Browsing by Author "Jacques, Filipa"
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- Effects of methanolic and aqueous Carica Papaya leaf extracts in transcriptional and miRNA-mediated regulation of fetal hemoglobinPublication . Jacques, Filipa; Delgadinho, Mariana; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Introduction: Hemoglobinopathies are genetic blood disorders characterized by abnormal hemoglobin that disrupts oxygen transport and leads to diverse health complications. Raising fetal hemoglobin (HbF) levels is one possible therapeutic approach. But the current main therapy based on the use of hydroxyurea (HU), faces limitations related to cost and safety, especially in underserved areas. To overcome these constraints, researchers are investigating natural alternatives like Carica papaya (CP) leaf extracts to boost HbF production with fewer side effects. Methodology: We conducted a comparative study using aqueous (AECP) and methanolic (MECP) CP leaf extracts to evaluate their impact on HbF regulation in K562 myeloid cells. Gene expression changes were assessed using RT-qPCR after 24-hour exposure to varying extract concentrations (0,05 μg/ml, 0,5μg/ml, and 5μg/ml). Results: The transcriptional analysis of hemoglobin genes (HBA, HBB, and HBG1), as well as that of its epigenetic regulators (HDAC1, HDAC2, HDAC3, DNMT1 and DNMT3b), transcription inhibitors (BCL11A and KLF1), transcription activators (MYC and HIC2) and miRNA-mediated regulators (miR-30a-3p, miR-29C-3p and miR-148b-3p), has revealed distinct expression patterns in K562 cells exposed to AECP when compared to MECP. While the expression levels of some genes were affected by one extract, other genes were affected by the other extract and some remained unaffected suggesting that the concentration and specific composition of AECP or MECP have a different impact on hemoglobin expression. Discussion: The differential effects of MECP and AECP exposure on hemoglobin-related genes expressed in K562 cells indicate that complex regulatory mechanisms underlie their action. The concentration-dependent responses observed emphasize the need for further research to comprehend their implications in hemoglobinopathies. Overall, this study highlights the potential of extracts of CP leaf as modulators of HbF gene expression in K562 cells. Further investigation into their gene modulation mechanisms is warranted.
- Effects of methanolic and aqueous Carica Papaya leaf extracts in transcriptional and miRNA-mediated regulation of fetal hemoglobinPublication . Jacques, Filipa; Canteiro, Beatriz; Mendes, Maria; Delgadinho, Mariana; Oliveira, Ketlyn; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Sickle cell disease (SCD) is a genetic blood disorder caused by mutations in β-globin gene that affect the shape and transport of red blood cells in blood vessels, leading to various clinical complications. The pharmacological reactivation of Fetal Hemoglobin (HbF) through compounds such as Hydroxyurea (HU), is one of the available treatments, however, their safety concerns and expensive cost in low- and middle-income countries limit their use. In this context, it is essential to study novel HbF-inducing compounds that have scarcer adverse effects and can be widely available, such as Carica papaya extracts, a medicinal plant with anti-oxidant and anti-inflammatory properties. Therefore, the main aim of this work is to evaluate the effects of Carica Papaya leaf extracts (CPLE) in HbF reactivation. More specifically, we started by evaluating the effect of a methanolic CPLE extract in K562 cells (human immortalized myeloid leukemia cell line) at the proliferation rate and viability of the K562 cell line, compared to HU exposure. Subsequently, we analyzed the expression levels of HBG1 and HBG2 genes and that of their transcriptional and miRNA-mediated regulators. To achieve these goals, the K562 cell line was first exposed for 72 hours to CPMLE at 500 μg/mL and for 24 hours to EMFCP (0.5; 50, and 100 μg/mL) and to HU (25 μg/mL). After exposure to natural compounds, the effects of gene expression were quantified from total RNA using RT-qPCR. The results have indicated that cell proliferation and viability were affected by CPMLE only at the concentration of 500 μg/ml, with no effects being observed at the lower concentrations analyzed. Upon analysis of the expression levels of globins (HBA, HBB, HBG1, and HBG2), HbF regulatory genes (MYB, KLF1, BCL11A, and BGLT3), and miRNAs involved in the regulation of HbF we could observe more significant differences for the lower concentrations of extracts used, namely at the concentration of 0.5 μg/ml. As such we have decided to titrate down the concentration of methanolic leaf extracts used and test the exposure to aqueous leaf extracts which possess different biological compounds that might lead to a differential regulation of the genes under analysis. Our preliminary results have revealed that at the concentrations of 0,05 ug/ml; 0,5 ug/ml and 5 ug/ml the cell viability and proliferation rates were not affected either for methanolic or aqueous extracts. We are currently analyzing the expression levels of target and regulatory genes to determine the effect of both types of leaf extracts on regulators of HbF expression.
- Effects of Quercetin in transcriptional and post-transcriptional regulation of fetal hemoglobinPublication . Canteiro, Beatriz; Mendes, Maria; Jacques, Filipa; Delgadinho, Mariana; Oliveira, Ketlyn; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Hemoglobinopathies are a group of inherited blood disorders that primarily affect red blood cells. The most common type is known as sickle cell anemia (SCA). It is characterized by mutations in the HBB gene, which encodes the β-subunit of human hemoglobin, giving rise to hemoglobin S (HbS). When deoxygenated, HbS polymerizes in the red blood cell, giving it a sickle shape and making it rigid and fragile. Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Currently, therapies that induce HbF are promising, such as hydroxyurea (HU). However, due to high costs for underdeveloped countries and the adverse side effects, it is important to test alternative products and develop new compounds, such as Quercetin, a natural flavonoid present in plants that has antioxidant and anti-inflammatory properties.
- Effects of quercetin in transcriptional and post-transcriptional regulation of fetal hemoglobinPublication . Canteiro, Beatriz; Mendes, Maria; Jacques, Filipa; Delgadinho, Mariana; Oliveira, Ketlyn; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Sickle Cell anemia (SCA) is a hereditary hemoglobinopathy with the formation of hemoglobin S, associated with severe health outcomes. Currently, induction of fetal hemoglobin (HbF) is one of the most promising therapeutic strategies. Here we aimed to assess the potential of the natural compound Quercetin, in transcriptional expression of globin and HbF regulatory/silencing genes. In this study, the K562 cell line was used as an SCA model. Cells were exposed to Quercetin at final concentrations of 0.2 and 20 µM, and Hydroxyurea (25 µg/mL) was used as a positive control. Cell viability and proliferation were assessed through trypan blue exclusion assay. Transcriptional expression was performed by RT-qPCR using specific primers. Significant differences were analyzed using a t-test. No cytotoxic effects were observed following exposure to Quercetin. The transcriptional analysis demonstrated that Quercetin affects mRNA levels of HbF regulatory/silencing genes with associated downregulation of BCL11A, MYB, KLF1, and HBB and upregulation of HBG and BGLT3, as well as alterations in the expression of miRNAs involved in HbF post-transcriptional regulation. Our results sustain Quercetin's potential as an HbF inducer with associated upregulation of HbF-activators and decreased expression of HbF-inhibitors. These data support the need for further studies in order to confirm the potential of this compound as a new therapeutic option for β -hemoglobinopathies in the future.