We examined the effect of E2 and selective estrogen receptor modulators (SERMs) on the proliferation and estrogen receptor (ER) activities in normal human prostate cells. SERMs such as toremifene, raloxifene and tamoxifen suppressed the proliferation of prostate epithelial and stromal cells whereas anti-androgens did not. In prostate stromal cells, the transactivation activities of ER were enhanced by adding E2 and reduced remarkably by toremifene. The results indicate that the ER-mediated pathway plays a central role in the growth of normal prostate cells.
Although the most striking characteristic of the prostate gland is its androgen dependence, estrogen and the estrogen receptors have been reported to be involved in the development of the prostate gland and the pathogenesis of prostatic diseases.1 Maternal estradiol, for example, stimulates squamous metaplasia within the developing prostatic epithelium in a male offspring.2 With regard to localized prostate cancer, expression levels of the estrogen receptor β (ERβ) decline with increasing grade from prostatic intraepithelial neoplasia through low to high Gleason scores, suggesting that ERβ is a putative tumor suppressor.2 In contrast, estrogens seem to induce prostate stromal cell proliferation; it is well known that the administration of estrogens combined with androgens has been used to induce BPH experimentally in dogs.2
Since the discovery of the ERβ,3 expression of estrogen receptors α and β has been extensively studied using RT-PCR or immunohistochemical technique in several prostate cancer cell lines as well as in normal and malignant prostatic tissues, those authors associating the expression of each estrogen receptor (ER) in various cells with its possible function and role.4, 5, 6, 7, 8, 9 Effect of ligands and selective estrogen receptor modulators (SERMs) on the growth of several prostate cancer cells has also been investigated; the results indicate the possible involvement of estrogen receptors (ERs) in prostate cancer cell proliferation.5, 10
BPH is the most common prostatic disease that will affect many men as they age. One of the hypothesized pathogeneses of BPH is that the stromal hyperplasia is related to an enhanced estrogenic status after middle age, with a decreased androgen level and an increased conversion of adrenal androgens to estrogen by aromatase.11 Recently, key studies on the role of estrogen in the pathogenesis of BPH have been carried out using normal human prostate stromal and epithelial cells. King et al.12 reported that an increased estrogen/androgen ratio stimulated the proliferation of prostate stromal cells and enhanced the growth of prostate epithelial cells co-cultured with stromal cells. Zhong et al.13 focused on ERK, which was shown to be activated by estradiol with the proliferation of prostate stromal cells.
While those investigations support that ERs α and β may directly modulate the prostatic cell growth, it is necessary to investigate whether the ERs in prostatic cells are actually activated by ligands with the corresponding transcription of the downstream genes causing changes in cell proliferation.
Primary cultures of normal human prostatic epithelial and stromal cells are commercially available and have been used as useful models for the normal prostate epithelium and normal stroma in many studies. They exhibit limited lifespan with cellular senescence in culture.14, 15 In cultured prostatic stromal cells, mRNA of androgen receptor (AR) is expressed,16, 17, 18 whereas reduced expression of AR protein in late passages has been reported.18 The commercially available prostatic epithelial cells possess the features of basal epithelia that lack the expression of AR protein;15, 19, 20 those cells are not fully differentiated to be the secretory epithelial cells.15
Currently, we show for the first time that endogenous estrogen receptors were activated by estrogen in the primary cultures of normal human prostate cells, and that their activities were modulated by selective estrogen receptor modulators (SERMs) accompanied by changes in cell growth.
Materials and methods
The normal human prostate stromal and epithelial cells, PrSC and PrEC, were purchased from LonzaWalkersville (Walkersville, MD, USA). The prostate cancer cell line, LNCaP cells, was purchased from American Type Culture Collection (Rockville, MD, USA). Dual-luciferase reporter assay system was obtained from Promega (Madison, WI, USA) and FuGENE 6 Transfecton Reagent from Roche (Mannheim, Germany). A reporter gene for ER, pGL3-ERE-X3tK luciferase (pERE-luc), was a generous gift from Dr. Shigeaki Kato, Tokyo University, Tokyo, Japan. An anti-androgen, flutamide and an anti-estrogen, toremifene, were generous gifts from Nippon Kayaku Corp. (Tokyo, Japan). Raloxifene, tamoxifen, genistein and 5α-dihydrotestosterone (DHT) were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and a pure anti-estrogen faslodex (ICI182,780) was from Tocris (Ellisville, MO, USA). β-Estradiol and bisphenol A (BPA) were from Wako Pure Chemical (Osaka, Japan).
Cell culture and transient transfection
The normal human prostate stromal and epithelial cells, PrSC and PrEC, were maintained in Prostate Stromal Cell Growth Medium (PrSCGM) supplemented with PrSCGM SingleQuots (Lonza) and Prostate Epithelial Cell Growth Medium (PrEGM) supplemented with PrEGM SingleQuots (Lonza), respectively according to the manufacturer's instruction. LNCaP cells were maintained in RPMI-1640 supplemented with 10% FCS, 100 U ml−1 of penicillin and 100 μg ml−1 of streptomycin.
Cells (2 × 105) for LNCaP or 1 × 105 cells for PrEC and PrSC per each well were plated onto six-well culture plates, incubated for 24 h and used for assay. The PrEC and PrSC plated onto six-well culture plates were transiently transfected using FuGENE transfection reagent (Roche) with 1.05 μg of DNA mixture containing pERE-luc reporter (1 μg) and pRL-TK (50 ng) in accordance with the manufacturer's instructions and further grown for 24 h in PrECGM for PrEC or in PrSCGM for PrSC. LNCaP cells plated onto six-well culture plates were transiently transfected using FuGENE transfection reagent with pERE-luc (1 μg) and pRL-TK (50 ng) according to the manufacturer's instruction and further grown for 24 h in RPMI-1640 with 10% fetal bovine serum dialyzed (10 000 MW cutoff, Sigma), penicillin and streptomycin. Then, β-estradiol or SERM was added to the final concentration at 10 nM for β-estradiol or at 10 μM for SERM, and cells were further cultured for another 24 h followed by luciferase assay.
The luciferase assay was carried out using Dual-luciferase reporter assay system (Promega) according to the methods recommended by the manufacturer. In short, cells were rinsed with PBS and harvested following the addition of 250 μl of the lysis buffer per well by scraping with a rubber policeman. The cells were subjected to two freeze/thaw cycles to accomplish complete lysis, and the lysate was cleaned by centrifugation for 1 min. After preparation of the Luciferase Assay Reagent II and the Stop & Glo Reagent supplied in the kit, 20 μl of the cell lysate was added to 100 μl of the Luciferase Assay Reagent II in a luminometer tube and mixed. The firefly luciferase activity was measured using a luminometer with the program of a 2-s premeasurement delay followed by a 10-s measurement period for each reporter assay. Then, 100 μl of Stop & Glo Reagent was further added and the second measurement of the renilla luciferase activity was performed. The results of the luciferase assay were normalized using the activity of the renilla expression vector pRL-TK co-transfected together with the reporter plasmid.
The cell proliferation was measured using the water-soluble tetrazolium salt assay system (WST-1 Cell Proliferation System: TaKaRa Bio, Otsu, Shiga, Japan). In short, PrSC or PrEC cells were seeded at 1 × 105 cells per well onto 6-well plates and incubated for 24 h. Then, ligands were added and the cells were further grown for 48 h and subjected to WST-1 assay. WST-1 reagent was added to each well, cells were incubated at 37 °C for 2 h and the absorbance was measured at 450 nM.
The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay was carried out using APO-DIRECT Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instruction. After labeling DNA breaks with FITC-dUTP, samples were subjected to flow cytometric analysis using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
Differences between treatment groups were assessed by Student's paired t-test. P<0.05 was considered as statistically significant.
Effect of hormones and an anti-estrogen toremifene on proliferation of normal human prostate epithelial and stromal cells
To access the importance of the estrogenic signaling in normal human prostate cells, DHT, estradiol (E2), estrogenic chemicals and an anti-estrogen were administrated to prostate epithelial and stromal cells. As a control, hormone-dependent prostate cancer cells, LNCaP, were used. In prostate epithelial cells, the proliferation was enhanced by adding E2, genistein (a phyto-estrogen) or bisphenol A (BPA) compared with the control cells. An anti-estrogen toremifene suppressed the proliferation of epithelial cells (Figure 1a). In stromal cells, only DHT enhanced the proliferation. Suppression of the ER by toremifene reduced the growth of the cells remarkably in both prostate epithelial and stromal cells indicating that the ER activation is necessary in the growth of normal prostate cells (Figures 1a and b). In LNCaP cells, on the other hand, estrogenic stimulation seems to reduce cell proliferation: suppression of ER by toremifene did not reduce cell growth strongly (Figure 1c).
Effect of varying toremifene concentration on growth suppression of prostatic stromal cells
As toremifene reduced the growth of prostatic stromal cells remarkably, we varied its concentration and carried out cell-proliferation assays to define the optimum concentration. Toremifene suppressed the growth of stromal cells dose-dependently. The suppressive growth effect of toremifene was reduced to some extent by the longer incubation time (Figure 2).
To validate whether or not the negative growth effect of toremifene is due to apoptosis or blockade of the cell proliferation, we carried out TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay with FITC-dUTP followed by flow cytometric analysis. After incubation of prostatic stromal cells with 10 μM of toremifene for 48 h, no peak of apoptotic nuclei was detected by flow cytometric analysis: a single peak of the normal DNA which was the same pattern as the control was observed (data not shown).
Effect of SERMs on proliferation of normal human prostate cells
As shown in Figures 3a and b, SERMs such as toremifene, tamoxifen and raloxifene suppressed the proliferation of prostate epithelial and stromal cells. Faslodex did not reduce cell proliferation albeit tended to increase the epithelial cell growth; the possible explanation is the agonistic property of faslodex in epithelial cells. Anti-androgens, however, did not suppress the cell growth in normal prostate cells. The observation that the suppression of ER by SERMs, rather than that of AR by anti-androgens, reduced the growth of normal prostate stromal cells emphasizes the importance of estrogenic signaling in normal prostate. In LNCaP cells, SERMs did not reduce cell proliferation strongly. Because LNCaP cells have a mutated AR at the codon 877 that is activated by flutamide and suppressed by bicalutamide, the result that flutamide enhanced the growth of LNCaP cells and bicalutamide seemed to suppress its growth indicates the relevance of AR-mediated signaling in LNCaP cells.
Effect of E2 and SERMs on ER activities in human normal prostate cells and LNCaP cells
To demonstrate the functionality of the endogenous ERs in normal prostate cells, the transactivation activities of ERs were measured using pERE-luc as a reporter gene. As pERE-luc is a reporter plasmid for both ERs α and β, the activities measured with each cell line represent the sum of those of ERsα and β. The activities of ERs seemed to be enhanced by adding E2 and suppressed by toremifene and tamoxifen in prostate epithelial cells (Figure 4a). In prostate stromal cells, the activities of ERs were enhanced by adding E2 and reduced remarkably by adding toremifene, and seemed to be suppressed by raloxifene whereas they were not suppressed by tamoxifen nor faslodex (Figure 4b). In LNCaP cells, the ER activities were increased by E2 and tended to be suppressed by all of the SERMs used (Figure 4c).
Recently, much attention has been paid to the ERs α and β in terms of their involvement in prostatic diseases. Contrary to the ERα that is expressed at a higher level in stromal cells and seems to be involved in the pathogenesis of BPH,2, 11 the ERβ, through activation by its putative ligand 5α-androstane-3β, 17β-diol, was reported to suppress the growth of the ventral prostate and to decrease the AR content in rodents.21, 22 Accordingly, there have been several studies describing that a loss of ERβ expression is seen in high-grade prostate intraepithelial neoplasia and high-grade dysplasia compared with normal prostate epithelium expressing ERβ.23, 24, 25 Recent experimental studies have demonstrated that ERβ behaves as a tumor suppressor gene. Hurtado et al.26 for example showed that overexpression of ERβ1 (ERβ) induced a cell cycle arrest in the early G1 phase in LNCaP cells. Walton et al.27 showed that re-expression of ERβ using a DNA demethylating agent and a histone deacetylase inhibitor caused increased apoptosis in prostate cancer cells. Thus, carcinogenesis in prostate seems to be characterized by a loss of ERβ expression. However, conflicting results have been reported regarding the frequency of the ERβ expression and its correlation to the grade in prostate cancer.24, 25, 28, 29 With regard to ERα, recent investigations have emphasized the important role of ERα in prostatic carcinogenesis.30, 31
Although those studies seem to illustrate the role of ERs in the prostatic gland, an issue still remains whether the endogenous ERs will actually be activated by their ligands followed by the ER-mediated induction of the relevant genes.
In this study, we demonstrated for the first time that endogenous ERs in normal human prostate stromal cells were activated by estrogen and suppressed by SERMs. We also showed that the growth of normal prostate cells was suppressed by SERMs and not by anti-androgens, the weight of evidences indicating that the ER-mediated signaling is more relevant to the growth of normal prostate cells than the AR-mediated one in the primary culture model.
Concerning the effect of hormones on proliferation of human normal prostate cells, there have been several reports showing variable results. Some reported that DHT or estradiol stimulated the growth of stromal cells whereas others published that they did not.18, 32, 33, 34, 35, 36 In our study, the proliferation of prostate epithelial cells was significantly enhanced by adding E2, and that of stromal cells by DHT although the effect was marginal. This may be attributed to the serum and other additives in the culture medium of these slow-growing cells. The suppression study using SERMs and anti-androgens seemed to yield a good understanding of the relevance of the ER-mediated signaling in normal human prostate cells.
The downstream genes of which ERs activate the transcription have not been studied well in normal prostate cells. Zhang et al.13 showed that ERK was activated by estradiol with the proliferation of prostate stromal cells and that a small interfering RNA of ERα blocked the estrogen-mediated activation of ERK and the proliferation of stromal cells. Chen et al.37 showed that ERα activity was enhanced by overexpressing ERAP75, a co-activator of ERα, with upregulation of the ERα target gene, stromal-derived factor-1, in normal prostate stromal cells transfected with ERα. Stromal-derived factor-1 has been reported to be associated with the metastasis and progression of prostate cancer.38
The ERβ, originally isolated from rat prostate, has a variety of its isoforms, that is, splicing variants. Those isoforms are expressed in an organ-specific manner, but their function has not been well investigated, which may account for the complexity of the ERβ signaling.39 Accordingly, in our experiments, some SERMs functioned as an antagonist whereas others tended to act as an agonist in normal human prostate stromal and epithelial cells.
A few studies demonstrated the association of ERβ and the downstream genes regulated by ERβ in prostate cancer cells. Leng et al.40 reported that ICI182,780 induced several key genes by activating ERβ that bound to the upstream NFκ consensus sequences. Matsumura et al.41 showed that genistein, a phyto-estrogen from soybean, induced p21 and ERβ expression in PC-3 prostate cancer cells. Silencing ERβ by siRNA suppressed the p21 expression and the p21 promoter activity. Further study is necessary to elucidate the roles of the ERs and the genes of which transcriptions are regulated by ERs in normal and malignant prostate cells.
Of particular interest is a case report that a 56-year-old male-to-female transsexual who had undergone sex-changing surgery 25 years ago followed by continuous estrogen substitution presented with obstructive voiding symptoms. The patient was diagnosed as BPH and treated with TUR-P with the histopathology showing an ER expression and a strong AR expression.42 Probably, the gene amplification of AR has been caused by the low androgen level. One can imagine that continuous stimulation of ER in stromal cells played an important role in the pathogenesis of BPH in the case.
From a chemopreventive point of view, Price et al.43 performed a clinical trial using toremifene for the prevention of developing prostate cancer in men with high-grade prostatic intraepithelial neoplasia, a precancerous lesion. They concluded that toremifene decreased the incidence of developing prostate cancer in men with high-grade prostatic intraepithelial neoplasia. In our study, toremifene showed a strong property of suppressing ER activity and cell proliferation in prostatic stromal cells. The ability of toremifene to decrease the incidence of prostate cancer may be attributed to the suppression of the stromal paracrine that stimulates the precancerous lesion. The possible use of SERMs for treating BPH may be of further study.
In conclusion, endogenous estrogen receptors were functional in normal human prostate stromal cells, and their activities were reduced by toremifene accompanied by suppression in cell growth.
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We thank Dr Shigeaki Kato, Tokyo University, for providing us with pGL3-ERE-X3tK luc (pERE-luc). We also thank Nippon Kayaku Corp. (Tokyo, Japan) for flutamide and toremifene. This work was supported in part by grants from Osaka City University Medical Research Foundation.
Conflict of interest
The authors declare no conflict of interest.
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Nomura, H., Kawashima, H., Masaki, S. et al. Effect of selective estrogen receptor modulators on cell proliferation and estrogen receptor activities in normal human prostate stromal and epithelial cells. Prostate Cancer Prostatic Dis 12, 375–381 (2009). https://doi.org/10.1038/pcan.2009.20
- selective estrogen receptor modulator (SERM)
- estrogen receptors a and b
- normal prostate stromal cells
- normal prostate epithelial cells
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