Endocrine-Related Cancer (2010) 17 757–770

Differential expression of 5a-reductase and association with disease severity and angiogenic predict their biological role in cancer

Kakoli Das1,2, Pia D N Lorena2, Lai Kuan Ng2, Diana Lim2, Liang Shen3, Woei Yun Siow4, Ming Teh2, Juergen K V Reichardt 5 and Manuel Salto-Tellez1,2

1Cancer Science Institute, 2Department of Pathology, 3Biostatistics Unit and 4Department of Urology, National University Health System, National University of Singapore, 28 Medical Drive, Singapore, Singapore 117456 5Plunkett Chair of Molecular Biology (Medicine), University of Sydney, Sydney, New South Wales 2006, Australia (Correspondence should be addressed to K Das at Cancer Science Institute, National University of Singapore; Email: [email protected]; M Salto-Tellez at Cancer Science Institute, National University of Singapore; Email: [email protected])

Abstract The biological role of steroid 5a-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (nZ62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and , followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (PZ0.02), higher cancer stage (PZ0.01), and higher serum prostate specific antigen (PSA) levels (PZ0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (PZ0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor b 1(TGFB1), endothelin (EDN1), TGFa (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 mM). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors. Endocrine-Related Cancer (2010) 17 757–770

Introduction types I and II, encoded by the SRD5A1 and SRD5A2 et al Prostatic carcinogenesis and growth are largely genes respectively (Bruchovsky . 1988, Bonkhoff dependent on the vital male hormone dihydrotestoster- et al. 1996). Angiogenesis is regulated by several one (DHT), the most bioactive , and the stimulating and inhibiting growth factors, the most process of neovascularization called angiogenesis. potent stimulatory factor being vascular endothelial Production of in prostate cells occurs by growth factor (VEGF; Leung et al. 1989, Boddy et al. the action of two isozymes, steroid 5a-reductase 2005). Recently, a third SRD5A, encoded by the

Endocrine-Related Cancer (2010) 17 757–770 Downloaded from Bioscientifica.comDOI: 10.1677/ERC-10-0022 at 09/30/2021 11:49:40AM 1351–0088/10/017–757 q 2010 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access K Das et al.: Steroid 5a-reductase in prostate cancer

SRD5A3 , has been suggested to be associated isozymes in metastatic PC (Habib et al. 2003). Others, with DHT production and androgen receptor (AR) acti- however, have shown higher expression levels of vation in hormone refractory prostate cancer (PC; SRD5A1 and SRD5A2 in both types of PC (Nakamura Uemura et al.2008). Its role, however, remains obscure. et al. 2005), and enhanced expression of SRD5A1 in SRD5A1 and SRD5A2 are membrane-associated PC compared with BPH tissues (Bonkhoff et al. 1996, , composed of 259 and 254 amino acids Thomas et al. 2003, Wako et al. 2008). respectively. The genes encoding them, SRD5A1 and Prostate carcinogenesis is a slow, stepwise process, SRD5A2, map to bands 5p15 and 2p23 and medical approaches are necessary to either delay respectively, and are mainly expressed in the , skin the natural history of the disease or prevent it (Thigpen et al. 1993, Russell & Wilson 1994), and PC altogether. The 5a-reductases offer a mechanism for tumors (Thigpen & Russell 1992, Reichardt et al. 1995, reducing androgen stimulation of the prostate without Nakamura et al. 2005). Polymorphisms in the SRD5A2 lowering circulating levels (Rittmaster gene were previously demonstrated to be associated et al. 2009). Although SRD5A2 is known to be the with PC susceptibility (Makridakis et al. 1999), predominant gene in the prostate and a prominent although the same group recently revisited their therapeutic target for BPH, its potential as a therapeutic multi-ethnic cohort data and reported no association agent for PC is currently unknown. , an between the alanine-49 to threonine (A49T) missense SRD5A2 inhibitor that prevents the conversion of variant of the SRD5A2 gene and PC risk (Pearce et al. testosterone to DHT, is a widely used drug for the 2008). We also studied the valine-89 replaced by treatment of BPH. It is also used for BPH-related leucine (V89L) single nucleotide polymorphism of hematuria, since expression of VEGF and microvessel the SRD5A2 gene reported in a Singapore hospital. density are affected by SRD5A2 activity (Pareek et al. However, further study failed to find any such 2003). Finasteride has been shown to stimulate VEGF correlation between this variant and PC risk (Das expression in other cells as well, e.g. dermal papilla et al. 2008a). cells, which are targets for many circulating hormonal Both SRD5A isozymes are thought to be important and growth factors (Lachgar et al. 1999). Dutasteride, in the local production and action of DHT in normal another inhibitor of 5a-reductase, blocks the activity of and neoplastic prostatic tissue. Cellular interactions both isozymes, and is currently being investigated for in the prostate allow 5a-reductase to metabolize both PC risk reduction and treatment (Rittmaster et al. testosterone to DHT, which forms a complex with 2009). Higher androgen levels, as noted in primary AR, which is then translocated to the nucleus. In and metastatic PC, increase VEGF and AR expression contrast, androgen target cells in peripheral tissues use (Joseph et al. 1997), but have the opposite effect in testosterone directly to activate ARs. The DHT–AR androgen-deprived PC (Cheng et al. 2004, Aslan et al. complex then binds to nuclear DNA sequences known 2005). However, whether or not VEGF, or other as androgen response elements, which promote angiogenic genes, is regulated by 5a-reductase transcription of the target genes involved in prostate remains uncertain. cell homeostasis, angiogenesis, differentiation, and In order to understand the relationship between apoptosis (Russell & Wilson 1994, Gelmann 2002). SRD5A1 and SRD5A2 and their effects on the PC cell Testosterone activates AR in the same manner as DHT. angiogenic response, we investigated the expression of However, DHT dissociates from AR more slowly than SRD5A1 and SRD5A2, AR, and the key angiogenic testosterone (Russell & Wilson 1994); hence, most molecule VEGF in PC tissue microarrays (TMAs), and ARs in the prostate are occupied by DHT under steady correlated them with clinico-pathological charac- conditions. A major role of 5a-reductase, therefore, is teristics of PC patients. Furthermore, expression of a to enhance the androgen effect by allowing tighter panel of genes in the angiogenesis pathway was also binding to the AR. examined in selected tissues by quantitative PCR. The expression level of SRD5A is elevated in benign Malignant and non-malignant prostate cells were also prostatic hyperplasia (BPH) and PC, resulting in treated with finasteride and dutasteride in order to enhanced production of DHT and overexpression of analyze the expression of these angiogenesis pathway AR (Thomas et al. 2003, 2005, Nakamura et al. 2005, genes. Our results showed an increase in SRD5A1 Wako et al. 2008). However, the biological role of the expression in PC tissues, which was correlated with two 5a-reductase isoforms in PC and BPH is not disease aggressiveness and upregulation of certain clearly understood. Some studies have shown a switch genes in the angiogenesis pathway in response to both from SRD5A2 to SRD5A1 expression in recurrent PC finasteride and dutasteride, despite a reduction in (Titus et al. 2005) and loss of expression of both tumor cell viability.

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Materials and methods antibodies for SRD5A2 (dilution 1:50) (Abnova Corporation, Taipei City, Taiwan) were used, followed Patient tissues by incubation with biotinylated anti-mouse/rabbit PC and matched benign tissues were consecutively secondary HRP-conjugated streptavidin (DAKO obtained from 62 patients who had undergone radical Real, Cambridgeshire, UK). Peroxidase activity was prostatectomy (nZ25) and transurethral prostatectomy then visualized using diaminobenzidine substrate. As a (nZ37) without prior treatments such as hormone negative control, normal rabbit or mouse IgG was used therapy, chemotherapy, or radiotherapy at the Division instead of the primary antibodies, with no specific of Surgery, National University Hospital (NUH), immunoreactivity observed in these tissue sections. Singapore, between 2000 and 2007. All prostate TMA-immunostained sections were visually scored specimens were acquired in compliance with the by two observers (P D and K D) who were blinded to guidelines of the Institutional Review Board and ethics the clinical outcome of the patients. Scoring was then committee including patients’ informed consent confirmed by an experienced pathologist (M S T). (DSRB-B/09/141). Table 1 shows the characteristics Within each tissue sample, expression in the tumor of the patients included in the study. Histopathological cells was relatively homogenous. The staining was staging of PC was based on the 1997 TNM-staging semi-quantitatively assessed on a scale ranging from 0 system (Sobin & Fleming 1997). Patients were followed to 3, termed as ‘expression scores’ (0, no expression; from the time of diagnosis until death. Alternatively, 1, weak expression; 2, moderate expression; 3, strong those patients with a minimum of 3 years of followup expression), following our previously published pro- were included for the survival analysis. tocols (Zhang et al. 2003). To determine the protein expression correlation between BPH and PC by Immunohistochemistry immunohistochemistry, expression scores 0 and 1 were grouped together and assessed as ‘negative Immunohistochemistry was performed on TMA con- expression’, and expression scores 2 and 3 were structed from formalin-fixed paraffin-embedded grouped together and assessed as ‘positive expression’, (FFPE) prostate tumors and matched benign tissues as described previously (Das et al. 2009). Tumors that following the protocol described previously (Zhang could not be scored due to insufficient tissue cores in et al.2003, Salto-Tellez et al. 2007, Das et al. the TMA were discarded from the study. 2008b). Sections (4 mm) were deparaffinized and rehydrated using graded alcohols before heating with antigen retrieval solution (pH 6) at 120 8C for 5 min, followed by peroxidase blocking for 10 min at room Two established human PC cell lines, LNCaP and PC3 temperature. Primary polyclonal antibodies for (ATCC, Manassas, VA, USA), were maintained in human VEGF (dilution 1:20) (sc-152, Santa Cruz RPMI 1640 and F-12 media (Gibco), supplemented Biotechnology, Santa Cruz, CA, USA), AR (dilution with 10% heat-inactivated fetal bovine serum and 1% 1:1500) (Chemicon International, Temecula, CA, L-glutamine respectively. Another prostate cell line USA), and SRD5A1 (dilution 1:100), and monoclonal derived from normal prostate cells, RWPE-1 (ATCC), was maintained in keratinocyte serum-free medium Table 1 Prostate cancer patient characteristics (NZ62) (Gibco) supplemented with 0.05 mg/ml bovine pitu- itary extract and 5 ng/ml epidermal growth factor. Cell Characteristics N lines were seeded in 12-well plates at a density of 5 Age, median (range), in years 70 (52–95) 1!10 cells/ml and incubated for 24 h. They were Pre-operative PSA, 19.4 (1.49–4864) then treated with finasteride (Sigma–Aldrich) and median (range), in mg/l dutasteride (GlaxoSmithKline) at the following Gleason score concentrations: 0, 0.5, 1, 4, 8, 13, 15, 25, 35, and 4–6 19 (31%) 7 16 (26%) 50 mM, and incubated for 72 h. Each treatment was 8–10 26 (43%) carried out in triplicate for the given time point. Group stage I 6 (10%) MTT assay II 14 (23%) III 22 (36%) The Methylthiazol Test (MTT), an in vitro toxicology IV 19 (31%) assay (Sigma–Aldrich), was performed to detect cell Lymph node metastasis 1 viability after 72 h of incubation at 37 8C. Following Bony metastasis 18 the finasteride treatment and incubation described

Downloaded from Bioscientifica.com at 09/30/2021 11:49:40AM via free access www.endocrinology-journals.org 759 K Das et al.: Steroid 5a-reductase in prostate cancer above, reconstituted MTT solution (15 mg/vial) equal was centrifuged to separate the supernatant and the to 10% of the culture medium volume was added to pellet. The supernatant was resuspended in 500 mlof each well of the microtiter plate, and incubated for 4 h 1! hypotonic buffer and incubated in ice for 15 min. at 37 8C until the yellow MTT dye was reduced to This was followed by the addition of 25 ml detergent purple formazan solubilized with acidified iso- and centrifuged for 30 s at 4 8C. This served as the propanol. The experiment was performed in triplicate. cytoplasmic extract. The pellet represented the nuclear The microtiter plate was then read at an absorbance of fraction. This nuclear pellet was lysed in 150 ml 570 nm, and cell viability was examined by dividing complete lysis buffer, incubated for 30 min with the optical density (OD) of the drug-containing wells constant shaking, and centrifuged at 4 8C. Nuclear by the OD of the drug-free control. Appropriate cut off and cytoplasmic extracts were then used for western values were determined for the cell lines after the blotting as described below. background subtraction at the reference wavelength, 690 nm. The percent cell viability was then calculated Western blotting as OD(treated)KOD(blank)/OD(control)KOD(blank)!100%. Cell lysates extracted from the finasteride- and dutaste- ride-treated and untreated LNCaP, PC3, and RWPE-1 Quantitative real-time PCR cell lines were used for detecting the expression of Quantitative real-time PCR (Q-PCR) was performed SRD5A1, SRD5A2, VEGF, AR, TGFB1, and b-actin in FFPE prostate tissue samples (nZ3) and finasteride- proteins by western blot analysis. Protein samples were treated, dutasteride-treated, and untreated (control) separated using a 12% SDS-PAGE gel and transferred groups using low-density arrays consisting of to nitrocellulose membranes. The membranes were genes involved in angiogenesis (each in duplicate) pre-treated with 3% BSA and incubated with primary (Applied Biosystems, Foster City, CA, USA). Each antibodies, namely polyclonal goat anti-human VEGF Q-PCR reaction constituted of 2! TaqMan Universal (dilution 1:800) (Santa Cruz), SRD5A1 (dilution PCR master mix (10 ml) and cDNA template (15 ng) in 1:1000) (Santa Cruz), SRD5A2 (dilution 1:2000) a final reaction volume of 20 ml, and was carried out (Abnova), AR (dilution 1:2000) (Millipore, Temecula, using the ABI Prism 7900HT thermocycler CA, USA), TGFB1 (dilution 1:200) (Cell Signaling, (Applied Biosystems) under standard cycling con- Danvers, MA, USA), or b-actin (1:5000) (Santa Cruz) ditions. Each of the cell lines and tissues was for 1 h. The membranes were then washed in PBS-T normalized using an endogenous reference, glycer- (0.1% Tween 20 in PBS) and incubated at room aldehyde-3-phosphate dehydrogenase. For each temperature for 30 min with the secondary antibodies analysis, a negative control was prepared using all of specific for each primary antibody. The blots were then the reagents except the cDNA template. All reactions visualized with enhanced chemiluminescence with an were conducted in triplicate. Relative expression of ECL detection kit (Santa Cruz Biotechnology). each gene (RQ) was calculated by the comparative cycle threshold method using the formula K ð DDCtÞ Z tumor tissue or treated cellsK ELISA ð2 Þ,whereDDCt DCt benign tissue or untreated cells Z DCt and DCt DCt (target Expression of VEGF and other proteins in the gene)KDCt (endogenous reference). Taking the angiogenesis pathway was measured using the Quanti- standard error and the RQ range into consideration, body Human Angiogenesis Array 2 (RayBiotech Inc., we established a cut off RQ value or fold change of Norcross, GA, USA) in dutasteride-treated and R1.5 as ‘upregulation’ of . For a untreated cells (seven treated and three untreated few genes in the treated cells, gene expression could cells). The antibody array is a glass-chip-based not be determined. This was most likely due to multiplexed sandwich ELISA system designed to experimental error, and such genes were designated determine the expression of 30 cytokines simul- as ‘undetermined’. taneously. Each chip has 16 wells (6 for standards and 10 for samples) with 120 spots per well representing 30 cytokines in quadruplicate. Briefly, Nuclear–cytoplasmic extraction from cell lines samples and standards were added to the wells of the Sub-cellular components (i.e. nucleus and cytoplasm) chip array and incubated for 3 h at 4 8C. This was were separated using a nuclear extraction kit (Active followed by three to four washing steps and the Motif, Tokyo, Japan). Briefly, the cell suspension addition of primary antibody and HRP-conjugated from the prostate cell lines LNCaP, PC3, and RWPE-1 streptavidin to the wells. The chip was then scanned

Downloaded from Bioscientifica.com at 09/30/2021 11:49:40AM via free access 760 www.endocrinology-journals.org Endocrine-Related Cancer (2010) 17 757–770 using a Genepix scanner and quantified using Quanti- adjustment was applied to compare different inhibitor body Analyzer software (Ray Biotech Inc). Relative concentrations with the untreated control in the three expression levels of the angiogenic proteins in the types of prostate cell lines. A P value of !0.05 was treated samples were analyzed by comparing the considered to be significant. intensities of the signals with respect to the positive and negative controls provided on the chip. Results Statistical analyses Expression of target proteins in prostate tissues Statistical analysis was performed using SPSS version by immunohistochemistry 17.0 for Windows (SPSS Inc., Chicago, IL, USA). The We used immunohistochemistry to examine the two-sided c2 test or Fisher’s exact test was used to expression of the androgen genes SRD5A1, SRD5A2, evaluate the correlation between two categorical and AR, as well as one of the major angiogenic variables, i.e. for group stages, Gleason scores (GS), proteins, VEGF, in PC and BPH tissues. Whereas both and metastasis. The Mann–Whitney U test was used to nuclear and cytoplasmic expression in epithelial cells determine the correlation between pre-operative serum was observed for the isozymes encoded by SRD5A1 PSA levels and target proteins in the immunohisto- (Fig. 1a) and SRD5A2 (Fig. 1b), and for AR protein chemical analysis. Survival analysis was performed (Fig. 1c), only cytoplasmic expression was detected for according to the Kaplan and Meier method. The Cox VEGF protein (Fig. 1d) in both PC and BPH tissues. proportional hazards regression model was used to The positive cytoplasmic expression of SRD5A1 was analyze the prognostic factors for the clinical and higher in PC compared with BPH (80 vs 65%; biological characteristics determined at the time of PZ0.048), whereas positive nuclear expression was diagnosis. Disease-specific deaths were defined as all higher in BPH compared with PC (79 vs 58%; deaths presumably caused directly by PC. A P value of PZ0.012; Table 2). However, the magnitude of the !0.05 was considered to be significant. effects was relatively small. Both the 5a-reductase The general linear model was used to evaluate the types also showed stromal cell expression overall effects of different concentrations of the two (Fig. 1a and b). inhibitors, finasteride and dutasteride, on percent cell Endogenous levels of SRD5A enzymes in the viability. Multiple comparison with Bonferroni nuclear and cytoplasmic components of the prostate

(a) (b) (e)

SRD5A1 C 26 kDa

SRD5A1 N

SRD5A2 N 30 kDa

AR N 108 kDa

β-Actin C 43 kDa

β-Actin N

PC3 LNCaP RWPE-1 (c) (d)

Figure 1 Protein expression in PC tissues of (a) SRD5A1, (b) SRD5A2, (c) AR, and (d) VEGF at low (4!) and high (40!) magnification. (e) Immunoblots showing nuclear (N) and cytoplasmic (C) protein expression of SRD5A1, SRD5A2, and AR in prostate malignant LNCaP and PC3 cells, and non-malignant RWPE-1 cells.

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Table 2 Protein expression in the epithelial cells of benign prostatic hyperplasia (BPH) and prostate cancer (PC)

Nuclear Cytoplasmic

Expression of proteins BPH (%) PC (%) P value BPH (%) PC (%) P value

SRD5A1 NZ48 NZ103 NZ48 NZ103 K 21 42 0.01* 35 20 0.048* C 79 58 65 80 SRD5A2 NZ46 NZ106 NZ46 NZ106 K 61 74 0.12 35 29 0.50 C 39 26 65 71 VEGF NZ52 NZ111 K 44 30 0.07 C 56 70 AR NZ52 NZ108 NZ52 NZ108 K 71 57 0.09 83 63 0.01* C 29 43 17 37

*P value !0.05 is significant. malignant and non-malignant cells were also assessed expression, and the expression of these proteins by western blot. SRD5A1 was highly expressed in both occurred more frequently in PC compared with BPH nuclear and cytoplasmic fractions in LNCaP cells, cases. This is consistent with previous findings (Cheng whereas its level was more cytoplasmic than nuclear et al. 2004, Aslan et al. 2005, Boddy et al. 2005). in PC3 cells and was nuclear only in RWPE-1 cells. The expression of SRD5A2, however, was almost Protein expression correlation with undetectable in LNCaP and RWPE-1 nuclear and clinico-pathological parameters cytoplasmic fractions, and was very low in the PC3 PC group stage nuclear fraction (Fig. 1e). The expression scores of the proteins under investi- Correlation between hormonal components and the gation, SRD5A1, SRD5A2, AR, and VEGF, in PC angiogenic factor showed a statistically significant (nZ62) were analyzed in two groups, stages I and II association between nuclear SRD5A2 and VEGF (primary stage cancer) and stage III (locally advanced expression in PC. Tissues that were positive for cancer). Nuclear versus cytoplasmic expression of VEGF and nuclear SRD5A2 were 95 vs 5% respect- SRD5A1 in tumors was significantly associated with ively in PC (PZ0.012), and 89 vs 11% respectively in stage III PC (40 vs 80%, PZ0.01, and 60 vs 95%, BPH (PZ0.001). No such association, however, was PZ0.02 respectively) as shown in Table 3. Overall observed between SRD5A1 and VEGF in the two expression of SRD5A2, AR, and VEGF showed no tissue types. VEGF expression was significantly significant difference across the two groups, suggesting associated with nuclear AR expression in PC (69 vs that expression of these proteins was maintained 39%; PZ0.027) and AR expression in BPH (80 vs throughout early and advanced cancer stages. The 20%; PZ0.02). In other words, tumors that showed target proteins also did not show any significant expression of AR also showed higher levels of VEGF association with distant metastasis.

Table 3 Clinical characteristics compared with nuclear and cytoplasmic protein expression

SRD5A1 expression % SRD5A2 expression % VEGF expression % AR expression %

Clinical characteristic NZ62 Nu Cyt Nu Cyt Nu Cyt Nu Cyt

Group stages (I and II/III) 40/80 60/95 35/27 65/91 – 84/77 42/45 21/45 P value 0.01* 0.02* 0.59 0.06 0.70 0.83 0.10 Metastasis (M0/M1) 62/80 79/87 29/20 77/73 79/62 44/25 37/44 P value 0.20 0.71 0.74 0.76 0.31 0.18 0.65 Gleason score (4–6/7/8–10) 42/73/83 63/93/87 26/37/21 63/100/71 83/87/60 33/37/44 33/44/39 P value 0.02* 0.05 0.51 0.03* 0.08 0.78 0.82 Pre-operative serum PSA level 66 79 25 76 71 35 39 Median (mg/l) 23 20 10 20 89 23 29 P value 0.01* 0.002* 0.25 0.32 0.25 0.87 0.12

*P value !0.05 is significant (Leung et al. 1989).

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PC grade been analyzed immunohistochemically. The samples Protein expression, when analyzed in three groups of were of the same grade (GSZ7) but different stages GS, showed increased SRD5A1 nuclear expression (T2, T3, and T4, one of each). The fold changes of the in GS 8–10 as compared with GS 7 and GS 4–6 of genes in PC tissues are shown in Table 4. Gene PC, which was statistically significant (83 vs 73 vs expression was upregulated in the metastatic stage (T4) 42%, PZ0.02). SRD5A2 cytoplasmic expression compared with the other two stages (T2 and T3) in the was, however, more frequent in the GS 7 group than PC tissues, confirming their expression. in the GS 4–6 and GS 8–10 groups (71 vs 100 vs 63%, PZ0.03; Table 3). Cell viability assay in finasteride and dutasteride treatment groups Pre-operative serum PSA levels We next performed an in vitro analysis to study the SRD5A1 nuclear and cytoplasmic expression (median molecular effects of SRD5A inhibitors in the angio- 23 and 20 mg/l respectively, range 2.0–4864 mg/l) genesis pathway. For this, we first examined whether showed significant association with elevated pre- or not inhibition of 5a-reductase activities could operative serum PSA levels (Table 3). The other attenuate the proliferation of prostate malignant and three proteins, SRD5A2, VEGF, and AR, did not non-malignant cells. We treated the malignant LNCaP correlate with PSA levels. Survival analysis did not and PC3, and non-malignant RWPE-1, cells with indicate any statistical significance among these finasteride and dutasteride for 72 h. Since a time- and biomarkers (data not shown). dose–response experiment for dutasteride had already been conducted by Schmidt et al. (2004), demonstrat- Expression of angiogenesis pathway genes in PC ing that viability and morphology of the androgen- tissues by Q-PCR dependent LNCaP cells was affected after 48 h of treatment, we chose a longer time period of 72 h We also carried out Q-PCR using low-density arrays treatment for our experiments. to examine the expression of SRD5A1, SRD5A2, AR, The MTT assay demonstrated a significant difference and a panel of genes in the angiogenesis pathway in in cell viability between finasteride-treated cells and prostate tumor FFPE samples (nZ3) that had already untreated controls in the three cell types. The LNCaP Table 4 Expression of genes in the angiogenesis and cells showed a reduction in cell viability as the treatment androgen-signaling pathway in prostate cancer (PC) tissues concentration increased. The cell viability was reduced to 65% at 4 mM, and was reduced to 8% at 13 mM. This Fold change was statistically significant when compared with GS7 T2 GS7 T3 GS7 T4 untreated controls (P!0.0005; Fig. 2a). In PC3-treated Z Z Z Gene symbols N 1 N 1 N 1 cells, cell numbers were also reduced noticeably at CDH1 8.7 16.4 186.3 concentrations between 4 and 50 mM. Cell viability EDN1 57.3 282 6571.7 at 4 mM was 57%, and dropped to 36% at 25 mM. IL-15 60 115.5 9822.7 In RWPE-1 cells, growth was inhibited significantly at IL-6 146.8 913 1869.8 concentrations between 35 and 50 mM compared with IL-7R 25.1 45.2 96.6 ! SERPINB5 K4.5 2.6 5.7 untreated control cells (P 0.0005). Though not SERPINB8 2.2 7.8 11.5 statistically significant, cell viability reductions of up HIF-1 25.6 161.9 1481.6 to 81% were observed at 4–50 mM finasteride, with a TGFA 1.7 3.9 9.8 65% reduction at the 25 mM concentration. TGFB1 236.1 607 592.6 In the dutasteride-treated LNCaP group, a reduction TGFB2 24.4 114.9 4833.6 TGFB3 2816 7823.2 2.80!104 in cell number was evident starting at the first dosage TGFBR1 12.4 44.5 375.6 of 0.5 mM dutasteride; at this dose, cell viability TGFBR2 27.8 43.1 2988.8 was reduced to 88%. Then, at 1 mM, cell viability was TGFBR3 256.1 493.7 483.7 further reduced to 79%, and at 25 mM, viability was VEGF 70.7 1073.4 6962.1 VEGFR1 2744 21 956.8 2.90!104 reduced to 24%, a statistically significant reduction VEGFR2 175.5 264.1 2.90!105 (P!0.0005) compared with the untreated controls. SRD5A1 6 21.1 65.1 Cell numbers were also reduced in PC3 cells; at SRD5A2 5.5 15.1 20.6 0.5 mM dutasteride, cell viability was reduced to 90%. AR 1.60!104 1.06!107 UD As with LNCaP cells, higher doses of dutasteride UD, undetermined. resulted in more significant reductions in viability: at

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(a) (b) PC3 0 µM1 µM 25 µM 200.00

LNCaP cells

150.00

100.00 PC3 cells % cell viability

50.00

0.00

0.00 10.00 20.00 30.00 40.00 50.00 Inhibitor concentration (µM)

Figure 2 Inhibition of 5a-reductase activity by finasteride and dutasteride treatment attenuates viability of PC cells. (a) Percent viability as a function of inhibitor concentration in LNCaP, PC3, and RWPE-1 cells treated at various concentrations of inhibitor for 72 h as described in Materials and methods, with 0.00 concentration indicating untreated controls. (b) Phase contrast microscopy image of LNCaP and PC3 cells, untreated (0 mM) and treated with dutasteride at 1 and 25 mM for 72 h. 4 mM, viability was reduced to 58%, and at 25 mM, for both inhibitors. However, the expression of the viability was reduced to 2%. In RWPE-1 cells, genes was not statistically significant. dutasteride was also effective in inhibiting cell growth In finasteride-treated PC3 cells, the expression of the at doses ranging from 0.5 to 50 mM(P!0.0005), with genes did not follow that of the androgen-responsive a reduction to 55% cell viability at 0.5 mM and 11% LNCaP cells. At the 1 mM concentration, most of the at 25 mM concentrations. genes were downregulated, with the exception of We also compared the overall effects of finasteride interleukin (IL)-7R. However, at a concentration of and dutasteride treatment at different concentrations 25 mM finasteride, at least 7 of the 21 genes tested were on cell viability in the three cell types (Fig. 2a and b). upregulated (Table 5). In dutasteride-treated PC3 cells, No statistically significant differences were observed downregulation of all of the genes was observed at both between the two inhibitors in LNCaP-treated cells 1 and 25 mM concentrations, with the exception of (PZ0.520), but in PC3 and RWPE-1 cells, dutasteride TGFB1, although the expression was not statistically was significantly more effective in inhibiting cell significant (PZ0.33) probably due to smaller number growth than finasteride (P!0.0005). of the samples. In RWPE-1 cells treated with 1 mM finasteride, Expression of angiogenesis pathway genes in the expression of hypoxia-inducible factor 1 a (HIF-1A), treated PC cells IL-6, IL-15, serine peptidase inhibitor (SERPINB5), TGFA TGFB TGFB2 b Using Q-PCR, we next examined the effect of , , , transforming growth factor m receptor 1 (TGFBR1), TGFBR3, VEGFA,andSRD5A1 finasteride and dutasteride at 1 and 25 M on the % expression level of different genes in the angiogenesis genes showed a fold change 1.4-fold. Furthermore, pathway. By examining RNA isolated from treated and the expression of these genes was lower in cells treated m m untreated cells, expression of our gene set was with 25 M finasteride doses compared with 1 Mdoses. compared using low-density arrays. The two doses In the dutasteride-treated cells, the expression of all of were chosen, based on the results of the viability assay, the genes was lower than that in the untreated cells for m to show the molecular effects of treatment at a low both 1 and 25 M concentrations. concentration of 1 mM and at a relatively high concentration of 25 mM. Expression of angiogenesis pathway proteins in In LNCaP cells treated with 1 and 25 mM finasteride treated PC cells and dutasteride, the angiogenic genes endothelin Using ELISA with a human angiogenesis antibody (EDN1), VEGFR1, TGFA,andTGFB1 showed increased array, we also examined the expression of proteins expression compared with the untreated controls involved in the angiogenesis pathway. Though (Table 5). The expression of AR was also increased at expression of VEGF was reduced at all concentrations the 1 mM dose, but was reduced (!1.5-fold) at 25 mM, (0.5–50 mM) of finasteride in LNCaP cells, the

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Table 5 Expression of genes in the angiogenesis and androgen-signaling pathways in LNCaP- and PC3-treated cells

LNCaP cells PC3 cells

Finasteride Dutasteride Finasteride Dutasteride

1 mM25mM1mM25mM1mM25mM1mM25mM

Genes Fold change CDH1 1.3 1.4 1.1 1.0 K2.6 K3 K1.1 1.2 EDN1 2.7* 1.9* 2.0* 1.8* 1.0 1.4 K1.4 1.2 VEGFR1 1.7* 1.9* 1.5* 1.8* K100 UD UD K5.3 HIF-1A 1.1 1.0 K1.2 K1.1 1.1 1.7* K1.1 K1.3 IL-6 K2.9 K1.2 K4.3 K12.5 1.0 3.7* 1.2 K1.2 IL-7R K1.2 K1.1 K1.5 K2.2 1.5* UD K1.5 K1.8 IL-15 K5 K1.8 K2.2 K4.5 UD UD K2 K2.8 VEGFR2 K10 1.0 1.8* 1.12 1.0 2.6* K1.1 K1.5 SERPINB5 K14.3 K3.1 1.3 K1.1 K1.9 1.4 K1 1.0 SERPINB8 1.1 K1.8 K1.8 K1.3 K1.5 1.4 K1.3 K2 TGFA 1.5* 1.5* 2.1* 1.7* K1.2 K1.1 K1.1 K1 TGFB1 2.2* 2.1* 2.1* 2.7* K1.1 1.7* 1.6* 1.7* TGFB2 K1.0 0.7 K2.2 K1.9 K1.2 1.7* K1.1 1.1 TGFB3 K1.1 0.4 K1.3 K1.7 1.1 2.5* K1.1 1.3 TGFBR1 1.1 1.0 K1.1 K1.3 K2 K1.1 K1.3 K1.2 TGFBR2 1.2 K1.1 K1.4 K1.0 K1.4 1.3 K1.2 1.0 TGFBR3 1.1 1.5* 1.6* 1.0 K1.6 K1.5 K1.1 1.0 VEGFA 1.4 K1.1 K5.5 K1.2 K1.1 1.6* K1.3 K2 AR 1.5* 1.2 1.5* 1.3 K4 K2.3 K1.1 K4.2 SRD5A1 1.2 1.2 K1.1 K16.7 K1.3 K2 K2.3 UD SRD5A2 UD UD UD UD K1.3 K25 K1.8 UD

UD, undetermined; * denotes upregulation. difference between control and treatment groups was (LNCaP, PC3, and RWPE-1). These proteins included not statistically significant (PZ0.085; Fig. 3a). In PC3 a neuropeptide, agouti-related protein; a vasculo- cells, however, VEGF levels increased at higher genesis protein, angiopoietin-2 (ANG-2); a cytokine, finasteride concentrations (35–50 mM) compared with IL1-A; growth factors basic fibroblastic growth factor untreated controls, and the difference between the and insulin-like growth factor 1; and the tissue treated and control group was statistically significant inhibitor of metalloproteinase 2 (TIMP-2). The (P!0.0005; Fig. 3a). In RWPE-1 cells, VEGF levels expression of these proteins, however, was lower were considerably increased with finasteride doses than that in the untreated controls. Expression of the ranging from 1 to 50 mM, but there was no statistical chemokines epithelial cell-derived neutrophil-activating difference between different dosages compared with peptide (ENA-78) and growth-related chemokine the controls (PZ0.094). (GRO) and the collagenase inhibitor TIMP-1 was In the dutasteride treatment group, the expression observed only in the PC3 cells. Expression of the C-C level of VEGF in the LNCaP and PC3 cells was motif chemokine monocyte chemotactic protein-1 reduced compared with the untreated controls. The (MCP-1) and the growth factors heparin-binding difference was statistically significant in PC3 cells EGF-like growth factor (HB-EGF) and platelet-derived (P!0.0005) and nearly significant in LNCaP cells growth factor b polypeptide (PDGF-BB), on the other (PZ0.059; Fig. 3a). Western blot analysis indicated a hand, was observed only in the LNCaP cells, and the similar pattern of expression of VEGF protein in expression of these proteins was lower in the treated finasteride- and dutasteride-treated LNCaP and PC3 cells compared with the untreated controls. However, cells (Fig. 3b). In RWPE-1 cells, however, no expression of the transforming growth factor TGFB1 statistical difference was observed between VEGF at was higher in dutasteride-treated LNCaP cells than in 25–50 mM and the untreated controls. controls (Fig. 3c). This was also confirmed by western The Quantibody array analysis detected expression blot (Fig. 3b). Similarly, in PC3 cells, TGFB1 and IL-6 of several proteins essential for hormone-mediated expression was also increased in the treated groups signaling in treated groups of all three cell types versus the controls, but IL-8 expression was

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(a) Leptin, leukemia inhibitory factor, PIGF, CD8-positive

6000 LNCaP T lymphocytes (RANTES), tumor necrosis growth 5000 PC3 factor a (TNFA), TNFB, thyroid peroxidase, and 4000 interferon g) could be detected in the dutasteride- 3000 treated groups in any of the three cell lines 2000 investigated. 1000

VEGF concentration (pg/ml) VEGF concentration 0 0 0.5 1 4 8 13 15 25 35 50 Finasteride concentration (µM) Discussion 5000 LNCaP It has previously been demonstrated that the two 4000 PC3 important isoforms of SRD5A, encoded by the 3000 SRD5A1 and SRD5A2 genes, which convert testos- 2000 terone into the more bioactive DHT in the prostate, are 1000 required in greater amounts to suppress the activity of

VEGF concentration (pg/ml) VEGF concentration 0 DHT in malignant versus non-malignant prostatic 0 0.5 1 4 8 13 15 25 35 50 Dutasteride concentration (µM) epithelial cells (Ellis & Isaacs 1985, Lamb et al. 1992). However, which of the two isoforms plays the (b) LNCaP PC3 0115350 1 15 35 major role in PC is still debatable. Because there are VEGF discrepancies regarding the presence and significance 42 kDa of SRD5A1 in the prostate due to variability in its SRD5A1 expression (Habib et al. 2003, Thomas et al. 2003, β -Actin 2005, Nakamura et al. 2005, Titus et al. 2005, Wako Finasteride concentration (µM) LNCaP PC3 et al. 2008), we decided to investigate the sub-cellular 0115350 1 15 35 expression level of the isozymes in BPH versus PC VEGF in human tissues of different pathological grade SRD5A1 and stage. TGFB1 SRD5A2 In the present study, we show distinct nuclear and 45 kDa β-Actin cytoplasmic expression of SRD5A1 in human PC

Dutasteride concentration (µM) tissues and a significant correlation between nuclear (c) SRD5A1 expression and higher stage, GS, and pre- 4000 IL-6 operative serum PSA levels, suggesting that this is the 3500 IL-8 TGFB1 predominant during PC progression. Although 3000 2500 increased expression of SRD5A1 in prostate tumor 2000 tissue has been reported previously (Thomas et al. 1500 2005, Wako et al. 2008), the importance of its sub- 1000 500 cellular localization in PC has not been clearly

Protein concentration (pg/ml) Protein concentration 0 demonstrated. The activity of DHT in the prostate is 0 µm1µm35µm350 µm1µm1µm350 µm µm µm based on nuclear and cytoplasmic localization of PC3 cells LNCaP cells RWPE-1 cells Dutasteride concentration SRD5A. Considering an earlier finding that approxi- mately half of the 5a-reductase enzyme responsible for Figure 3 (a) VEGF protein expression levels by ELISA assay in the transformation of testosterone to DHT is located in LNCaP and PC3 cells treated with finasteride and dutasteride the nuclei of the prostate cell (Gustafsson & Pousette as described in Materials and methods. (b) Immunoblots of total protein lysates of LNCaP and PC3 cells treated with 1, 15, and 1974, Enderle-Schmitt et al. 1986), it was necessary to 35 mM finasteride and dutasteride, and untreated controls investigate the nuclear presence of the enzyme in PC (0 mM), using anti-VEGF, anti-SRD5A1, anti-SRD5A2, anti- TGFB1, and anti-b-actin antibodies. (c) Quantibody array graph cells. Although SRD5A was shown to be localized in showing increased levels of TGFB1 in LNCaP, PC3, and the nuclear membrane (an integral membrane protein; RWPE-1 cells both untreated and treated with dutasteride. Enderle-Schmitt et al.1986), we also observed localization of the enzyme within the nucleus. This comparable to that of the controls. None of the other could be due to the fact that the prostatic enzyme is proteins (Activin A, Angiogenin, angiopoietin-like 4 dependent on both polar and non-polar portions of the (ANGPTL4), hepatocyte growth factor (HGF), IL-2, surrounding phospholipids, and that modifications of IL-17, chemokine (C-X-C motif) ligand 10 (IP-10), the phospholipids surrounding this enzyme could have

Downloaded from Bioscientifica.com at 09/30/2021 11:49:40AM via free access 766 www.endocrinology-journals.org Endocrine-Related Cancer (2010) 17 757–770 resulted in alterations in enzymatic activity, resulting the role of SRD5A in PC cell angiogenic responses in localization to the nucleus. in our experimental in vitro model by using Since DHT formed in the cytoplasm by cytoplasmic SRD5A inhibitors. SRD5A could be subject to several other reductive and When the effects of the inhibitors on cell viability oxidative metabolic pathways, the less-varied nuclear were compared in the three treated cell lines, dutasteride DHT formed by nuclear SRD5A is much more readily was more effective at overall inhibition of cell growth available to be transported to the intranuclear target in PC3 and RWPE-1 cells compared with LNCaP cells, site (Gustafsson & Pousette 1974). Therefore, our although LNCaP cells were inhibited at a lower finding of an association between nuclear SRD5A1 and concentration (0.5 mM) of dutasteride. Schmidt et al. advanced PC suggests that this isozyme may activate (2004) demonstrated a reduction in LNCaP cell large amounts of androgens during its transport from viability and cell numbers only at 1 mM of dutasteride the cytoplasm to the nucleus, affecting transcriptional after 48 h of treatment, in agreement with our events in the cell to bring about highly aggressive observation at 72 h of treatment. Dutasteride was, tumors. We also confirmed expression of SRD5A1 in however, shown to be more effective at inhibiting human prostate malignant and non-malignant cell LNCaP cell growth compared with finasteride in lines. This was in accordance with a previous report another study (Lazier et al. 2004). The discrepancy (Xu et al. 2006), although we examined sub-cellular in the results could be due to the passage number, as our protein expression, whereas they examined mRNA cells were at an early passage of !20. levels of SRD5A1 in these cells. Our low-density array data revealed continuously Conversely, we were unable to establish a strong increased expression of TGFB1 in both finasteride- and association between SRD5A2 expression and GS or dutasteride-treated LNCaP and PC3 cells, although the stage in PC tissues. This likely reflected the biological expression was not statistically significant. Expression function of SRD5A2 in regulating DHT level during was, however, lower in PC3 than in LNCaP cells, and tumorigenesis, so that continued expression of this expression was confirmed by western blot in LNCaP isoform once cancer was initiated did not have to cells (Fig. 3b). In addition, Quantibody array analysis correlate with histological appearances. SRD5A2 showed greater amounts of TGFB1 with higher expression in the PC tissues did not correlate with concentrations of dutasteride treatment in LNCaP and that in cell lines, either, with almost undetectable levels PC3 cells compared with untreated controls (Fig. 3c). of SRD5A2 in the cell lines. The presence of stromal TGFB1 is known to be frequently upregulated in tumor cells in the tissues, but not in the cell lines, may explain cells. Increased expression of this gene has been this discrepancy, as also suggested by Xu et al. (2006). suggested to affect the tumor microenvironment, It is now widely accepted that PC growth and resulting in tumor growth promotion, invasion, and progression are characterized not only by high DHT angiogenesis (Ueki et al. 1992, Hanahan & Weinberg levels in the prostate but also by a high degree of 2000, Derynck et al. 2001). On the other hand, vascularization (Folkman 1995). We were interested in stimulation of TGFB1 is also known to suppress studying both hormonal and vascular components to tumor growth (Moses et al. 1990). Thus, besides determine the relationship between and PC participating in decreased cell viability in our cell angiogenic responses. We chose VEGF as a experimental model, increased expression of TGFB1 marker of angiogenesis, as it is considered the most at higher concentrations of the inhibitors could also be important regulator of angiogenesis (Ferrara et al. suggestive of tumor resistance to the inhibitors. 1992). We verified the pattern of distribution of VEGF We also observed increased expression of other in PC in vivo, and then correlated the VEGF expression genes, including EDN1, TGFA, and the VEGF receptor level with levels of the SRD5As and AR. A statistically FLT1, in the treated LNCaP cells, and VEGF, HIF-1, significant association between VEGF, SRD5A2, and and IL-6 in the treated PC3 cells. It has been reported AR was evident in the PC tissues, but not between that growth factors such as VEGF and TGF and their VEGF and SRD5A1, suggesting that the type II receptors induce other signaling pathways (PI3K, isoform may regulate the expression of VEGF Smad, etc.) that result in endothelial cell proliferation, mediated by AR during tumorigenesis. increased vascular permeability, and cell migration Q-PCR results examining different cancer stages, (Karkkainen & Petrova 2000). The increased but of the same GS, indicated that expression of a expression of VEGF in our study could be either due panel of genes in the angiogenesis pathway to increased TGFB1 expression inducing VEGF to increased according to disease aggressiveness. Conse- act directly on endothelial cells and stimulate cell quently, these data were used to further understand proliferation and migration (Pertovaara et al. 1994),

Downloaded from Bioscientifica.com at 09/30/2021 11:49:40AM via free access www.endocrinology-journals.org 767 K Das et al.: Steroid 5a-reductase in prostate cancer or due to increased expression of AR, as AR has been Declaration of interest shown to regulate VEGF in PC (Zhu & Kyprianou The authors declare that there is no conflict of interest that 2008). Another possible reason could be the extended could be perceived as prejudicing the impartiality of the or longer duration of treatment, since increased research reported. expression levels of the above genes were not reported by Schmidt et al. (2004). The exposure of the tumor Funding cells to the inhibitors for 72 h might have changed the tumor microenvironment, resulting in evasive This work was supported by an A*Star Singapore Cancer resistance to these inhibitors by the tumors, which Syndicate Grant (MN05-R) and a Cancer Science Institute has also been reported recently for VEGF inhibitors Grant (R-713- 002-015-271) to M Salto-Tellez. (Loges et al. 2009, Paez-Ribes et al. 2009). Our study also demonstrated upregulation of AR gene expression References in LNCaP cells, which might have been the result of the sudden depletion in the level of DHT; this Aslan G, Cimen S, Yorukoglu K, Tuna B, Sonmez D, Mungan U & Celebi I 2005 Vascular endothelial growth depletion could have stimulated AR to bind with other factor expression in untreated and androgen-deprived available ligands. patients with prostate cancer. Pathology, Research and Our experimental findings in the LNCaP cells for Practice 201 593–598. the two inhibitors may not be therapeutically Boddy JL, Fox SB, Han C, Campo L, Turley H, Kanga S, promising. However, differential expression of the Malone PR & Harris AL 2005 The androgen receptor is angiogenesis pathway genes at the same concentration significantly associated with vascular endothelial growth of the two inhibitors may suggest that the effects of the factor and hypoxia sensing via hypoxia-inducible factors two SRD5A isozymes on angiogenesis genes are HIF-1a, HIF-2a, and the prolyl hydroxylases in human similar. Of course, dutasteride was able to suppress prostate cancer. Clinical Cancer Research 11 7658–7663. tumor growth more efficiently than finasteride, as Bonkhoff H, Stein U, Aumuller G & Remberger K 1996 described in our viability assay, possibly also due to Differential expression of 5 alpha-reductase isoenzymes in the human prostate and prostatic carcinomas. Prostate SRD5A1 inhibition. 29 261–267. Since the aim of the present study was to Bruchovsky N, Rennie PS, Batzold FH, Goldenberg SL, demonstrate the biological role of the 5a-reductase Fletcher T & McLoughlin MG 1988 Kinetic parameters of isozymes and identify their relationship with the genes 5 alpha-reductase activity in stroma and epithelium of and proteins in the angiogenesis pathway, we therefore normal, hyperplastic, and carcinomatous human . conclude that we have observed the predominance of Journal of Clinical Endocrinology and 67 SRD5A1 in the more advanced tumor tissues and cell 806–816. lines, and its inhibition along with SRD5A2 was more Cheng L, Zhang S, Sweeney CJ, Kao C, Gardner TA & effective in reducing cell numbers in the prostate tumor Eble JN 2004 Androgen withdrawal inhibits tumor growth cells than SRD5A2 inhibitor alone. However, the and is associated with decrease in angiogenesis and expression level of our target angiogenic genes and VEGF expression in androgen-independent CWR22Rv1 human prostate cancer model. Anticancer Research 24 proteins in the presence of SRD5A isoform, SRD5A1 2135–2140. (inhibiting SRD5A2), and in the absence of SRD5A1 Das K, Cheah PY, Lim PL, Zain YB, Stephanie FC, Zhao Y, and SRD5A2 did not show any significant difference, Cheng C & Lau W 2008a Shorter CAG repeats in and few of the target genes were still upregulated for androgen receptor and non-GG genotypes in prostate- which duration of treatment and tumor resistance to the specific antigen loci are associated with decreased risk of inhibitors could not be ruled out. benign prostatic hyperplasia and prostate cancer. Cancer Since endogenous levels of SRD5A2 expression Letters 268 340–347. were undetectable in the PC cells, and since treatment Das K, Mohd Omar MF, Ong CW, Bin Abdul Rashid S, with the inhibitor of the type II isozyme showed Peh BK, Putti TC, Tan PH, Chia KS, Teh M & Shah N molecular effects both before and after treatment, we 2008b TRARESA: a tissue microarray-based hospital think a better model than the cell lines should be used system for biomarker validation and discovery. Pathology 40 441–449. for further analysis. Therefore, future studies may Das K, Leong DT, Gupta A, Shen L, Putti T, Stein GS, consider using a different experimental model, such as van Wijnen AJ & Salto-Tellez M 2009 Positive associa- a transgenic mouse model, to study the efficacy of these tion between nuclear Runx2 and oestrogen–progesterone inhibitors and also to understand the relationship receptor gene expression characterises a biological between angiogenesis and androgen signaling subtype of breast cancer. European Journal of Cancer 45 pathways in tumor progression and metastasis. 2239–2248.

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Derynck R, Akhurst RJ & Balmain A 2001 TGF-beta Makridakis NM, Ross RK, Pike MC, Crocitto LE, Kolonel signaling in tumor suppression and cancer progression. LN, Pearce CL, Henderson BE & Reichardt JK 1999 Nature Genetics 29 117–129. Association of mis-sense substitution in SRD5A2 gene Ellis WJ & Isaacs JT 1985 Effectiveness of complete versus with prostate cancer in African–American and Hispanic partial androgen withdrawal therapy for the treatment of men in Los Angeles, USA. Lancet 354 975–978. prostatic cancer as studied in the Dunning R-3327 system Moses HL, Yang EY & Pietenpol JA 1990 TGF-beta of rat prostatic adenocarcinomas. Cancer Research 45 stimulation and inhibition of cell proliferation: new 6041–6050. mechanistic insights. Cell 63 245–247. Enderle-Schmitt U, Volck-Badouin E, Schmitt J & Nakamura Y, Suzuki T, Nakabayashi M, Endoh M, Aumuller G 1986 Functional characteristics of nuclear Sakamoto K, Mikami Y, Moriya T, Ito A, Takahashi S & 5 alpha-reductase from rat ventral prostate. Journal of Yamada S 2005 In situ androgen producing enzymes in Steroid Biochemistry 25 209–217. human prostate cancer. Endocrine-Related Cancer 12 Ferrara N, Houck K, Jakeman L & Leung DW 1992 101–107. Molecular and biological properties of the vascular Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, endothelial growth factor family of proteins. Endocrine Vinals F, Inoue M, Bergers G, Hanahan D & Casanovas O Reviews 13 18–32. 2009 Antiangiogenic therapy elicits malignant pro- Folkman J 1995 Angiogenesis in cancer, vascular, gression of tumors to increased local invasion and distant rheumatoid and other disease. Nature Medicine 1 27–31. metastasis. Cancer Cell 15 220–231. Gelmann EP 2002 Molecular biology of the androgen Pareek G, Shevchuk M, Armenakas NA, Vasjovic L, receptor. Journal of Clinical Oncology 20 3001–3015. Hochberg DA, Basillote JB & Fracchia JA 2003 The Gustafsson JA & Pousette A 1974 Properties of nuclear 5 effect of finasteride on the expression of vascular alpha-reductase in rat liver. Biochemistry 13 875–881. endothelial growth factor and microvessel density: a Habib FK, Ross M, Bayne CW, Bollina P, Grigor K & possible mechanism for decreased prostatic bleeding in Journal of Urology 169 Chapman K 2003 The loss of 5alpha-reductase type I and treated patients. 20–23. Pearce CL, Van Den Berg DJ, Makridakis N, Reichardt JK, type II mRNA expression in metastatic prostate cancer to Ross RK, Pike MC, Kolonel LN & Henderson BE 2008 bone and lymph node metastasis. Clinical Cancer No association between the SRD5A2 gene A49T Research 9 1815–1819. missense variant and prostate cancer risk: lessons learned. Hanahan D & Weinberg RA 2000 The hallmarks of cancer. Human Molecular Genetics 17 2456–2461. Cell 100 57–70. Pertovaara L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Joseph IB, Nelson JB, Denmeade SR & Isaacs JT 1997 Saksela O & Alitalo K 1994 Vascular endothelial growth Androgens regulate vascular endothelial growth factor factor is induced in response to transforming growth content in normal and malignant prostatic tissue. Clinical factor-beta in fibroblastic and epithelial cells. Journal of Cancer Research 3 2507–2511. Biological Chemistry 269 6271–6274. Karkkainen MJ & Petrova TV 2000 Vascular endothelial Reichardt JK, Makridakis N, Henderson BE, Yu MC, growth factor receptors in the regulation of angiogenesis Pike MC & Ross RK 1995 Genetic variability of the Oncogene 19 and lymphangiogenesis. 5598–5605. human SRD5A2 gene: implications for prostate cancer Lachgar S, Charveron M, Sarraute J, Mourard M, Gall Y & risk. Cancer Research 55 3973–3975. Bonafe JL 1999 In vitro main pathways of steroid action Rittmaster RS, Fleshner NE & Thompson IM 2009 in cultured hair follicle cells: vascular approach. Pharmacological approaches to reducing the risk of Journal of Investigative Dermatology. Symposium prostate cancer. European Urology 55 1064–1073. Proceedings 4 290–295. Russell DW & Wilson JD 1994 Steroid 5 alpha-reductase: Lamb JC, Levy MA, Johnson RK & Isaacs JT 1992 two genes/two enzymes. Annual Review of Biochemistry Response of rat and human prostatic cancers to the novel 63 25–61. 5 alpha-reductase inhibitor, SK&F 105657. Prostate 21 Salto-Tellez M, Nga ME, Han HC, Wong AS, Lee CK, 15–34. Anuar D, Ng SS, Ho M, Wee A & Chan YH 2007 Tissue Lazier CB, Thomas LN, Douglas RC, Vessey JP & microarrays characterise the clinical significance of a Rittmaster RS 2004 Dutasteride, the dual 5alpha- VEGF-A protein expression signature in gastrointestinal reductase inhibitor, inhibits androgen action and promotes stromal tumours. British Journal of Cancer 96 776–782. cell death in the LNCaP prostate cancer cell line. Prostate Schmidt LJ, Murillo H & Tindall DJ 2004 Gene expression in 58 130–144. prostate cancer cells treated with the dual 5 alpha- Leung DW, Cachianes G, Kuang WJ, Goeddel DV & reductase inhibitor dutasteride. Journal of Andrology 25 Ferrara N 1989 Vascular endothelial growth factor is a 944–953. secreted angiogenic mitogen. Science 246 1306–1309. Sobin LH & Fleming ID 1997 TNM Classification of Loges S, Mazzone M, Hohensinner P & Carmeliet P 2009 Malignant Tumors, fifth edition (1997). Union Inter- Silencing or fueling metastasis with VEGF inhibitors: nationale Contre le Cancer and the American Joint antiangiogenesis revisited. Cancer Cell 15 167–170. Committee on Cancer. Cancer 80 1803–1804.

Downloaded from Bioscientifica.com at 09/30/2021 11:49:40AM via free access www.endocrinology-journals.org 769 K Das et al.: Steroid 5a-reductase in prostate cancer

Thigpen AE & Russell DW 1992 Four-amino acid segment transforming growth-factor beta 1 can play an in steroid 5 alpha-reductase 1 confers sensitivity to important role in the development of tumorigenesis by finasteride, a competitive inhibitor. Journal of Biological its action for angiogenesis: validity of neutralizing Chemistry 267 8577–8583. antibodies to block tumor growth. Biochimica et Thigpen AE, Silver RI, Guileyardo JM, Casey ML, Biophysica Acta 1137 189–196. McConnell JD & Russell DW 1993 Tissue Uemura M, Tamura K, Chung S, Honma S, Okuyama A, distribution and ontogeny of steroid 5 alpha-reductase Nakamura Y & Nakagawa H 2008 Novel 5 alpha-steroid isozyme expression. Journal of Clinical Investigation 92 reductase (SRD5A3, type-3) is overexpressed in hormone- 903–910. refractory prostate cancer. Cancer Science 99 81–86. Thomas LN, Douglas RC, Vessey JP, Gupta R, Fontaine D, Wako K, Kawasaki T, Yamana K, Suzuki K, Jiang S, Umezu Norman RW, Thompson IM, Troyer DA, Rittmaster RS & H, Nishiyama T, Takahashi K, Hamakubo T & Kodama T Lazier CB 2003 5alpha-reductase type 1 immunostaining 2008 Expression of androgen receptor through androgen- is enhanced in some prostate cancers compared with converting enzymes is associated with biological aggres- benign prostatic hyperplasia epithelium. Journal of siveness in prostate cancer. Journal of Clinical Pathology Urology 170 2019–2025. 61 448–454. Thomas LN, Lazier CB, Gupta R, Norman RW, Troyer DA, Xu Y, Dalrymple SL, Becker RE, Denmeade SR & Isaacs JT O’Brien SP & Rittmaster RS 2005 Differential alterations 2006 Pharmacologic basis for the enhanced efficacy of in 5alpha-reductase type 1 and type 2 levels during dutasteride against prostatic cancers. Clinical Cancer development and progression of prostate cancer. Prostate Research 12 4072–4079. 63 231–239. Zhang D, Salto-Tellez M, Putti TC, Do E & Koay ES 2003 Titus MA, Gregory CW, Ford OH III, Schell MJ, Maygarden Reliability of tissue microarrays in detecting protein SJ & Mohler JL 2005 Steroid 5alpha-reductase isozymes expression and gene amplification in breast cancer. I and II in recurrent prostate cancer. Clinical Cancer Modern Pathology 16 79–84. Research 11 4365–4371. Zhu ML & Kyprianou N 2008 Androgen receptor and growth Ueki N, Nakazato M, Ohkawa T, Ikeda T, Amuro Y, factor signaling cross-talk in prostate cancer cells. Hada T & Higashino K 1992 Excessive production of Endocrine-Related Cancer 15 841–849.

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