Análise quantitativa da proliferação celular no epitélio seminífero humano

Mestrado em Biologia Molecular e CelularApesar de décadas de investigação básica na espermatogénese humana, pouco se sabe acerca da proliferação e diferenciação das células germinais humanas em termos quantitativos. Com este trabalho pretendeu-se estudar quantitativamente os efeitos temporais e específicos de cada estadio celular, da Hormona Foliculo Estimulante (FSH) e da testosterona (T) na capacidade proliferativa do epitélio seminífero humano normal sob condições de cultura de órgão. Para tal, fragmentos de túbulos seminíferos humanos foram mantidos em cultura durante 28 dias, usando-se a incorporação da 5- bromo-2’-deoxiuridina (BrdU) e a sua detecção por imunohistoquímica para aceder à proliferação celular. Os resultados quantitativos deste estudo mostraram uma perda gradual das células germinais meióticas e pós-meióticas ao longo do período de cultura, com uma boa manutenção da arquitectura geral dos túbulos e sem diminuição do número das células de Sertoli. Verificou-se que a taxa de proliferação das espermatogónias A dark era bastante baixa independentemente do suplemento hormonal. A proliferação das espermatogónias A, espermatogónias B e pré-leptótenos, foi máxima durante a primeira semana de cultura atingindo valores mais elevados quando induzida pelas hormonas (FSH e testosterona). Ao final da primeira semana houve diferenciação das espermatogónias e pré-leptótenos em espermatócitos em zigóteno e paquíteno e espermatócitos secundários, promovendo as hormonas uma aceleração da passagem de leptóteno para paquíteno em especial na concentração 50 U/L de FSH e 1μmol/L de testosterona. A FSH + T aumentou a sobrevida das células germinais, estimulou a proliferação das espermatogónias e a diferenciação das células germinais, durante a primeira semana de cultura. Com este estudo, foi possível quantificar pela primeira vez a proliferação e diferenciação das células germinais, utilizando uma metodologia bastante reprodutível e com resultados semelhantes aos in vivo. ABSTRACT: In spite of decades in study of human spermatogenesis almost anything is known about germinal cell proliferation and differentiation in quantitative ways. The aim of this project was to study quantitatively the temporal and stage specific effects of Follicle Stimulating Hormone and testosterone on the proliferative capacity of the normal human seminiferous epithelium under organ culture conditions. Seminiferous tubules fragments were kept in culture during 28 days, and 5-bromo-2’-deoxyuridine (BrdU) incorporation was used followe by immunohistochemistry detection to achieve cellular proliferation. Quantitative data of this study demonstrated a gradual loss of meiotic and pos-meiotic germinal cells during the culture period, no decrease on Sertoli cells number and a good maintenance of the seminiferous tubules general architecture. The rate of A dark spermatogonia proliferation was very low and was independently of hormonal supplement. Spermatogonia A, spermatogonia B and pre-leptotene spermatocytes proliferation was higher during the first week of culture, and reached its highest values in response to hormonal stimulation. At the end of the first week occurred differentiation of BrdU-labelled pre-leptotene spermatocytes into zygotene and pachytene spermatocytes and the presence of FSH and testosterone promoted an acceleration of leptotene spermatocyte into pachytene spermatocyte, especially when the 50 U/L of FSH and 1μmol/L of testosterone concentration were used. FSH + T increased germ cell survival, spermatogonial proliferation and germinal cell differentiation, during the first week of culture. In this study it was possible to quantify, for the first time germinal cell proliferation and differentiation, using a high reproducible methodology which gave results similar to in vivo conditions

Behavior of prostate cancer cells in a nanohydroxyapatite/collagen bone scaffold

Prostate cancer (PCa) is the second leading cause of death among men in Europe and U.S. The metastatic dissemination pattern of PCa is unique, developing bone metastasis as the only site of progression, consequently with a prognosis very poor. The cancer cells interactions within the surrounding bone environment are critical for tumor growth and progression. Secreted protein, acidic and rich in cysteine (SPARC) is described to be involved in PCa cells migration and invasion into bone. Three-dimensional (3D) in vitro systems that are able to closely resemble the in vivo microenvironment are recently taking importance in cancer research. Original nanohydroxyapatite/collagen scaffolds were designed to resemble bone microenvironment in order to be applied as substitutes in bone defects and as potential biomaterials to mimic skeletal tumors. In fact, these 3D structures were cytocompatible and able to support osteoblast (MC3T3-E1) colonization and to promote bone ingrowth. Additionally, SPARC adsorption onto the scaffolds affected PC3 and LNCaP PCa cell lines behavior. PC3 cells were found to adapt and colonize the scaffolds, differing from LNCaP where cells underwent morphogenic changes and grew as clusters. Furthermore, for the tested SPARC concentration, SPARC plays a role in retaining LNCaP cells at the latter time points while with PC3 cells no significant differences were observed. This characterization study is required to establish a bone model to provide new insights into the poorly understood PCa mechanisms of metastasis to bone and the generation of improved therapies.info:eu-repo/semantics/publishedVersio

Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cells

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA re-expression was only attained GSTP1 and APC. Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy

Regulation of histone H2A.Z expression is mediated by sirtuin 1 in prostate cancer

Histone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes’ upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2’-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patient

Enoxacin inhibits growth of prostate cancer cells and effectively restores microRNA processing

Prostate cancer (PCa) is one of the most incident malignancies worldwide. Although efficient therapy is available for early-stage PCa, treatment of advanced disease is mainly ineffective and remains a clinical challenge. microRNA (miRNA) dysregulation is associated with PCa development and progression. In fact, several studies have reported a widespread downregulation of miRNAs in PCa, which highlights the importance of studying compounds capable of restoring the global miRNA expression. The main aim of this study was to define the usefulness of enoxacin as an anti-tumoral agent in PCa, due to its ability to induce miRNA biogenesis in a TRBP-mediated manner. Using a panel of five PCa cell lines, we observed that all of them were wild type for the TARBP2 gene and expressed TRBP protein. Furthermore, primary prostate carcinomas displayed normal levels of TRBP protein. Remarkably, enoxacin was able to decrease cell viability, induce apoptosis, cause cell cycle arrest, and inhibit the invasiveness of cell lines. Enoxacin was also effective in restoring the global expression of miRNAs. This study is the first to show that PCa cells are highly responsive to the anti-tumoral effects of enoxacin. Therefore, enoxacin constitutes a promising therapeutic agent for PCa

Phenotypic impact of deregulated expression of class I histone deacetylases in urothelial cell carcinoma of the bladder

Deregulated expression of histone deacetylases (HDACs) has been implicated in tumorigenesis. Herein, we investigated class I HDACs expression in bladder urothelial cell carcinoma (BUCC), its prognostic value and biological significance. Significantly increased transcript levels of all HDACs were found in BUCC compared to 20 normal mucosas, and these were higher in lower grade and stage tumors. Increased HDAC3 levels were associated with improved patient survival. SiRNA experiments showed decrease cell viability and motility, and increased apoptosis. We concluded that class I HDACs play an important role in bladder carcinogenesis through deregulation of proliferation, migration and apoptosis, constituting putative therapeutic targets.info:eu-repo/semantics/publishedVersio

Relatório estágio profissional

Relatório final do estágio profissionalizante do 6º ano

Histone Posttranslational Modifications and Chromatin Remodelers in Prostate Cancer

Prostate cancer (PCa) is one of the most incident and prevalent cancers in men worldwide and a leading cause of cancer-related morbidity and mortality. Epigenetics plays an important role in prostate carcinogenesis, namely abnormal expression of histone modifier enzymes and related chromatin modifications. Notwithstanding, the limited knowledge of the specific role of deregulated activity/expression of histone modifiers and respective histone modifications hinders their use in clinical management, particularly for PCa detection and prognostication. Due to the limitations in the analysis of global patterns of histone modifications, few studies reported on the biomarker potential of these epigenetic marks. Importantly, the reversible nature of histone modifications makes them a promising therapeutic option for this malignancy. Indeed, over the past years, molecules with inhibitory activity upon the epigenetic machinery have been developed and are currently under evaluation in clinical trials to test their effectiveness.info:eu-repo/semantics/publishedVersio

Anti-neoplastic properties of hydralazine in prostate cancer

Prostate cancer (PCa) is a major cause of cancer-related morbidity and mortality worldwide. Although early disease is often efficiently managed therapeutically, available options for advanced disease are mostly ineffective. Aberrant DNA methylation associated with gene-silencing of cancer-related genes is a common feature of PCa. Therefore, DNA methylation inhibitors might constitute an attractive alternative therapy. Herein, we evaluated the anti-cancer properties of hydralazine, a non-nucleoside DNA methyltransferases (DNMT) inhibitor, in PCa cell lines. In vitro assays showed that hydralazine exposure led to a significant dose and time dependent growth inhibition, increased apoptotic rate and decreased invasiveness. Furthermore, it also induced cell cycle arrest and DNA damage. These phenotypic effects were particularly prominent in DU145 cells. Following hydralazine exposure, decreased levels of DNMT1, DNMT3a and DNMT3b mRNA and DNMT1 protein were depicted. Moreover, a significant decrease in GSTP1, BCL2 and CCND2 promoter methylation levels, with concomitant transcript re-expression, was also observed. Interestingly, hydralazine restored androgen receptor expression, with upregulation of its target p21 in DU145 cell line. Protein array analysis suggested that blockage of EGF receptor signaling pathway is likely to be the main mechanism of hydralazine action in DU145 cells. Our data demonstrate that hydralazine attenuated the malignant phenotype of PCa cells, and might constitute a useful therapeutic tool