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Keywords:

  • prostate;
  • senescence;
  • hormonal therapy;
  • growth factors;
  • angiogenesis

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The influence of senescence and hormone replacement on the onset of pathologic processes in the prostate is not yet fully understood. The aim was to identify the immunoreactivity and protein levels of molecules involved in cell proliferation, tissue remodeling and angiogenesis in the ventral prostate of elderly rodents following hormonal replacement. Male Sprague–Dawley rats were separated into one Young group (4-months old), treated with peanut oil (5 mL kg−1, s.c.), and six Senile groups. The senile rats (10-months old) were subdivided into: Senile group (SEN) (5 mL kg−1 peanut oil, s.c.); Testosterone group (TEST) (5 mg kg−1 testosterone cipionate, s.c.); Estrogen group (EST) (25 µg kg−1 17β-estradiol, s.c.); castrated group (CAS) (surgical castration); castrated-testosterone group (CT) (same treatment as CAS and TEST groups); and castrated-estrogen group (CE) (same treatment as CAS and EST groups). After 30 days, samples of the ventral prostate were harvested for analyses of insulin-like growth factor-1 receptor (IGFR-1), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and endostatin features. IGFR-1 and MMP-9 showed increased protein levels and epithelial immunolabeling both after hormonal replacement and castration. Increased VEGF levels and reduced endostatin were verified in the SEN group. Hormonal therapy and castration led to a higher increase of VEGF, especially in the EST, CAS, and CE groups. Endostatin increased mainly in the TEST and CT groups. Hormonal therapy in senescence generated a reactive microenvironment characterized by the increase of mitogenic and tissue remodeling factors and by the imbalance of angiogenesis, which possibly compromised organ function and predisposed toward glandular disorders. Anat Rec, 296:1758–1767, 2013. © 2013 Wiley Periodicals, Inc.

Senescence is a period of life characterized by hormonal imbalance, which negatively affects prostate structure and function, in both men and experimental animals (Srinivasan et al., 1995; Banerjee et al., 2000; Lau et al., 2003). Also, during senescence the organism accumulates senescent cells whose transcriptional program is altered, in such a way that growth factors and enzymes involved in tissue remodeling and angiogenic processes are upregulated (Krtolica and Campisi, 2002; Sprenger et al., 2008).

Elderly men present a partial androgen deficiency, showing an age-related decline in total and free testosterone plasma levels (Tenover, 1999; Algarté-Génin et al., 2004; Hellstrom et al., 2012). This hormone deficiency leads to late onset hypogonadism, which is associated to decreased libido, increased erectile dysfunction, loss of muscle mass and strength, osteoporosis, decreased cognitive ability and depression (Tenover, 1999; Algarté-Génin et al., 2004; Drewa and Chlosta, 2010; Hellstrom et al., 2012). Thus, testosterone supplementation emerged as a clinical alternative for the treatment of these multiple deregulations associated to the loss of these androgens, improving life quality and decreasing symptoms of hypogonadism in aging men (Morales, 2002; Algarté-Génin et al., 2004; Drewa and Chlosta, 2010). However, despite the increase of the male population which could receive this hormone treatment, there is a common concern that higher testosterone levels may also increase the risk of developing clinically significant prostate cancer from a pre-existing subclinical lesion, due to the well-known androgen dependence of prostatic diseases (Drewa and Chlosta, 2010; Hellstrom et al., 2012). Considering that prostate cancer is a disease with a long natural history and that the observation time, until now, following testosterone treatment in elderly men is limited, there is the necessity of additional long-term studies to evaluate this issue (Tenover, 1999; Hellstrom et al., 2012). On the other hand, some authors suggest that testosterone supplementation can contribute with a possible protective role against prostate cancer (Algarté-Génin et al., 2004; Hellstrom et al., 2012). These data indicate that the relationship between androgen replacement and prostatic diseases is still very controversial, especially taking into consideration the lack of studies concerning the influence of this treatment on molecules, which interfere in the prostatic microenvironment dynamics.

The prostate is an accessory sex gland, which is essential to the reproductive process and its secretion plays a fundamental role in spermatozoid capacitation and survival (Bull et al., 2001; Marker et al., 2003). Because of its well-known androgen dependence, the rodent ventral prostate is widely used in different investigations concerning prostatic biology. This interest is related to the pathological conditions affecting this organ, such as prostate cancer (Slayter et al., 1994). The incidence of this malignancy increases with age, representing the second most frequently detected cancer and the sixth leading cause of cancer death among men worldwide (Algarté-Génin et al., 2004; Jemal et al., 2011).

The bidirectional interaction between the epithelium and stroma plays a major role in the development and maintenance of prostatic structure and function. In addition, the imbalance of this interaction could lead to the occurrence of prostate cancer and other lesions in this organ (Cunha et al., 2002; Zhao et al., 2002; Chung et al., 2005; Reynolds and Kyprianou, 2006). In this context, several growth factors in the prostate microenvironment act as mediators of the epithelium-stroma crosstalk (Reynolds and Kyprianou, 2006).

Insulin-like growth factor-1 (IGF-1) acts as a mitogenic factor and also blocks the apoptotic pathway in several cell types (Djavan et al., 2001; Gennigens et al., 2006). IGF-1 is synthesized by the prostatic stroma and plays a paracrine action over the secretory epithelium by means of the IGFR-1 receptor (Djavan et al., 2001). High IGF-1 and IGFR-1 levels have been related to increased cell proliferation and reduced apoptosis, highlighting this molecule as a potential contributing factor for the development of benign prostatic hyperplasia (BPH) and prostate cancer (Pandini et al., 2005; Gennigens et al., 2006; Meinbach and Lokeshwar, 2006).

Matrix metalloproteinases (MMPs) are important enzymes involved in extracellular matrix degradation and also in the regulation of a wide range of cellular functions due to their capacity to release molecules, such as angiogenic and growth factors, from the cell surface (Lynch and Matrisian, 2002). Despite participating in tissue remodeling in physiological conditions, MMP overexpression has been related to several pathologic processes, including cancer (Tuxhorn et al., 2001; Lynch and Matrisian, 2002; London et al., 2003). MMPs 2 and 9 are directly involved in prostatic diseases, specifically degrading collagen IV and the basal membrane (Stearns and Stearns, 1996).

Angiogenesis is another important aspect of prostatic microenvironment dynamics and is defined as the development of new blood vessels from pre-existing vasculature, showing important roles in development, wound healing and tumorigenesis (Van Moorselaar and Voest, 2002; Shibuya and Claesson-Welsh, 2006). The angiogenic process is controlled by the relative balance between inducing factors and inhibitors (Van Moorselaar and Voest, 2002; Shibuya and Claesson-Welsh, 2006). Vascular endothelial growth factor (VEGF) is the most powerful mediator of angiogenesis and endothelial cell functions, stimulating the proliferation, differentiation and migration of these cells, as well as promoting the increase of vascular permeability (Van Moorselaar and Voest, 2002; Delongchamps et al., 2006). On the other hand, endostatin, a C-terminal portion of collagen XVIII, is an endogenous inhibitor of angiogenesis, which blocks endothelial cell migration and proliferation as well as inducing apoptosis in these cells, and also in the tumor microenvironment, characterizing this molecule as a powerful anti-tumoral agent (O'Reilly et al., 1997; Schmidt et al., 2005).

Thus, the aim of this study was to evaluate the influence of steroid hormone replacement during senescence on molecules involved in tissue remodeling and angiogenesis in the prostate, which may be associated to lesions in this organ.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Animals and Experimental Procedures

A total of 35 male rats (Sprague–Dawley) were used and divided into one young group and six senile groups (five animals per group). The young group (YNG), 4-months old, received a 5 mL kg−1 dose of peanut oil subcutaneously (s.c.). The rats in the senile groups (10-months old) were submitted to the following treatments: the Senile group (SEN): 5 mL kg−1 peanut oil (s.c.); the testosterone group (TEST): 5 mg kg−1 testosterone cipionate (Deposteron-Novaquímica, São Paulo, SP, Brazil) diluted in 5 mL peanut oil (s.c) (altered Franck-Lissbrant et al., 1998; altered Sáttolo et al., 2004); the Estrogen group (EST): 25 µg kg−1 17β-estradiol (Sigma Chemical, St. Louis, USA) diluted in 25 µL peanut oil (s.c.) (altered Prins and Birch, 1997; Montico et al., 2011); the castrated group (CAS): rats were anesthetized with a 0.25 mL/100 g body weight dose of Francotar/Virbaxyl (1:1, Vibra®, Roseira, SP, Brazil) and castrated by surgical incision; the Castrated–Testosterone group (CT): surgical castration was carried out and after 30 days the rats received the same treatment as the TEST group; the Castrated-Estrogen group (CE): surgical castration was carried out followed by the same treatment as the EST group after 30 days.

All rats received water and solid ration ad libitum (Nuvilab, Colombo, PR, Brazil). After 30 days of treatment every other day, the animals were weighed on a semi-analytical scale (Marte AS 5500) and euthanized. Samples from the prostatic ventral lobe were collected and processed for immunohistochemistry and Western Blotting analysis.

This study was approved by the institutional Committee for Ethics in Animal Research (University of Campinas—Unicamp, protocol no. 1748-1) and the experiments were carried out in agreement with the Ethical Principles for Animal Research established by the Brazilian College for Animal Experimentation (COBEA).

Immunohistochemistry

Samples of the ventral prostate were collected from all the animals in each group and fixed in Bouin's solution for 24 hr. After fixation, the tissues were washed in 70% ethanol, dehydrated in an increasing alcohol series, diaphanized in xylene for 2 hr and subsequently embedded in paraplast (Paraplast Plus, St. Louis, USA). The materials were then cut into 5-μm-thick sections using a microtome (Zeiss Hyrax M60) and the sections were deparaffinized in xylene, hydrated in a decreasing alcohol series and rinsed under distilled water. Antigens were retrieved by boiling the sections in 10 mM citrate buffer (pH 6.0) three times for 5 min in a microwave oven. After that, the sections were incubated in H2O2 (0.3% in methanol) for 15 min to block endogenous peroxidase. Nonspecific binding was blocked by incubating the sections in a blocking solution (3% BSA in TBS-T buffer) for 1 hr at room temperature. The IGFR-1, MMP-9, VEGF antigens and endostatin were detected, respectively, using the following antibodies: rabbit N-20 (sc-712) (Santa Cruz Biotechnology, CA), mouse Ab-3 (IM37) (Calbiochem, USA), mouse VG-1 (sc-53462) (Santa Cruz Biotechnology, CA) and mouse 4i37 (ab64569) (Abcam, USA). Primary antibodies were diluted in 1% BSA (1:50) and applied to the sections overnight at 4°C. The sections were washed for 15 min with TBS-T and secondary labeled polymer from the Envision HRP Kit (Dako Cytomation, Carpenteria, USA) was applied for 40 min at room temperature. After washing in TBS-T, peroxidase activity was detected using a diaminobenzidine (DAB) chromogen from Envision HRP Kit (Dako) for 10 min. Sections were lightly counterstained with Methyl Green or Harris Hematoxylin according to antigen distribution in the prostatic tissue. The sections were dehydrated in an increasing ethanol series and xylene, mounted in Entellan (Merck, Darmstadt, Germany) and photographed in the photomicroscope (Nikon Eclipse E-400). Sections from the ventral prostate that were not stained with primary antibody were used as negative controls. The intensity of antigen immunoreactivity in the epithelial and stromal compartments was graded as intense (+++), moderate (++), and weak (+), according to the antigens distribution in the sectioned tissue (altered Markopoulos et al., 2000).

Western Blotting

Ventral prostate samples from all the animals in each experimental group were collected and frozen. The samples were weighed and homogenized in a Polytron homogenizer (Kinematica, Lucerne, Switzerland) for 1 min in a 50 µL mg−1 lysis buffer. The tissue homogenates were centrifuged at 14,000 rpm for 20 min at 4°C and a sample of each extract was used for protein quantification with Bradford reagent (Bio-Rad Laboratories, Hercules, USA). The supernatants were mixed (1:1) with 3× sample buffer and transferred to a dry bath at 100°C for 5 min. Aliquots containing 75 μg of protein were separated by electrophoresis in SDS-polyacrylamide gels under reducing conditions. After electrophoresis, proteins were transferred to Hybond-ECL nitrocellulose membranes (Amersham, Pharmacia Biotech, Arlington Heights, USA) at 70 V for 3 hr. The membranes were blocked with 3% BSA in TBS-T for 60 min and incubated at 4°C overnight with the following primary antibodies: rabbit N-20 (sc-712) (Santa Cruz Biotechnology, CA) for IGFR-1, mouse Ab-3 (IM37) (Calbiochem, USA) for MMP-9, mouse VG-1 (sc-53462) (Santa Cruz Biotechnology, CA) for VEGF, mouse 4i37 (ab64569) (Abcam, USA) for endostatin, and mouse ACTBD11B7 (sc-81178) (Santa Cruz Biotechnology, CA) for β-actin diluted in 1% BSA (1:500). The membranes were then incubated for 2 hr with rabbit or mouse secondary HRP-conjugated antibody (Promega Corporation, USA) diluted 1:2500 in 1% BSA. After washing in TBS-T, peroxidase activity was detected by incubation with a diaminobenzidine (DAB) chromogen (Sigma Chemical Company, St Louis, USA) for 10 min. β-actin quantification was used as an endogenous control for comparison among groups. The intensity of antigen bands in each experimental group was determined by densitometry using the Nis-Elements: Advanced research (USA) software and was expressed as the mean ratio in relation to β-Actin band intensity.

Statistical Analysis

The comparative statistical analysis of IGFR-1, MMP-9, VEGF, and endostatin protein levels among the different experimental groups was carried out by analysis of variance (ANOVA) and Tukey multiple range test, with the level of significance set at 5% (Montgomery, 1991; Zar, 1999). The results were expressed as the mean ± standard deviation.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Immunohistochemistry and Western Blotting

The young group (YNG)

Weak IGFR-1, MMP-9, and VEGF immunoreactivities were verified in the epithelium as well as in the prostatic stroma (Fig. 1A–C; Table 1), showing 57.6, 46.5, and 39.3% protein levels, respectively, in relation to the β-Actin standard (Fig. 3). On the other hand, intense endostatin immunolabeling was seen in both the prostatic compartments, with 109.3% protein levels (Figs. 1D and 3; Table 1).

image

Figure 1. IGFR-1, MMP-9, VEGF and endostatin immunoreactivities in the ventral prostatic lobe of rats from YNG (A-D), SEN (E-H), TEST (I-L) and EST (M-P) groups. Epithelial (arrows) and stromal (asterisks) reactivities were graded as weak (+), moderate (++) and intense (+++), according to Table 1. Bars = 10 µm. Ep = epithelium; St = stroma; L = lumen.

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Table 1. Distribution of insulin-like growth factor receptor-1 (IGFR-1), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), and endostatin reactivities in the ventral prostate of the experimental groups
MoleculesGlandular compartmentsExperimental groups
YNGSENTESTESTCASCTCE
IGFR-1Epithelium++++++++++++++++
Stroma++++++++++++++
MMP-9Epithelium++++++++++++++
Stroma+++++++++
VEGFEpithelium+++++++++++++++++
Stroma++++++++++++
EndostatinEpithelium++++++++++++++++
Stroma+++++++++++++
The senile group (SEN)

IGFR-1 and MMP-9 showed weak epithelial and stromal immunoreactivities, and 56.6 and 44.0% protein levels respectively, which showed statistical similarity to those found in the YNG group (Figs. 1E,F and 3; Table 1). VEGF presented moderate immunolabeling in the epithelial cells and weak immunolabeling in the stromal compartment, with significant increase of protein concentrations (60.9%) in relation to the young animals (Figs. 1G and 3; Table 1). Decreased levels of endostatin (60.5%) were verified in comparison to the YNG group, with moderate epithelial and weak stromal reactivities for this molecule (Figs. 1H and 3; Table 1).

The testosterone group (TEST)

IGFR-1 immunoreactivity was intense in the luminal edge of the epithelium and moderate in the glandular stroma (Fig. 1I; Table 1). This molecule showed 138.2% protein levels, which were statistically higher than the values registered for the senile animals (Fig. 3). Moderate epithelial and weak stromal immunoreactivities were seen for MMP-9, presenting 78.2% protein levels, which were significantly higher when compared to the SEN group (Figs. 1J and 3; Table 1). Also, VEGF immunolocalization was intense in the luminal border of the epithelium and weak in the stroma, with statistically raised protein levels (107.1%) in comparison to senile rats (Figs. 1K and 3; Table 1). Similarly, endostatin levels (113.9%) were also increased in relation to the SEN group, whereas intense immunoreactivity was observed for this molecule in both the epithelium and stroma (Figs. 1L and 3; Table 1).

The estrogen group (EST)

Intense IGFR-1 reactivity in the luminal surface of the epithelium and moderate stromal reactivity were verified (Fig. 1M; Table 1). The protein levels (127.7%) were increased in comparison to the SEN group and showed no statistical difference in relation to the TEST group (Fig. 3). MMP-9 presented intense epithelial and weak stromal immunoreactivities, showing 119.1% protein levels, which were statistically higher than the values observed in the SEN group (Figs. 1N and 3; Table 1). Moreover, a significant increase of the VEGF protein levels (166.1%) was observed, demonstrating intense reactivity in the luminal border of the acini and moderate stromal labeling (Figs. 1O and 3; Table 1). In terms of endostatin, 83.3% protein levels were detected, which were higher than in the SEN group, but statistically lower when compared to the TEST group, characterizing intense epithelial immunolocalization and weak stromal reactivity (Figs. 1P and 3; Table 1).

The castrated group (CAS)

Moderate IGFR-1 and MMP-9 reactivities were verified in both glandular compartments (Fig. 2A,B; Table 1). Protein levels for these molecules were 90.1 and 132.9%, respectively, characterizing a significant increase compared to the SEN group (Fig. 3). VEGF showed intense immunolocalization in both the epithelium and stroma, with 242.0% protein levels, which were also greater than those verified in the SEN group (Figs. 2C and 3; Table 1). Weak immunolabeling was seen for endostatin in the epithelium and stroma, showing significant reduction of its protein levels when compared to the aged control animals (Figs. 2D and 3; Table 1).

image

Figure 2. IGFR-1, MMP-9, VEGF, and endostatin immunoreactivities in the ventral prostatic lobe of rats from CAS (A–D), CT (E–H), and CE (I–L) groups. Epithelial (arrows) and stromal (asterisks) reactivities were graded as weak (+), moderate (++) and intense (+++), according to Table 1. Bars = 10µm. Ep = epithelium; St = stroma; L = lumen.

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image

Figure 3. Western blotting and semiquantitative analysis of IGFR-1, MMP-9, VEGF, and endostatin protein levels in the ventral prostatic lobe of the different groups. Data is shown as the mean percentage ± standard deviation in relation to the endogenous control β-Actin.

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The castrated-testosterone group (CT)

IGFR-1 showed intense reactivities in both the epithelium and stroma and 204.2% protein levels, which were statistically higher in comparison to the TEST and CAS groups (Figs. 2E and 3; Table 1). MMP-9 immunolabeling was moderate in the epithelium and weak in the stroma, whereas VEGF presented moderate reactivity in both glandular compartments (Figs. 2F,G; Table 1). The protein levels for these molecules were 82.5 and 118.0%, respectively, representing statistical similarity when compared to the TEST group and a decrease in relation to the castrated animals (Fig. 3). On the other hand, endostatin protein levels (123.1%), despite also being similar to the value registered for the TEST group, were significantly higher in comparison to the CAS group, whereas its reactivities were intense in the epithelium and moderate in the stroma (Figs. 2H and 3; Table 1).

The castrated–estrogen group (CE)

Intense IGFR-1 reactivity was observed not only in the epithelial cells but also in the glandular stroma, with 206.5% protein levels, characterizing a significant rise in comparison to the EST and CAS groups, as well as statistical similarity with the CT group (Figs. 2I and 3; Table 1). Intense epithelial and moderate stromal immunoreactivities were observed for MMP-9 and VEGF (Figs. 2J,K; Table 1). In addition, VEGF immunolocalization was located in the luminal surface of the epithelium (Fig. 2K). Protein levels of 174.5 and 292.6% were verified for MMP-9 and VEGF, respectively, and they were statistically higher than the values verified for the CAS, EST and mainly the CT groups (Fig. 3). The epithelium showed weak endostatin reactivity whereas its stromal immunolabeling was moderate, representing 42.6% protein levels, which were lower compared to the EST and CT groups, but similar to the concentration found in the CAS group (Figs. 2L and 3; Table 1).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The results herewith demonstrated increased IGFR-1 and MMP-9 following hormonal replacement and castration, considering the weak reactivity and low protein levels of these molecules in the prostatic microenvironment of senile control rats. IGFR-1 presented intensified immunolabeling in both the prostatic epithelium and stroma, especially in the CT and CE groups. On the other hand, intense MMP-9 immunolabeling occurred mainly in the epithelial compartment, and the higher protein levels were verified particularly in the groups submitted to androgen ablation and/or estradiol treatment. The contrary was verified for VEGF and endostatin in senile rat prostatic tissue, highlighting the increase of the angiogenic stimulator in contrast to the inhibitor during the aging process. After hormonal replacement and surgical castration, increased VEGF protein levels were also detected, especially in the EST, CAS, and CE groups. On the other hand, endostatin levels were higher mainly in the TEST and CT groups.

The effects of the hormone therapy and senescence on serum steroid hormone levels in senile rats have been previously described by our group, using the current experimental protocol and diet, which contained soybean meal (Montico et al., 2011; Cândido et al., 2012). To summarize, we found a significant decrease of plasma testosterone levels in senescence, however the administration of this hormone led to increased concentrations in the TEST group, to a similar extent to those verified in young rats. On the other hand, elevated estradiol levels were registered following its administration and/or rat castration, especially in the CAS and CE groups (Montico et al., 2011). In that study, we also investigated the effects of senescence and hormonal replacement on the ventral prostate histoarchiteture in senile rats. Despite showing positive effects over important adhesion molecules involved in the maintenance of epithelial–stromal interaction, we concluded that the hormonal therapy applied led to the enhancement of structural changes associated to senescence, probably due to the increased imbalance between androgens and estrogens in the prostatic microenvironment (Montico et al., 2011). In another series of experiments, we observed lobe-specific steroid hormone receptor responses in senile rats submitted to hormonal replacement, which could be guided by the imbalanced estrogen/androgen ratio in the prostate (Cândido et al., 2012). In agreement with our studies, it has previously been shown that the increase in the estrogen/androgen ratio in elderly men can be seen as an explanation for the idea that estrogens, in addition to androgens, are fundamental factors in prostatic carcinogenesis (Vermeulen et al., 2002).

Many rodent diets include soybean and its derived isoflavones, which present similar structure to the steroid hormone estradiol as well as weak estrogenic-activity (Thompson et al., 2006; Andres et al., 2011). The main soy-derived isoflavones are genistein, daidzein, and glycitein (Andres et al., 2011). According to Fritz et al. (2003), high dietary genistein (1,000 ppm) reduced testicular aromatase activity, which may have caused reduced estrogen concentrations and contributed to the suppressed development of prostate cancer in rats. In addition, Lee et al. (2004) registered that a 2.5 mg/kg/day genistein treatment during 5 weeks did not cause any lesion in the testis, epididymis and prostate after a 6-months exposure to a soy-bean based chow. Moreover, Cardoso-Báo (2009) verified that rabbits exposed to soy-containing diet and soy isoflavones during the perinatal period did not present any alterations in the testis, epididymis and prostate weight and gross morphology. Both the above results indicated that exposure to soy-containing diet and soy-derived isoflavones in adult and perinatal life periods may not adversely affect the reproductive development and function of male rabbits and rodents (Lee et al., 2004; Cardoso-Báo, 2009). In the present study, the diet used in the different experimental groups contained soybean meal content mixed with other important nutrients, such as vitamins, mineral microelements and aminoacids. However, taking into consideration the literature above, we indicated that the results were not distorted due to the animal diet used in the present study.

Considering proliferative molecules, Wang and Wong (1998) registered higher IGF-1 and IGFR-1 reactivities in the prostatic epithelium after testosterone and estradiol administration, indicating an autocrine action of this growth factor on the glandular epithelium and its possible association to the glandular disorders observed after hormonal therapy. Other studies showed that both androgens and estrogens per se are able to up-regulate IGFR-1 expression in prostate cancer cell lines (Plymate et al., 2004; Pandini et al., 2009). Moreover, different authors suggested the existence of a signaling interplay between IGFR-1 and androgen receptor pathways, in which IGFR-1 could induce AR transactivation and nuclear translocation, starting androgen-activated pathways even in the absence of these hormones (Gennigens et al., 2006; Wu et al., 2006). Ohlson et al. (2006) found that androgens stimulated IGF-1 synthesis in the mouse ventral prostate, especially in the stroma, and that castration led to reduction not only in the IGF-1 stromal secretion but also in the glandular responses to its biological signals, despite the increase in the IGFR-1 epithelial reactivity following androgen ablation (Ohlson et al., 2006). Moreover, it is known that IGF-1 is able to up-regulate MMP expression, pointing out that increased IGF-1 bioavailability accelerates tumoral progression not only through its proliferative actions but also increasing extracellular matrix degradation and therefore tumor invasion potential (Lynch and Matrisian, 2002; Saikali et al., 2008).

Sprenger et al. (2008) verified increased MMP activity in senescence due to the loss of their regulatory mechanisms. In addition, Li et al. (2001) demonstrated that long term estrogen and androgen treatment led to increased MMP-2 and MMP-9 immunolabeling and proteolytic activity in the different prostatic lobes. These authors identified intense MMP reactivity especially in the stroma adjacent to areas of epithelial neoplasia induced by hormonal treatment, suggesting the involvement of these enzymes in the process of tumor invasion towards the stroma (Li et al., 2001). On the other hand, Kanagaraj et al. (2007) verified reduced MMP-2 and MMP-9 activities and protein levels in prostatic tumoral cells submitted to estrogen treatment.

The influence of MMPs in tumor invasiveness is due to direct and indirect mechanisms. Direct effects occur based on extracellular matrix degradation, creating a permissive microenvironment for cellular migration. On the other hand, indirect actions include the activation of other pro-MMPs, the cleavage of regulatory molecules in the cellular surface and the enzymatic activation of chemokines, angiogenic and newly produced growth factors (Sprenger et al., 2008). Moreover, several studies highlighted MMP-9 participation in tumor angiogenesis, with proangiogenic effects, through VEGF solubilization from an extracellular reservatory, and also displaying anti-angiogenic roles, with the cleavage of matrix elements and production of angiogenic inhibitors such as endostatin (Bergers et al., 2000; Nilsson and Dabrosin, 2006; Bendrik et al., 2010).

VEGF is secreted by epithelial and smooth muscle cells in the prostate. The latter source is considered the major angiogenic stimulus in the organ, due to the fact that VEGF secretion from epithelial cells occurs mainly in the apical region towards the lumen, making it unavailable to stromal blood vessels (Wang and Wong, 1998; Doll et al., 2001; Richard et al., 2002). Even though senescence compromises the cellular production of proangiogenic factors such as VEGF, studies with senescent fibroblasts in cell culture demonstrated that these cells expressed increased levels of this molecule, giving them a higher capacity to stimulate vascularization and tumor growth in vivo (Rivard et al., 1999; Coppé et al., 2006). Thus, despite senescence being related to reduced VEGF body levels, the expression of this factor may increase in organs such as the prostate, considering the accumulation of senescent cells in the tissue (Sprenger et al., 2008). Different authors verified that androgens induced VEGF expression in the prostatic gland (Levine et al., 1998; Haggström et al., 1999; Montecinos et al., 2012). It is possible that this regulation mechanism occurs indirectly through androgen effects on other growth factors, such as IGF-1, which is able to upregulate VEGF expression in different cell types (Levine et al., 1998; Miele et al., 2000). In the same way, studies in healthy and neoplastic mammary tissue indicated that estrogens can also positively regulate VEGF levels in the glandular microenvironment (Nakamura et al., 1999; Dabrosin et al., 2003; Garvin et al., 2005).

Androgen and estrogen treatments together led to increased VEGF reactivity in the rodent prostate, especially in the secretory epithelial cells and smooth muscle of the blood vessel wall, in progressive lesions induced by hormonal treatment, including cancer (Wang and Wong, 1998). In addition, increased VEGF reactivity was verified in BPH, high-grade PIN and cancer, in both tumoral cells and glandular stroma (Ferrer et al., 1997; Jackson et al., 1997; Mazzucchelli et al., 2000; Doll et al., 2001). Studies also demonstrated that castration were able to significantly reduce VEGF levels in the normal prostate and in androgen-dependent tumor xenografts (Joseph et al., 1997; Haggström et al., 1998). However, Burchardt et al. (2000) verified a biphasic response of this angiogenic factor after androgen ablation, characterized by decreased VEGF expression only on the second day following castration, returning to levels similar to or even greater than those of controls on the third day.

In addition, some studies have observed an interaction between pathways involving endostatin and AR, demonstrating that cells presenting elevated AR expression were more sensitive to endostatin anti-tumoral effects in comparison to androgen-independent cells (Isayeva et al., 2009). Also, studies carried out on mammary and ovarian tumors demonstrated that tamoxifen, an anti-estrogenic drug, promoted increase of endostatin levels leading to reduced vascularization and tumor growth, whereas estradiol administration alone had opposite effects (Nilsson and Dabrosin, 2006; Bendrik et al., 2010). Moreover, these authors verified that endostatin increase was due to higher levels of MMPs 2 and 9 expression and activity, pointing to the anti-angiogenic role of these enzymes upon the blockade of estrogenic pathways in the tumor microenvironment (Nilsson and Dabrosin, 2006; Bendrik et al., 2010).

On the basis of these findings, we concluded that prostatic angiogenesis was stimulated in senescence and its regulation was influenced by the testosterone and estrogen imbalance during hormone replacement therapy. On the basis of this, we verified an estrogen-dependent upregulation of VEGF, whereas endostatin was more sensitive to testosterone-activated pathways. Furthermore, our data showed that the hormonal imbalance after testosterone and estrogen treatments was crucial to upregulate proliferative and tissue remodeling molecules such as IGFR-1 and MMP-9, especially in secretory epithelial cells. Finally, MMP-9 levels showed themselves to be more responsive to estrogen replacement, while IGFR-1 responded to both testosterone and estrogen. Summarizing, the imbalance between androgens and estrogens, due to hormonal replacement in senescence, led to molecular disorders, generating a reactive prostatic microenvironment characterized by increased mitogenic and tissue remodeling factors and angiogenesis disturbance.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED