular auto-regulation9 mediated by caspase-depen- Necrotic cells are characterized by loss of cell membrane dent10 and caspase-independent11 pathways. Defining integrity and they allow PI to enter the cell. Unlike apoptotic the precise cellular mechanism by which bicalutamide cells, DNA cleavage does not occur, but owing to membrane induces apoptosis would enhance our understanding of disruption, necrotic cells show higher PI staining. In this its role in prostate cancer. Promoting apoptosis in work, necrosis was quantified as the percentage of cells with advanced disease with bicalutamide, either as a single increased PI uptake. Cells (1 Â105) were trypsinized for agent or as an adjunct to multimodality therapy, would 5 min and centrifuged at 1100 r.p.m. for 5 min. Following represent a major therapeutic breakthrough for prostate this, they were resuspended in 400 mlofhypotonic cancer research. 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 Materials and methods 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. PC-3 cells were maintained in RPMI-1640, which was supplemented with 0.5% glucose, 10% heat-inactivated fetal bovine serum, 50 U mlÀ1 penicillin, 50 mgmlÀ1 Viability streptomycin and 2 mML-glutamine. DU-145 cells were To determine cell viability, crystal violet staining was cultured and stored identically. PWR-1E cells were used. Briefly, following treatment with different concen- grown in keratinocyte-free medium supplemented trations of bicalutamide, cells were fixed with 500 mlof with 50 U mlÀ1 penicillin and 50 mgmlÀ1 streptomycin, 2% glutaraldehyde per well and left at room temperature 150 ml of epidermal growth factor (from a stock concen- for 30 min. Following the removal of glutaraldehyde, 1% tration of 2.0 ng mlÀ1) and 25 mg mlÀ1 of bovine pituitary crystal violet solution was added to each well. The plates extract. All three cell lines were grown routinely in a were then left on an orbital shaker at 4000 r.p.m. for 1 h. 2 humidified atmosphere of 5% CO2 in T-75 cm vented The crystal violet was then washed off and solubilized tissue culture flasks. All cell lines were purchased from using 400 ml of 1% Triton X-100 (per well) and again left the American Type Tissue Collection (LGC Promochem, on an orbital shaker at 4000 r.p.m. for 1 h. Finally, 100 ml Teddington, UK). of lysate from each well was transferred to a 96-well

Prostate Cancer and Prostatic Diseases Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al 27

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. plate and absorbance read using a spectrophotometer Po0.05. All experiments were performed on a minimum (absorbance 590 nm; Tecan UK Ltd, Reading, UK). of four occasions unless stated otherwise (n ¼ number of independent experiments).

Statistical methods Statistical analysis was carried out using the Student’s Results t-test (independent, two-tailed, type 3 (two-sample unequal variance)), using the Excel package on Microsoft As hypothesized, bicalutamide induced apoptosis at Office XP edition. Significance was assumed for values of 24 h in the androgen-dependent PWR-1E cells in a

Prostate Cancer and Prostatic Diseases Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al

28 dose-dependent manner independent of growth condi- remained unaffected after 24 h of bicalutamide treatment tions. However, bicalutamide also induced apoptosis at in both cell lines (data not shown). 24 h in the androgen-independent PC-3 cells, but the Growth factor withdrawal for 24 h before bicalutamide effects were more pronounced in the PWR-1E cell line at treatment was undertaken to determine if cells in a G0 all doses (Figure 2a). Cell membrane integrity (viability) phase of cell growth were more or less susceptible to the apoptotic effects of bicalutamide. No significant altera- tion in apoptotic potential was demonstrated (Figure2b; 30 PWR-1E with growth factor bovine pituitary extract (BPE) þ ;10mM 11.56±4.42%, 50 mM 15.28±6.0% and ± PWR BPE+ * 100 mM 20.43 3.89%) vs PWR-1E without growth factor 25 PC3 10% FCS (BPEÀ;10mM 9.02±4.13%, 50 mM 12.62±2.7% and 100 mM 21.38±4.25%)). In the androgen-independent cell line again, no 20 significant alteration in apoptotic potential was demon- * strated (Figure 2c; PC-3 with growth factor (10% fetal calf 15 serum (FCS); 10 mM 5.86±3.8%, 50 mM 9.38±3.79% and 100 mM 11.61±5.06%) vs PC-3 without growth factor (1% FCS; 10 mM 2.37±0.5%, 50 mM 5.34±1.9% and 100 mM

% Mean Apoptosis 10 11.18±6.3%)). To determine if prolonged exposure to bicalutamide had an effect on apoptotic potential, 5 treatment time was extended to 48 h up to a maximum dose of 50 mM bicalutamide only. These experiments were performed only under full growth conditions. In the 0 androgen-dependent PWR-1E cell line at 48 h, there was 0 10 50 100 no appreciable increase in the amount of apoptosis Bicalutamide (µM) induced at the higher dose of 50 mM bicalutamide (Figure 3a) (50 mM 15.28±6.0% apoptosis at 24 h vs 30 50 mM 14.2±3.85% 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 demon- 25 BPE+ strated (Figure 3b) (50 mM 9.39±3.79% apoptosis at 24 h BPE- vs 50 mM 21.75±6.01% apoptosis at 48 h). Having 20 identified an increase in apoptosis at 48 h in an androgen-independent cell line, we sought to verify this 15 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 mM 20.43±4.73%).

% Mean Apoptosis 10 At 48 h, no differences in viability were detected in PWR- 1E, PC-3 or DU-145 cell lines (data not shown). 5 To specifically determine if bicalutamide-induced apoptosis was caspase dependent, the pan-caspase inhibitor zVAD-FMK was used. Bicalutamide-induced 0 apoptosis was successfully inhibited by pan-caspase 0 10 50 100 inhibition in the androgen-dependent PWR-1E cells Bicalutamide (µM) (Figure 4a). However, in the androgen-independent PC-3 cells (Figure 4b), no significant inhibition was

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10% FCS Figure 2 (a) Effects of bicalutamide on PWR-1E and PC-3 cell 25 5 1% FCS apoptosis. PWR-1E and PC-3 cells (1 Â10 ) were cultured under normal growth conditions for 24 h before bicalutamide treatment (0, 20 10, 50 and 100 mM). Following trypsinization, cells were stained with * * PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. *Po0.05 vs untreated controls (n ¼ 5). (b) Effects of 15 bicalutamide on PWR-1E cell apoptosis under both conditions. PWR-1E cells (1 Â105) were cultured with growth factor (BPE þ )or without (BPEÀ) for 24 h before bicalutamide treatment (0, 10, 50 and 10 100 mM). Following trypsinization, cells were stained with PI and % Mean Apoptosis Triton X-100 to assess apoptosis and then analysed by flow cytometry. *Po0.05 vs untreated controls (n ¼ 5). (c) Effects of 5 bicalutamide on PC-3 cell apoptosis under both conditions. PC-3 cells (1 Â105) were cultured in 10% FCS or 1% FCS for 24 h before m 0 bicalutamide treatment (0, 10 and 50 M). Following trypsinization, 0 10 50 100 cells were stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. *Po0.05 vs untreated controls Bicalutamide (µM) (n ¼ 5). FCS, fetal calf serum; PI, propidium iodide.

Prostate Cancer and Prostatic Diseases Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al 29 noted using the higher dose of 50 mM bicalutamide the role of calpains in bicalutamide function. To this (P ¼ 0.06). effect we used a calpain 2 inhibitor. In the PWR-1E cells, Thus, having demonstrated disparities in caspase bicalutamide-induced apoptosis was not inhibited by dependency between androgen-dependent and andro- calpain 2 blockade (Figure 5a). The PC-3 cell line showed gen-independent cells, our next step was to determine a partial yet significant (Po0.05) inhibition of apoptosis by calpain 2 blockade (Figure 5b). Viability remained unaffected in both cell lines following calpain blockade 30 (data not shown). 24 Hours BPE+ 48 Hours BPE+ 25 Discussion

20 How bicalutamide induces prostate cell apoptosis * definitively remains the focus of much research. It is known that bicalutamide does not prevent nuclear 15 translocation and as the bicalutamide/receptor complex is in the nucleus, it can bind androgen-responsive elements. The complex, however, remains transcription- % Mean Apoptosis 10 ally 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 5 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 0 5 01050receptor, and thus elicits an antiapoptotic effect. There is Bicalutamide (µM) emerging evidence to show that bicalutamide exerts its proapoptotic effects independent of mitochondrial mem- 30 brane 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 25 24 Hours 10% FCS bicalutamide in advanced disease may be agonistic in 48 Hours 10% FCS nature. Some authors have reported that bicalutamide 20 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, 15 we observed that low-dose bicalutamide (10 mm) con- sistently exhibited lower rates of induced apoptosis when compared to control (Figures 2a, c, 3b, c and 5b).

% Mean Apoptosis 10 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 5 therapy, raised levels of Bcl-2 are found, reflecting the changes that occur in early hormone-refractory disease.7 Bicalutamide, via promoting extracellular matrix pro- 0 tease formation may facilitate cellular invasiveness and 01050aid the metastatic process.8 In addition, in the presence Bicalutamide (µM)

30 Figure 3 (a) Effects of bicalutamide on PWR-1E cell apoptosis at 5 48 hrs 10% FCS * 48 h under normal growth conditions. PWR-1E cells (1 Â10 ) were 25 cultured with pituitary extract (BPE) for 24 h before bicalutamide treatment (0, 10 and 50 mM) for 24 and 48 h. Following trypsiniza- tion, cells were stained with PI and Triton X-100 to assess apoptosis 20 and then analysed by flow cytometry. *Po0.05 vs untreated controls (n ¼ 3). (b) Effects of bicalutamide on PC-3 cell apoptosis at 48 h 5 15 under normal growth conditions. PC-3 cells (1 Â10 ) were cultured in 10% FCS for 24 h before bicalutamide treatment (0, 10 and 50 mM) for 24 and 48 h. Following trypsinization, cells were stained with PI 10 and Triton X-100 to assess apoptosis and then analysed by flow % Mean Apoptosis cytometry. *Po0.05 vs untreated controls (n ¼ 3). (c) Effects of bicalutamide on DU-145 cell apoptosis at 48 h. DU-145 cells (1 Â105) 5 were cultured in 10% FCS for 24 h before bicalutamide treatment (0, 10 and 50 mM) for 48 h only. Following trypsinization, cells were 0 stained with PI and Triton X-100 to assess apoptosis and then analysed by flow cytometry. *Po0.05 vs untreated controls (n ¼ 3). 01050 FCS, fetal calf serum; PI, propidium iodide.

Prostate Cancer and Prostatic Diseases Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al

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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 5 (b) cells (1 Â10 ) were grown to confluence and preincubated with 100 mM of zVAD-FMK for 1 h and then treated with bicalutamide (0, 10 and 50 mM) for 48 h under normal growth conditions. Following trypsinization, cells were stained with PI and Triton X-100 to assess apoptosis and w then analysed by flow cytometry (n ¼ 4). (a)**Po0.005 vs 10 mM bicalutamide, *Po0.05 vs 50 mM bicalutamide (n ¼ 4). (b) P40.05 vs 50 mM bicalutamide (n ¼ 4). PI, propidium iodide; zVAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.

of increased androgen receptor levels (which has been androgenic component of bicalutamide (the R enantio- shown to be the most consistent change in the develop- mer) has a plasma half-life of approximately 7 days. In ment of prostate cancer), bicalutamide switches from clinical practice, doses up to 150 mg of bicalutamide an antagonist to an agonist and serves to assemble exhibit a linear dose-related curve, and correspondingly coactivators to act on androgen-responsive genes.14 maximum prostate specific antigen (PSA) reduction is Prostate cancer is a slow growing disease, which in the seen at these doses.16 In this study, we did not perform advanced stages is androgen independent. In comparing intracellular assays of bicalutamide but we did employ the amount of apoptosis induced by bicalutamide, in two doses of 50 and 100 mg to mirror the clinically relevant different prostate cell lines the clinical scenario was doses, although in vivo the exact intraprostatic concen- mirrored. Following androgen ablation, androgen- trations of bicalutamide remain undetermined. dependent cells activate a cell death pathway (apoptosis) In our initial experiments, at 24 h, the androgen- ensuring that daily cell death exceeds proliferation.15 independent PC-3 cells showed a less marked apoptotic It is important, briefly, to comment on the dosing response to bicalutamide as compared to the androgen- regimen of bicalutamide and the clinical correlation dependent PWR-1E cell line (Figure 2). Growth factor (where a 150-mg dose can be used as monotherapy and withdrawal (simulating cellular stress) had no significant where 50 mg is used in conjunction with luteinizing effect on either cell line in terms of the amount of hormone-releasing hormone analogues). The active anti- apoptosis induced by bicalutamide. However, after 48 h,

Prostate Cancer and Prostatic Diseases Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al

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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 5 (1 Â10 ) were grown to confluence and preincubated with 5 mM of the calpain 2 inhibitor for 1 h and then treated with bicalutamide (0, 10 and 50 mM) 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)*Po0.05 vs 50 mM bicalutamide (n ¼ 4). (b)*Po0.05 vs 50 mM bicalutamide (n ¼ 4). PI, propidium iodide. the apoptotic rate of the PC-3 cell was increased growth curves in androgen receptor-negative PC-3 cells, (Figure 3b). Thus, the malignant cancer cell, represented although the method of cell death was thought to be here by the PC-3 cell, may require extra time to create the cytotoxic and also receptor independent. The same necessary apoptotic machinery, and therefore it is not as authors also showed similar effects in DU-145 cells. sensitive to apoptosis as the receptor-bearing cell, Furthermore, it was effectively demonstrated that represented here by the PWR-1E cell. This may account altering the cellular environment to slow proliferation for the more pronounced rate of apoptosis at 48 h in the of the cell had minimal effect on apoptotic susceptibility PC-3 cell. However, the PC-3 cell line does not express an and did not differ significantly in terms of an apoptotic androgen receptor and we demonstrated that bicaluta- response when compared to cells grown in normal mide, a receptor antagonist, induced apoptosis in this media. cell line. As we had shown that bicalutamide induces apoptosis This represented an interesting finding in that a drug in cells with no apparent androgen receptor, the (bicalutamide) that ostensibly acts as a receptor antago- mechanism that is responsible for causing cell death is nist induced cell death in a receptor-negative cell line. At not yet clear. Our next step was to determine the role 48 h, we also showed that bicalutamide induced appreci- of the caspases in bicalutamide function and to able apoptosis in a different malignant androgen- examine specifically if there were disparities in caspase independent cell line (DU-145). Vicentini et al.17 also dependency between the two cell lines. To this effect, demonstrated that high-dose bicalutamide inhibited we assessed the role of pan-caspase inhibition

Prostate Cancer and Prostatic Diseases Bicalutamide-induced apoptosis in prostate cell lines M St John Floyd Jr et al

32 (using zVAD-FMK) on bicalutamide-induced apoptosis cell death pathway. Although we did not directly assess in vitro. the impact of serine protease inhibition in this work, we Differences in caspase dependency between androgen- did demonstrate that pan-caspase inhibition did affect dependent (PWR-1E) cells and androgen-independent bicalutamide action in androgen-dependent cells. cells (PC-3) with respect to bicalutamide-induced apop- Recent data have demonstrated a differential effect of tosis were noted. In the androgen-dependent PWR-1E bicalutamide on different cell lines. Telomerase activity is cell, bicalutamide function is caspase dependent. In the altered after 3 days of bicalutamide therapy in androgen- androgen-independent PC-3 cell line, pan-caspase inhi- sensitive cells (accompanied by decreased levels of bition had minimal effect (P ¼ 0.06) on bicalutamide- androgen receptors and chaperone proteins), and yet it 25 induced apoptosis at the higher dose 50 mM. Thus, remains unaffected in insensitive cells. This same study bicalutamide-induced apoptosis was a predominantly also illustrated a caspase-independent apoptosis by caspase-independent event with respect to the PC-3 cell. bicalutamide in androgen-insensitive cells. This gave rise to two fundamental questions: (a) why is In conclusion, these results demonstrate the proapop- there a disparity between the cell lines (PWR-1E and totic effect of bicalutamide on benign androgen-depen- PC-3) using the same drug? and (b) if it is only partially dent and malignant androgen-independent prostatic caspase dependent, what cell death pathway does epithelial cells. In keeping with the work by Vicentini bicalutamide use to induce PC-3 cell apoptosis? From et al.,17 we demonstrated that bicalutamide can induce our results, it is evident that bicalutamide can induce cell death in receptor-negative cells. apoptosis of androgen receptor blockade. Expression of This apoptotic event is partially reliant on calpain antiapoptotic Bcl-2 members such as Bcl-xl (preventing activity in androgen-independent cells. In addition, cytochrome c release, mitochondrial incapacitation and given our specific aim at apoptotic pathway delineation, subsequent caspase activation) has been implicated in we demonstrated that bicalutamide, in androgen-inde- the resistance of PC-3 cells to apoptosis.18 pendent cells, acts in a predominantly caspase-indepen- As we had determined that bicalutamide-induced dent manner and yet is also partially reliant on calpains apoptosis may be a partially caspase-independent event to induce apoptosis. in PC-3 cells, we next determined the role of calpains in Thus, this study provides evidence that bicalutamide bicalutamide-induced apoptosis. persists in providing a proapoptotic effect on prostate Calpains are -dependent cysteine proteases cancer cells in vitro, even in androgen-independent cells. found in all eukaryotic species. Structurally two forms Further work at defining specific apoptotic pathways exist, namely m and m calpain, and they are produced will help to determine the exact role of how bicalutamide with their endogenous cytosolic inhibitor, calpastatin,19 induces apoptosis in androgen-independent cells. Ex- which regulates calpain activity in vivo. They have a tending the use of antiandrogens in hormone-refractory broader range of targets than the caspases, with regards disease will broaden the spectrum of prostate cancer to substrate cleavage, and are involved in functions such treatment available. as cytoskeleton remodelling of proteins (such as fodrin), receptor removal, protein kinase degradation and processing of calcium ATPase.19 In HL-60 cells, pan- caspase inhibition affects calpain cleavage of Bax, Acknowledgements whereas use of calpeptin, a calpain inhibitor, does not Michael St John Floyd Jr was a British Urological affect caspase activity, highlighting a possible temporal Foundation Scholar during the course of this work. role between caspase and subsequent calpain activa- tion.20 Inhibition of calpain activity, which can affect growth factor signalling, has been demonstrated to reduce prostate tumour cell invasiveness in vitro.21 References Inhibition of calpain function in prostate cancer cells yields conflicting results. Calpain inhibition did not 1 Saad F. Treatment of complications in advanced prostate affect the benign PWR-1E cell line. In androgen- cancer: rationale for bisphosphonate use and results of a phase independent PC-3 cells, bicalutamide acts in a partially III trial with zoledronic acid. Semin Oncol 2002; 29 (6 Suppl 21): calpain-dependent manner. In CPTX prostate epithelial 19–27. cells, thapsigargin induces apoptosis with a concomitant 2 Blackledge G. Casodex—mechanisms of action and opportu- 72 reduction in insulin receptor substrate-1 protein levels. nities for usage. Cancer 1993; (12 Suppl): 3830–3833. 3 Anderson J. The role of antiandrogen monotherapy in the Use of a calpain 2 inhibitor prevents this, highlighting a treatment of prostate cancer. BJU Int 2003; 91: 455–461. role for calpains in thapsigargin-mediated prostate cell 4 Lekas E, Bergh A, Damber JE. Effects of and 22 apoptosis. This supports our finding with the PC-3 cell bicalutamide on prostatic blood flow in the rat. BJU Int 2000; line, as CPTX cells are derived from a primary prostate 85: 962–965. carcinoma and are thought not to express androgen 5 Nickerson T, Pollak M. Bicalutamide. (Casodex)-induced pros- receptors. Specific to PC-3 cells, calpain 2 inhibition alone tate regression involves increased expression of genes encoding has been shown not to be cytotoxic, but in malignant insulin-like growth factor binding proteins. Urology 1999; 54: lymphoid cells, caspase inhibition inhibits calpain 2, 1120–1125. again highlighting a possible sequential, dependent 6 Culig Z, Hoffmann J, Erdel M, Hobisch A, Hittmair A, Bartsch G. Switch from antagonist to agonist of the androgen receptor relationship between the caspase and calpain families.23 24 bicalutamide is associated with prostate tumour progression in a Lu and Mellgren demonstrated that caspase inhibi- new model system. Br J Cancer 1999; 81: 242–251. tion prevented apoptosis induced by serine protease 7 Madarova J, Lukesova M, Hlobilkova A, Rihakova P, Murray inhibition, pointing towards an involvement of the serine PG, Student V. Androgen sensitivity related proteins in proteases in apoptosis with caspases being the ultimate hormone-sensitive and hormone-insensitive prostate cancer cell

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