Tempol effect on oxidative and mitochondrial markers in preclinical models for prostate cancer.

Background: Tempol is a redox-cycling nitroxide considered a potent antioxidant. The present study investigated the tempol effects on oxidative stress and mitochondrial markers on prostate cancer (PCa). Methods: PC-3 and LnCaP cells were exposed to tempol. Cell viability test, western blot and Amplex Red analyses were performed. In vivo, five experimental groups evaluated tempol effects in the early (CT12 and TPL12 groups) and late stages (CT20, TPL20-I, and TLP20-II) of PCa development. The TPL groups were treated with 50 or 100 mg/kg tempol doses. Control groups received water as the vehicle. The ventral lobe of the prostate and the blood were collected and submitted to western blotting or enzymatic activity analyses. Results: In vitro, tempol decreased cell viability and differentially altered the H2O2 content for PC-3 and LNCaP. Tempol increased SOD2 levels in both cell lines and did not alter Catalase protein levels. In vivo, tempol increased SOD2 levels in the early stage and did not change Catalase levels in the different PCa stages. Systemically, tempol decreased SOD2 levels in the late-stage and improved redox status in the early and late stages, which was confirmed by reduced LDH in tempol groups. Alterations on energetic metabolism and oxidative phosphorylation were observed in TRAMP model. Conclusion: Tempol can be considered a beneficial therapy for PCa treatment considering its antioxidant and low toxicity properties, however the PCa progression must be evaluated to get successful therapy.

Differential tempol effects in prostatic cancer: angiogenesis and short- and long-term treatments.

Prostate cancer (PCa) is the second cause of cancer death among men worldwide. Several processes are involved in the development and progression of PCa such as angiogenesis, inflammation and oxidative stress. The present study investigated the effect of short- or long-term Tempol treatment at different stages of prostate adenocarcinoma progression, focusing on angiogenic, proliferative, and stromal remodeling processes in TRAMP mice. The dorsolateral lobe of the prostate of TRAMP mice were evaluated at two different stages of PCa progression; early and late stages. Early stage was again divided into, short- or long-term. 50 mg/kg Tempol dose was administered orally. The results demonstrated that Tempol mitigated the prostate histopathological lesion progressions in the TRAMP mice in all treated groups. However, Tempol increased molecules involved in the angiogenic process such as CD31 and VEGFR2 relative frequencies, particularly in long-term treatment. In addition, Tempol upregulated molecule levels involved in angiogenesis and stromal remodeling process VEGF, TGF-ß1, VE-cadherin and vimentin, particularly, in T8-16 group. Thus, it was concluded that Tempol treatment delayed prostatic lesion progression in the dorsolateral lobe of the TRAMP mice. However, Tempol also led to pro-angiogenic effects and glandular stromal microenvironment imbalance, especially, in the long-term treatment.

Lobe-specific responses of TRAMP mice dorsolateral prostate following celecoxib and nintedanib therapy.

Delayed cancer progression in the ventral prostate of the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model has been previously reported upon celecoxib and nintedanib co-administration. Herein, we sought to further investigate the effects of these drugs association in some of their direct molecular targets (COX-2, VEGF and VEGFR-2) and in reactive stroma markers (TGF-ß, αSMA, vimentin and pro-collagen 1) in the dorsolateral prostate, looking for lobe-specific responses. Male TRAMP mice were treated with celecoxib (10 mg/Kg, i.o.) and/or nintedanib (15 mg/Kg, i.o.) for 6 weeks and prostate was harvested for morphological and protein expression analyses. Results showed that combined therapy resulted in unique antitumor effects in dorsolateral prostate, especially due to the respective stromal or epithelial antiproliferative actions of these drugs, which altogether led to a complete inversion in high-grade (HGPIN) versus low-grade (LGPIN) premalignant lesion incidences in relation to controls. At the molecular level, this duality in drug action was paralleled by the differential down/upregulation of TGF-ß signaling by celecoxib/nintedanib, thus leading to associated changes in stroma composition towards regression or quiescence, respectively. Additionally, combined therapy was able to promote decreased expression of inflammatory (COX-2) and angiogenesis (VEGF/VEGFR-2) mediators. Overall, celecoxib and nintedanib association provided enhanced antitumor effects in TRAMP dorsolateral as compared to former registers in ventral prostate, thus demonstrating lobe-specific responses of this combined chemoprevention approach. Among these responses, we highlight the ability in promoting TGF-ß signaling and its associated stromal maturation/stabilization, thus yielding a more quiescent stromal milieu and resulting in greater epithelial proliferation impairment.

Tempol differential effect on prostate cancer inflammation: In vitro and in vivo evaluation.

BACKGROUND: Tempol is a redox-cycling nitroxide that acts directly on inflammation. However, few studies have reported the use of tempol in prostate cancer (PCa). The present study investigated the effects of tempol on inflammation related to NF-κB signaling, using hormone-dependent or hormone-independent cell lines and the transgenic adenocarcinoma of the mouse prostate PCa animal model in the early and late stages of cancer progression. METHODS: PC-3 and LnCaP cells were exposed to different tempol doses in vitro, and cell viability assays were performed. The optimal treatment dose was chosen for subsequent analysis using western blotting. Five experimental groups were evaluated in vivo to test for tempol effects in the early (CT12 and TPL12 groups) and late stages (CT20, TPL20-I, and TLP20-II) of PCa development. The TPL groups were treated with 50 or 100 mg/kg tempol. All control groups received water as the vehicle. The ventral lobe of the prostate was collected and subjected to immunohistochemical and western blot analysis. RESULTS: Tempol treatment reduced cellular proliferation in vitro and improved prostatic morphology in vivo, thereby decreasing tumor progression. Tempol reduced inflammation in preclinical models, and downregulated the initial inflammatory signaling through toll-like receptors, not always mediated by the MyD88 pathway. In addition, it upregulated iκB-α and iκB -ß levels, leading to a decrease in NF-κB, TNF-α, and other inflammatory markers. Tempol also influenced cell survival markers. CONCLUSIONS: Tempol can be considered a beneficial therapy for PCa treatment owing to its anti-inflammatory and antiproliferative effects. Nevertheless, the action of tempol was different depending on the degree of the prostatic lesion in vivo and hormone reliance in vitro. This indicates that tempol plays a multifaceted role in the prostatic tissue environment.

Association of anti-inflammatory and antiangiogenic therapies negatively influences prostate cancer progression in TRAMP mice.

BACKGROUND: Chronic inflammation has been implicated in cancer etiology and angiogenesis is stimulated in this disease. In prostate, the crosstalk between malignant epithelial cells and their microenvironment is an essential step of tumorigenesis during which glandular stroma undergo changes designated as reactive stroma. Thus, the aim herewith was to evaluate the effects of associating anti-inflammatory and antiangiogenic therapies on cancer progression, correlating them with steroid hormone receptor (AR and ERα), reactive stroma (vimentin, αSMA, and TGF-ß), and cell proliferation (PCNA) markers expression in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. METHODS: TRAMP mice (12-week old) were divided into the groups: Control (TRCON): received the vehicles used for drug dilution; Celecoxib (TRCEL): received oral doses of the anti-inflammatory drug celecoxib (15 mg/kg) twice daily; Nintedanib (TRNTB): received oral doses of the antiangiogenic drug nintedanib (10 mg/kg) daily; Nintedanib+Celecoxib (TRNTCEL): received the combination of drugs. After 6 weeks, mice were euthanized and ventral prostate samples were harvested for morphological, immunohistochemical, and Western blotting analyses. RESULTS: While celecoxib led to fibromuscular hypertrophy attenuation, nintedanib significantly reduced the incidence of well-differentiated adenocarcinoma (WDAC) foci in relation to controls, both when administered per se or in association to celecoxib. Furthermore, drug combination was associated with unique effects, including lower incidence of HGPIN lesions; lower AR stromal distribution; changes in ERα localization from epithelial nuclei to stroma as well as significant decrease of TGF-ß levels and associated angiogenesis. In parallel, all treatments applied resulted in reduced inflammatory marker and vimentin (VIM) expression. CONCLUSIONS: Celecoxib plus nintedanib is an effective antitumor combination against prostate cancer progression in TRAMP mice, showing remarkable efficacy in relation to isolated therapies. Importantly, this efficacy might be due to drug association effect on driving AR and mainly ERα distribution in the prostatic tissue towards benign patterns. In addition, celecoxib and nintedanib impaired the development of a stromal reaction by reducing the recruitment of reactive stroma cells and maintaining a normal smooth muscle cell-rich prostate stroma in TRAMP mice. Collectively, these findings pointed to the beneficial effects of combining anti-inflammatory and antiangiogenic strategies to prevent or delay prostatic tumorigenesis.

Steroidal hormone and morphological responses in the prostate anterior lobe in different cancer grades after Celecoxib and Goniothalamin treatments in TRAMP mice.

Prostate cancer is the second most diagnosed cancer in the world, and alternative methods to prevent and treat different lesion grades need to be evaluated. The objective was to evaluate the morphological, hormonal, and inflammatory responses in the prostate anterior lobe in transgenic adenocarcinoma of the mouse prostate (TRAMP), following Celecoxib and Goniothalamin (GTN) treatments. All animals were treated for 4 weeks, from 8 weeks of age and euthanized either immediately after treatment (12-week-old mice: immediate response) or later (22-week-old mice: late response). The results showed a significant increase of high-grade prostatic intraepithelial neoplasia (HGPIN) and well-differentiated adenocarcinoma (WDA), according to the age in the control groups. Celecoxib treatment decreased the WDA incidence in the late response group. GTN led to a significant healthy tissue increase, and an LGPIN and HGPIN decrease in the immediate response group. In the late response group, GTN led to healthy area increase and there was no occurrence of WDA. AR and ERα immunoexpressions were reduced by both treatments in the immediate response groups. However, only GTN was able to decrease the ERα level in the late response group. Regarding COX-2 immunoreactivity, both treatments reduced the frequency of this enzyme. We can conclude that the prostate anterior lobe is a good model to study prostate cancer, considering its slow progression. Both treatments led to cancer delay in the prostate anterior lobe. However, GTN pointed towards a better treatment spectrum in the signaling pathways in the prostate microenvironment, particularly in ERα.

Nintedanib treatment delays prostate dorsolateral lobe cancer progression in the TRAMP model: contribution to the epithelial-stromal interaction balance.

Prostate cancer (PCa) progression mechanism has been linked to epithelial proliferation, tumor invasion ability, and growth factors. Nintedanib (BIBF 1120) has been reported as being FGF and VEGF pathway inhibitors, exhibiting antitumor activity. Thus, the objective herein was to characterize the early Nintedanib treatment effects on the structure and molecules involved in the basal membrane, the extracellular matrix (ECM) maintenance, in addition to the angiogenesis and mitogenic processes at different grades of prostatic tumor development in TRAMP mice. Therefore, 45 male TRAMP mice were divided into control groups: 8-week-old mice (TC8), 12-week-old mice (TC12), and 16-week-old mice (TC16); and treated groups with 10 mg/kg/day Nintedanib dose for 4 weeks. The treated groups were euthanized at 12 (TN12) and 16 (TN16) weeks of age. Samples from the dorsolateral lobe were collected and processed for light microscopy, immunohistochemistry, Western blotting, and microvessel density analysis. The results showed that early Nintedanib treatment led to an increase of healthy epithelium frequency and a reduction of LGPIN and a maximum vascularization density in the TN12 group. Also, treatment led to a well-differentiated adenocarcinoma decrease and an α and ß dystroglycan and also laminin 1 increase in the TN16 group. IGFR1 decreased in the TN16 group. To conclude, early Nintedanib treatment led to a reduction in cancer severity, interfering in both ECM compounds and angiogenesis process to then contribute to a balance, not only in the prostatic epithelium and stroma, but also in the epithelial-stromal interaction during PCa progression.

Nintedanib antiangiogenic inhibitor effectiveness in delaying adenocarcinoma progression in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP).

BACKGROUND: In recent times, anti-cancer treatments have focused on Fibroblast Growth Factor (FGF) and Vascular-Endothelial Growth Factor (VEGF) pathway inhibitors so as to target tumor angiogenesis and cellular proliferation. One such drug is Nintedanib; the present study evaluated the effectiveness of Nintedanib treatment against in vitro proliferation of human prostate cancer (PCa) cell lines, and growth and progression of different grades of PCa lesions in pre-clinical PCa transgenic adenocarcinoma for the mouse prostate (TRAMP) model. METHODS: Both androgen-independent (LNCaP) and androgen-dependent (PC3) PCa cell lines were treated with a range of Nintedanib doses for 72 h, and effect on cell growth and expression of angiogenesis associated VEGF receptors was analyzed. In pre-clinical efficacy evaluation, male TRAMP mice starting at 8 and 12 weeks of age were orally-fed with vehicle control (10% Tween 20) or Nintedanib (10 mg/Kg/day in vehicle control) for 4 weeks, and sacrificed immediately after 4 weeks of drug treatment or sacrificed 6-10 weeks after stopping drug treatments. At the end of treatment schedule, mice were sacrificed and ventral lobe of prostate was excised along with essential metabolic organ liver, and subjected to histopathological and extensive molecular evaluations. RESULTS: The total cell number decreased by 56-80% in LNCaP and 45-93% in PC3 cells after 72 h of Nintedanib treatment at 2.5-25 µM concentrations. In pre-clinical TRAMP studies, Nintedanib led to a delay in tumor progression in all treatment groups; the effect was more pronounced when treatment was given at the beginning of the glandular lesion development and continued till study end. A decreased microvessel density and VEGF immunolocalization was observed, besides decreased expression of Androgen Receptor (AR), VEGFR-1 and FGFR-3 in some of the treated groups. No changes were observed in the histological liver analysis. CONCLUSIONS: Nintedanib treatment was able to significantly decrease the growth of PCa cell lines and also delay growth and progression of PCa lesions to higher grades of malignancy (without inducing any hepatotoxic effects) in TRAMP mice. Furthermore, it was observed that Nintedanib intervention is more effective when administered during the early stages of neoplastic development, although the drug is capable of reducing cell proliferation even after treatment interruption.

Goniothalamin and Celecoxib Effects During Aging: Targeting Pro-Inflammatory Mediators in Chemoprevention of Prostatic Disorders.

BACKGROUND: Prostate is highly affected by aging, which lead to inflammatory disorders that can predispose to cancer development. Chemoprevention has emerged as a new therapeutic approach, intensifying studies evaluating the biological properties of new compounds. The aim of this study was to characterize the inflammatory responses in the prostate ventral lobe from senile mice treated with Goniothalamin (GTN), a promising natural compound with anti-inflammatory and antiproliferative properties. Its activity was compared to Celecoxib, an established nonsteroidal anti-inflammatory drug (NSAID). METHODS: The animals were divided into: Control groups; Young (18-week-old FVB), Senile (52-week-old FVB). Treated groups: Senile-Goniothalamin (150 mg/kg orally), Senile-Celecoxib (10 mg/kg orally). The ventral lobe was collected after 4 weeks for light microscopy, immunohistochemistry, ELISA, and Western blotting analysis. RESULTS: Both treatments were efficient in controlling the inflammatory process in the prostate from senile mice, maintaining the glandular morphology integrity. GTN reduced all inflammatory mediators evaluated (TNF-α, COX-2, iNOS) and different from Celecoxib, it also decreased the protein levels of NF-kB and p-NF-kB. CONCLUSIONS: Finally, GTN and Celecoxib controlled inflammation in the prostate, and sensitized the senescent microenvironment to anti-inflammatory stimuli. Thus, both treatments are indicated as potential drugs in the prostatic diseases prevention during senescence. Prostate 77:838-848, 2017. © 2017 Wiley Periodicals, Inc.

Morphology and MMP-9, AR and IGFR-1 responses of the seminal vesicle in TRAMP mice model.

Seminal vesicles are important hormone-dependent accessory sex glands. Transgenic adenocarcinoma of the mouse prostate (TRAMP) model has been used to evaluate malignant diseases in the prostate and in other sexual glands. The aim of this study was to characterize structural and molecular features of the seminal vesicle in different life periods of the TRAMP mice. Groups: Control Group (5 FVB/12 week old mice), TRAMP 12 and 22 Groups (10 TRAMP 12 and 22 week old mice, respectively). Seminal vesicles were evaluated by morphological and immunohistochemical parameters; androgenic receptor (AR), Insulin-like growth factor 1 (IGFR-1) and metalloproteinase 9 (MMP-9). The TRAMP mice showed frequent epithelial proliferation, including cellular stromal invasion, especially in the TRAMP 22 group. Intense AR reactivity was seen in both stroma and epithelial regions in the TRAMP 22 group. Intense IGFR-1 and MMP-9 stromal immunolabeling was identified in both TRAMP groups. Thus, there were structural and molecular changes in the seminal vesicle in TRAMP mice, compromising not only the structure but also the stromal signaling, damaging thus the function and leading to glandular lesions. TRAMP mice could be indicated as a good model to study alterations of the seminal vesicle in association to prostate cancer.

Prolactin, EGFR, vimentin and α-actin profiles in elderly rat prostate subjected to steroid hormonal imbalance.

The aim of this study was to characterize and relate the prolactin (PR), epidermal growth factor receptor (EGFR), α-actin and vimentin immunoreactivity in the prostate of elderly rats subjected to steroid hormonal imbalance. Senile and young rats were divided into the young group (YNG), the senile group (SE), the castrated group (CAS), the estrogen-deficient group (ED), the castrated+estrogen group (CASE), and the estrogen-deficient+androgen group (EDTEST). PR and EGFR increased in the estrogen and androgen ablation groups. In addition, EGFR influenced the immunolocalization by changing it from the prostatic stroma to the epithelium in elderly rats. Hormone ablation in elderly rats, not only related to androgen but also estrogen, led to increased stromal EGFR immunolocalization. The α-actin pattern decreased in the groups with estrogenic imbalance. Moreover, vimentin increased in the senile and estrogen deficient group. To conclude, we can suggest that EGFR contributed towards the proliferative process in the prostate, by means however, of different mechanisms, considering the androgenic and estrogenic pathways. Also, our results indicated that prolactin could be activated not only in an androgen-independent pathway but also in an estrogen independent pathway. Finally, PR and vimentin immunolocalization increase, in the prostatic stroma in the group showing estrogenic ablation, could be one of the factors which contribute to the reactive stroma formation.

Anti-inflammatory therapies in TRAMP mice: delay in PCa progression.

The aim of this study was to characterize the structural and molecular biology as well as evaluate the immediate and late responses of prostatic cancer in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model after treatment with goniothalamin (GTN) and celecoxib. The treated mice received GTN (150 mg/kg, gavage) or celecoxib (10 mg/kg, gavage) from 8 to 12 weeks of age. They were killed at different ages: the immediate-response groups at 12 weeks and the late-response groups at 22 weeks. The ventral prostate was collected for light microscopy, immunohistochemistry, western blotting, TUNEL, and ELISA. Morphological analyses indicated that GTN treatment delayed the progression of prostatic adenocarcinoma, leading to a significant decrease of prostatic lesion frequency in both experimental period responses to this treatment, mainly high-grade prostatic intraepithelial neoplasia and well-differentiated adenocarcinoma. Also, the celecoxib treatment showed a particular decrease in the proliferative processes (PCNA) in both the experimental periods. Despite celecoxib diminishing the COX2 and IGFR1 levels, GTN presented higher action spectrum considering the decrease of a greater molecular number involved in the proliferative and inflammatory processes in prostatic cancer. Goniothalamin attenuated the pro-inflammatory response in TRAMP prostatic microenvironment, delaying prostate cancer (PCa) progression. Celecoxib treatment was efficient in the regulation of COX2 in the TRAMP mice, mainly in the advanced disease grade. Finally, we concluded that inflammatory process control in early grades of PCa was crucial for the downregulation of the signaling pathways involved in the proliferative processes in advanced cancer grades.

Reactive stroma in the prostate during late life: The role of microvasculature and antiangiogenic therapy influences.

BACKGROUND: Prostate cancer is associated to a reactive stroma microenvironment characterized by angiogenic processes that are favorable for tumor progression. Senescence has been identified as a predisposing factor for prostate malignancies. In turn, the relationships between aging, reactive stroma, and the mechanisms that induce this phenotype are largely unknown. Thus, we investigated the occurrence of reactive stroma in the mouse prostate during advanced age as well as the effects of antiangiogenic and androgen ablation therapies on reactive stroma recruitment. METHODS: Male mice (52-week-old FVB) were treated with two classes of angiogenesis inhibitors: direct (TNP-470; 15 mg/kg; s.c.) and/or indirect (SU5416; 6 mg/kg; i.p.). Androgen ablation was carried out by finasteride administration (20 mg/kg; s.c.), alone or in association to both inhibitors. The Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model was used as a paradigm of cancer-associated reactive stroma. The dorsolateral prostate was collected for α-actin (αSMA), vimentin (VIM), and transforming growth factor-beta (TGF-ß) immunohistochemical and Western blotting analyses as well as for CD34/αSMA and CD34/VIM colocalization. RESULTS: Senescence was associated with increased αSMA, VIM, and TGF-ß expression as well as with the recruitment of CD34/αSMA and CD34/VIM dual-positive fibroblasts. These observations were similar to those verified in TRAMP mice. Antiangiogenic treatment promoted the recovery of senescence-associated stromal changes. Hormonal ablation, despite having led to impaired CD34/αSMA and CD34/VIM dual-positive cell recruitment, did not result in decreased stimulus to reactive stroma development, due to enhanced TGF-ß expression in relation to the aged controls. CONCLUSIONS: Reactive stroma develops in the prostate of non-transgenic mice as a result of aging. The periacinar microvasculature is a candidate source for the recruitment of reactive stroma-associated cells, which may be derived either from perivascular-resident mesenchymal stem cells (MSCs) or from an endothelial-to-mesenchymal transition (EndMT) process. Thus, antiangiogenic therapy is a promising approach for preventing age-associated prostate malignancies by means of its negative interference in the development of reactive stroma phenotype from the vascular wall.

Prostatic angiogenic responses in late life: antiangiogenic therapy influences and relation with the glandular microenvironment in the transgenic adenocarcinoma of mouse prostate (TRAMP) model.

BACKGROUND: Aging is considered one of the main predisposing factors for the development of prostate malignancies. Angiogenesis is fundamental for tumor growth and its inhibition represents a promising therapeutic approach in cancer treatment. Thus, we sought to determine angiogenic responses and the effects of antiangiogenic therapy in the mouse prostate during late life, comparing these findings with the prostatic microenvironment in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. METHODS: Male mice (52 week-old FVB) were submitted to treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c.). Finasteride was administered (20 mg/kg; s.c.), alone or in association to both inhibitors. The dorsolateral prostate was collected for VEGF, HIF-1α, FGF-2 and endostatin immunohistochemical and Western Blotting analyses and for microvessel density (MVD) count. RESULTS: Senescence led to increased MVD and VEGF, HIF-1α and FGF-2 protein levels in the prostatic microenvironment, similarly to what was observed in TRAMP mice prostate. The angiogenic process was impaired in all the treated groups, demonstrating significantly decreased MVD. Antiangiogenic and/or finasteride treatments resulted in decreased VEGF and HIF-1α levels, especially following TNP-470 administration, either alone or associated to SU5416. The combination of these agents resulted in increased endostatin levels, regardless of the presence of finasteride. CONCLUSIONS: Prostatic angiogenesis stimulation during senescence favored the development of neoplastic lesions, considering the pro-angiogenic microenvironment as a common aspect also observed during cancer progression in TRAMP mice. The combined antiangiogenic therapy was more efficient, leading to enhanced imbalance towards angiogenic inhibition in the organ. Finally, finasteride administration might secondarily upregulate the expression of pro-angiogenic factors, pointing to the harmful effects of this therapy.

Antiangiogenic and finasteride therapies: responses of the prostate microenvironment in elderly mice.

AIMS: The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. MAIN METHODS: 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. KEY FINDINGS: The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-ß and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-ß reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-ß protein levels. SIGNIFICANCE: The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-ß.

Antiangiogenic therapy effects on age-associated matrix metalloproteinase-9 (MMP-9) and insulin-like growth factor receptor-1 (IGFR-1) responses: a comparative study of prostate disorders in aged and TRAMP mice.

Senescence is associated with hormonal imbalance and prostatic disorders. Angiogenesis is fundamental for the progression of malignant lesions and is a promising target for prostate cancer treatment. The aim was to characterize matrix metalloproteinase-9 (MMP-9) and insulin-like growth factor receptor-1 (IGFR-1) responses in the prostate during senescence and following antiangiogenic and/or androgen ablation therapies, comparing them to cancer progression features in TRAMP mice. Aged male mice (52-week-old FVB) were submitted to antiangiogenic treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c). Finasteride (20 mg/kg; s.c.) was administered alone or associated to both inhibitors. Dorsolateral prostate was collected for light microscopy, and immunohistochemistry and Western blotting collected for MMP-9 and IGFR-1. Senescence led to inflammation and different proliferative lesions in the prostate, as well as to increased MMP-9 and IGFR-1, resembling TRAMP mice prostatic microenvironment. Antiangiogenic therapies promoted recovery and/or interruption of age-associated alterations, presenting differential effects on the molecules studied. SU5416 acted mainly on MMP-9, whereas TNP-470 showed its best influence on IGFR-1 levels. Finasteride administration, alone or in combination with antiangiogenic agents, also resulted in regression of inflammation and neoplastic lesions, besides having a negative modulatory effect on both MMP-9 and IGFR-1. We concluded that stimulated tissue remodeling and proliferative processes during senescence predisposed the prostate to malignant disorders. The combination of different agents was more effective to minimize prostatic imbalance during this period, probably due to the differential action of each drug on factors involved in cell proliferation and extracellular matrix remodeling, resulting in a broader spectrum of effects following the combined treatment.

Angiogenic and tissue remodeling factors in the prostate of elderly rats submitted to hormonal replacement.

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) 17ß-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.

Fibroblast growth factor, estrogen, and prolactin receptor features in different grades of prostatic adenocarcinoma in elderly men.

The objective was to characterize and associate the receptor reactivities of fibroblastic growth factor (FGF)-2, FGF-7, FGF-8, epidermal growth factor (EGF), α-actin and vimentin in relation to the androgen receptor (AR), α and ß estrogen receptors (ERα and ERß), and prolactin receptor in the prostate of elderly men showing low- and high-grade adenocarcinoma. Thirty prostatic samples were taken from 60- to 90-year-old patients without prostatic lesions and with low-grade cancer and high-grade cancer, from the University Hospital, School of Medicine, the State University of Campinas. The results showed that increased FGF-2, FGF-7, and FGF-8 receptor reactivities and decreased AR reactivity were verified in both high- and low-grade cancer. However, the FGF-8 receptor showed greater involvement at the beginning of the malignancy alterations. Increased EGF receptor (EGFR) reactivity and diminished α-actin immunohistochemistry were identified in both cancer groups. Also, increased ERα, PR, and vimentin receptors were verified in both cancer groups. To conclude, the ERα involvement in the reactive stroma activation led to a microenvironment, which was favorable to cancer progression, due to maximizing stromal imbalance. The prolactin could be related to cancer progression due to its interaction with ERα action, indicating that this hormone could be a relevant target to prevent the estrogenic effects in the prostatic lesions. Both FGF receptor (FGFR)-2 and FGFR-8 play a fundamental role in the early stages of prostate cancer, suggesting that these molecules could be a promising therapeutic target. The differential localization of the fibroblastic factors between the prostatic epithelium and stroma of elderly men, who presented prostate cancer, could indicate a favorable distinction for tumoral progression.

Senescence and steroid hormone receptor reactivities in accessory sex glands of elderly rats (Sprague-Dawley) following exogenous hormonal therapy.

The aim of this study was to characterize the stromal and epithelial distribution of AR, ERα and ERß reactivities in the different accessory sex glands of elderly rats and during strong hormonal changes. Ten month old male rats were divided into six senile groups and submitted to treatment: Senile/Control group (SC); Senile/Testosterone group (ST): Senile/Estrogen group (SE); Castrated group (CA); Castrated/Testosterone group (CT); Castrated/Estrogen group (CE). After a 30-day treatment, the prostatic ventral lobe (VL), dorsal lobe (DL) and coagulating gland (CG) samples were processed for immunohistochemistry and Western Blotting. The results showed that AR immunoreactivity was characterized in the epithelium of VL and DL in senile/control rats and senile rats submitted to exogenous hormonal therapy. AR reactivity in the coagulating gland was verified predominantly in the stromal cells in the different experimental groups. ERα reactivity occurred predominantly in the stromal compartment in all accessory sex glands. In the DL and CG, ERα immunoreactivities were intense in the groups which received testosterone (ST) and estrogen (SE). ERß immunoreactivity in the CG was verified in the stromal compartment in the different experimental groups, showing a positive response to both increased testosterone and estrogen levels. ERß reactivity, in the DL, was intensified in the stroma of senile rats with higher serum testosterone levels, and in senile rats with increased serum estrogen levels, especially in the glandular epithelium. Thus, the results revealed different distribution pattern of steroid hormone receptors in each one of the prostatic lobes in senescence, especially in the prostate dorsal lobe and coagulating gland, which is a fundamental factor due to the fact that major prostatic diseases occur in a later period of life.

Hormonal therapy in the senescence: Prostatic microenvironment structure and adhesion molecules.

Hormonal replacement has been utilized to minimize the harmful effects of hormonal imbalance in elderly men. The development and progression of prostatic diseases and their relation to hormone therapy is still unclear. Thus, the aim herewith was to characterize the structure and dystroglycan molecule (DGs) reactivities in the ventral prostatic lobe from elderly rats submitted to steroid hormone replacement. Male rats (Sprague-Dawley) were divided into one Young group and six senile groups. The Young group (YNG) (4 months old) received peanut oil (5mL/kg, s.c.). The senile rats (10 months old) were submitted to the following treatments: Senile group (SEN) (5mL/kg peanut oil, s.c.); Testosterone group (TEST) (5mg/kg testosterone cipionate, s.c.); Estrogen group (EST) (25µg/kg 17ß-estradiol, s.c.); Castrated group (CAS) (surgical castration); Castrated-Testosterone (CT) (surgical castration and treatment similar to TEST group); and Castrated-Estrogen (CE) (surgical castration and treatment similar to EST group). After 30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples were processed for light and transmission electron microscopies, morphometrical analysis, immunohistochemistry and Western Blotting. The results showed decreased serum testosterone levels in the senescence and increased testosterone and estrogen plasmatic levels after hormone administration in the TEST and EST groups, respectively, highlighting the therapy efficiency. Hypertrophied stroma and inflammatory cells were verified in the SEN group. After hormone replacement in the senescence or following castration, atrophic epithelium, epithelial cells with clear cytoplasmic halo around the nucleus, microacini and maintenance of hypertrophied stroma were seen. Decreased DG levels were verified in the senescence. After hormonal therapy, increased protein levels of these molecules were observed, especially in those groups which received estradiol. Thus, the occurrence of inflammatory cells, stromal hypertrophy and the presence of cells with clear halo around the nucleus after hormonal therapy probably indicated prostatic paracrine signaling imbalance, suggesting a stromal reactive microenvironment favorable to the development of glandular lesions. However, the increase of DG levels characterized positive effect of steroid hormone replacement on the prostate in the senescence. Thus, it could be concluded that despite having positive effects on important molecules involved in the maintenance of epithelial-stromal interaction and glandular cytoarchitecture, such as DGs, hormonal therapy enhanced structural changes associated with senescence, probably due to increased hormonal imbalance between androgens and estrogens in the prostatic tissue.