Epigallocatechin-3-gallate reduces the proliferation of benign prostatic hyperplasia cells via regulation of focal adhesions

Life Sciences 191 (2017) 74–81

Contents lists available at ScienceDirect

Life Sciences journal homepage: www.elsevier.com/locate/lifescie

Epigallocatechin-3-gallate reduces the proliferation of benign prostatic hyperplasia cells via regulation of focal adhesions

MARK

Burcu Erbaykent Tepedelena, Elif Soyab, Mehmet Korkmazb,⁎ a b

Department of Molecular Biology and Genetic, Faculty of Arts and Science, Uludağ University, Bursa 16059, Turkey Department of Medical Biology, Faculty of Medicine, Manisa Celal Bayar University, Manisa 45030, Turkey

A R T I C L E I N F O

A B S T R A C T

Keywords: EGCG BPH Cytoskeleton F-Actin FAK Paxillin

Aims: Benign prostatic hyperplasia (BPH) is the most common urological disease that is characterized by the excessive growth of prostatic epithelial and stromal cells. Pharmacological therapy for BPH has limited use due to the many side effects so there is a need for new agents including natural compounds such as epigallocatechin3-gallate (EGCG). This study was undertaken to assess the role of EGCG, suppressing the formation of BPH by reducing inflammation and oxidative stress, in cytoskeleton organization and ECM interactions via focal adhesions. Main methods: We performed MTT assay to investigate cell viability of BPH-1 cells, wound healing assay to examine cell migration, immunofluorescence assay for F-actin organization and paxillin distribution and finally immunoblotting to investigate focal adhesion protein levels in the presence and absence of EGCG. Key findings: We found that EGCG inhibits cell proliferation at the concentration of 89.12 μM, 21.2 μM and 2.39 μM for 24, 48 and 72 h, respectively as well as inhibitory effects of EGCG on BPH-1 cell migration were observed in a wound healing assay. Furthermore, it was determined by immunofluorescence labeling that EGCG disrupts F-actin organization and reduces paxillin distribution. Additionally, EGCG decreases the activation of FAK (Focal Adhesion Kinase) and the levels of paxillin, RhoA (Ras homolog gene family, member A), Cdc42 (cell division cycle 42) and PAK1 (p21 protein-activated kinase 1) in a dose-dependent manner. Significance: For the first time, by this study, we found evidence that BPH-1 cell proliferation could be inhibited with EGCG through the disruption of cytoskeleton organization and ECM interactions. Consequently, EGCG might be useful in the prevention and treatment of diseases characterized by excessive cell proliferation such as BPH.

1. Introduction BPH is the most common urological disease affecting approximately half of men over 50 years of age that involves pathologically and clinically phases of nodular hyperplasia, prostatis and prostate hypertrophy [1–3]. Likewise BPH, which begins as a simple micronodular hyperplasia, usually results in bladder outlet obstruction and lower urinary tract symptoms (LUTS) with subsequent macroscopic nodular enlargement [4,5]. BPH is histologically characterized by the excessive and uncontrolled growth of prostatic epithelial and stromal cells and also it occurs due to complex cellular changes which include alterations in proliferation, differentiation and senescence [3,6–9]. The pathological changes that lead to the observed phenotype as well as specific mechanisms that regulate the abnormal growth in BPH pathogenesis remain incompletely understood. However, many factors such as

⁎

hormonal (androgens, estrogens, adrenergic receptors), nutritional, environmental and inflammatory mediators play a role in the etiology of BPH while chronic inflammation and oxidative stress represent the major factors in the progression of BPH [10–13]. To date, treatments for BPH include surgical procedures or pharmacological therapy that used α-blockers and Type II 5α-reductase inhibitors [6,14–15]. The αblockers relax the smooth muscles in the prostate and the Type II 5αreductase inhibitors prevent the formation of dihydrotestosterone (DHT) which causes the prostate enlargement through overgrowth of stromal and epithelial cells. However the application of these drugs are very limited due to the side effects such as orthostatic hypotension, diarrhea, headache, nasal congestion and sexual dysfunction furthermore, they cannot completely prevent the BPH [15–16]. Therefore there is a necessity for the development of new agents that can alleviate the symptoms of BPH. In recent years, due to the side effects and consequences, there has been an increasing tendency towards

Corresponding author. E-mail address: [email protected] (M. Korkmaz).

http://dx.doi.org/10.1016/j.lfs.2017.10.016 Received 7 August 2017; Received in revised form 29 September 2017; Accepted 11 October 2017 Available online 12 October 2017 0024-3205/ © 2017 Published by Elsevier Inc.

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

Fig. 1. Effects of EGCG on viability of BPH-1 cells were assessed by MTT assay for 24, 48 and 72 h. (A) Increasing concentrations of EGCG lead to a significant decrease in proliferation of BPH-1 cells compared with untreated controls. The absorbance of control cells was considered as 100% and cell viability of the applied concentrations was calculated. (B) GI50 values of EGCG were calculated by GraphPad Prism 5.0 program and represented in the table. The MTT assay was performed by triplicate samples in at least three independent experiments. Standard deviation and p values were determined by two-tailed equal variance Student's t-test or two-way ANOVA and values of p ≤ 0,005 were considered significant.

been shown to suppress the migration and adhesion of many cell types and decrease the phosphorylation of FAK (Focal Adhesion Kinase) in MDA-MB-231 breast cancer cells [26–29]. In terms of prostate cancer, EGCG has been associated with lower prostate cancer incidence [30–31] and reduced risk of progression to advanced disease [31–33]. EGCG has been shown to inhibit cellular proliferation and induce apoptosis in the prostate cancer cell lines through inhibiting the activation of EGFR (epidermal growth factor receptor), decreasing the expression of pAkt and reducing NF-κB nuclear localization [34–36]. In a recent study conducted by Jinglou and Hongping [11]; it was determined that continuous administration of EGCG (100 or 50 mg/kg/ day) for four weeks decreased prostate growth and hyperplasia compared to untreated control group by significantly reducing glucose, total cholesterol and triglyceride levels. It was also shown that the suppression of oxidative damage, reduction of inflammatory markers such as IL-Iβ (Interleukin 1-Beta), IL-6 (Interleukin-6) and TNF-α (tumor necrosis factor-alpha), inhibition of IGF-I (insulin-like growth factor I) and IGF-II (insulin-like growth factor II) and upregulation of PPAR-α/-γ (peroxisome proliferator activated receptors) were play important role in the reduction of hyperplasia [11]. However, the changes in these molecules have led to the necessity of evaluating the extracellular

phytotherapy using natural substances in the management of this disease [5]. Recent studies by Udensi and Paul [17] showed that prostatic hyperplasia significantly reduced antioxidant levels of the prostate tissue. Therefore, oxidative stress is thought to be one of the mechanisms that trigger the chain of reaction that plays a role in the development and progression of prostatic hyperplasia. This circumstance is especially acceptable as human prostate is vulnerable to oxidative DNA damage due to faster cell cycle and fewer DNA repair enzymes [12]. Green tea, one of the most important drink, is widely consumed nowadays. Green tea is believed to have antiangiogenic, antiproliferative and antiinflammatory effect as well as improving lipid profiles, increasing body fat loss and impairing insulin resistance due to its potential antioxidant property and capacity to modulate various cell signaling pathways [11,18]. The main active polyphenols of green tea are catechins and it was thought that Epigallocatechin-3-gallate (EGCG), which has been shown to prevent the development of proliferative diseases [19], is responsible for the beneficial therapeutic effects of green tea [11,20]. The effects of EGCG are widely examined in epidemiological, animal, clinical and cell culture studies including prostate, breast and pancreatic cancer cells due to its ability to inhibit cell growth, arrest cell cycle and induce apoptosis [21–25]. Also, EGCG has 75

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

Fig. 2. Inhibitory effects of EGCG on BPH-1 cell migration in a wound healing assay. (A) BPH-1 cells were seeded on 12-well plates and a wound was scratched in a confluent cell layer. Cells were treated with EGCG at 10 μM, 25 μM, 50 μM, 75 μM and 100 μM and wound closure was examined at 4× magnification and documented by photography at 0 h, 12 h and 24 h. (B) The percentage of wound closure that was analyzed with ImageJ software is depicted in graphs. Images represent results obtained from three independent experiments; significance was determined with unpaired student's t-test (*p ≤ 0,01; **p ≤ 0,005).

suppressing the formation of BPH by reducing inflammation and oxidative stress, in cytoskeleton organization and ECM interactions via focal adhesions. For the first time, by this study, we showed that EGCG suppressed the proliferation and migration of BPH-1 cells through the regulation of focal adhesions. Eventually EGCG might be useful in the treatment of LUTS and BPH where FAK activation is critical for the pathogenesis of these disorders.

matrix interactions and cytoskeleton, which may be effective in the formation of BPH pattern. As known, benign and/or premalignant lesions can be caused by changes in the interaction of epithelial cells with the tissue microenvironment including stromal cells and extracellular matrix (ECM). Adhesion of the fibroblast, endothelial and prostate epithelial cells to ECM is required for not only structural support but also vital signals of a cell [2]. Additionally, ECM is important in mediating the effects of changing chemokine/cytokine ratio on the development of epithelial hyperplasia in BPH [7]. However, smooth muscle contraction that is critical for LUTS and BPH requires attachment of the cytoskeleton to membranes and membranes to the ECM. These connections are accomplished by activation of focal adhesion kinase (FAK) through its phosphorylation, which plays an important role in assembly of focal adhesion proteins, focal adhesion function and regulation of cell growth and migration [37–40]. Focal adhesions that include integrins, paxillin, talin, and Src (proto-oncogene tyrosine-protein kinase) occur at the site of interaction between actin cytoskeleton and ECM [37–38,40–41]. Paxillin is a cytoskeletal protein involved in actin membrane attachment to ECM and the interaction between FAK and paxillin are pivotal for the regulation of cell morphology and motility [26,38,42–43]. Additionally, Rho subfamily of small GTPases (Rho, Rac and Cdc42) also controls actin filament dynamics and focal adhesions assembly through formation of stress fibers, assembly of lamellipodia and membrane ruffles and regulation of filopodial protrusions, while the serine/ threonine kinase PAK is involved in Rac- and Cdc42-stimulated reorganization of the actin cytoskeleton [44–45]. In this context, this study was undertaken to assess the role of EGCG,

2. Materials and methods 2.1. Cell culture Human benign prostate hyperplasia cell line BPH-1 was purchased from German Collection of Microorganisms and Cell cultures (Leibniz Institute DSMZ, Germany). BPH-1 cells were propagated in RPMI 1640 (Invitrogen, UK) medium supplemented with 10% FBS (Fetal bovine Serum) (Invitrogen, UK), 1% L–glutamine (Invitrogen, UK), and 1% penicillin–streptomycin (Invitrogen, UK) in a humidified incubator at 37 °C and 5% CO2. Cells were removed from culture plates with trypsinEDTA solution (0.25%, Invitrogen, UK) and subcultured for further experiments. 2.2. Cell treatment A stock solution of EGCG (Sigma, UK) was prepared at a concentration of 1 M in ddH2O and stored at +4 °C until the analysis. The concentrations (1, 2.5, 5, 7.5, 10, 20, 30, 40, 50, 60, 80, 100 μM) used in MTT assay were freshly prepared by diluting the stock solution in 76

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

Fig. 3. EGCG disrupts organization of actin cytoskeleton and reduces the paxillin distribution (A) EGCG-induced changes in F-actin and Paxillin distribution following 48 h exposure at increasing concentrations in BPH-1 cells. Cells were stained with AlexaFlour-594 for Paxillin (red), 488phalloidin for F-actin (green) and DAPI for the nucleus (blue). Cells were imaged at a × 60 objective at identical exposure settings with immunofluorescence microscope. Images of EGCG-exposed cells show structural alterations in shape and size. a.b.c. Untreated control cells with a smooth actin structure and robust paxillin distribution. d-s. Images from cells treated with 10 μM, 25 μM, 50 μM, 75 μM and 100 μM EGCG, respectively that were shown reduced paxillin distribution. h.n. Loss of stress fibers. h.k. Diffuse Factin staining. r. Disrupted actin organization. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.4. MTT assay

culture medium and cells were incubated for 24, 48 and 72 h in 37 °C and 5% CO2. For wound healing, immunofluorescence and immunoblotting experiments, BPH-1 cells were treated with increasing concentrations of EGCG for 48 h.

The effect of EGCG on BPH-1 cell viability was investigated by MTT assay. Cells (8 × 103/well) were plated in 96-well plates and incubated for 24 h without EGCG. After administration of EGCG, cells were incubated for 24, 48, and 72 h. MTT solution (0.5 mM/well) was added to cells in 96-well plates followed by incubation for 4 h at 37 °C. Formazan crystals were dissolved with 200 μL DMSO (Dimethyl sulfoxide) and read spectrophotometrically at 570 nm by a microplate reader (BioRad, Coda, Richmond, CA). The viability of the cells was calculated as the percentage of MTT reduction. The absorbance of control cells was assumed as 100% and cell viability of the applied concentrations was represented as graphs. The GI50 values (the concentration of drug to cause 50% reduction in proliferation of cells) were calculated by the GraphPad Prism 5.0 program. The MTT assay was performed by triplicate samples in at least three independent experiments. Standard deviation and p values were determined by two-tailed equal variance Student's t-test or two-way ANOVA and values of p ≤ 0,005 were considered significant.

2.3. Antibodies The polyclonal human antibodies against RhoA (pı-ma1011; 1/ 1000), Cdc42 (pı-pa1092; 1/2000) and PAK1 (pı-pa534696; 1/1000) were purchased from Pierce (Thermo Scientific, US). The polyclonal human anti-phospho-FAK(Tyr397) (8556S; 1/1000) and anti-FAK (13009S; 1/2000) antibody were purchased from Cell Signaling Technology (CST, US). The monoclonal anti-β-actin (A5316; 1/ 100.000) antibody was obtained from Sigma (UK). The anti-mouse and anti-rabbit HRP-conjugated antibodies (1706516-1706515; 1/5000) were purchased from Bio-Rad (US).

77

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

Fig. 4. EGCG decreases the level of focal adhesion proteins (A) BPH-1 cells were treated with 10, 25, 50, 75 and 100 μM EGCG for 48 h, then the expression level of focal adhesion proteins were determined by immunoblotting. Actin was used as the loading control. EGCG administration remarkably decreases the pFAK(Tyr397), paxillin, RhoA, Cdc42 and PAK1 expression level in a dose-dependent manner in BPH-1 cells. (B) Alterations in the expression level of pFAK(Tyr397), paxillin, RhoA, Cdc42 and were measured by the ImageJ software program, normalized to β-Actin levels and represented as graphs.

2.5. Wound closure assay for cell migration

images were taken at identical exposure settings.

For wound healing assay, cells were grown to confluency on 12-well culture plates. When the cells completely covered the surface, a scratch was made on cells with the pipette tip. After washing with PBS, fresh culture medium containing EGCG at a concentration of 10, 25, 50, 75 and 100 μM was added for examining the effect of EGCG on cell migration. Cells were visualized at 4× magnification by Leica DMIL Inverted Microscope and wound size was measured by ImageJ software program (NIH, Bethesda, MD, USA, ImageJ 1.47v, http://rsb.info. nih. gov/ij/). Standard deviation and p values were determined by twotailed equal variance Student's t-test or two-way ANOVA and provided in figure legends.

2.7. Immunoblotting Cells were lysed with ice-cold modified RIPA buffer (1% Nonidet P40, 50 mM Tris HCl pH 7.4, 0.25% Na-deoxycholate, 1 mM EDTA, 150 mM NaCl) with NaF, PMSF and Na3VO4 (1 mM each) and complete protease and phosphatase inhibitor cocktails (Roche, Germany). The immunoblots were carried out by separating the proteins on 8–15% SDS-PAGE gels and immobilizing the proteins onto PVDF membranes (Bio-Rad, US) by a wet transfer. Briefly, membranes were blocked using TBS-T (Tris-Base-Saline containing 0.1% Tween 20) containing either 5% skim milk (w/v). The primary and secondary antibody incubations were carried out in TBS-T containing 0.5% dry milk at RT for 1 h or at + 4 °C overnight. Membranes were developed using 2 mL Clarity Western ECL Substrate (Bio-Rad, US) for 5 min and were photographed using Kodak X-ray films in a dark room. The immunoblot assay was repeated two times. Alterations in the expression of investigated proteins were measured by the imageJ software program, normalized with respect to β-Actin levels and the fold changes observed relative to control cells represented as graphs.

2.6. Immunofluorescence labeling and microscopy BPH-1 cells were grown on cover slips and treated with increasing concentrations of EGCG for 48 h. Cells were fixed with 4% paraformaldehyde for 1 h at room temperature then permeabilized with 0.2% Triton X-100-containing PBS (Phosphate-buffered Saline) and blocked for 5 min by using 1% BSA in PBS buffer. Firstly, cells were labeled with paxillin (1/300; 12065S, CST, US) primary antibody for 1 h and washed with PBS four times. Then cells were co-labeled with DyLight 488 Phalloidin Conjugate for F-actin (1/500; pı-21833, Pierce, Thermo, US) and Alexa-Flour 594 (1/750; A11012, Invitrogen, US) for paxillin. Cells were incubated at 37 °C 20 min and washed with PBS for four times, mounted with DAPI (1 μg/mL) and analyzed using a Leica DMIL fluorescent microscope (Leica, Germany). Immunofluorescence experiments were performed twice, for each treatment at least 20

3. Results 3.1. EGCG exhibits antiproliferative effect on BPH-1 cells The potential effect of EGCG on BPH-1 cell viability was investigated through the colorimetric MTT assay. Standard deviation and p values were determined by two-tailed equal variance Student's t-test 78

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

or two-way ANOVA and values of p ≤ 0,005 were considered significant. The results showed that cell proliferation and viability decreased with EGCG treatment compared to control cells. As shown in Fig.1A, proliferation was significantly reduced with EGCG treatment especially at 48 h (Fig.1A). GI50 values of EGCG were calculated by GraphPad Prism 5.0 program and determined as 89.12 μM, 21.2 μM and 2.39 μM for 24, 48 and 72 h, respectively (Fig.1B). Eventually, a time- and dose-dependent antiproliferative effect was shown in BPH-1 cells treated with EGCG.

The prostate, which is the requirement of the immune system, normally populated by small numbers of inflammatory cells such as T/B lymphocytes, macrophages and mast cells [46–48]. Chronic inflammation can create a local vicious cycle through triggering tissue damage, activating cytokine release and increasing the concentration of growth factors. In this respect, upregulation of pro-inflammatory cytokines has been widely reported in prostate tissues of BPH patients [13,49–53]. Local hypoxia may play a role in the pathophysiology of BPH by neovascularization following release of ROS and growth factors such as VEGF, IL-8, FGF-7, FGF-2 and TGFβ [54]. These growth factors may interact not only inflammatory cells but also with the stromal and epithelial cells of the prostate and may lead prostate growth. Although the role of chronic prostatic inflammation in the pathogenesis of BPH is still poorly understood, there are many studies investigating the effect of inflammation in BPH progression [55–56]. In the light of this information, Jiglou and Hongping [11] reports that EGCG reduces the release of proinflammatory cytokines and oxidative damage thereby suppressing the induction of BPH. Our results about the impairment of ECM interactions and cytoskeletal regulation are thought to be consistent with the initial results of decreased inflammation with EGCG administration in mice [11]. Some recent studies showed that epigallocatechin-3-gallate (EGCG) suppressed the migration and adhesion of many cell types and could prevent the development of proliferative diseases [19,26,57–59]. On the other hand, adhesion of the fibroblast, endothelial and prostate epithelial cells to ECM plays an important role in the development of benign and/or premalignant lesions such as BPH and LUTS in terms of smooth muscle contraction. These connections are accomplished by activation of focal adhesion kinase (FAK) and assembly of focal adhesion proteins [2,26,37–40]. Therefore, in this study, we investigated the antiproliferative and antimigratory effect of EGCG on benign prostatic hyperplasia cell line BPH-1 through the organization of actin cytoskeleton and the formation of focal adhesions. Initially, we have shown that EGCG inhibits BPH-1 cell proliferation and viability in a time and dose-dependent manner. Then, we found that EGCG suppressed the migration and motility of BPH-1 cells in vitro due to decreased healing capacity. Finally to determine whether EGCG inhibits cell migration by preventing cytoskeleton organization and formation of focal adhesions, we investigated F-actin and paxillin distribution and the level of FAK phosphorylation and other related proteins. We found that EGCG-treated cells showed structural alterations that are evidenced by shorter stress fibers, diffuse actin staining and disruption of F-actin structure (Fig.3). Therefore we suggested that EGCG contributes to the inability of cells to proliferate and reduced the capacity of migration due to these structural alterations. FAK is highly expressed in the early stages of prostate carcinoma and in the proliferative compartment of the normal prostate epithelium [39]. On the other hand, FAK plays an important role in integrin signaling that regulate cell adhesion and migration through its Tyr397 phosphorylation. The interaction of ECM with integrins at the cell surface leads to increase of FAK phosphorylation which in case results in activation of Rho family members such as RhoA, Rac1 and Cdc42 [19,26,60]. RhoA and Cdc42 induce different actin reorganization events and they provide cellular extensions for migration of cells. Also, it was recently reported that RhoA is likely involved in the smooth muscle contraction and plays important role in the Ca+ 2 sensitizing mechanisms [61]. Furthermore, it was shown that RhoA regulates the expression of genes related to cell proliferation and growth such as cJun and c-Fos [19]. We observed that EGCG treatment significantly inhibited the FAK phosphorylation in a dose-dependent manner and especially at higher doses EGCG leads to the reduced level of RhoA, Cdc42 and PAK1 (Fig.4). Recently, the cell surface receptor of EGCG has been described as the 67kD laminin receptor [19]. Therefore one possible mechanism may be that EGCG disrupts the ECM-integrin interaction leading to a decrease in levels of adhesion molecules through inhibiting their

3.2. EGCG suppresses cell migration To evaluate the migratory potential of BPH-1 cells treated with EGCG in increasing concentrations, the wound healing migration assay was performed in response to artificial wound produced on a cell monolayer (Fig.2). The time of wound closure was examined at 4× magnification after the specified time in the presence and absence of EGCG using an inverted phase-contrast microscope. As shown in Fig.2A, the suppression of migration was observed in a dose-dependent manner. Wound size was measured by ImageJ software program and the percentage of wound closure inhibition was represented as graphs (Fig.2B). It was determined that compared with control cells, treatment with EGCG reduced cell migration and only 44, 39, 37, 28 and 15% wound closure was observed for 24 h at 10, 25, 50, 75 and 100 μM, respectively (*p ≤ 0,01; **p ≤ 0,005). These results showed that EGCG suppressed the migration and motility of BPH-1 cells in vitro. EGCG reduces the level of focal adhesion proteins and disrupts organization of actin cytoskeleton. We investigated the effect of EGCG on actin cytoskeleton organization and the levels of focal adhesion proteins. Firstly, immunofluorescence labeling was performed by staining F-actin with Alexafluor 488-conjugated phalloidin and paxillin by Alexafluor 594 to determine intracellular actin and paxillin distribution after 48 h EGCG treatment. As shown in Fig.3A, control cells exhibited a developed actin cytoskeleton which was clearly visible and had a smooth structure as well as a robust paxillin distribution. However, we observed that cell morphology, organization of actin cytoskeleton and paxillin distribution changed with EGCG treatment. Cells exposed to EGCG showed a reduced paxillin distribution. Furthermore many cells reduced intercellular contacts which had shorter stress fibers (Fig.3A; h–n) and showed diffuse actin staining (Fig.3A; h–k) as well as disrupted F-actin structure (Fig.3A; r). Eventually, it was suggested that EGCG may have an effect on cell migration due to the flattened and angular morphology of BPH-1 cells observed with EGCG treatment. To determine whether EGCG effects on cell motility and migration via FAK regulation, we analyzed the levels of FAK and other focal adhesion proteins by western-blot (Fig.3B). Firstly, we examined the activation of FAK by its Tyr397 phosphorylation and observed that it was significantly decreased with EGCG treatment especially after at 50 μM concentration while total FAK levels were unchanged. Furthermore, paxillin levels have showed a rapid loss consistent with immunofluorescence results. Both FAK and paxillin are important regulators of cell shape and interactions with other cells, thus we suggested that EGCG inhibits cell migration through regulation of FAK and paxillin or their interaction with each other. Additionally, we investigated the levels of RhoA and Cdc42 due to decreased formation of stress fibers and we found that both protein levels were reduced in a dose-dependent manner. Finally, it was concluded that EGCG inhibits Cdc42 stimulated reorganization of the actin cytoskeleton because of the reduced level of PAK1. 4. Discussion In recent years, the effect of chronic inflammation has emerged prominently among several suggested hormonal and vascular mechanism that play a role in the pathogenesis and progression of BPH. 79

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

[4] A. Ryl, I. Rotter, T. Miazgowski, M. Slojewski, B. Dolegowska, A. Lubkowska, M. Laszczynska, Metabolic syndrome and benign prostatic hyperplasia: association or coincidence? Diabetol. Metab. Syndr. 7 (2015) 94. [5] C. Eleazu, K. Eleazu, W. Kalu, Management of Benign Prostatic Hyperplasia: could dietary polyphenols be an alternative to existing therapies? Front. Pharmacol. 8 (2017) 234. [6] J. Lin, J. Zhou, X. Zhong, Z. Hong, J. Peng, Inhibition of the signal transducer and activator of transcription 3 signaling pathway by Qianliening capsules suppresses the growth and induces the apoptosis of human prostate cells, Mol. Med. Rep. 11 (3) (2015) 2207–2214. [7] I.G. Schauer, D.R. Rowley, The functional role of reactive stroma in benign prostatic hyperplasia, Differentiation 82 (4–5) (2011) 200–210. [8] P. Castro, C. Xia, L. Gomez, D.J. Lamb, M. Ittmann, Interleukin-8 expression is increased in senescent prostatic epithelial cells and promotes the development of benign prostatic hyperplasia, Prostate 60 (2) (2004) 153–159. [9] T.M. Hällström, M. Laiho, Genetic changes and DNA damage responses in the prostate, Prostate 68 (8) (2008) 902–918. [10] A. Siejka, A.V. Schally, N.L. Block, N. Barabutis, Mechanisms of inhibition of human benign prostatic hyperplasia in vitro by the luteinizing hormone-releasing hormone antagonist cetrorelix, BJU Int. 106 (9) (2010) 1382–1388. [11] J. Chen, H. Song, Protective potential of epigallocatechin-3-gallate against benign prostatic hyperplasia in metabolic syndrome rats, Environ. Toxicol. Pharmacol. 45 (2016) 315–320. [12] P.L. Minciullo, A. Inferrera, M. Navarra, G. Calapai, C. Magno, S. Gangemi, Oxidative stress in benign prostatic hyperplasia: a systematic review, Urol. Int. 94 (3) (2015) 249–254. [13] T. Castelli, G.I. Russo, G. Reale, S. Privitera, M. Chisari, E. Fragal, V. Favilla, S. Cimino, G. Morgia, Metabolic syndrome and prostatic disease: potentially role of polyphenols in preventive strategies. A review, Int. Braz. J. Urol. 42 (3) (2016) 422–430. [14] M.M. Norström, E. Rådestad, B. Sundberg, J. Mattsson, L. Henningsohn, V. Levitsky, M. Uhlin, Progression of benign prostatic hyperplasia is associated with pro-inflammatory mediators and chronic activation of prostate-infiltrating lymphocytes, Oncotarget (2016), http://dx.doi.org/10.18632/oncotarget.8051. [15] M. Sarbishegi, M. Khani, S. Salimi, M. Valizadeh, F. Sargolzaei Aval, Antiproliferative and antioxidant effects of Withania coagulans extract on benign prostatic hyperplasia in rats, Nephrourol Mon. 8 (1) (2016) e33180. [16] N. Zhang, N. Ji, W.M. Jiang, Z.Y. Li, M. Wang, J.M. Wen, Y. Li, X. Chen, J.M. Chen, Hypoxia-induced autophagy promotes human prostate stromal cells survival and ER-stress, Biochem. Biophys. Res. Commun. 464 (4) (2015) 1107–1112. [17] K.U. Udensi, B.T. Paul, Oxidative stress in prostate hyperplasia and carcinogenesis, J. Exp. Clin. Cancer Res. 35 (1) (2016) 139. [18] S. Yadav, P. Gupta, M. Goyal, M. Bansal, Benign prostatic hyperplasia: current and future therapeutics, Urology 24 (2) (2013) 179–182. [19] N. Higashi, M. Kohjima, M. Fukushima, S. Ohta, K. Kotoh, M. Enjoji, N. Kobayashi, M. Nakamuta, Epigallocatechin-3-gallate, a green-tea polyphenol, suppresses rho signaling in TWNT-4 human hepatic stellate cells, J. Lab. Clin. Med. 145 (6) (2005) 316–322. [20] D.G. Nagle, D. Ferreira, Y.D. Zhou, Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives, Phytochemistry 67 (17) (2006) 1849–1855. [21] N. Khan, H. Mukhtar, Multitargeted therapy of cancer by green tea polyphenols, Cancer Lett. 269 (2008) 269–280. [22] D.S. Albrecht, E.A. Clubbs, M. Ferruzzi, J.A. Bomser, Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/ 2 activation, Chem. Biol. Interact. 171 (1) (2008) 89–95. [23] R. Yamauchi, K. Sasaki, K. Yoshida, Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549, Toxicol. in Vitro 23 (2009) 834–839. [24] B.N. Singh, S. Shankar, R.K. Srivastava, Green tea catechin, epigallocatechin-3gallate (EGCG): mechanisms, perspectives and clinical applications, Biochem. Pharmacol. 82 (12) (2011) 1807–1821. [25] F. Zhou, H. Zhou, T. Wang, Y. Mu, B. Wu, D.L. Guo, X.M. Zhang, Y. Wu, Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro, Acta Pharmacol. Sin. 33 (1) (2012) 120–126. [26] K.K. Ganguly, T. Sen, S. Pal, J. Biswas, A. Chatterjee, Studies on focal adhesion kinase in human breast cancer cell MDA-MB-231, Adv. Biol. Chem. 2 (2012) 29–42. [27] Y.J. Chae, C.H. Kim, T.S. Ha, J. Hescheler, H.Y. Ahn, A. Sachinidis, Epigallocatechin-3-O-gallate inhibits the angiotensin II-induced adhesion molecule expression in human umbilical vein endothelial cell via inhibition of MAPK pathways, Cell. Physiol. Biochem. 20 (2007) 859–866. [28] S. Hazgui, A. Bonnomet, B. Nawrocki-Raby, M. Milliot, C. Terryn, J. Cutrona, M. Polette, P. Birembaut, J.M. Zahm, Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells, Respir. Res. 9 (2008) 33. [29] C.M. Chan, J.H. Huang, H.S. Chiang, Wu WB, H.H. Lin, J.Y. Hong, C.F. Hung, Effects of (−)-epigallocatechin gallate on RPE cell migration and adhesion, Mol. Vis. 16 (2010) 586–595. [30] J. Zheng, B. Yang, T. Huang, Y. Yu, J. Yang, D. Li, Green tea and black tea consumption and prostate cancer risk: an exploratory meta-analysis of observational studies, Nutr. Cancer 63 (2011) 663–672. [31] S. Erdrich, K.S. Bishop, N. Karunasinghe, D.Y. Han, L.R. Ferguson, A pilot study to investigate if New Zealand men with prostate cancer benefit from a Mediterraneanstyle diet, PeerJ 3 (2015) e1080. [32] A. Modernelli, V. Naponelli, M. Giovanna Troglio, M. Bonacini, I. Ramazzina, S. Bettuzzi, F. Rizzi, EGCG antagonizes Bortezomib cytotoxicity in prostate cancer cells by an autophagic mechanism, Sci Rep 5 (2015) 15270. [33] N. Kurahashi, S. Sasazuki, M. Iwasaki, M. Inoue, S. Tsugane, J.S. Group, Green tea

activity, particularly FAK. Thus, we thought that loss of FAK signals and decrease in the level of Rho family proteins may lead to slow down prostate cell growth and prevent the contraction of smooth muscle and prostate enlargement. Consequently, EGCG leads to the inability of cells to proliferate and migrate via disrupting the cell morphology and its interactions with other cells through suppressing activation of FAK and other related proteins. Otherwise it is thought that EGCG may prevent the transformation of BPH cells since these cells are shown to be capable of acquire an invasive phenotype through ectopic expression of LIMK1 or interaction with carcinoma-associated fibroblasts [31,62]. On the other hand, it has been reported that oxidative stress leads to activation of FAK and Src and at the same time rapidly increase in the level of paxillin [38,63]. Furthermore, we found that paxilin levels were shown a rapid decrease at 10 μM EGCG treatment and this reduction level is unchanged dependent on the dose of EGCG (Fig.4). In this context, another possible mechanism is that EGCG may alter the level and/or signaling of focal adhesion proteins by reducing oxidative damage via antioxidant activity and may reduce prostate growth and BPH formation. As well, EGCG could disrupt the interaction between FAK and paxillin that is critical for the regulation of cell morphology and motility. Additionally, considering paxillin functions as a coactivator for androgen receptor [64], we thought that paxillin is more often related with proliferative signals of BPH-1 cells and contributes the inhibition of proliferation via androgens. Over the past few decades, EGCG have been recognized as a multifunctional bioactive molecule responsible for antitumorigenic, antiinflammatory, antioxidative, antiproliferative, antibacterial and antiviral effects. In addition to having these many functions, our study has provided new insights into how EGCG interfere with ECM interactions and cytoskeletal organization that can prevent the smooth muscle contraction and prostate enlargement. In this context, if we consider our antiproliferation based results at the molecular level obtained in current study, it is easily noticed that EGCG is of vital importance in the prevention or treatment of BPH. 5. Conclusions We once again showed that EGCG suppresses the proliferation of BPH-1 cells but also we found evidence that proliferation could be inhibited through the loss of FAK signals, disruption of cytoskeleton organization and ECM interactions. These results indicate that EGCG may play a potential role in the prevention and treatment of BPH. It is necessary to increase the number of in vivo studies in this area and achieve more detailed results. Conflict of interest The authours declared that they have no conflict of interest. Acknowledgements This research was supported with grants [TUBITAK 113S700] from the Turkish Scientific and Technological Research Council. References [1] O. Allkanjari, A. Vitalone, What do we know about phytotherapy of benign prostatic hyperplasia? Life Sci. 126 (2015) 42–56. [2] M.T. Sutton, M. Yingling, A. Vyas, H. Atiemo, A. Borkowski, S.C. Jacobs, N. Kyprianou, Finasteride targets prostate vascularity by inducing apoptosis and inhibiting cell adhesion of benign and malignant prostate cells, Prostate 66 (11) (2006) 1194–1202. [3] G. Penna, B. Fibbi, S. Amuchastegui, E. Corsiero, G. Laverny, E. Silvestrini, A. Chavalmane, A. Morelli, E. Sarchielli, G.B. Vannelli, M. Gacci, E. Colli, M. Maggi, L. Adorini, The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA/rho kinase and NF-kappaB pathways, Prostate 69 (5) (2009) 480–493.

80

Life Sciences 191 (2017) 74–81

B.E. Tepedelen et al.

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45] [46]

[47] [48]

[49]

106–117. [50] J.E. König, T. Senge, E.P. Allhoff, W. König, Analysis of the inflammatory network in benign prostate hyperplasia and prostate cancer, Prostate 58 (2) (2004) 121–129. [51] G.E. Steiner, U. Stix, A. Handisurva, M. Wilheim, A. Haitel, F. Reithmayr, D. Paikl, R.C. Ecker, K. Hrachowitz, G. Kramer, C. Lee, M. Marberger, Cytokine expression pattern in benign prostatic hyperplasia infiltrating T cells and impact of lymphocytic infiltration on cytokine mRNA profile in prostatic tissue, Lab. Investig. 83 (8) (2003) 1131–1146. [52] A. Handisurya, G.E. Steiner, U. Stix, R.C. Ecker, S. Pfaffeneder-Mantai, D. Langer, G. Kramer, N. Memaran-Dadgar, M. Marberger, Differential expression of interleukin-15, a pro-inflammatory cytokine and T-cell growth factor, and its receptor in human prostate, Prostate 49 (2001) 251–262. [53] D. Giri, M. Ittmann, Interleukin-8 is a paracrine inducer of fibroblast growth factor 2, a stromal and epithelial growth factor in benign prostatic hyperplasia, Am. J. Pathol. 159 (1) (2001) 139–147. [54] L. Wang, J.R. Yang, L.Y. Yang, Z.T. Liu, Chronic inflammation in benign prostatic hyperplasia: implications for therapy, Med. Hypotheses 70 (5) (2008) 1021–1023. [55] G. Robert, A. Descazeaud, N. Nicolaiew, S. Terry, N. Sirab, F. Vacherot, P. Maille, Y. Allory, A. de la Taille, Inflammation in benign prostatic hyperplasia: a 282 patients' immunohistochemical analysis, Prostate 69 (16) (2009) 1774–1780. [56] E.H. Kim, J.A. Larson, G.L. Andriole, Management of benign prostatic hyperplasia, Annu. Rev. Med. 67 (2016) 137–151. [57] Y.J. Chae, C.H. Kim, T.S. Ha, J. Hescheler, H.Y. Ahn, A. Sachinidis, Epigallocatechin-3-O-gallate inhibits the angiotensin II-induced adhesion molecule expression in human umbilical vein endothelial cell via inhibition of MAPK pathways, Cell. Physiol. Biochem. 20 (2007) 859–866. [58] S. Hazgui, A. Bonnomet, B. Nawrocki-Raby, M. Milliot, C. Terryn, J. Cutrona, M. Polette, P. Birembaut, J.M. Zahm, Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells, Respir. Res. 9 (2008) 33. [59] C.M. Chan, J.H. Huang, H.S. Chiang, Wu WB, H.H. Lin, J.Y. Hong, C.F. Hung, Effects of (−)-epigallocatechin gallate on RPE cell migration and adhesion, Mol. Vis. 16 (2010) 586–595. [60] C.F. Hung, T.F. Huang, H.S. Chiang, W.B. Wu, (−)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms, J. Cell. Biochem. 96 (2005) 183–197. [61] A. Tsujimura, S. Fukuhara, T. Soda, K. Takezawa, H. Kiuchi, T. Takao, Y. Miyagawa, N. Nonomura, S. Adachi, Y. Tokita, T. Nomura, Histologic evaluation of human benign prostatic hyperplasia treated by dutasteride: a study by xenograft model with improved severe combined immunodeficient mice, Urology 85 (1) (2015) 274.e1–274.e8. [62] Yu HM, D.E. Frank, J. Zhang, X. You, W.G. Carter, B.S. Knudsen, Basal prostate epithelial cells stimulate the migration of prostate cancer cells, Mol. Carcinog. 41 (2) (2004) 85–89. [63] S. Basuroy, M. Dunagan, P. Sheth, A. Seth, R.K. Rao, Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers, Am. J. Physiol. Gastrointest. Liver Physiol. 299 (1) (2010) G186–95. [64] M. Kasai, J. Guerrero-Santoro, R. Friedman, E.S. Leman, R.H. Getzenberg, D.B. DeFranco, The group 3 LIM domain protein paxillin potentiates androgen receptor transactivation in prostate cancer cell lines, Cancer Res. 63 (16) (2003) 4927–4935.

consumption and prostate cancer risk in Japanese men: a prospective study, Am. J. Epidemiol. 167 (2008) 71–77. D.W. Eom, J.H. Lee, Y.J. Kim, G.S. Hwang, S.N. Kim, J.H. Kwak, G.J. Cheon, K.H. Kim, H.J. Jang, J. Ham, K.S. Kang, N. Yamabe, Synergistic effect of curcumin on epigallocatechin gallate-induced anticancer action in PC3 prostate cancer cells, BMB Rep. 48 (8) (2015) 461–466. D.S. Albrecht, E.A. Clubbs, M. Ferruzzi, J.A. Bomser, Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/ 2 activation, Chem. Biol. Interact. 171 (1) (2008) 89–95. E.C. Stuart, M.J. Scandlyn, R.J. Rosengren, Role of epigallocatechin gallate (EGCG) in the treatment of breast and prostate cancer, Life Sci. 79 (2006) 2329–2336. C.A. Garces, J. Chang, R.I. Wilson, E.V. Kurenova, V.M. Golubovskaya, W.G. Cance, Focal adhesion kinase inhibitory peptides induce apoptosis in human breast cancer cells, Ann. Surg. Oncol. 11 (2) (2004) S68–S69. L.C. Chen, Tu SH, C.S. Huang, C.S. Chen, C.T. Ho, H.W. Lin, C.H. Lee, H.W. Chang, C.H. Chang, Wu CH, W.S. Lee, Y.S. Ho, Human breast cancer cell metastasis is attenuated by lysyl oxidase inhibitors through down-regulation of focal adhesion kinase and the paxillin-signaling pathway, Breast Cancer Res. Treat. 134 (3) (2012) 989–1004. J.D. Rovin, H.F. Frierson Jr., W. Ledinh, J.T. Parsons, R.B. Adams, Expression of focal adhesion kinase in normal and pathologic human prostate tissues, Prostate 53 (2) (2002) 124–132. T. Kunit, C. Gratzke, A. Schreiber, F. Strittmatter, R. Waidelich, B. Rutz, W. Loidl, K.E. Andersson, C.G. Stief, M. Hennenberg, Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate, Am. J. Physiol. Ren. Physiol. 307 (7) (2014) F823–32. A. Scibelli, D. d'Angelo, A. Pelagalli, S. Tafuri, L. Avallone, R. Della Morte, N. Staiano, Expression levels of the focal adhesion-associated proteins paxillin and p130CAS in canine and feline mammary tumors, Vet. Res. 34 (2) (2003) 193–202. Z. Meng, R. Cao, Z. Yang, T. Liu, Y. Wang, X. Wang, Inhibitor of 5-lipoxygenase, zileuton, suppresses prostate cancer metastasis by upregulating E-cadherin and paxillin, Urology 82 (6) (2013) 1452.e7–1452.e14. E.M. Posadas, H. Al-Ahmadie, V.L. Robinson, R. Jagadeeswaran, K. Otto, K.E. Kasza, M. Tretiakov, J. Siddiqui, K.J. Pienta, W.M. Stadler, C. Rinker-Schaeffer, R. Salgia, FYN is overexpressed in human prostate cancer, BJU Int. 103 (2) (2009) 171–177. M. Davila, A.R. Frost, W.E. Grizzle, R. Chakrabarti, LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer, J. Biol. Chem. 278 (38) (2003) 36868–36875. C.E. Turner, Paxilin and focal adhesion signalling, Nat. Cell Biol. 2 (12) (2000) E231–6. G.E. Steiner, M.E. Newman, D. Paikl, U. Stix, N. Memaran-Dagda, C. Lee, M.J. Marberger, Expression and function of pro-inflammatory interleukin IL-17 and IL-17 receptor in normal, benign hyperplastic, and malignant prostate, Prostate 56 (3) (2002) 171–182. M.J. Ribal, The link between benign prostatic hyperplasia and inflammation, Eur. Urol. Suppl. 12 (2013) 103109. G. Steiner, A. Gessl, G. Kramer, A. Schöllhammer, O. Förster, M. Marberger, Phenotype and function of peripheral and prostatic lymphocytes in patients with benign prostatic hyperplasia, J. Urol. 151 (1994) 480–484. C. De Nunzio, G. Kramer, M. Marberger, R. Montironi, W. Nelson, F. Schröder, A. Sciarra, A. Tubaro, The controversial relationship between benign prostatic hyperplasia and prostate cancer: the role of inflammation, Eur. Urol. 60 (1) (2011)

81