Profile of cell proliferation and apoptosis activated by the intrinsic and extrinsic pathways in the prostate of aging rats.

BACKGROUND: Estrogens acting through the receptors ERα and ERß participate in prostate normal growth and cancer. ERß is highly expressed in the prostate epithelium, playing pro-apoptotic, anti-proliferative, and pro-differentiation roles. Apoptosis is activated by the intrinsic pathway after castration and by the extrinsic pathway after ERß agonist treatment. This differential activation of apoptotic pathways is important since a major problem in the treatment of prostate cancer is the recurrence of tumors after androgen withdrawal. However, a comprehensive study about the pattern of apoptosis in the aging prostate is lacking, a knowledge gap that we aimed to address herein. METHODS: Cellular age-related proliferative and apoptotic profiles of prostate tissue obtained from aging Wistar rats were evaluated. Cell death (caspase-3, -8, -9, TNFα) was assessed by immunohistochemistry, immunofluorescence, and TUNEL. Cell proliferation (MCM7) and cell survival factors (ERK1/2, p-ERK1/2, p-Akt, and NF-κB) were determined by immunohistochemistry. RESULTS: As the rats aged, the number of proliferating cells gradually reduced in the normal epithelium of all prostate lobes, while increasing in focal areas of intraepithelial proliferation. Interestingly, in areas of intraepithelial proliferation, we observed a reduction in the number of cells positive for caspase-3, -8, and -9. Regardless the animal's age, few prostate epithelial cells were positive for caspase-3, caspase-9, and TUNEL. In contrast, a progressive increase was seen in the positivity for caspase-8, especially in the atrophic epithelium of ventral prostate, which coincided with a reduction in TNFα immunoreaction. However, morphology of most caspase-8 positive cells suggests that they were not apoptotic. We also found reduced ERß expression in the same areas. Possibly, low levels of the pro-apoptotic inductors TNFα and ERß direct caspase-8 activity to an alternative pro-survival role in the atrophic epithelium. This hypothesis is supported by the increased expression of the key survival factors (ERK1/2, p-ERK1/2, p-Akt, and NF-κB) in these areas. CONCLUSIONS: Our findings reveal that, as the animals age, there is an increase of proliferation in restricted areas of the prostate epithelium, and a concomitant reduction of the apoptosis rate with an increase in cell survival induced by caspase-8, indicating a focused and spontaneous disruption of tissue homeostasis.

Altered expression of the vitamin D metabolizing enzymes CYP27B1 and CYP24A1 under the context of prostate aging and pathologies.

Low circulating levels of vitamin D are common at older ages and have been linked to an increased risk of prostate disease, including cancer. However, it has not yet been determined whether aging affects the ability of prostate cells to locally metabolize vitamin D into its active metabolite calcitriol and thus mediate the vitamin D signaling in autocrine and paracrine ways. By using a suitable rat model to interrogate spontaneous prostatic modifications over the course of aging, here we showed that both CYP27B1 and CYP24A1 enzymes, which are key players respectively involved with calcitriol synthesis and deactivation, were highly expressed in the prostate epithelium. Furthermore, as the animals aged, a drastic reduction of CYP27B1 levels was detected in total protein extracts and especially in epithelial areas of lesions, including tumors. On the other hand, CYP24A1 expression significantly increased with aging and remained elevated even in altered epithelia. Such intricate unbalance in regard to vitamin D metabolizing enzymes was strongly associated with reduced bioavailability of calcitriol in the senile prostate, which in addition to decreased expression of the vitamin D receptor, further limits the protective actions mediated by vitamin D signaling. This evidence was corroborated by the increased proliferative activity exactly at sites of lesions where the factors implicated with calcitriol synthesis and responsiveness had its expression inhibited. Taken together, our results emphasize a set of modifications over the course of aging with a high potential to hamper vitamin D signaling on the prostate. These findings highlight a crosstalk between vitamin D, aging, and prostate carcinogenesis, offering new potential targets in the prevention of malignancies and other aging-related disorders arising in the gland.

Tumor-Associated Macrophages (TAM) are recruited to the aging prostate epithelial lesions and become intermingled with basal cells.

BACKGROUND: Prostate cancer remains one of the most common cancers in men. Macrophages are thought to be important regulators in cancers, and their potential involvement in prostate cancer should not be overlooked. Therefore, the association between macrophages and the pre-tumorous changes in prostate epithelium during aging deserves further investigation. OBJECTIVES: We sought to investigate whether macrophages would be recruited into the prostate epithelium that display pathological lesions commonly found during aging. MATERIALS AND METHODS: Prostates of aging rats, with and without treatment with a combination of testosterone and estradiol, were examined for premalignant and malignant epithelial lesions. For comparison, prostates of castrated rats were also investigated. RESULTS: Intraepithelial macrophages were found restricted to areas of premalignant and malignant lesions. An unprecedented interaction between macrophages and basal cells was observed in the aging pathological lesions. The intraepithelial macrophages were associated with autophagy, in contrast to those found after castration. In prostate lesions, the intraepithelial macrophages had TAM phenotype (CD68+/iNOS+/CD206+/ARG+), denoting a possible involvement in cancer progression. However, M2 macrophages (CD68+/CD163+) were recruited into the epithelium after castration, possibly to phagocytize cells undergoing apoptosis. DISCUSSION AND CONCLUSION: In conclusion, macrophages were recruited into the prostate epithelium and presented diverse phenotypes and morphology, consistent with changes reflected in the hormonal environment. Macrophages with the TAM phenotype were found restricted to areas of premalignant and malignant lesions in aging prostates, denoting a possible involvement in cancer progression. In contrast, M2 macrophages were found in the regressed epithelium after castration.

Targeting Wistar rat as a model for studying benign, premalignant and malignant lesions of the prostate.

AIMS: The purpose of this study was to describe a suitable experimental model for studying aging-related prostate disorders including cancer. MATERIALS AND METHODS: 12-month old Wistar rats were kept in control conditions (n = 12) or treated (n = 16) for 6 months with Silastic implants filled with testosterone (T) and estradiol (E2). After the experiment period (at 18 months of age), animals were euthanized and the prostate and other organs were harvested, dissected, weighed, and processed for morphological, ultrastructural and molecular analyses. KEY FINDINGS: We demonstrated that male rats of Wistar strain nicely recapitulate the carcinogenesis process taking place in the aging prostate through the arising of benign, precancerous and malignant lesions, and above all yields a modest incidence of spontaneous PCa (~36%). Moreover, our results highlight that 100% incidence of PCa and precancerous lesions such as prostatic intraepithelial neoplasia and proliferative inflammatory atrophy were achieved in this rat strain after T + E2 treatment, without changing the broad spectrum of changes that naturally emerge in the prostate at advanced ages. Such enhancement of precancerous lesions and tumors was linked to a decreased expression of E-cadherin and ß-catenin in parallel with an increase in Vimentin and N-cadherin, hallmark modifications of epithelial-mesenchymal transition. SIGNIFICANCE: Our findings provide solid evidence that aged Wistar rats may be an excellent model for studies regarding human prostate biology and related disorders including cancer.

Basal Cells Show Increased Expression of Aromatase and Estrogen Receptor α in Prostate Epithelial Lesions of Male Aging Rats.

Besides androgens, estrogen signaling plays a key role in normal development and pathologies of the prostate. Irreversible synthesis of estrogens from androgens is catalyzed by aromatase. Interestingly, animals lacking aromatase do not develop cancer or prostatitis, whereas those with overexpression of aromatase and, consequently, high estrogen levels develop prostatitis and squamous metaplasia via estrogen receptor 1 (ERα). Even with this evidence, the aromatase expression in the prostate is controversial. Moreover, little is known about the occurrence of age-dependent variation of aromatase and its association with histopathological changes commonly found in advanced age, a knowledge gap that is addressed herein. For this purpose, the immunoexpression of aromatase was evaluated in the prostatic complex of young adult to senile Wistar rats. ERα was also investigated, to extend our understanding of estrogen responsiveness in the prostate. Moderate cytoplasmic immunoreactivity for aromatase was detected in the glandular epithelium. Eventually, some basal cells showed intense staining for aromatase. The expression pattern for aromatase appeared similar in the normal epithelium when young and senile rats were compared; this result was corroborated by Western blotting. Conversely, in senile rats, there was an increase in the frequency of basal cells intensely stained for aromatase, which appeared concentrated in areas of intraepithelial proliferation and prostatitis. These punctual areas also presented increased ERα positivity. Together, these findings suggest a plausible source for hormonal imbalance favoring estrogen production, which, by acting through ERα, may favor the development of prostatic lesions commonly found in advanced age.