Is Transforming Growth Factor-β Signaling Activated in Human Hypertrophied Prostate Treated by 5-Alpha Reductase Inhibitor?

Background and Aim. It is well known that androgen deprivation relates to penile fibrosis, so we hypothesize that long-term treatment with 5-alphareductase inhibitors (5ARIs) may increase the risk of fibrosis of prostate. Patients and Methods. Thirty-two BPH patients who underwent transurethral resection of the prostate were enrolled. The patients were divided into two groups: group one, 16 patients underwent TURP who had been treated with tamsulosin for 2 years; group two, 16 patients underwent TURP who had been treated with combination of tamsulosin and dutasteride for at least 1 year. We evaluated the expressions of nNOS, iNOS, eNOS, TGF-β1, TGF-β2, phosphorylated-Smad2/3 (p-Smad2/3), E-cadherin, N-cadherin, and α-smooth muscle actin in the resected prostate tissues by western blotting, and the TGF-β concentration was determined by ELISA kit. Results. The expressions of 3 isoforms of NOS were significantly increased in group 2 except of eNOS in lateral prostate, and the expressions of TGF-β1, TGF-β2, and p-Smad2/3 increased about 2-fold compared with group 1. In group 2, the E-cadherin expression decreased while N-cadherin expression increased significantly. Conclusions. The overexpression of nNOS may contribute to prostate smooth muscle relaxation; however, long-time treatment with 5 ARI increases the risk of fibrosis of prostate.


Introduction
The 5-alpha reductase (5 AR) inhibitor (5 ARI) was developed to treat the patients with symptomatic benign prostatic hyperplasia (BPH) and decrease the frequency and risk of BPH-related morbidities [1,2]. Presently available orally administered 5 ARIs, finasteride and dutasteride, inhibit 5 AR, an enzyme that catalyzes the irreversible reduction of testosterone (T) to dihydrotestosterone (DHT), with NADPH as the hydrogen donor [3]. Dutasteride is a selective inhibitor of 5 AR type 1 (5 AR1) and 5 AR type 2 (5 AR2). Finasteride is considered mainly an inhibitor of 5 AR2 and is approximately 50 times weaker in inhibiting 5 AR1 than 5 AR2 [4]. The development of BPH is an androgen-dependent process, and androgen suppression causes regression primarily of the epithelial elements of the prostate, resulting in a reduction in the size of the gland and improvement in symptoms [5,6].
As we know, androgen deprivation by surgical or medical castration is associated with penile cavernosal fibrosis resulting in penile tissue atrophy, alterations in dorsal nerve structure, alterations in endothelial morphology, reductions in trabecular smooth muscle content, increases in deposition of extracellular matrix, and increases in accumulation of adipocytes in the subtunical region of the corpus cavernosum  PSA means prostate specific antigen. T-zone means transitional zone. [7]. Transforming growth factor-(TGF-) signaling pathway plays the most important role in the fibrotic process.
TGF-reduces collagenase production and stimulates the expression of tissue inhibitor of metalloproteinases, resulting in an overall inhibition of extracellular matrix (ECM) degradation and leading to excessive matrix accumulation [8,9]. As androgen deprivation by surgical or medical castration induces cavernosal fibrosis, it may also affect the prostate tissue. In the present study, we investigated the effect of longtime treatment with 5 ARI on TGF-signaling pathway in the prostate of BPH patients.  underwent transurethral resection of the prostate (TURP) were enrolled in the present study. The patients were divided into two groups: group 1, 16 patients underwent TURP who had been treated with alpha blocker (tamsulosin) for 2 years; group 2, the other 16 patients were treated with combination of alpha blocker (tamsulosin) and 5 ARI (dutasteride) for at least 1 year before the operation (Table 1).

Tissue Preparation. Approximately 1 g of each lateral
and anterior portion of the prostate tissue resected from each patient was obtained during the TURP. The tissues were immediately frozen at −80 ∘ C. For western blot analysis, the removed tissues were placed in protease-inhibitor buffer containing 1% Nonidet P-40, 50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 10 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 100 mM sodium fluoride and were homogenized. The homogenate was centrifuged at 13,000 rpm for 30 min. The supernatant was collected and the volume was recorded.

Western
Blotting. The samples were normalized for total protein content with a pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 30 g aliquots were suspended in an equal volume of 2× Laemmli buffer containing 2-mercaptoethanol and boiled for 5 min before performing SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA), blocked with skim milk (5%), and incubated with various primary antibodies

Human TGF-Immunoassay.
The samples were normalized for total protein content with a BCA kit and tested immediately or preserved at −80 ∘ C. TGF-levels were analyzed using a commercially available ELISA (R&D Systems, Minneapolis, MN, USA) per the manufacturer's protocol. Briefly, this assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TGF-1 has been precoated onto a microplate. Standard, controls, and samples are pipetted into the wells and any TGF-1 present is bound by the immobilized antibody. After washing away any unbound substances, an enzymelinked polyclonal antibody specific for TGF-1 is added to the wells to sandwich the TGF-1 immobilized during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TGF-1 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Statistical
Analyses. Data were presented as means ± SD. The statistical significances of the differences were calculated by one-way ANOVA followed by Bonferroni's multiple comparison. The values were compared using Student's pairedtest. A value < 0.05 was considered statistically significant.

The Expression of nNOS, iNOS, and eNOS.
The expressions of nNOS and iNOS were significantly increased in group 2. However, the expression of eNOS in lateral portion of prostate was decreased (Figure 1, < 0.05). Figure 2. The upregulated expressions of TGF-1 and TGF-2 were observed in group 2. The result showed that lateral portion of prostate expressed more TGF-1, TGF-2 than anterior wall. P-Smad2/3 expression was also increased in group 2, but there was no difference between the lateral portion and anterior wall (Figure 2, < 0.05).

The Expression of TGF-. Western blot of TGF-was shown in
The TGF-level was determined by ELISA, and the results indicated that TGF-was significantly increased in group 2 ( Figure 3, < 0.05).

The Expression of E-Cadherin and N-Cadherin.
The western blotting results of E-cadherin and N-cadherin were shown in Figure 4. The E-cadherin level decreased and Ncadherin increased significantly in group 2. Alpha smooth muscle actin was markedly increased in group 2 and expressed more in lateral portion of the prostate.

Discussion
TGF-participates in the pathogenesis of multiple cardiovascular diseases, including hypertension, atherosclerosis, cardiac hypertrophy, and heart failure [10][11][12]. Also, it has been shown to be a regulator of stromal proliferation and differentiation in the prostate [13]. It is well known that androgen plays an important role in the pathogenesis of BPH. The action of androgen is mediated through actions of a host of soluble growth factors, among which TGFis the most versatile in its ability to regulate proliferation, growth arrest, differentiation, and apoptosis of prostatic stromal cells [14]. It has been demonstrated that the effect of 5 ARI on BPH was associated with suppression of the insulin like growth factor 1 gene and with an increase in TGFgene expression [15]. TGF-predominantly transmits the signals through cytoplasmic proteins called Smads, which translocates into the cell nucleus acting as transcription factors. Eight different members of the Smad family have been identified in mammals, in which Smad2 and Smad3 are specific mediators of TGF-/activin pathways [10,11]. In this study, the results showed that long-term treatment with 5 ARI significantly increased the expression of TGF-1, TGF-2, and p-Samd2/3 compared with the control group, suggesting 5 ARI-induced cell death was regulated by TGF--Smad pathway. Decreased expression of E-cadherin accompanied by increased expression of N-cadherin resulted in a loss of epithelial characteristics and indicated that the epithelial cell underwent epithelial to mesenchymal transition (EMT). TGF--Smad pathway is very important stream also involved in fibrotic process. In vascular smooth muscle cells, endothelial cells, and fibroblasts, TGF-1 increases the synthesis of ECM proteins, such as fibronectin, collagens, and activator inhibitor-1 (PAI-1), even at low concentrations [8,9]. TGF-induces expression of fibronectin which is required for enhancement of -smooth muscle actin and collagen type I expression [8]. PAI-1 is a serpin class protease inhibitor, important in tissue remodeling by modulating thrombosis, inflammation, migration, and ECM. TGF-reduces collagenase production and stimulates the expression of tissue inhibitor of metalloproteinases (TIMP), resulting in an overall inhibition of ECM degradation and leading to excessive matrix accumulation [8,9]. The mechanisms involved in TGF-mediated vascular fibrosis are complex, including activation of Smad proteins, protein kinases, production of mediators, and crosstalk between pathways. TGF-also acts as a mediator of vascular fibrosis induced by several agents involved in cardiovascular diseases, including mechanical stress, angiotensin II, high glucose, and advanced glycation products [16][17][18]. In Peyronie's disease, the fibrosis is characterized by an increase in collagen over the intracellular compartment and is associated with the production of profibrotic factors, such as TGF-1, PAI-1, and reactive oxygen species during oxidative stress [19][20][21]. This fibrosis is accompanied by the induction of iNOS, which acts as an endogenous antifibrotic mechanism in response to the profibrotic processes [19,22].
The presence of NOS has been demonstrated in the human prostate that the eNOS is related to the maintenance of local vascular perfusion, whereas the nNOS is mainly involved in the control of smooth muscle tone and glandular function, including proliferation of epithelial and subepithelial cells [23,24]. Until today, the inducible form of NOS, the iNOS, has not been found in normal prostate tissue; however, there are hints that the enzyme is expressed in hyperplastic and malignant tissue [25,26]. The reduction in glandular tissue by 5 ARI is achieved by the induction of apoptosis, which is histologically manifested by ductal atrophy. Inhibition also diminishes the number of blood vessels in the prostate because of a reduction in vascularderived endothelial growth factor which downregulates the eNOS expression in lateral portion of prostate [27].

Conclusions
The overexpression of nNOS may contribute to prostate smooth muscle relaxation and improve the symptoms; however, long-time treatment with 5 ARI increases the risk of fibrosis by the TGF--p-Smad signaling pathway.

What is Known?
(1) The 5 ARIs have been successfully used for treatment of BPH by inhibiting the conversion of T to DHT, resulting in a reduction in the size of the gland and improvement in symptoms. (2) Androgen deprivation by surgical or medical castration is associated with penile cavernosal fibrosis.

What is New?
(1) Androgen deprivation induced by long-term treatment with 5 ARI increases the risk of fibrosis via the TGF--p-Smad signaling pathway. (2) The upregulated nNOS expression may contribute to prostate smooth muscle relaxation and improve the symptoms.