Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10990
RESEARCH ARTICLE
Modulation of inflammatory and hormonal parameters in response to testosterone therapy: Effects on the ventral prostate of adult rats Leonardo O. Mendes 1*, Anthony C. S. Castilho1, Cristiane F. Pinho2, Bianca F. GonSc alvez2, Eduardo M. Razza3, Luiz Gustavo A. Chuffa4, Janete A. Anselmo-Franci5, Wellerson R. Scarano2 and Francisco E. Martinez4 1 Graduate Program in Animal Science, University of Western S~ ao Paulo, Campus II, RodoviaRaposo Tavares, Km 572, BairroLimoeiro CEP 19067–175, Presidente Prudente, S~ ao Paulo, Brazil 2 Department of Morphology, Institute of Biosciences, S~ ao Paulo State University (Botucatu campus), Botucatu, S~ao Paulo, Brazil 3 Department of Pharmacology, Institute of Biosciences, S~ ao Paulo State University (Botucatu campus), Botucatu, S~ ao Paulo, Brazil 4 Department of Anatomy, Institute of Biosciences, S~ ao Paulo State University (Botucatu campus), Botucatu, S~ ao Paulo, Brazil 5 Department of Morphology, Stomatology and Physiology, S~ ao Paulo University (Ribeir~ aoPreto campus), Ribeir~ ao Preto, S~ ao Paulo, Brazil
Abstract Testosterone is often recommended in the treatment of several aging-related conditions. However, there are still questions about the consequences of this therapy in terms of hormonal and inflammatory parameters that are crucial for prostate homeostasis. Thus, we investigate if the testosterone therapy (TT) modulates the hormone receptors and inflammatory cytokines in the ventral prostate of adult rats. Wistar rats aging 150 days were divided into two experimental groups (n ¼ 10/ group): T: received subcutaneous injections of testosterone cypionate (5 mg/kg body weight) diluted in corn oil every other day for 4 weeks; and C: received corn oil as vehicle. Animals were euthanized at 180 days old by decapitation. Blood was collected to obtain hormone and cytokines concentrations. The ventral prostate was dissected and processed for light microscope and molecular analyses. Relative ventral prostate weight and epithelial compartment were increased after TT. The number of intact and degranulated mast cells was reduced in the T group. Plasma testosterone, DHT and intraprostatic testosterone concentrations were higher in the T group. TT leads to an increase in cell proliferation and up-regulation of AR, ERb, PAR-4, and NRF2. Importantly, plasma concentration and tissue expression of IL-10 and TNF-a were higher after TT. In summary, these results indicate that TT can regulate inflammatory response, with impacts in cytokines and mast cell population, and modulates steroids receptors, important parameters for prostatic homeostasis. Keywords: androgen therapy; androgen receptor; estrogen receptors; interleukin-10; mast cells
Introduction Androgens are steroid hormones characterized by complex physiological actions in several target tissues such as bones, muscles, bone marrow, central nervous system, and sexual function (Mooradian et al., 1987). The action of androgenic hormones in male reproductive organs is measured by their ability to induce the differentiation and maturation of these organs, with a distinct action on the prostate, a hormone
sensitive or hormone-dependent organ (Scarano et al., 2006). Within the prostatic microenvironment, specific cell types respond differently to androgens (Isaacs, 1999). The epithelial compartment consists of two main cell types: a basal layer of androgen-independent cuboidal cells and androgen-dependent columnar secretory cells (luminal cells). Testosterone can diffuse passively or actively across the luminal cells remaining in the cytoplasm as free
Corresponding author: e-mail:
[email protected] Abbreviations: AR, androgen receptor; BSA, bovine serum albumin; DACH-1, dachshund family transcription factor 1; DHT, dihydrotestosterone; E2, 17b-estradiol; ER-a, estrogen receptor a; ERb, estrogen receptor b; HE, hematoxylin-eosin; IHC, immunohistochemistry; IL-6, interleukin 6; IL-10, interleukin 10; NRF2, nuclear factor erythroid-related factor 2; PAR-4, prostate apoptosis response four protein; RT, room temperature; TGF-b1, transforming growth factor-b1; TNF-a, tumor necrosis factor-a; TNFR-1, tumor necrosis factor receptor-1; TNFR-2, tumor necrosis factor receptor-2; VP, ventral prostate
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testosterone or being converted to dihydrotestosterone (DHT) by the 5a-reductase (Rosner et al., 1999). After binding to the androgen receptor (AR), these hormones are implicated in the control of prostatic homeostasis, but also exert influence in the pathological processes related to aging, including benign prostatic hyperplasia and cancer (Coffey and Pienta, 1987). Their action extends to the modulation of the immune system, playing an important role in the inflammatory responses (Cutolo et al., 2002; Chen and Parker, 2004; Page et al., 2006). Testosterone has become one of the most widely used medications worldwide. The increased life expectancy of the world population has also increased the incidence of pathologies related to the aging process; this can be associated with low testosterone levels, such as those found in diabetes/metabolic syndrome, cardiovascular diseases, and osteoporosis (Khera, 2015). In the last years, the use of testosterone therapy (TT) has been proposed to relieve the symptoms related to such pathologies (Amory et al., 2004; Wang et al., 2004; Yassin and Saad, 2007; Reyes-Vallejo et al., 2007). However, both physicians and part of the scientific community are still skeptical about the relative safety of TT, especially concerned with its possible association with the induction or establishment of the prostate cancer (Gooren et al., 2007). In the past decade, researchers have demonstrated the lack of scientific support indicating testosterone as being able to induce or even stimulate the growth of prostate cancer by recruiting preexisting malignant lesions (Isbarn et al., 2009; Drewa and Chlosta, 2010). In addition, Morgentaler and Schulman (2009) raised some questions of whether it would be reasonable to avoid TT due unproven evidence of its association with greater prostate cancer risk, despite its known beneficial effects in hypogonadal men. In view of the issues concerning the safety of TT, this study aims to clarify the effects of TT on the healthy prostate microenvironment, focusing on sex hormone-receptor status and inflammatory response, important parameters for the gland homeostasis. Material and methods
Animal and experimental design Twenty adult Wistar male rats were obtained from the Central Animal Facility of Bioscience Institute of Botucatu, UNESP—Univ Estadual Paulista and maintained at the Department of Anatomy. Rats aging 150 days were divided into two experimental groups: T group (n ¼ 10) receiving subcutaneous injections of testosterone cypionate (Deposteron1, 5 mg/kg body weight) diluted in corn oil, every other day over 4 weeks at the same time (8:00–8:30 am) (Sattolo et al., 2004; Scarano et al., 2006) and C group (n ¼ 10) 2
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receiving corn oil only. All rats were housed in polypropylene cages with laboratory-grade pine shavings as bedding, and were maintained under a controlled temperature (23 1 C) and day/night cycle (12/12 h), and provided filtered tap water and rat chow ad libitum. Experimental protocols followed the ethical principles in animal research of the Brazilian College of Animal Experimentation (208CEEA).
Body and prostate weights The rats were weighed at the beginning and end of the treatment to obtain the body weight gain. At the end of treatment, the ventral prostate was dissected and weighed using an analytical balance (Owa Labor, Oschatz, Germany).
Sex hormone assay Plasma testosterone, dihydrotestosterone (DHT) and 17bestradiol (E2) At 180 days old, rats were euthanized in a CO2 chamber followed by decapitation. Blood samples were collected from the trunks of decapitated rats into heparinized tubes at the time of death (between 9:00 and 11:30 am). Then, plasma was obtained by centrifugation at 1,200g for 15 min at 4 C and stored at 20 C until it was assayed. Testosterone and DHT levels were determined by a double-antibody radioimmunoassay using Coat-A-Count1 (Diagnostics Products Corporation, Los Angeles, USA). All samples were dosed in the same assay to avoid inter-assay variations. The intra-assay variation was 1.75%, and the results were in ng/mL. 17b-estradiol levels were assayed by chemiluminescence (Elecsys Kit—Roche1, Basileia, Swiss; Estradiol E2 II, test sensitivity: 5 pg/mL, linearity: 4.300 pg/mL). The assays were performed at the Neuroendocrinology Laboratory, Dental School of Ribeir~ao Preto, University of S~ao PauloUSP. Intraprostatic testosterone After euthanasia, the ventral prostate was rapidly removed, and tissue samples of 150 mg were immediately frozen in liquid nitrogen and stored at 80 C. The tissue fragments were homogenized (9500 rpm) with phosphate buffer saline (PBS) for 2 min in “tube A,” and diethyl ether was added. The tissues were then placed on the vortex for 2 min. The homogenate was incubated at room temperature and the liquid phase transferred into “tube B.” The precipitate from “tube A” was resuspended with diethyl ether, homogenized, and incubated in dry ice, and the liquid phase transferred to “tube B.” “Tube B” was left in the fume hood overnight to evaporate all of the volatile components. The next day, PBS was added, the specimen was aliquoted and the testosterone levels were determined by double antibody Cell Biol Int 9999 (2018) 1–12 © 2018 International Federation for Cell Biology
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radioimmunoassay using Coat-A-Count1 (Diagnostics Products Corporation, Los Angeles, CA, USA). The protocol was according Mendes et al. (2015) and the assays were performed at the Neuroendocrinology Laboratory, Dental School of Ribeir~ao Preto, University of S~ao Paulo—USP. Plasma cytokines Plasma levels of interleukin (IL)-6, IL-10, tumor necrosis factor-a (TNF-a), and transforming growth factor-b1(TGFb1) were measured by specific enzyme-linked immunoassay (ELISA) using Quantikine1 kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Plasma cytokines levels were expressed as absolute concentrations (pg/mL). Specifically for TGF-b1 measurement, the samples were activated before the assay. Sample activation basically comprised biochemical steps (acidification followed by neutralization of the pH) in order to activate latent TGF-b1 to immune-reactive TGF-b1 detectable by the Quantikine1 TGF-b1 immunoassay, as recommended by the manufacturer. The minimum detection limits were 21 pg/mL (IL-6), 10 pg/mL (IL-10), 5 pg/mL (TNF-a), and 4.6 pg/mL (TGF-b1). The protocol was standardized according to Mendes et al. (2014).
Light microscopy Histological analysis At 180 days of age, rats were euthanized by decapitation, and their ventral prostate was removed and weighed. The ventral prostate was chosen as the study model by the fact that, in rodents, it is the lobe that first responds to morphological and inflammatory changes (Roy-Burman et al., 2004; Scarano et al., 2009). The fragments of the intermediate segment of the ventral prostate were rapidly fixed by immersion in methacarn (6 methanol: 3 chloroform: 1 acetic acid) for 2 h and kept in 70% ethanol. Next, the tissues were dehydrated in graded ethanol and were embedded in paraplast (Oxford Labware, St. Louis, MO, USA). The blocks were sectioned at 4-mm thicknesses and the slides were stained with hematoxylin–eosin (HE) for general studies and toluidine blue (1% v/v) to identify mast cells. The slides were analyzed and images captured using an Axiophot II (Zeiss-Jenaval, Jena, Germany) digital photomicroscope. Morphometric–stereological analysis Using an imaging analysis system (Image Pro Plus version 4.5 for Windows software), all of the slides stained by HE were evaluated. For morphometric-stereological method, images were randomly captured and studied using 50 histological fields per experimental group (n ¼ 5 ventral prostates/group). Five histological sections per ventral prostate were evaluated and 2 histological fields per section chosen randomly were photographed, resulting in 10 fields/ Cell Biol Int 9999 (2018) 1–12 © 2018 International Federation for Cell Biology
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animal. These sections were separated approximately 50 mm far one each other. Stereological analysis were performed using Weibel’s multipurpose test with 120 points and 60 test lines (Weibel, 1963) to compare the relative proportions among the prostate compartments (epithelium, stroma, and lumen) in the experimental groups according Brandt et al. (2014). Mast cells score Because the granules within mast cells contain heparin and sulfated glycosaminoglycan, they were stained metachromatically using toluidine blue 1% to evaluate and quantify the intact and degranulated mast cells. These dynamics of mast cells were achieved by the absence or presence of metachromatic granules outside the mast cells in the surrounding connective tissue, as described by Moron et al. (2000) and Keith et al. (2001). The number of intact and degranulated mast cells was measured in five animals of each experimental group and the values were given in cell/mm2. Random images of 50 histological fields per experimental group (10 fields/rat as described in morphometric–stereological analysis) of were captured by digital photomicroscope Axiophot II—Zeiss using 40 objective, with each field covering an area of 0.085 mm2, according to Mendes et al. (2014). Immunohistochemistry (IHC) The primary antibodies used for IHC were anti-ki67 (ab16667, Abcam, 1:200) and anti-e-cadherin (sc-7870, Santa Cruz Biotechnology, Dallas, TX, USA, 1:200). After the removal of paraffin and rehydration of the ventral prostate sections, antigens were retrieved at high temperature (100 C) for 50 min. Endogenous peroxidase was quenched with 3% H2O2 diluted in 50% methanol for 30 min, and nonspecific proteins were blocked by the incubation of the slides in bovine serum albumin (BSA) diluted to 3% in PBS plus 0.1% NP-40. The primary antibody was diluted in 1% BSA in PBS plus 0.1% NP-40, and the slides were incubated overnight at 4 C. For the immunoperoxidase assay, the slides were rinsed in PBS, incubated with biotinylated secondary antibodies followed by a VECTASTIN ABC Kit (Vector Laboratories Ltd.) and visualized with diaminobenzidine. The protocol used was standardized by Mendes et al. (2015). The sections were counter stained with Harris hematoxylin. Negative controls were obtained by omitting the primary antibody-incubation step. Proliferation index determination The number of Ki67 positive epithelial cells were counted in 50 random fields at 40 magnification using five different ventral prostate histological samples (10 fields/ventral prostate according methodology used in morphometric– stereological analysis and mast cell score) in each 3
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experimental group and expressed as a percentage of the total cells counted. All image acquisition and quantitative measurements were performed by an investigator blinded to both the animal identity and experimental conditions.
Western blotting analyses and protein quantification The ventral prostate of the experimental animals (n ¼ 5 samples/group) was rapidly removed and stored at 80 C. The samples were mechanically homogenized with RIPA lysis buffer (Millipore, CA, USA), 10 (0.5 M Tris–HCl, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, pH 7.4) and protease inhibitor cocktail (Sigma Chemical Co., St Louis, MO, USA), using a homogenizer (IKA1 T10 basic Ultra, Staufen, Germany). Aliquots containing 1:10 (v/v) of Triton-X-100 were added to homogenates, and samples were placed on dry ice under agitation for 2 h to improving extraction. These suspensions were centrifuged at 21,000g for 20 min at 4 C and were measured by the Bradford method. Total proteins were dissolved in 2 sample buffer as previously described by Laemmli and were used for SDS–PAGE (Bio-Rad Laboratories, Hercules, CA, USA). The same amount of protein (100 mg) of each sample was loaded per well onto preformed gradient gels, 4–15% (Bio-Rad Laboratories), and electrophoresis/transfer was performed according to Chuffa et al. (2016). Subsequently, the membranes were blocked with a TBS-T solution containing 5% BSA or 5% non-fat dry milk at room temperature (RT) for 1 h and were then incubated at 4 C overnight with the following primary antibodies: antiandrogen receptor (AR, sc-816, Santa Cruz Biotechnology, 1:300), anti-estrogen receptor alpha (ERa, Ab37438, Abcam, Cambridge, MA, USA, 1:300), anti-estrogen receptor beta (ERb, sc-8974, Santa Cruz Biotechnology, 1:1000), dachshund family transcription factor 1 (DACH-1, 10914- AP, Proteintech, Chicago, IL, USA, 1:500), prostate apoptosis response 4 protein (PAR4, sc-1666, Santa Cruz
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Biotechnology, 1:200) IL-6 (Ab1692, Abcam, 1:500), IL-10 (Ab34843, Abcam, 1:1000), TNF-a (Ab1793, Abcam, 1:500), TGF-b1 (Ab9758, Abcam, 1:500), nuclear factor erythroidrelated factor 2 (NRF2, sc-722, Santa Cruz Biotechnology, 1:1000). This was followed by washing six times for 5 min with TBS-T solution and then incubating for 2 h at RT with rabbit or mouse HRP-conjugated secondary antibodies (diluted between 1:5,000 and 1:20,000 in 1% BSA or 1% nonfat dry milk; Abcam). Bands were detected using a chemiluminescence substrate kit (Pierce ECL Western Blotting Substrate—GE Healthcare1, Pittsburgh, PA, USA), according to the manufacturer’s instructions. The substrates were removed from the membranes, and ECL signals were captured by CCD camera (G:BOXChemi, Syngen1, Sacramento, CA, USA). The integrated optical density (IOD) of the bands of target proteins was measured using the ImageJ software downloaded from the NIH website (http://rsb.info.nih.gov.ij/) to compare proteins levels. b-actin was used as an endogenous control.
Statistical analysis All statistical analyses were carried out using GraphPad Prism software (version 5.0). Mann–Whitney test was used to examine the significance of any difference between groups. The results were presented as the mean SEM. Differences were considered statistically significant when the P-value was 0.05. Results
Testosterone therapy stimulates epithelial development while reducing the activation of mast cells in the prostate stroma While no differences were observed in the body weight gain, there was an increase in absolute and relative prostate weights of animals that were submitted to TT (Figure 1).
Figure 1 Body weight gain (a) and relative ventral prostate weight (b) of Wistar rats submitted or not to testosterone therapy. P 0.05. All results are expressed as the mean SEM. Mann–Whitney test.
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The prostate morphology of the C group showed acini with simple cubic epithelium and polarized nuclei in the basal region of the cells (Figures 2a and 2b); conversely, animals of T group presented prostate epithelium varying from simple high cubic to cylindrical epithelium (Figures 2d and 2e), without histopathological changes that could indicate the appearance of future pre-neoplasic lesions. Reinforcing this data, stereological analysis indicated that the epithelial compartment was increased, and the luminal compartment was decreased in the prostate of treated animals (Table 1). We did not observe any morphological or structural differences in the stromal compartment (Figures 2d and 2e; Table 1). Importantly, the number of intact and degranulated mast cells in stromal compartment was decreased after TT (Figures 2c, f–h).
Testosterone and DHT increased together with IL-10 and TNF-a following TT We observed an increase in the plasma levels of testosterone (Figure 3a) and DHT (Figure 3d) after TT, which was accompanied by increased levels of intraprostatic testosterone as well (Figure 3b). Conversely, the levels of 17bestradiol were unchanged after TT (Figure 3c). As to the inflammatory response, we observed a distinct pattern of cytokine secretion following the treatment. While plasma IL-10 (Figure 3f) and TNF-a (Figure 3g) were augmented after TT, the levels of IL-6 (Figure 3e) and TGFb1 (Figure 3h) remained unchanged.
Testosterone induces high expression of sex steroid receptors and differentially regulates the expression of pro- and anti-inflammatory cytokines in the prostate Testosterone therapy increased the proliferation index in the prostate acini (Figure 4a) and the expression of PAR4 was also higher in the T group than the control (Figures 4b and 4c). The cell–cell adhesion does not appear to be modified by testosterone action, since the T group showed a homogeneity e-cadherin immunostaining (Figures 4f and 4g). Some areas in the C group presented weak immunoreactivity in the epithelial cells (Figures 4d and 4e). There was increased expression of prostate AR (Figures 5a and 5b), ERb (Figures 5e and 5f) and DACH1 (Figures 5g and 5h) after TT, while the expression of ERa showed the same pattern in both groups (Figures 5c and 5d). Regarding the cytokines panel, similar to plasma concentrations, we observed an increased expression of IL-10 (Figures 5k and 5l) and TNF-a (Figures 5o and 5p) in the prostate of T group, but the expression of IL-6 (Figures 5i and 5j) and TGF-b1 (Figures 5m and 5n) was unchanged. In addition, TT was responsible for increasing NRF2 expression (Figures 5q and 5r). Cell Biol Int 9999 (2018) 1–12 © 2018 International Federation for Cell Biology
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Discussion The safe use of testosterone as a treatment for men with hormonal deficiency has been a matter of debate (Brand et al., 2007; Schwartz and Woloshin, 2013), despite the risk of developing prostatic cancer (Warburton et al., 2015). However, testosterone alone is unable to induce malignant prostatic lesions (Bosland, 2014), thus requiring the action of other hormones, such as E2, the most potent estrogen in men (Nicholson and Ricke, 2011), that remained unchanged after TT in our study. Increased plasma testosterone and DHT followed the higher expression of AR in the T group. The androgen signaling pathway involves the expression of several proteins that regulate prostatic growth (Bennett et al., 2010), which might explain the higher proliferation index observed after TT. The growth and maintenance of the prostatic microenvironment relies on a sensitive balance between proliferation and cell death. Any disturbance in the molecular mechanisms that control this homeostasis can induce the appearance of prostatic pathologies (Xie et al., 2000). The increased number of proliferating cells in the T group was compensated by the greater expression of PAR4, a strong apoptotic activator is poorly expressed in a variety of cancers, including prostate tumors (Gurumurthy and Rangnekar, 2004). The close relationship between AR and testosterone supplementation can be explained by the prostate saturation theory. The AR activation increases until reaches a threshold and, when all receptors are saturated, the testosterone increasing will not have an effect (Warburton et al., 2015). Support for this theory comes from studies such as those by Khera et al. (2011) and Kang and Li (2015) where hypogonadal men did not show difference in PSA levels after testosterone therapy. In addition to being a potent natural ligand for AR, DHT is also a precursor of the hormone 3b,17b-diol (3b-diol), the most abundant estrogenic hormone in the prostatic environment with strong affinity for ERb (Dey et al., 2013), that could explain its up-regulation after TT. On the other hand, the non-activation of ERa corroborates with data from the literature that E2 is the major ligand of this receptor (Prins and Korach, 2008), since E2 plasma levels remained unchanged in our study. Recent data presented by Vignozzi et al. (2012) has associated the increased inflammatory process in elderly men with low testosterone levels, suggesting that DHT plays a central role in the immune regulation of prostatic epithelial cells. IL-10 and NRF2 up-regulation and decreased number of mast cells were found in the present study and corroborate the anti-inflammatory role of androgens (Yatkin et al., 2009), probably via ERb (Wu et al., 2017), due to its apoptotic and anti-inflammatory roles, besides reduces oxidative stress, as reviewed by Christoforou et al. (2014). 5
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Figure 2 Histological sections and mast cells quantification of Wistar rats submitted or not to testosterone therapy (a–h). General features of distal segment of the gland. C group (a–c) shows simple cuboidal epithelium unlike the T group, where the epithelium was higher (d–f). A decreased number of intact (thick arrow) and degranulated (thin arrow) mast cells was observed in the T group (g and h). bc, basal cells; ep, epithelium; lc, luminal cells; lu, lumen; st, stroma; bv, blood vessels. Bars: 50 mm. P 0.05. All results are expressed as the mean SEM. Mann–Whitney test.
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Table 1 Stereological analysis (%) of ventral prostate of Wistar rats submitted or not to TT Parameters
C
T
Epithelium Lumen Stroma
21.61 (13.83–28.57) 53.69 (42.41–65.35) 24.65 (17.41–29.76)
29.33 (26.19–32.58) 41.16 (29.76–53.86) 29.51 (19.64–39.88)
Values represent median (Q1–Q3) (n ¼ 5). P 0.05 versus C group. Mann–Whitney test.
IL-10, an anti-inflammatory cytokine, is characterized by inhibiting the production of proinflammatory cytokines (Hamidullah et al., 2012). A shift in cytokines balance was observed in hypogonadal men submitted to androgen replacement, with increase of IL-10 e reduction of total
cholesterol, improving the inflammatory status (Malkin et al., 2004). Regarding the mast cells, they have recently been postulated as an important marker for the prognosis of prostate cancer and a possible target for immune therapies (Johansson et al., 2010; Taverna et al., 2013). Despite the scarce data evaluating the influence of testosterone on mast cells, Felix-Patrıcio et al. (2017) showed that castration induced an increase of 528% in mast cell number in rat ventral prostate, which was knocked down after testosterone replacement. Associated with inflammation, oxidative stress in an essential factor for the initiation of prostate cancer (Gupta-Elera et al., 2012). The NRF2 increased expression following TT is related to improved conditions of oxidative stress through reduced production of reactive oxygen species (Cheung et al., 2014). In prostate tumor cells, NRF2 upregulation is able to inhibit proliferation and invasion (Liu
Figure 3 Hormonal and cytokines assays of Wistar rats submitted or not to testosterone therapy. Plasma testosterone (a), intraprostatic testosterone (b), plasma 17b-estradiol (c), plasma DHT (d), plasma IL-6 (e), plasma IL-10 (f), plasma TNF-a (g), plasma TGF-b1 (h). P 0.05. All results are expressed as the mean SEM. Mann–Whitney test.
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Figure 4 Proliferation index (a) and Western blotting analysis of PAR4 (b) in the ventral prostate of Wistar rats submitted or not to testosterone therapy. Densitometry values for PAR4 levels (c) were studied following normalization to the housekeeping protein (b-actin). E-cadherin immunoreactivity of C (d and e) and T (f and g) groups. E-cadherin immunostaining in the T group was homogenous (f and g) and in the with C group some areas showed weak immunostaining (arrow). P 0.05. All results are expressed as the mean SEM. Mann–Whitney test.
et al., 2015; Li et al., 2016), and becomes a new promising target for treating prostate cancer (Xue et al., 2016). TNF-a is a cytokine known for its paradoxical role in prostate cancer (Tse et al., 2012). By its ability to bind to two different receptors, termed TNFR-1 and TNFR-2, the TNFa activates different signaling pathways that can either trigger inflammation, apoptosis or increase cell proliferation and survival (Balkwill, 2009). Chopra et al. (2004) reported the TNF-a is able to mediate apoptosis in human prostate epithelial cells and the mechanism behind this process probably involves PAR4, that is cleaved by caspase 8 (Treude et al., 2014), corroborating with the PAR4 up-regulation after TT. 8
DACH-1, a tumor suppression protein (Wu et al., 2009) and e-cadherin, a cell–cell adhesion molecule (GonSc alves et al., 2013), are important markers of prostatic homeostasis. The up-regulation of DACH-1 in our study was a positive response of TT since Chen et al. (2015) reported the arrest of prostate tumor growth and migration and inhibition of inflammatory cytokines as role of DACH-1. Similarly, e-cadherin is inversely correlated with tumor aggressiveness (reviewed by Jeanes et al., 2008). Areas with loss of e-cadherin immunostaining and irregular distribution were observed in gerbils with prostate cancer chemicallyinduced (GonSc alves et al., 2013), showing an inverse patterning when compared with animals submitted to TT. Cell Biol Int 9999 (2018) 1–12 © 2018 International Federation for Cell Biology
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Figure 5 Western blotting analysis of AR (a), ERa (c), ERb (e), DACH1 (g), IL-6 (i), IL-10 (k) TGF-b1 (m), TNF-a (o), NFR2 (q). Densitometry values for AR (b), ERa (d) and ERb (f), DACH1 (h), IL-6 (j), IL-10 (l), TGF-b1 (n), TNF-a (p), and NFR2 (r) were studied following normalization to the housekeeping protein (b-actin). P 0.05. All results are expressed as the mean SEM. Mann–Whitney test.
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Collectively, the results presented in our experimental study suggest that TT modulates inflammatory and hormonal parameters that are important to prostate homeostasis and are inversely correlated with prostate cancer. Epidemiological studies that consider a greater number of subjects and longer periods of treatment are still required in order to prove the effectiveness of TT. It should be emphasized that although the study has been conducted with healthy adult animals, this type of treatment should be recommended for patients with low levels of testosterone, particularly avoiding its use for non-medical purposes.
Acknowledgments and funding We would like to thank Ruither de Oliveira Gomes Carolino for the technical support in the hormonal analysis. This work was supported by a grant from the FundaSc ~ao de Amparo a Pesquisa do Estado de S~ao Paulo—FAPESP (Grants Numbers: 2009/54606-1, 2011/05936-9, 2012/00917-9) and CoordenaSc ~ao de AperfeiSc oamento de Pessoal de Ensino Superior—CAPES. Conflict of interest The authors declare that they have no conflict of interest. References Amory JK, Watts NB, Easley KA, Sutton PR, Anawalt BD, Matsumoto AM, Bremner WJ, Tenover JL (2004) Exogenous testosterone or testosterone with finasteride increases bone mineral density inolder men with low serum testosterone. J Clin Endocrinol Metab 89: 503–10. Balkwill F (2009) Tumour necrosis factor and cancer. Nat Rev Cancer 9: 361–71. Bennett NC, Gardiner RA, Hooper JD, Johnson DW, Gobe GC (2010) Molecular cell biology of androgen receptor signalling. Int J Biochem Cell Biol 42: 813–27. Bosland MC (2014) Testosterone treatment is a potent tumor promoter for the rat prostate. Endocrinol 155: 4629–33. Brand TC, Canby-Hagino E, Thompson IM (2007) Testosterone replacement therapy and prostate cancer: A word of caution. Curr Urol Rep 8: 185–9. Brandt JZ, Silveira LTR, Grassi TF, Anselmo-Franci JA, Favaro WJ, Felisbino SL, Scarano WR (2014) Indole-3-carbinol attenuates the deleterious gestational effects of bisphenol A exposure on the prostate gland of male F1 rats. Reprod Toxicol 43: 56–66. Chen CC, Parker CR, Jr (2004) Adrenal androgens and the immune system. Semin Reprod Med 22: 369–77. Chen K, Wu K, Jiao X, Wang L, Ju X, Wang M, Di Sante G, Xu S, Wang Q, Li K, Sun X, Xu C, Li Z, Casimiro MC, Ertel A, Addya S, McCue PA, Lisanti MP, Wang C, Davis RJ, Mardon G, Pestell RG (2015) The endogenous cell-fate factor dachshund restrains
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