Mmp-2 And Mmp-9 activities and Timp-1 and Timp-2 expression in the prostatic tissue of two ethanol-preferring rat models

We investigated whether chronic ethanol intake is capable of altering the MMP-2 and MMP-9 activities and TIMP-2 and TIMP-1 expression in the dorsal and lateral prostatic lobes of low (UChA) and high (UChB) ethanol-preferring rats. MMP-2 and MMP9 activities and TIMP-1 and TIMP-2 expression were significantly reduced in the lateral prostatic lobe of the ethanol drinking animals. Dorsal prostatic lobe was less affected showing no significant alterations in these proteins, except for a reduction in the TIMP-1 expression in UChA rats. These important findings demonstrate that chronic ethanol intake impairs the physiological balance of the prostate extracellular matrix turnover, through downregulation of MMPs, which may contribute to the development of prostatic diseases. Furthermore, since these proteins are also components of prostate secretion, the negative impact of chronic ethanol intake on fertility may also involve reduction of MMPs and TIMPs in the seminal fluid2015COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/03394-4; 2011/13713-

Prostate telocytes change their phenotype in response to castration or testosterone replacement

Telocytes are CD34-positive cells with a fusiform cell body and long, thin cytoplasmic projections called telopodes. These cells were detected in the stroma of various organs, including the prostate. The prostate is a complex gland capable of undergoing involution due to low testosterone levels; and this condition can be reversed with testosterone replacement. Telocyte function in the mature prostate remains to be dermined, and it is not known whether telocytes can take place in tissue remodeling during prostate involution and regrowth. The present study employed structural, ultrastructural and immunohistochemical methods to investigate the telocyte's phenotypes in the ventral prostate (VP) from control (CT), castrated (CS) and testosterone replacement (TR) groups of adult male Wistar rats. Telocytes were found in the subepithelial, perimuscular and interstitical regions around glandular acini. Telocytes from CT animals have condensed chromatin and long and thin telopodes. In CS group, telocytes appeared quiescent and exhibited layers of folded up telopodes. After TR, telocytes presented loose chromatin, abundant rough endoplasmic reticulum and enlarged telopodes, closely associated with bundles of collagen fibrils. We called these cells "telocytes with a synthetic phenotype". As testosterone levels and glandular morphology returned toward to the CT group parameters, after 10 days ofTR, these telocytes progressively switched to the normal phenotype. Our results demonstrate that telocytes exhibit phenotypic plasticity upon androgen manipulation and interact with fibroblast and smooth muscle cells to maintain glandular architecture in control animals and during tissue remodeling after hormonal manipulation9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305391/2014-3; 306900/2016-5; 305840/2015-0; 310663/2018-02002/11102-4; 2014/26660-0; 2017/01063-

Finasteride effects on epithelium-stroma interaction in the different rat prostatic lobes

Orientador: Sergio Luis FelisbinoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As células do câncer prostático são geralmente dependentes, para o seu crescimento e sobrevivência, do estímulo androgênico mediado pelos receptores de andrógeno (RA). Conseqüentemente, a manipulação hormonal, como a castração e/ou o uso de antagonistas de RA, resulta na regressão do câncer. Entretanto, esses tratamentos raramente levam à cura da doença em estágios mais avançados e o câncer, conseqüentemente, torna-se independente de andrógeno. Importantes evidências sustentam a hipótese de que as metaloproteinases de matriz (MMPs) têm papel fundamental nos múltiplos passos da progressão tumoral, incluindo a promoção tumoral, angiogênese, invasão e metástase. As MMPs constituem uma ampla família de endopeptidases dependentes de zinco e cálcio e atuam na degradação da matrix extracelular e da membrana basal. Dois importantes membros da família das MMPs, MMP-2 e MMP-9, pertencentes à subfamília das gelatinases, são conhecidas por degradarem especificamente o colágeno do tipo IV, principal componente das membranas basais. Recentemente, o PCPT (Prostate Cancer Prevention Trial) mostrou que a finasterida, um inibidor da enzima 5_-redutase amplamente usado no tratamento da hiperplasia prostática benigna, diminuiu significativamente a incidência do câncer prostático quando comparado ao grupo que recebeu apenas placebo. Porém, tumores com gradação de Gleason entre 7 e 10 foram significativamente mais comuns no grupo tratado com finasterida que no grupo placebo. Nesse sentido, o papel da finasterida na quimioprevenção do câncer prostático necessita de mais investigação. Assim, este trabalho examinou se as alterações induzidas pela finasterida na morfologia e na fisiologia prostáticas têm impacto na expressão e atividade das MMPs 2 e 9. Ratos machos Wistar (90 dias de idade) foram tratados diariamente com injeções subcutâneas de finasterida (25mg/Kg) durante 3, 7, 30 e 90 dias. Um grupo de animais controle recebeu diariamente injeções subcutâneas do veículo farmacológico (óleo de milho). Os lobos ventrais (PVs), dorsolaterais (PDLs) e anteriores (PAs) foram dissecados, pesados e processados para as análises citoquímicas, imunocitoquímicas, morfométrico-estereológicas e de zimografias. A finasterida promoveu involução progressiva no compartimento epitelial e luminal, redução na altura e na proliferação das células epiteliais e aumentou o índice apoptótico e a área estromal dos três lobos prostáticos. A análise das zimografias mostrou que o tratamento com finasterida altera de modo lobo específico o padrão gelatinolítico da próstata do rato, com o aumento da pró- MMP-9 no dia 30 e da forma ativa da MMP-9 nos dias 30 e 90 na PV, aumento da pró- MMP-9 na PDL nos dias 7 e 30 e o aumento da forma intermediária da MMP-2 na PA no dia 7. Porém, a atrofia induzida pela finasterida não promoveu um aumento significativo na atividade das MMPs, em contraste com o que é encontrado na ablação hormonal pela castração. Isso se deve, provavelmente, ao fato das alterações promovidas pela finasterida na próstata constituírem um processo mais lento em comparação com a castração. Assim, os nossos resultados sugerem que a finasterida é eficaz no tratamento das afecções prostáticas sem promover um ambiente favorável para o crescimento de tumores invasivos e metástaseAbstract: Prostate cancer cells are generally dependent on androgen stimulation mediated by the androgen receptor (AR) for growth and survival. Therefore, hormonal manipulation, such as castration and/or the use of AR antagonists, results in a regression of the cancer. However, these treatments very rarely lead to the ''cure¿¿ of advanced disease and cancer eventually become androgen-independent. A substantial amount of evidence supports the hypothesis that Matrix Metalloproteinases (MMPs) play a key role in multiple steps of tumor progression, including tumor promotion, angiogenesis, invasion, and metastasis. MMPs constitute a broad family of zinc-binding endopeptidases that play a key role in degradation of the extracellular matrix and basement membrane. Two important members of the MMP family, MMP-2 and MMP-9, belonging to the gelatinase subfamily, are known to specifically cleave type IV collagen, the major component of basement membranes. Recently, The Prostate Cancer Prevention Trial (PCPT) showed that finasteride, a 5 alphareductase inhibitor widely employed in the treatment of benign prostatic hyperplasia, significantly decreased the incidence of prostate cancer versus placebo. However, Gleason score 7-10 tumors were significantly more common in the finasteride versus the placebo group. In this sense, the role of finasteride for prostate cancer chemoprevention needs further examination. Thus, this study examined whether the finasteride-induced changes on prostate morphology and phisiology has an impact on MMP-2 and MMP-9 expression and activity. Male Wistar rats (90 days of age) were treated daily with finasteride subcutaneously injected (25mg/Kg) during 3, 7, 30 and 90 days. A control group of animals received daily doses of vehicle (corn oil). The ventral (VPs), dorsolaterals (DLPs) and anterior (APs) lobes had been dissected, weighed and processed for citochemistry, immunocitochemistry, morphometric-stereological, and zymography analyses. Finasteride promoted a progressive involution in the epithelium and lumen compartments, reduced of the epithelial cell heights and proliferation and increased apoptosis and the stromal area in the three prostatic lobes. Gelatin zymography analysis showed that finasteride treatment changed the gelatinolitic profile in the prostatic lobes, with an increase in the pro-MMP-9 at day 30 and in the active-MMP-9 at days 30 and 90 in VP, increase in the pro-MMP-9 inDLP at days 7 and 30 and an increase in the intermediate-MMP-2 in PA at day 7. However, finasteride induced-atrophy did not cause a significant increase in MMPs activity, in contrast with is found in androgen ablation by castration. This is probably due to the fact that finasteride changes in prostate are a slow process in comparison with castration. Take together, our results suggest that finasteride is effective in treating prostate diseases without creating a most favorable environment for tumor growth invasion and metastasisMestradoBiologia CelularMestre em Biologia Celular e Estrutura

Finasteride and doxazosin effects on rat prostate : ultrastructural and type Iand type III and TGF-1 expression analyses

Orientador: Sérgio Luis FelisbinoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A finasterida e a doxazosina são drogas usadas no tratamento da hiperplasia prostática benigna (HPB) e, mais recentemente, estão sendo também usadas na quimioprevenção do câncer de próstata (CaP), principalmente devido aos seus efeitos na indução de apoptose das células epiteliais prostáticas. Entretanto, pouca atenção tem sido dispensada aos efeitos destes fármacos sobre o estroma glandular. Assim, o objetivo deste estudo foi analisar os efeitos da finasterida e da doxazosina no arranjo estromal e em componentes específicos, como o colágeno do tipo I, colágeno do tipo III e o TGF-Beta 1. Para isso, foram empregadas técnicas morfológicas, bioquímicas e moleculares. O tratamento com finasterida promoveu apoptose epitelial, alterações na membrana basal epitelial e na membrana basal das células musculares lisas, além da diminuição na expressão dos colágenos I e III. O bloqueio ?-adrenérgico com doxazosina aumentou a quantidade de fibras do sistema elástico, promoveu apoptose nas células epiteliais, além de alterar a expressão dos colágenos I e III. Os dois fármacos influenciaram o aumento da expressão do TGF-Beta 1, que parece estar mais relacionado com o evento da apoptose no tratamento com a finasterida e com as alterações das fibras colágenas e a ativação de fibroblastos no tratamento com a doxazosina. Conclui-se que as alterações estromais pós tratamento com finasterida e doxazosina podem contribuir para a regressão prostática esperada no tratamento da HPB e na ruptura de eventos parácrinos responsáveis pela evolução tumoral no CaPAbstract: Finasteride and doxazosin are drugs used in the benign prostatic hyperplasia (BPH) treatment and, more recently, they're have been used in the prostate cancer (PCa) chemoprevention, mainly because their apoptotic effect in the prostatic epithelial cells. However, few attentions have been given to the effects of these drugs in the glandular stroma. Thus, the objective of this study was to analyze the effects of the finasteride or doxazosin treatment on stroma arrangement and on specific components, like collagen type I, collagen type III and TGF-Beta 1. For this aim, we have used morphological, biochemical and molecular approaches. Finasteride treatment provoked epithelial apoptosis, epithelial basal membrane and smooth muscle cells basal membrane alterations, besides decrease the type I and type III collagen fibers expression. The alpha-adrenergic blockade increased the elastic fibers system, promoted apoptosis in epithelial cells, as well altered the type I and type III collagen fibers expression. Both drugs influenced the TGF-Beta 1 up-regulation that show to be related with apoptosis in finasteride treatment and with collagen fibers alterations and fibroblasts activation in doxazosin treatment. In conclusion, the stromal alterations promoted by finasteride and doxazosin treatments can contribute to the prostate regression expected in the BPH treatment and to the rupture of paracrine events responsible by tumoral evolution in the PCaDoutoradoBiologia CelularDoutor em Biologia Celular e Estrutura

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of ∼20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role. © 2007 Springer-Verlag

Fibrosis-related gene expression in the prostate is modulated by doxazosin treatment

Aims To gain new insights into the molecular mechanisms of action of doxazosin, we investigated the prostatic stroma ultrastructure and the expression of genes involved with fibrosis, such as collagen type I and III (COL1A1 and COL3A1, respectively) and TGF-beta 1, in the rat ventral prostate. Main methods Adult Wistar rats were treated with doxazosin (25 mg/kg/day), and the ventral prostates were excised at 7 and 30 days after treatment. Untreated rats were controls. Ventral prostates were subjected to ultrastructural, immunohistochemical, biochemical and molecular analyses. Key findings Doxazosin-treated prostates showed thickened bundles of collagen fibrils, activated fibroblasts, enlarged neurotransmitter vesicles and increased tissue immunostaining for collagen type I and type III when compared to untreated prostates. After 7 and 30 days of doxazosin treatment mRNA expression of COL1A1 and COL3A1 was significantly increased and reduced, respectively, compared to the control group. TGF-beta 1 mRNA and protein levels were increased after 7 days of doxazosin treatment, whereas only mRNA levels remained increased after 30 days of treatment. Significance Our data suggest that relaxation of smooth muscle cells by alpha-blockers interferes with the mechanical dynamics of the prostatic stroma extracellular matrix components, generating a pro-fibrotic effect probably via the TGF-beta 1 signaling pathway. Long term treatment with doxazosin may also lead to a reduced turnover of extracellular matrix components. Our results add to a better understanding of the molecular mechanisms behind the effects of alpha-blockade on prostatic histoarchitecture and the response to treatment for benign prostatic hyperplasia9125-2612811287CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação06/60114-6; 06/60115-2The authors thank the Electron Microscopy Center (Institute of Biosciences, UNESP, Botucatu, SP) for the transmission electron microscopy protocol procedures and for the use of Phillips electron microscope. This work was funded by FAPESP (São Paulo State Research Foundation; grants 06/60114-6, to S.L.F. and 06/60115-2, to F.K.D); by FUNDUNESP (Foundation for Development of Univ Estadual Paulista), by CNPq, and by CAPES do Brazil. This paper represents part of the PhD thesis presented by F.K.D. to the University of Campinas—UNICAMP, Brazi

Metalloproteinase inhibition protects against reductions in circulating adrenomedullin during lead-induced acute hypertension

Intoxication with lead (Pb) results in increased blood pressure by mechanisms involving matrix metalloproteinases (MMPs). Recent findings have revealed that MMP type two (MMP-2) seems to cleave vasoactive peptides. This study examined whether MMP-2 and MMP-9 levels/activities increase after acute intoxication with low lead concentrations and whether these changes were associated with increases in blood pressure and circulating endothelin-1 or with reductions in circulating adrenomedullin and calcitonin gene-related peptide (CGRP). Here, we expand previous findings and examine whether doxycycline (a MMPs inhibitor) affects these alterations. Wistar rats received intraperitoneally (i.p.) 1st dose 8g/100g of lead (or sodium) acetate, a subsequent dose of 0.1g/100g to cover daily loss and treatment with doxycycline (30mg/kg/day) or water by gavage for 7days. Similar whole-blood lead levels (9g/dL) were found in lead-exposed rats treated with either doxycycline or water. Lead-induced increases in systolic blood pressure (from 143 +/- 2 to 167 +/- 3mmHg) and gelatin zymography of plasma samples showed that lead increased MMP-9 (but not MMP-2) levels. Both lead-induced increased MMP-9 activity and hypertension were blunted by doxycycline. Doxycycline also prevented lead-induced reductions in circulating adrenomedullin. No significant changes in plasma levels of endothelin-1 or CGRP were found. Lead-induced decreases in nitric oxide markers and antioxidant status were not prevented by doxycycline. In conclusion, acute lead exposure increases blood pressure and MMP-9 activity, which were blunted by doxycycline. These findings suggest that MMP-9 may contribute with lead-induced hypertension by cleaving the vasodilatory peptide adrenomedullin, thereby inhibiting adrenomedullin-dependent lowering of blood pressure.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC50 values ranging from 22.53 mu M to 50.18 mu M, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)] PF6 complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Impact of gestational diabetes and lactational insulin replacement on structure and secretory function of offspring rat ventral prostate

Clinical and experimental studies have shown that exposure to adverse conditions during the critical stages of embryonic, fetal or neonatal development lead to a significantly increased risk of later disease. Diabetes during pregnancy has been linked to increased risk of obesity and diabetes in offspring. Here, we investigated whether mild gestational diabetes mellitus (GDM) followed or not by maternal insulin replacement affects the ventral prostate (VP) structure and function in male offspring at puberty and adulthood. Pregnant rats were divided into the following 3 groups: control (CT); streptozotocin (STZ)-induced diabetes (D); and D plus insulin replacement during lactation (GDI). The male offspring from different groups were euthanized at postnatal day (PND) 60 and 120. Biometrical parameters, hormonal levels and prostates were evaluated. Mild-GDM promoted reduction in the glandular parenchyma and increased collagen deposition. Insulin replacement during lactation restored the VP morphology. Most importantly, mild-GDM decreased the androgen-induced secretory function as determined by prostatein expression, and insulin replacement reversed this effect. Our results demonstrated that mild GDM impairs VP parenchyma maturation, which is associated with an increase in the fibromuscular stroma compartment. Functionally, the reduction in the VP parenchyma decreases the glandular secretory activity as demonstrated by low expression of prostatein, a potent immunosuppressor factor that protects sperm from immunologic damage into the feminine reproductive tract. This change could lead to impairment of reproductive function in male offspring from diabetic mothers. Maternal insulin replacement during the weaning period apparently restores the prostate function in male offspring. (C) 2014 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

The Anti-Inflammatory Effects of the Methanolic Extract and Fractions from Davilla elliptica St. Hil. (Dilleniaceae) on Bothrops jararaca Envenomation

Inflammation and haemorrhage are the main characteristics of tissue injury in botropic envenomation. Although some studies have shown that anti-venom prevents systemic reactions, it is not efficient in preventing tissue injury at the site of the bite. Therefore, this work was undertaken to investigate the anti-inflammatory effects of the methanolic extract and fractions from D. elliptica and to evaluate the role of matrix metalloproteinases (MMPs) in this process. Effects of the extract and fractions from D. elliptica were evaluated using a carrageenan-induced paw oedema model in rats, and leukocyte rolling was visualized by intravital. The quantification of MMPs activities (MMP-2 and MMP-9) extracted from the dermis of mice treated with extract and fractions alone or incubated with venom was determined by zymographic analyses. Our results show that intraperitoneal (i.p.) injection of fractions significantly reduced paw oedema after the carrageenan challenge. Treatment with the tannins fraction also resulted in considerable inhibition of the rolling of leukocytes and this fraction was able to decrease the activation of MMP-9. These results confirmed the anti-inflammatory activity of the methanolic extract and tannins fraction of D. elliptica and showed that the dermonecrosis properties of B. jararaca venom might be mediated through the inhibition of MMP-9 activity.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq