Effects of Dutasteride on Prostate Carcinoma Primary Cultures: A Comparative Study With Finasteride and MK386

Effects of Dutasteride on Prostate Carcinoma Primary Cultures: A Comparative Study With Finasteride and MK386 Claudio Festuccia,* Giovanni Luca Gravina,* Paola Muzi, Roberto Pomante, Adriano Angelucci, Carlo Vicentini and Mauro Bologna From the Departments of Experimental Medicine (CF, PM, AA, MB), Surgery (GLG, CV) and Basic and Applied Biology (MB), University of L Aquila, L Aquila and Pathology Department, G. Mazzini Hospital (RP), Teramo, Italy Purpose: The profound decrease in serum dihydrotestosterone observed with the dual -reductase 5 inhibitor dutasteride makes it an attractive agent for prostate cancer therapy. To our knowledge we compared for the first time the antitumor effe of dutasteride with that of the specific -reductase-1 5 inhibitor MK386 and the specific -reductase-2 5 inhibitor finasteride in human prostate primary cultures. Materials and Methods: Biochemical markers of the cellular response to -reductase 5 inhibitors were evaluated in primary cultures of prostate epithelial cancer cells from 54 patients with prostate carcinoma. Results: In our cohort of 54 patients prostate cancer cell growth decreased with dutasteride in 42 (about 78%), whereas i 21 (39%) it decreased with finasteride or MK386 alone. We observed a relationship between the levels -reductase of 5 enzymes in cell culture extracts and those revealed by immunohistochemistry in sections of samples from which we established primary cultures. Finasteride effects depended on -reductase-2 5 levels and they were higher when the reductase-1:2 ratio was low. However, dutasteride effects were related -reductase-1 to 5 and 2 levels, and were not influenced 5 by the 5 -reductase-1:2 ratio. Conversely the effects of MK386 were related -reductase-1 to 5 levels and they were higher when the 5 -reductase-1:2 ratio was high. Conclusions: Our data may provide a rationale for the use of a dual -reductase 5 inhibitor rather than a mono specific inhibitor for the prevention or treatment of early prostate cancer. This finding appears to confirm some preliminary clinica results and it could be due to the simultaneous presence of each -reductase 5 isoenzyme in prostate tumor cells. Key Words: prostate; prostatic neoplasms; cholestenone 5 alpha-reductase; receptors, androgen; dutasteride Dihydrotestosterone is the major intracellular growth nant type in benign prostate tissue, expression of the type 1 factor for normal and neoplastic prostatic epithelial enzyme seems to be increased in localized and more advanced prostate cancers. 2 4 cells due to its high affinity binding to AR. The intracellular DHT concentration determines the prostatic cell Finasteride, which is specific for SDRA2, was the first content via its ability to regulate the proportion of ligandsrd5a inhibitor tested as monotherapy for metastatic pros- cancer. 5 The clinical efficacy of finasteride monotherapy occupied AR. After a critical threshold of AR is occupied bytate DHT signal transduction pathways controlling the growth in metastatic cases has been modest. The reason for the and functional activities of the prostatic epithelium are ac-limitetivated. Thus, the intracellular DHT concentration is para- is a potent (ie IC 50 69 nmol/l), time dependent, effectiveness of finasteride may be that, although it irreversible mount and regulated by the supply of testosterone and otherinhibitor of human SRD5A2, it is not as potent (ie 50 360 IC precursors from the systemic circulation, and the complex nmol/l) and not an irreversible inhibitor of the human interplay between intracellular prostatic enzymes of andro- SRD5A1 isoform. gen metabolism, particularly the SRD5A family of reductive The SRD5A inhibitor dutasteride is identical to finasteride except in position 17. This modification shifts the enzymes that irreversibly convert testosterone into DHT. The SRD5A family includes 2 isoforms, of which each is serum half-life and increases the inhibition potency of dutasteride as a reversible SRD5A1 inhibitor and a time depen- 1 encoded by a distinct gene. SRD5A1 is expressed widely and it is the major isoform expressed in many tissues. 6,7 dent, irreversible SRD5A2 inhibitor. Due to its long serum SRD5A2, which is more restrictive in its expression, is the half-life and low IC major isoform expressed by male sex accessory tissues. Although the type 2 enzyme has been identified as the 50 for SRD5A1 and SRD5A2 dutasteride may effectively be used in clinical settings as a dual SRD5A domi- Submitted for publication September 24, 2007. Study received institutional review board approval. Supported by a grant from GlaxoSmithKline. * Equal study contribution. Correspondence: Department of Experimental Medicine, Via Vetoio, Coppito-2, 67100 L Aquila, Italy (telephone: 39 0862 433527; FAX: 39 0862 433523; e-mail: maurobo@univaq.it). inhibitor. We determined whether dutasteride would decrease cell proliferation and induce apoptosis in a series of prostate epithelial cancer specimens in primary culture. We also compared the effects of dutasteride with those of finasteride and the SRD5A1 specific inhibitor MK386 which are effective in prostate cancer cell lines and primary cultures, as previously demonstrated. 8,9 Dutasteride is also able to in- 0022-5347/08/1801-0367/0 367 Vol. 180, 367-372, July 2008 THE JOURNAL OF UROLOGY Printed in U.S.A. Copyright 2008 by AMERICAN UROLOGICAL ASSOCIATION DOI:10.1016/j.juro.2008.02.036

368 EFFECTS OF DUTASTERIDE ON PROSTATE CANCER PRIMARY CULTURES duce cell growth inhibition and apoptosis in prostate cancer cells and primary cultures. 10,11 MATERIALS AND METHODS Patient Selection From December 2005 to December 2006 men with histologically proven prostate cancer were recruited at the urology clinic of our institution. Patients were eligible if they had localized histologically confirmed adenocarcinoma of the prostate and did not undergo previous anti-hormonal or radiation therapy, chemotherapy or investigational agents. Our institutional review board approved the protocol and written informed consent was obtained from all human subjects. Eligible patients formed a cohort of 54 men. All tumors were graded according to the Gleason system. Histopathological tumor staging was assessed according to the TNM 2002 system. Immunohistochemical Analysis Type I and II 5 -reductase expression was evaluated in 4 m tissue sections cut from blocks selected for the presence of representative tumor tissue. AR antibody (clone N20, Santa Cruz Biotechnology, Santa Cruz, California), and type I and II 5 -reductase antibody were used. Scoring Methods All cases were histopathologically and independently interpreted by 2 investigators (RP and MB) without discrepancies in all interpretations. For statistic evaluations IR was classified as 0 no staining; 1 faint, 2 moderate and 3 strong. We also considered the percent of prostate cancer cells expressing IR as a score of 1 less than 10%, 2 10% to 50% and 3 greater than 50% of cells with IR. The IHC score was derived from the sum of the previous 2 parameters. An IHC score of greater than 4 was considered high expression. Primary Cultures Samples were obtained from radical prostatectomy and used to establish primary tumor cultures. After surgery a wedgeshaped specimen of fresh prostate was removed. The parts of tissue adjacent to those used for primary cultures underwent pathological examination to confirm prostatic origin, diagnosis and the absence of other diseases. Primary cultures were grown and characterized according to a previously described method. 12 Apoptosis Apoptosis was quantified as the percent of cells with hypodiploid DNA, as assessed using a HT titer TACS assay kit (Trevigen, Gaithersburg, Maryland), which is a colorimetric quantitative assay for detecting apoptosis. Western Blot Analysis Total protein was isolated from cell extracts in RIPA buffer and protein content was assayed using a DC protein assay kit (Bio-Rad ). Protein (50 g) was run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted. Blots were blocked with 1% bovine serum albumin/tris buffered saline and 0.1% Tween-20 for 1 hour at room temperature. Subsequently they were incubated in SDRA1, SDRA2, prostate specific antigen, AR or CK8/CK18 primary antibody. Blots were developed using an enhanced chemiluminescence substrate system (Amersham Biosciences, Newcastle-upon-Tyne, United Kingdom) for detecting horseradish peroxidase. Statistical Analysis Statistical analysis was performed using SPSS 11.0 software with p 0.05 considered significant. All statistical tests were 2-tailed. Differences in categorical variables were compared with the chi-square test. Continuous variables were analyzed using the Kruskal-Wallis test, followed by post hoc analysis (Tukey HSD) to compare the growth inhibition of primary tumor cultures after treatments. RESULTS SDR5 Levels in Prostate Cancer Tissues Table 1 lists patient clinical and pathological characteristics. The expression of SDR5A1 and was analyzed in tissue sections derived from 54 prostate tissue samples from radical prostatectomies used to establish primary cultures. Table 2 shows IHC results. We observed that 85% of samples (46 of 54) expressed at least 1 isoenzyme at high levels, that is 41% (22 of 54) showed high levels of SDR5A1, 32% (17 of 54) showed increased expression and 13% (7 of 54) had simultaneously high levels of the 2 SDR5 isoenzymes. We did not find statistically significant differences between SDR5A isoenzyme expression in our patient cohort (p 0.423). We observed also that the levels of SDR5A1 and did not significantly correlate with clinical and pathological parameters (data not shown). However, we noted a significant correlation between Gleason score and the percent of prostate cancers with high SDR5A1 and low expression (p 0.005, table 2). Pharmacological Treatment For pharmacological treatments primary cultures and cell 4 lines were seeded at a density of 2 cells 10 per dish in 50 mm Petri dishes. Cells were left to attach and grow in 5% fetal calf serum Dulbecco s modified Eagle s medium for 24 hours. All other cells were treated with finasteride, dutasteride and MK386 (5 and 10 M), always in the presence of testosterone (10 12 M). Cells trypsinized and resuspended in 20 ml saline were counted every 48 hours by a hemocytometer (LabRecyclers, Gaithersburg, Maryland) with 5 independent counts performed per dish. All experiments were done in triplicate. TABLE 1. Demographic and pathological characteristics in 54 study patients Variables Mean SD age 65 9 Mean SD prostate specific antigen at diagnosis (ng/ml) 8.4 3.1 No. prostatectomy Gleason score (%): 6 or Less 30/54 (56) 7 18/54 (33) 8 10 6/54 (11) No. pathological stage (%): pt2c 38 (70) pt3a 16 (30)

EFFECTS OF DUTASTERIDE ON PROSTATE CANCER PRIMARY CULTURES 369 TABLE 2. Expression of SDR5A1 and 2 in prostatectomy specimens and prostate cancer primary tumor cultures No./Total No. (%) High SDR5A1 High High SDR5A1/Neg/Low High SDR5A1/Neg/Low Neg/Low SDR5A1/High Prostatectomy high expression 22/54 (41) 17/54 (32) 7/54 (13) 15/54 (28) 10/54 (19) p Value (chi-square test) 0.423 Prostatectomy Gleason grade: 2 6 10/30 (33) 7/30 (23) 4/30 (13) 6/30 (20) 7/30 (23) 7 7/18 (39) 8/18 (44) 2/18 (11) 4/18 (22) 2/18 (11) 8 10 5/6 (83) 2/6 (33) 1/6 (17) 5/6 (83) 1/6 (17) p Value (chi-square test) 0.074 0.311 0.936 0.005 0.569 Prostate Ca high expression 36/54 (67) 26/54 (48) 12/54 (22) 24/54 (44) 14/54 (26) p Value (chi-square test) 0.080 SDR5 Levels in Prostate Cancer Tissue Primary Cultures Characterization of primary epithelial cancer cell cultures was done as previously described (see figure). 12 We also assessed the expression of SDR5A1 and SDRA2 by densitometric analysis of bands generated by Western blot analysis (part B of figure). Globally we observed that 67% of cultures (36 of 54) were SDR5A1 positive, whereas 48% (26 of 54) were positive (p 0.080). Only 4% of cultures (2 of 54) were doubly negative. Of the cases 44% (24 of 54) showed only SDR5A1 and 26% (14 of 54) showed only expression. Table 2 shows statistical analyses. Comparative Analysis of Dutasteride, Finasteride and MK386 Biological Effects When comparing drugs at the same doses (1 and at 5 M), we observed that dutasteride cell growth inhibition was higher with respect to finasteride and to MK386 (tables 3 to 5). Finasteride growth inhibition was higher compared to that of MK386 at 1 and 5 M. All previous comparisons were statistically highly significant. Using 1-way ANOVA we observed no statistically significant differences between cell growth inhibition and Gleason score for all pharmacological treatments. Apoptosis was measured at 72 hours of treatment. It was induced in 82% of primary cultures (44 of 54) treated with 5 M dutasteride. Finasteride (5 M) was able to induce apoptosis in 69% of primary cultures (37 of 54). Apoptosis was induced in 74% of primary cultures (40 of 54) treated with 1 M dutasteride. Instead, finasteride (1 M) was able to induce apoptosis in 57.4% of primary cultures (31 of 54). The difference between apoptosis induced by dutasteride and finasteride was statistically significant (tables 6 to 8). Comparisons between finasteride and MK386, and between dutasteride and MK386 also revealed a significant difference at the 2 drug concentrations. In addition, in this case apoptosis induced by the dual inhibition of SDR5A1/2 enzymes with dutasteride was similar to the expected inhibition of finasteride plus MK386. Comparisons Between Drug Effects, and SDR5A1 and 2 Expression We also performed correlation analysis between the effects of single drugs and the levels of SDR5 enzymes in primary TABLE 3. SRD5A inhibitor effects on tumor growth inhibition Mean SE % Inhibition* Prostate epithelial cells characterization in culture. A, light microscope shows isolated primary prostate epithelial cell culture from representative patient sample with characteristic cobblestone morphology associated with epithelial cell cultures. There was no morphological evidence of contaminating fibroblast/stromal cells. Reduced from 100. B, primary prostate cancer epithelial cells expressed cytokeratin 18 (K18), SDR5A1 (SR1) and SDRA2 (SR2) on Western blot. 1 24.0 2.0 5 42.1 2.7 1 4.6 0.9 5 12.3 2.3 1 32.2 1.4 5 55.9 2.7 * Vs primary tumor culture controls.

370 EFFECTS OF DUTASTERIDE ON PROSTATE CANCER PRIMARY CULTURES TABLE 4. SRD5A inhibitor effects on tumor growth inhibition cultures. We considered the percent of growth inhibition (decrease vs control) and the apoptotic rate measured at 5 M. Values were plotted vs the densitometric values of SDR5A enzymes. In this analysis we made certain observations. 1) The efficacy of finasteride significantly depended on levels present in primary cultures for cell growth inhibition and apoptosis (r 0.547, p 0.01 and r 0.836, p 0.001, respectively). 2) The efficacy of MK386 significantly depended on SDR5A1 levels present in primary cultures for cell growth inhibition and apoptosis (r 0.631, p 0.01 and r 0.756, p 0.005, respectively). 3) Dutasteride efficacy highly correlated with SDR5A1 expression (r 0.788, p 0.001 and r 0.844, p 0.001) and with expression (r 0.624, p 0.01 and r 0.788, p 0.001) for cell growth inhibition and apoptosis, respectively. When considering the ratio between SDRA1 and 2, finasteride was highly effective when this ratio was low for cell growth inhibition and apoptosis (r 0.835, p 0.01 and r 0.638, p 0.05, respectively), whereas dutasteride effects did not show any significant influence in relation to this ratio. DISCUSSION Mean SE % Tumor Growth* 1 M finasteride vs 1 M dutasteride 24.0 2.0 vs 32.2 1.4 1 M finasteride vs 1 M MK386 24.0 2.0 vs 4.6 0.9 5 M finasteride vs 5 M dutasteride 42.1 2.7 vs 55.9 2.7 5 M finasteride vs 5 M MK386 42.1 2.7 vs 12.3 2.3 1 M dutasteride vs 1 M MK386 32.2 1.4 vs 4.6 0.9 5 M dutasteride vs 5 M MK386 55.9 2.7 vs 12.3 2.3 For all comparisons p 0.01. * Vs primary tumor culture controls. During prostatic carcinogenesis molecular changes can determine a gain of function in the AR axis from a paracrine to an autocrine pathway, in which occupancy of AR by DHT in the nuclei of malignant cells directly controls the autocrine production of growth factors for the survival and proliferation of these malignant cells. Data from the Prostate Cancer Prevention Trial, in which the SRD5A2 specific inhibitor finasteride decreased the relative risk of biopsy proven prostate carcinoma by approximately 25%, 13 provide credible evidence of the effectiveness of such treatment in men with localized prostate cancer. Enhanced expression of the SRD5A1 isoform by prostate cancer cells can explain why previous clinical trials of finasteride for metastatic prostate TABLE 6. SRD5A inhibitor effects on apoptosis Mean SE % Apoptosis* 1 7.3 0.9 5 22.8 1.3 1 2.6 0.4 5 6.3 0.9 1 13.3 1.3 5 29.8 1.3 * Apoptosis (absolute value of apoptosis cell percent in treated primary cultures) (absolute value of apoptotic cell percent in untreated primary cultures). cancer treatment have had limited success when used as monotherapy, 5 or when combined with antiandrogens. Dutasteride is a dual SDR5A inhibitor. This drug is being studied to decrease the risk of prostate cancer in men at risk in the Reduction by Dutasteride of Prostate Cancer Events trial, 14 of which the results will be available in 2009. In addition, the Reduction by Dutasteride of Clinical Progression Events in Expectant Management trial is being done to assess the effect of dutasteride on decreasing disease progression in men with low risk, localized prostate cancer. 15 The underlying study hypothesis is that by decreasing androgenic stimulation to prostate cancer tissue dutasteride will inhibit the growth of such cancers, thereby decreasing disease progression and eliminating the need for or extending the time to the implementation of more aggressive therapy. Dutasteride may have higher chemopreventive effects than finasteride by lowering intraprostatic DHT concentrations more effectively. 16 The primary end point of our study was the comparison of the effects of dutasteride, finasteride and the specific SDR5A1 inhibitor MK386 using primary cultures from prostate cancer tissue samples. The study of prostate cancer biology is made difficult by the lack of appropriate in vitro and in vivo models. The most used cancer cell lines, which have been established from human metastatic lesions, do not accurately recapitulate the biological behavior of primary tumors compared to primary cultures generated from clinical prostate cancer specimens. A procedure to propagate human prostatic epithelial cells in vitro for a limited number of cell generations has been developed by and optimized for prostate cancer cells from primary tumors by our research group. 12 Previously we have reported that finasteride effects were higher compared to MK386 effects. 9 In the current study to our knowledge we report for the first time that dutasteride is significantly more effective than MK386 or TABLE 5. SRD5A inhibitor effects on tumor growth inhibition vs prostatectomy Gleason grade Inhibition Eta Correlation p Value 1 0.467 0.089 5 0.505 0.503 1 0.401 0.453 5 0.374 0.101 1 0.541 0.080 5 0.490 0.109 TABLE 7. SRD5A inhibitor effects on apoptosis Mean SE % Apoptosis* 1 M finasteride vs 1 M dutasteride 7.3 0.9 vs 13.3 1.3 1 M finasteride vs 1 M MK386 7.3 0.9 vs 2.6 0.4 1 M dutasteride vs 1 M MK386 13.3 1.3 vs 2.6 0.4 5 M finasteride vs 5 M dutasteride 22.8 1.3 vs 29.8 1.3 5 M finasteride vs 5 M MK386 22.8 1.3 vs 6.3 0.9 5 M dutasteride vs 5 M MK386 29.8 1.3 vs 6.3 0.9 For all comparisons p 0.0001. * Apoptosis (absolute value of apoptosis cell percent in treated primary cultures) (absolute value of apoptotic cell percent in untreated primary cultures).

EFFECTS OF DUTASTERIDE ON PROSTATE CANCER PRIMARY CULTURES 371 TABLE 8. SRD5A inhibitor effects on apoptosis vs prostatectomy Gleason grade Apoptosis Eta Correlation p Value 1 0.364 0.11 5 0.491 0.084 1 0.475 0.010 5 0.412 0.025 1 0.510 0.198 5 0.444 0.084 finasteride in vitro. This result confirms the clinical data. It may be due to the simultaneous presence of the 2 SDR5A isoenzymes in prostate tumor cells. We observed that 72 hours of treatment with dutasteride was able to induce apoptosis in almost all primary cultures. This finding is different from those of McCrohan et al, who analyzed apoptosis at 24 hours of culture, 11 and similar to those of Lazier et al, who analyzed dutasteride effects at 9 days of incubation. 10 McCrohan et al justified the choice of 24 hours by indicating that long-term treatment (9 days) could mask dutasteride effects due to cell overgrowth and cell death due to confluency. 11 However, we think that shortterm treatment (24 hours) can induce just the initial phases of apoptosis due to the time requirements of hormonal inhibition and, thus, underestimate the effect on apoptosis. Therefore, we analyzed pro-apoptotic effects at 72 hours, when these effects appear to be maximal. In addition, the method which we used for apoptosis assay was a colorimetric kit, which is able to discriminate apoptosis from post-apoptotic necrosis. Moreover, McCrohan et al invoked the increased percent of a basal phenotype in primary cultures as a potential factor for their negative results in low grade tumors. In fact, basal cells would not require DHT for survival. 17 This seems not to be the case in our cultures because they had an androgen sensitive phenotype. Additionally, the heterogeneous response in terms of efficacy observed in our cultures after all treatments could have been due to different expression in SDR5A isoenzymes as well as in phosphoinositide 30-kinase/Akt activity, which is increased after the down modulation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), as documented in about 30% of primary prostate cancers. 18 In this regard increased Akt activity seems to be associated with a decreased proapoptotic effect. 19 When analyzing the expression of SDR5A1 and 2, we observed a direct relationship between the levels of these enzymes in tissue cell culture extracts and those observed by IHC in tissue sections of samples from which we established primary cultures. This suggests that primary cultures maintain the biological properties of primary tumors and, therefore, they represent a good study model for evaluating drug efficacy. Additionally, we did not find statistically significant differences between SDR5A1 and 2 expression in our cohort in culture or in tissue sections. This is in agreement with the results of Nakamura et al, 20 although we found a lower percent of high SDR5A isoenzyme expression by IHC. This can be explained by considering differences in the clinical and pathological characteristics of patients, and in the scoring method. An interesting result is that finasteride effects depended on in vitro levels and they were higher when the SDR5A1:2 ratio was low, whereas dutasteride effects were related to SDR5A1 and 2 levels but were not influenced by the SDR5A1:2 ratio. Conversely the effects of MK386 were related to SDRA5A1 levels and they were higher when the SDR5A1:2 ratio was high. Moreover, the magnitude of cell growth inhibition and apoptosis induced by dutasteride was similar to the inhibition achieved by the combination of finasteride and MK386. In fact, in our patient cohort about 77.8% of tumors (42 of 54), which highly expressed either or both isoforms, could be effectively growth inhibited with dutasteride, whereas 38.9% (21 of 54), which highly expressed the SDRA5A2 isoform, could be growth inhibited with finasteride alone and 38.9% (21 of 54), which highly expressed the SDRA5A1 isoform, could be growth inhibited with MK386 alone (each p 0.043). CONCLUSIONS Our preclinical study suggests that a higher proportion of patients with prostate cancer could probably be treated advantageously with dutasteride compared to finasteride or MK386. We believe that this result may be due to the high expression of either or both of the SDR5A isoenzymes in prostate tumors. Nevertheless, clinical studies are needed to substantiate these preclinical results. ACKNOWLEDGMENTS Types I and II 5 -reductase antibody was provided by Lynn Thomas, Dalhousie University, Halifax, Nova Scotia, Canada. 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