Prostate Cancer 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 bicalutamide-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 antiandrogen used in the treatment of prostate cancer. Although widely accepted as an androgen receptor antagonist, 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 hormone-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 antiandrogens.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 dihydrotestosterone. This inhibits the androgen-depen- (luteinizing hormone-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-steroidal antiandrogen 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 insulin- 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 androgen receptor agonist. 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