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This study characterizes the clinical and morphofunctional effects of a 5α-reductase inhibitor on steroid hormone receptors in normal human prostate tissue, as potential mediators of the clinical effects of dutasteride. This work was a prospective, double-blind, and randomized study that evaluated 49 men aged between 45 and 70 years, with no alterations in a digital rectal examination and prostate-specific antigen measurements between 2.5 and 4.0 ng/mL. These patients underwent prostate biopsy guided by transretal ultrasound with prostate neoplasia being ruled out, and the patients were divided into two groups, with one group receiving dutasteride (n = 25) and one group receiving a placebo (n = 24). The patients were clinically assessed each quarter, and at the end of 12 months they underwent new laboratory tests, prostate rebiopsy, and histopathological, immunohistochemical and clinical analyses. The estrogen receptor-beta (ERβ) and androgen receptor immunoreactivities were higher, and the proliferation/apoptotic ratio was significantly lower with predominance of the apoptotic process, followed by a significant reduction in the prostate volume and the total serum prostate-specific antigen levels in the dutasteride group when compared with the placebo group, with a clear supremacy of ERβ. There were no significant variations in the serum estrogen and testosterone levels, in the body mass index, or in the ERα immunoreactivities in the dutasteride and placebo groups. The results demonstrated the importance of the ERβ pathway in the activation mechanisms of apoptosis, exerting a protective effect in the normal prostate, indicating that this receptor might be an important mediator of the clinical effects of dutasteride.
The prostate is a male sexual accessory gland located around the urethra and the inferior portion of the urinary bladder; there are three glandular regions in the prostate: the peripheral zone, the central zone, and the transitional zone with a thin fibromuscular layer (Marker, Donjacour, Dahiya, & Cunha, 2003; Untergasser, Madersbacher, & Berger, 2005). The morphogenesis, maintenance of functional activity, morphology, proliferation, and differentiation of prostate cells are regulated mainly by androgens (Cunha & Matrisian, 2002; Imamov, Lopatkin, & Gustafsson, 2004; Leav et al., 2001).
Testosterone and dihydrotestosterone (DHT) are the main androgens that act in the prostate. DHT is a testosterone resulting from conversion by the 5α-reductase enzyme (Prins, Birsch, & Greene, 1991). Although testosterone and DHT use the same androgen receptor (AR) in the prostate, these actions appear to be associated with different tissue functions (Prins et al., 1991). There is as much testosterone as DHT to maintain prostatic activity, but DHT is 10 times more potent than testosterone because of its dissociation from the AR is slower (Reis, 2012a).
The evidences for the use of 5α-reductase inhibitors in benign prostatic hyperplasia (BPH) are promising, but questions persist about the prostate effects on men with risk for developing cancer, as well as for the adoption of effective and safe therapeutic strategies in clinical practice.
The use of 5α-reductase inhibitors for prostate cancer prevention continues to be widely discussed within the scientific and medical communities, fueled by the findings of two important randomized, placebo-controlled trials, the prostate cancer prevention trial (PCPT) with finasteride (Thompson et al., 2003) and the reduction by dutasteride of prostate cancer events (REDUCE) trial (Andriole et al., 2004). The PCPT and REDUCE studies identified a decrease in the total number of prostate cancer cases compared with the placebo, but there was an increase in the high-grade tumor numbers identified by biopsy in the men treated with 5α-reductase inhibitors (Klotz et al., 2012).
The main objectives of this study were to characterize the clinical and morphofunctional effects of the 5α-reductase inhibitor Types 1 and 2, dutasteride, on steroid hormone receptors (SHRs) in normal human prostate tissue, as well as to verify the viability of these receptors as potential markers and mediators of the clinical effects of dutasteride.
After local ethics committee approval and signed consent, 49 patients in annual routine prostate evaluation at the Men’s Health Hospital in Paulinia county, São Paulo, Brazil, were randomized into the following two groups: the placebo group (n = 24) received orally excipient (placebo) once daily for 12 months; and the dutasteride group (n = 25) received orally 0.5 mg of dutasteride once daily for 12 months. The division of the groups occurred randomly and double blinded with the patient receiving capsules marked with A and B for identification without the patients and researchers knowing the random group assignment.
The patients were clinically evaluated by physical and laboratory examinations and biopsied prior to the initiation of treatment (time zero) and at the end of 12 months to rule out prostate cancer. The fragments of the biopsies (time zero and after 12 months) were evaluated pathologically and immunohistochemically; the clinical examinations consisted of (1) total and free prostate-specific antigen (PSA), total testosterone, and estradiol (quantitative chemiluminescent radioimmunoassay; Medlab DPC Immulite 2000, Malvern, PA), (2) digital rectal exam as standardized (Reis, Simao, Baracat, Denardi, & Gugliotta, 2013), anthropometric data, and body mass index, (3) prostate measurements by transretal ultrasound.
Inclusion criteria were the following: (1) agreeing to participate in the study, (2) aged between 45 and 70 years, and (3) PSA levels between 2.5 and 4.0 ng/dL.
Exclusion criteria were the following: (1) any hormonal manipulation, including the use of α-blockers and 5α-reductase inhibitor in the last 12 months; (2) first-degree family history (father or brother) of prostate cancer; (3) prostate cancer diagnosis at any time; and (4) urinary tract infection, prostatitis, or chronic urinary catheter use.
The biopsies were performed in the radiology section (Men’s Health Hospital in Paulinia county) under locoregional anesthesia by injecting 2% lidocaine without epinephrine in the periprostatic apical and basal neurovascular bundle, guided by Doppler ultrasound (7.5 MHz probe; Toshiba SSA-250-A, Tokyo, Japan). After the anesthetic block, 12 prostate fragments were obtained is sextant, six in each prostatic lobe (right and left). All the patients received ciprofloxacin one day before, maintained for two consecutive days at a dose of 500 mg orally for 12/12 hours.
The prostate samples were fixed by immersion in 10% buffered formaldehyde for 12 hours. After fixation, the tissues were washed in 70% ethanol, dehydrated, diaphanized in xylene, and included in plastic polymer (Paraplast Plus, St Louis, MO). Subsequently, the prostate samples were sectioned with a microtome (Leica RM 2165, Munich, Germany) for 5 µm thickness, stained with hematoxylin–eosin, and photographed under a Axiophot light microscope (Zeiss, Munich, Germany). The histopathological characterization of the samples was based on morphological criteria according to Mostofi and Price (1973).
All the prostate samples, the same as used for the histopathological analysis, were taken and cut into 5-µm thick sections. The sections were incubated in 0.3% H2O2 to block the endogenous peroxidase and nonspecific binding was blocked by incubating the sections in blocking solution at room temperature. Different protocols were used for the antigen retrieval. Primary antibodies, rabbit polyclonal sc-816 (Santa Cruz Biotechnology, Santa Cruz, CA) for the AR, rabbit monoclonal primary antibody 04-227 (Merck-Millipore, Temecula, CA) for the ERα, mouse monoclonal ab16813 (Abcam, Cambridge, MA) for the ERβ, and rabbit polyclonal ab66155 (Abcam) for the Ki-67, were diluted in 1% bovine serum albumin and incubated with the sections overnight at 4°C. The MACH 4 Universal HRP-Polymer kit (Biocare Medical, Concord, CA) was used for the detection of the antigens according to the manufacturer’s instructions. After washing with TBS-T buffer, pH 7.4, the sections were incubated with HRP conjugated secondary antibody from the MACH 4 kit for 40 minutes and subsequently revealed with diaminobenzidine, counterstained with Harris hematoxylin, and evaluated with a Nikon Eclipse light microscope (Nikon, Tokyo, Japan) fitted with a Nikon DS-IR-1 camera.
To evaluate the intensity of the immunoreactive antigens, the percentage of the positive epithelial and/or stromal was examined in 10 fields for each antibody at 400× magnification. The intensity of the marking was classified in a range from 0 to 3. The range 0 (no immunoreactivity) corresponded to 0% positive epithelial cells, 1 (weak immunoreactivity) corresponded to 1% to 35% positive epithelial cells, 2 (moderate immunoreactivity) corresponded to 36% to 70% positive epithelial cells, and 3 (intense immunoreactivity) corresponded to >70% positive epithelial cells (Fávaro et al., 2012).
The proliferative index was obtained by positive Ki-67 cells count; thus, 10 fields per section were examined at 1,000× magnification and the proliferative index was determined by dividing the number of positive Ki-67 cells by the number of total cells in the section analyzed.
The identical sections obtained for the anatomopathological and immunochemistry analyses were used to detect the DNA fragmentation reaction. The DNA fragmentation was detected using the detection system conjugated to peroxidase (Calbiochem, Madison, WI) according to the manufacturer’s instructions. The reagents supplied by the Calbiochem kit consist of marking the 3′-OH free extremities arising from the DNA fragmentation. The sections were stained with diaminobenzidine, counterstained with Harris hematoxylin, and evaluated with a Nikon Eclipse Ni-U light microscope fitted with a Nikon DS-IR-1 camera.
The microscopy was 10 fields per section at 1,000× magnification, and the apoptotic index was determined by dividing the number of apoptotic nuclei by the total number of nuclei in the analyzed section.
The quantitative descriptive analysis of the data was performed by separately calculating the mean and standard deviation for the placebo and dutasteride groups. The means were compared between the groups by t test for independent samples. It was considered statistically significant at p < .05. For the qualitative variables, contingency tables were constructed and Fisher’s exact test used to verify the dependence of the treatment of each variable.
All 49 patients completed the study and no significant adverse effects or complications were observed or reported by the patients.
There was a significant reduction of the serum total PSA in the dutasteride group compared with the placebo group (Figure 1A). This reduction was 38.64% in the patients who received dutasteride compared with the patients who received placebo, p = .001.
There were no statistically significant alterations in the serum estradiol levels in the dutasteride group compared with the placebo group (Figure 1B), but there was a slight increase in the serum estradiol levels in the dutasteride patients. There was no change in the serum testosterone between the analyzed groups (Figure 1C).
There was an increase in the immunoreactivity percentage for the estrogen beta receptor (ERβ) and AR in the dutasteride group compared with the placebo group. Compared with AR, the increase was dramatic for ERβ, from weak immunoreactivity (5.8%) to intense immunoreactivity (80.3% positive prostate cells); see Table 1 and Figure 2A, ,BB and andE,E, ,F.F. There was no significant change in the immunoreactivity percentage for the estrogen receptor alpha (ERα) of the dutasteride group compared with the placebo group; see Table 1 and Figure 2C and andDD.
The apoptotic and proliferative processes were significantly higher in the dutasteride group in relation to the placebo group, p < .001 (Figure 1D). The proliferation/apoptotic ratio was significantly lower in the dutasteride group when compared with the placebo group, indicating a predominance of the apoptotic process, p = .002 (Table 2).
There was a significant reduction in the prostate volume (cm3) of the dutasteride group compared with the placebo group; thus, the prostate volume in the dutasteride group was 27% smaller than that of the patients who received the placebo, p = .008 (Table 2). There were no statistically significant alterations in the body mass index of the dutasteride group compared with the placebo, p = .07 (Table 2).
The current study evaluated the histopathological, immunohistochemical, and clinical effects of dutasteride in healthy patients. The results demonstrate a significant reduction of serum PSA (38.64%) and prostate volume (27%) in the patients receiving dutasteride compared with those receiving the placebo. The action of this drug in reducing serum PSA levels and prostate volume has been previously reported (Klotz et al., 2012). There were no significant changes in the serum levels of estradiol and testosterone, though free testosterone may better reflect prostate tissue androgen levels than serum total testosterone concentration (Rove et al., 2014).
Also, these findings do not imply that there is no biological response triggered by modulation of the androgen and estrogen microenvironment, as a dramatic increase in the ERβ immunolocalization was observed, which correlated with a significant decrease in the proliferation/apoptotic ratio, reflecting the PSA and prostate volume decrease, compared with a less important AR increase and no alteration in the ERα. These results in normal human prostate are pioneer to the best of our knowledge.
The role of estrogens receptors in the prostate has received less attention than that of androgens, despite emerging recognition that estrogens—in addition to androgens—play an essential role (Ellem & Risbridger, 2010) including the BPH clinical scenario (Reis et al., 2014).
Given the central role that androgens play in prostate development and disease (BPH and cancer), prostatic androgen metabolism has been vigorously investigated, and consequently, androgen ablation therapy has been the mainstay of treatment for prostate cancer since the pioneering works of Huggins and Hodges more than 60 years ago (Huggins & Hodges, 1941; Reis, 2012b).
Despite the well-documented importance of androgens, illustrating the fact that our understanding of the pathophysiological processes involved in prostate disease remains incomplete, we have recently reported no correlation between total testosterone and PSA kinetics in the prostate cancer castration scenario (Reis, Denardi, Faria, & Silva, 2015). In particular, the androgens may be metabolized to estrogens by the action of the aromatase enzyme, and there is a growing body of evidence that implicates estrogens in the etiology of prostate disease (BPH and cancer). Significantly, the aberrant expression of aromatase has been implicated in the disease process in other tissues such as the breast and endometrium (Brodie, Lu, & Nakamura 1997; Watanabe et al., 1995).
The prostate estrogenic effects are the result of the binding of the hormones with specific estrogen receptors for α and β subunits (ERα, ERβ), which are predominantly expressed in the stroma and epithelium, respectively (Cunha & Matrisian, 2002; Risbridger et al., 2001).
Estrogens are essential for normal prostate development and differentiation, influenced not only by hormonal interactions but also by the alternate and temporal expression of each estrogen receptor during the different stages of life (Shapiro et al., 2005). ERα has an important role in the neonatal prostate until it is suppressed by the effects of physiologically increasing androgen levels, and when activated inappropriately, the cells of the prostate gland are permanently altered, resulting in late-life disease and the emergence of premalignant pathologies (Pylkkanen et al., 1993; Shapiro et al., 2005). In contrast, ERβ expression is initiated after ERα and appears to be important during later periods of development such as puberty and adulthood, acting to regulate cellular proliferation and differentiation in adult tissue (Prins & Birch, 1997).
In the current study, the dutasteride administration resulted in a dramatic increase of ERβ receptor immunolocalization, and consequently, there is a clear change in the prostatic steroid homeostasis environment, especially regarding the specific estrogen receptors balance.
The testosterone/DHT production classical pathway can undergo a shift because of a substrate percentage (of testosterone) increase and thereby increase the estrogen production by aromatase activation. In addition, unlike ERα, ERβ expression does not appear to be regulated by estrogens only, and there is some evidence suggesting that the expression of ERβ is also androgen-sensitive (Leav et al., 2001; Prins et al., 2001). In recent studies, it was reported that ERβ expression in the prostatic epithelial cells was suppressed following castration but restored when androgens (DHT or 3β-diol) but not estradiol were restored (Oliveira et al., 2007).
These data reinforce the concept that it is the fine balance between these receptors and the hormone interactions that is the critical determinant affecting prostatic pathology later in life. An antiproliferative role for ERβ within the prostate is supported by the results of the current study. The specific stimulation of ERβ might block the hyperplastic epithelial cell proliferation, preventing the onset of aberrant hyperplastic growth within the prostatic epithelium.
This antiproliferative role of ERβ concurs with earlier reports that the adult β-ERKO mouse develops aberrant proliferative lesions within the prostatic epithelium and has proliferative activity more than three times greater than that in normal mice (Imamov et al., 2004; McPherson et al., 2007). Although there is some doubt surrounding these data, this putative onset of aberrant proliferation in the absence of ERβ activation would be consistent with the antiproliferative role proposed for ERβ in the human prostate in the current study.
Given the limited studies conducted on regulation of the expression of estrogen receptors in the prostate, the understanding of the regulatory processes controlling the ERβ expression remains to be completely defined. While most of available data on the issue are based on animal models, the current study is focused on human tissue in a prospective, double-blind, randomized design. However, it is limited to serum hormonal quantification and tissue immunolocalization of SHRs, and further confirmatory studies are necessary to expand the analyses to protein concentration and by comparing the gene expression profiles in the prostate under different hormonal conditions (Rosa-Ribeiro et al., 2014).
Dutasteride treatment led to distinct SHRs reactivities demonstrating the importance of the ERβ pathway in the activation mechanisms of apoptosis, exerting a protective effect in the normal prostate and indicating that this receptor might be an important mediator of the clinical effects of dutasteride. While this is the first study focusing on ERβ in normal human prostate tissue under 5α-reductase inhibitor effect, future studies are necessary to determine if the current findings are causative or an association.
Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.