Mutações no gene da 21-hidroxilase e sua correlação fenotípica

Mestrado em Biologia Molecular e CelularA Hiperplasia Supra-renal Congénita (HSC) constitui um grupo de doenças hereditárias decorrentes da deficiência de enzimas necessárias para a biossíntese dos esteróides corticais. 90 a 95% dos casos de HSC decorrem da deficiência da 21-hidroxilase, codificada pelo gene CYP21. Mutações neste gene são correlacionadas com as diferentes formas clínicas da doença. O objectivo deste estudo foi determinar o espectro mutacional do CYP21 em 39 indivíduos e correlacioná-lo com o fenótipo apresentado. 17 indivíduos apresentam a forma não clássica (FNC) da HSC, enquanto os restantes 22 apresentam sinais suspeitos da referida forma clínica. Para tal, após optimização das técnicas utilizadas neste estudo, procedeu-se à amplificação do gene CYP21 por PCR a partir de DNA genómico isolado dos leucócitos do sangue periférico dos indivíduos em estudo. Os produtos de amplificação foram sequenciados e o espectro mutacional foi detectado e analisado tendo em consideração a sequência normal do gene CYP21. Foram identificadas 11 mutações, estando apenas 7 associadas a alterações funcionais na actividade da 21-hidroxilase. As mutações encontradas nos indivíduos do grupo da FNC podem ser correlacionadas com o fenótipo demonstrado, sendo a alteração V281L a mais frequente (64,7%). Na maioria dos casos do grupo com suspeita de HSC não foram detectadas mutações valorizáveis no CYP21 (95,45%). Estes resultados apontam para uma associação entre a mutação V281L em heterozigotia e a forma não clássica da HSC, sendo necessário aumentar amostra para uma possível confirmação. A maioria dos indivíduos com suspeita da forma não clássica da HSC não apresenta a referida mutação, resultado que sugere que outros factores estão envolvidos na manifestação deste fenótipo. ABSTRACT: Congenital Adrenal Hyperplasia (CAH) is a group of inheritable diseases associated with a lack of important enzymes in the biosynthesis of corticosteroids. 90 to 95% of the CAH cases are caused by 21-hydroxylase deficiency, an enzyme encoded by the CYP21 gene. CYP21 mutations are correlated with different phenotypic presentations of the disease. The aim of this study was to determine the mutational spectrum of CYP21 in 39 patients and correlate it with their clinical condition. 17 patients presented the non-classical form (NCF) of CAH and 22 had suspected symptoms of the same form. After optimization of the technical procedure, isolation of the genomic DNA from peripheral blood was made and the CYP21 gene was amplified by Multiplex-PCR. The amplification products were then sequenced to determine the mutational spectrum of the CYP21 gene. We detected 11 mutations, but only 7 were related to a decrease of the 21-hydroxylase activity. The patients of NCF group presented mutations that can be correlated with their phenotype, being V281L the most frequent (64,7%). The great majority of the suspected CAH cases did not present mutations with functional significance (95,45%). These results suggest an association of heterozigoty of V281L with the non-classical form of the CAH, but it will be necessary a larger sample for confirmation. The majority of patients with suspected non-classical form of CAH did not present this mutation, which points to other factors be involved

In vivo models in cancer research

Cancers figure among the leading causes of morbidity and mortality worldwide. Over the last decade there has been an extraordinary increase in our knowledge of the fundamental molecular processes that are involved in the development of cancer and its response to treatment. Studies in tissue culture have multiplied our acquaintance of cancer cell pathophysiology, mechanisms of transformation and strategies of survival of cancer cell lines, revealing therapeutically exploitable differences to normal cells. However, tumors are heterogeneous, structurally complex and result from an evolving crosstalk between different cell types and its surrounding tissue. A full elucidation of events occurring inside the cancer microenvironment is fundamental more effective therapies. Experiment in vivo models remains essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. This presentation will summarize currently available in vivo models of cancer, define the limitations and advantages of each modeling option and suggest the basis with which particular models should be used to answer a specific scientific question.info:eu-repo/semantics/publishedVersio

The Impact of [C16Pyr][Amp] on the Aggressiveness in Breast and Prostate Cancer Cell Lines

Breast (BrCa) and prostate (PCa) cancers are the most common malignancies in women and men, respectively. The available therapeutic options for these tumors are still not curative and have severe side e↵ects. Therefore, there is an urgent need for more e↵ective antineoplastic agents. Herein, BrCa, PCa, and benign cell lines were treated with two ionic liquids and two quinoxalines and functional experiments were performed—namely cell viability, apoptosis, cytotoxicity, and colony formation assays.info:eu-repo/semantics/publishedVersio

Natural and synthetic compounds as anti-cancer therapies for breast and prostate cancer

Breast Cancer (BCa) and Prostate Cancer (PCa) are the most prevalent tumors in females and males, respectively, being the main causes of cancer morbidity and mortality, worldwide. Currently, there is no available curative treatment for the hormone-resistant tumors, being the development of new therapeutic strategies using innovative anticancer agents imperative. Thus, the main goal of this work was to evaluate the anticancer properties of natural and synthetic compounds through in vitro assays.info:eu-repo/semantics/publishedVersio

Molecular impact of [C16Pyr][Amp] treatment on breast and prostate cancer cell lines

Prostate Cancer (PCa) and Breast Cancer (BCa) are the leading causes of cancer morbidity and mortality, worldwide, when diagnosed in advanced stages of the disease. Currently available therapies have limited curative effect, leading to the progression to highly aggressive hormone-resistant phenotypes. Thus, the development of new anti-tumor agents becomes imperative. Ionic liquids are organic salts with anti-neoplastic activity and have been studied in the pharmaceutical industry. Previous work of our team demonstrated that the ionic liquid [C16Pyr][Amp] has significant anti-tumor properties in PCa and BCa cell lines. However, the main cellular pathways affected were not characterized. Therefore, the aim of this work was to explore the molecular impact of [C16Pyr][Amp] treatment in order to identify relevant genes that have altered expression upon treatment and that can justify the anti-cancer effect observed in the in vitro assays.info:eu-repo/semantics/publishedVersio

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

Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication

Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of three genes-SMYD2, SETD3, and NO66-was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity (AUC=0.959), and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC: 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.This study was funded by research grants from Research Center of Portuguese Oncology Institute – Porto (CI-IPOP 4-2012) and European Community’s Seventh Framework Program – Grant number FP7-HEALTH-F5-2009-241783. ASP-L and FQV are and were supported by FCT-Fundação para a Ciência e a Tecnologia grants (SFRH/SINTD/94217/2013 and SFRH/ BD/70564/2010, respectively)

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

Deregulated expression of selected histone methylases and demethylases in prostate carcinoma

Prostate cancer (PCa), a leading cause of cancer-related morbidity and mortality, arises through the acquisition of genetic and epigenetic alterations. Deregulation of histone methyltransferases (HMTs) or demethylases (HDMs) has been associated with PCa development and progression. However, the precise influence of altered HMTs or HDMs expression and respective histone marks in PCa onset and progression remains largely unknown. To clarify the role of HMTs and HDMs in prostate carcinogenesis, expression levels of 37 HMTs and 20 HDMs were assessed in normal prostate and PCa tissue samples by RT-qPCR. SMYD3, SUV39H2, PRMT6, KDM5A, and KDM6A were upregulated, whereas KMT2A-E (MLL1-5) and KDM4B were downregulated in PCa, compared with normal prostate tissues. Remarkably, PRMT6 was the histone modifier that best discriminated normal from tumorous tissue samples. Interestingly, EZH2 and SMYD3 expression levels significantly correlated with less differentiated and more aggressive tumors. Remarkably, SMYD3 expression levels were of independent prognostic value for the prediction of disease-specific survival of PCa patients with clinically localized disease submitted to radical prostatectomy. We concluded that expression profiling of HMTs and HDMs, especially SMYD3, might be of clinical usefulness for the assessment of PCa patients and assist in pre-therapeutic decision-making