Browsing by Author "Henrique, Rui"
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- Anti-neoplastic properties of hydralazine in prostate cancerPublication . Pinho dos Santos Graça, Maria Inês; Sousa, Elsa Joana; Costa-Pinheiro, Pedro; Quintela Vieira, Ana Filipa; Torres-Ferreira, Jorge; Martins, Maria Gabriela; Henrique, Rui; Jeronimo, CarmenProstate 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.
- Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cellsPublication . Pinho dos Santos Graça, Maria Inês; Sousa, Elsa Joana; Baptista, Tiago; Almeida, Mafalda; Ramalho-Carvalho, João; Palmeira, Carlos; Henrique, Rui; Jerónimo, CarmenCurrent 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. 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. 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.
- Comprehensive Analysis of Secreted Protein, Acidic and Rich in Cysteine in Prostate Carcinogenesis: Development of a 3D Nanostructured Bone-Like ModelPublication . Ribeiro, Nilza; Costa-Pinheiro, Pedro; Henrique, Rui; Gomez-Lazaro, Maria; Pereira, Marisa P.; Mansur, Alexandra A. P.; Mansur, Herman S.; Jerónimo, Carmen; Sousa, Susana R.; Monteiro, Fernando J.Most aggressive prostate cancer (PCa) types tend to metastasize frequently to bone and SPARC, a matricellular protein, might participate in such biological processes. The objective of this study was to evaluate the effect of SPARC in prostate carcinogenesis and bone metastization. This was explored assessing the morphology, metabolic activity and SPARC expression of different PCa cell lines resembling different stages of carcinogenesis, using a 3D bone-biomimetic model (collagen nanofibers/nanohydroxyapatite) grafted with SPARC. Our findings highlight distinct cellular behavior depending on cell type and presence of exogenous SPARC. In fact, SPARC addition contributed to the survival and significant growth of a non-bone metastatic PCa cell line (LNCaP) on bone-like biomaterial. Moreover, SPARC expression levels were evaluated in a series of prostatic tissues, comparing normal prostate, pre-neoplastic prostate intraepithelial neoplasias and overtly malignant tumors, and also metastasis to its correspondent primary prostate tumors, ascertaining potential association between SPARC and clinicopathological data. Remarkably, SPARC was overexpressed in patients with higher Gleason Score, indicating tumors with poor prognosis, as well as in metastasis, particularly from bone sites, compared with their respective primary tumors. The results suggest a potential role of SPARC as a clinical target on PCa, due to its association with bone metastization.
- Deregulated expression of selected histone methylases and demethylases in prostate carcinomaPublication . Quintela Vieira, Ana Filipa; Costa-Pinheiro, Pedro; Ramalho-Carvalho, João; Menezes, Francisco Duarte; Antunes, Luis; Carneiro, Isa; Oliveira, Jorge; Henrique, Rui; Jeronimo, CarmenProstate 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.
- Diffuse reflectance and machine learning techniques to differentiate colorectal cancer ex vivoPublication . Fernandes, Luís; Carvalho, Sónia; Carneiro, Isa; Henrique, Rui; Tuchin, Valery V.; Oliveira, Hélder P.; Oliveira, LuísIn this study, we used machine learning techniques to reconstruct the wavelength dependence of the absorption coefficient of human normal and pathological colorectal mucosa tissues. Using only diffuse reflectance spectra from the ex vivo mucosa tissues as input to algorithms, several approaches were tried before obtaining good matching between the generated absorption coefficients and the ones previously calculated for the mucosa tissues from invasive experimental spectral measurements. Considering the optimized match for the results generated with the multilayer perceptron regression method, we were able to identify differentiated accumulation of lipofuscin in the absorption coefficient spectra of both mucosa tissues as we have done before with the corresponding results calculated directly from invasive measurements. Considering the random forest regressor algorithm, the estimated absorption coefficient spectra almost matched the ones previously calculated. By subtracting the absorption of lipofuscin from these spectra, we obtained similar hemoglobin ratios at 410/550 nm: 18.9-fold/9.3-fold for the healthy mucosa and 46.6-fold/24.2-fold for the pathological mucosa, while from direct calculations, those ratios were 19.7-fold/10.1-fold for the healthy mucosa and 33.1-fold/17.3-fold for the pathological mucosa. The higher values obtained in this study indicate a higher blood content in the pathological samples used to measure the diffuse reflectance spectra. In light of such accuracy and sensibility to the presence of hidden absorbers, with a different accumulation between healthy and pathological tissues, good perspectives become available to develop minimally invasive spectroscopy methods for in vivo early detection and monitoring of colorectal cancer.The application of machine learning methods to noninvasivelike diffuse reflectance spectra allowed us to reconstruct the absorption coefficient spectra of human healthy and pathological mucosa tissues from the colorectal wall. Consequently, we were able to obtain differentiated blood and pigment content in both tissues, which can be used for the development of new noninvasive diagnostic methods for colorectal cancer.
- DNA methylation as a triage marker for colposcopy referral in HPV-based cervical cancer screening: a systematic review and meta-analysisPublication . Salta, Sofia; Lobo, João; Magalhães, Bruno; Henrique, Rui; Jerónimo, CarmenScreening plays a key role in secondary prevention of cervical cancer. High-risk human papillomavirus (hrHPV) testing, a highly sensitive test but with limited specificity, has become the gold standard frontline for screening programs. Thus, the importance of effective triage strategies, including DNA methylation markers, has been emphasized. Despite the potential reported in individual studies, methylation markers still require validation before being recommended for clinical practice. This systematic review and meta-analysis aimed to evaluate the performance of DNA methylation-based biomarkers for detecting high-grade intraepithelial lesions (HSIL) in hrHPV-positive women. Hence, PubMed, Scopus, and Cochrane databases were searched for studies that assessed methylation in hrHPV-positive women in cervical scrapes. Histologically confirmed HSIL was used as endpoint and QUADAS-2 tool enabled assessment of study quality. A bivariate random-effect model was employed to pool the estimated sensitivity and specificity as well as positive (PPV) and negative (NPV) predictive values. Twenty-three studies were included in this meta-analysis, from which cohort and referral population-based studies corresponded to nearly 65%. Most of the women analyzed were Dutch, and CADM1, FAM19A4, MAL, and miR124-2 were the most studied genes. Pooled sensitivity and specificity were 0.68 (CI 95% 0.63–0.72) and 0.75 (CI 95% 0.71–0.80) for cervical intraepithelial neoplasia (CIN) 2+ detection, respectively. For CIN3+ detection, pooled sensitivity and specificity were 0.78 (CI 95% 0.74–0.82) and 0.74 (CI 95% 0.69–0.78), respectively. For pooled prevalence, PPV for CIN2+ and CIN3+ detection were 0.514 and 0.392, respectively. Furthermore, NPV for CIN2+ and CIN3+ detection were 0.857 and 0.938, respectively. This meta-analysis confirmed the great potential of DNA methylation-based biomarkers as triage tool for hrHPV-positive women in cervical cancer screening. Standardization and improved validation are, however, required. Nevertheless, these markers might represent an excellent alternative to cytology and genotyping for colposcopy referral of hrHPV-positive women, allowing for more cost-effective screening programs.
- Downregulation of miR-130b~301b cluster is mediated by aberrant promoter methylation and impairs cellular senescence in prostate cancerPublication . Ramalho-Carvalho, João; Pinho dos Santos Graça, Maria Inês; Gomez, Antonio; Oliveira, Jorge; Henrique, Rui; Esteller, Manel; Jerónimo, CarmenNumerous DNA-damaging cellular stresses, including oncogene activation and DNA-damage response (DDR), may lead to cellular senescence. Previous observations linked microRNA deregulation with altered senescent patterns, prompting us to investigate whether epigenetic repression of microRNAs expression might disrupt senescence in prostate cancer (PCa) cells. Differential methylation mapping in prostate tissues was carried using Infinium HumanMethylation450 BeadChip. After validation of methylation and expression analyses in a larger series of prostate tissues, the functional role of the cluster miR-130b~301b was explored using in vitro studies testing cell viability, apoptosis, invasion and DNA damage in prostate cancer cell lines. Western blot and RT-qPCR were performed to support those observations. We found that the miR-130b~301b cluster directs epigenetic activation of cell cycle inhibitors required for DDR activation, thus stimulating the senescence-associated secretory phenotype (SASP). Furthermore, overexpression of miR-130b~301b cluster markedly reduced the malignant phenotype of PCa cells. Altogether, these data demonstrate that miR-130b~301b cluster overexpression might effectively induce PCa cell growth arrest through epigenetic regulation of proliferation-blocking genes and activation of cellular senescence.
- Enhanced Ultraviolet Spectroscopy by Optical Clearing for Biomedical ApplicationsPublication . Carneiro, Isa; Carvalho, Sonia; Henrique, Rui; Selifonov, Alexey; Oliveira, Luís; Tuchin, Valery V.In this paper, we describe the combination of ultraviolet (UV) spectroscopy with the optical clearing technique to induce new tissue windows, evaluate their efficiency, study the diffusion properties of agents and discriminate cancer. The use of highly concentrated glycerol solutions has induced high efficiency clearing effects in the UV, both in human colorectal and gingival tissues. The protein dissociation rate obtained for colorectal tissues was approximately 3 times higher in pathological than in normal mucosa and the kinetics of diffuse reflectance in the UV allowed to estimate the diffusion coefficient for water in gingival mucosa at glycerol action as (1.78 ± 0.26) × 10 -6 cm 2 /s.
- Enoxacin inhibits growth of prostate cancer cells and effectively restores microRNA processingPublication . Sousa, Elsa Joana; Pinho dos Santos Graça, Maria Inês; Baptista, Tiago; Quintela Vieira, Ana Filipa; Palmeira, Carlos; Henrique, Rui; Jeronimo, CarmenProstate 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.
- Epigenetic disruption of miR-130a promotes prostate cancer by targeting SEC23B and DEPDC1Publication . Ramalho-Carvalho, João; Martins, João Barbosa; Cekaite, Lina; Sveen, Anita; Torres-Ferreira, Jorge; Pinho dos Santos Graça, Maria Inês; Costa-Pinheiro, Pedro; Eilertsen, Ina Andrassy; Antunes, Luís; Oliveira, Jorge; Lothe, Ragnhild A.; Henrique, Rui; Jerónimo, CarmenMicroRNAs (miRNAs) are small, non-coding RNAs that mediate post-transcriptional gene silencing, fine tuning gene expression. In an initial screen, miRNAs were found to be globally down-regulated in prostate cancer (PCa) cell lines and primary tumors. Exposure of PCa cell lines to a demethylating agent, 5-Aza-CdR resulted in an increase in the expression levels of miRNAs in general. Using stringent filtering criteria miR-130a was identified as the most promising candidate and selected for validation analyses in our patient series. Down-regulation of miR-130a was associated with promoter hypermethylation. MiR-130a methylation levels discriminated PCa from non-malignant tissues (AUC=0.956), and urine samples revealed high specificity for non-invasive detection of patients with PCa (AUC=0.89). Additionally, repressive histone marks were also found in the promoter of miR-130a. Over-expression of miR-130a in PCa cells reduced cell viability and invasion capability, and increased apoptosis. Putative targets of miR-130a were assessed by microarray expression profiling and DEPD1C and SEC23B were selected for validation. Silencing of both genes resembled the effect of over-expressing miR-130a in PCa cells. Our data indicate that miR-130a is an epigenetically regulated miRNA involved in regulation of key molecular and phenotypic features of prostate carcinogenesis, acting as a tumor suppressor miRNA.