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- 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.
- Top of page
- MATERIALS AND METHODS
- 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.