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Abstract(s)
Introdução: As β–hemoglobinopatias são uma das doenças autossómicas recessivas mais comuns em todo o mundo. A indução farmacológica da hemoglobina fetal (HbF) é uma estratégia terapêutica promissora e a hidroxiureia (HU) é um fármaco aprovado pela Agência Europeia do Medicamento para este efeito. No entanto, é necessária a identificação de novos agentes com maior atividade indutora, menor toxicidade celular e custo.
Mecanismos como a metilação do DNA e acetilação das histonas podem regular a expressão gênica e os compostos naturais são potentes moduladores epigenéticos. A genisteína (GN), um flavonoide da soja, e a epigalocatequina-3-galato (EGCG), principal polifenol do chá verde, foram associados a efeitos epigenéticos e previamente identificados como potenciais indutores da HbF sem efeitos citotóxicos em células K562. Objetivo: Avaliar a modulação epigenética da EGCG e GN na potencial indução da HbF. Material e Métodos: O RNA total de células da linhagem K562 previamente expostas durante 72 ou 96 horas à GN e EGCG em 3 concentrações (100, 250 e 500 ƞg/mL) e à HU a 25 μg/mL, foi quantificado e convertido a cDNA. Por sua vez, este foi usado para avaliar os seus efeitos transcricionais em moduladores epigenéticos (DNMT1, DNMT3A, DNMT3B e HDAC de classe I) pelo qRT-PCR com primers específicos. Resultados: A EGCG afeta maioritariamente a transcrição da HDAC2 e HDAC8 em ambos os tempos de exposição, enquanto a GN influencia os níveis de expressão génica da metilação às 72 horas (especialmente da DNMT1) e desacetilação nos dois tempos (HDAC1, HDAC3 e HDAC8). A análise transcricional de ambos revela uma dose resposta não
monotónica (DRNM). A HU aparenta ter efeitos divergentes, com impacto transcricional na metilação em ambos os tempos de exposição (DNMT3A) e da desacetilação às 96 horas (HDAC1 e HDAC8). Conclusão: Sucintamente, os nossos resultados apoiam a hipótese que a EGCG e a GN podem atuar como moduladores epigenéticos perante o seu potencial indutor da HbF e serem eventualmente, futuros candidatos a novos agentes terapêuticos nas hemoglobinopatias.
ABSTRACT - Introduction: β– hemoglobinopathies are one of the most common autossomic recessive diseases in the world. The pharmacologic induction of fetal hemoglobin (HbF) is a promising therapeutic strategy and hydroxyurea (HU) is an approved drug by the European Medicine Agency with this purpose. However, it’s necessary to identify new agents with higher HbF inducing activity, less toxic, and expensive. Mechanisms like DNA methylation and histone acetylation might regulate the genetic expression and natural compounds are powerful epigenetic modulators. Genistein (GN), a soy flavonoid, and Epigallocatechin-3-gallate (EGCG), a major green tea component, were associated with epigenetic effects and previously identified as potential HbF inductors without cytotoxic effects in K562 cells. Objective: Evaluate the epigenetic modulation by EGCG and GN in their potential HbF induction. Material and Methods: Total RNA from cells of the K562 lineage previously exposed for 72 or 96 hours to GN and EGCG in 3 concentrations (100, 250, and 500 ƞg/mL) and to HU at 25 μg/mL was quantified and converted to cDNA. In turn, this one was used to evaluate their transcriptional effects in epigenetic modulators (DNMT1, DNMT3A, DNMT3B e class I HDAC) by qRT-PCR using specific primers. Results: EGCG mostly affects transcription of HDAC2 and HDAC8 at both exposure times, while GN influences the genetic expression levels of methylation at 72 hours (especially DNMT1) and deacetylation at both times (HDAC1, HDAC3, and HDAC8). The transcriptional analysis of both reveals a non-monotonic dose-response (NMDR). HU appears to have divergent effects, with an impact on methylation transcription at both exposure times (DNMT3A) and deacetylation at 96 hours (HDAC1 and HDAC8). Conclusion: Succinctly, our results support the hypothesis that EGCG and GN might act as epigenetic modulators in relation to their potential HbF induction and are might be future candidates as new therapeutic agents in hemoglobinopathies.
ABSTRACT - Introduction: β– hemoglobinopathies are one of the most common autossomic recessive diseases in the world. The pharmacologic induction of fetal hemoglobin (HbF) is a promising therapeutic strategy and hydroxyurea (HU) is an approved drug by the European Medicine Agency with this purpose. However, it’s necessary to identify new agents with higher HbF inducing activity, less toxic, and expensive. Mechanisms like DNA methylation and histone acetylation might regulate the genetic expression and natural compounds are powerful epigenetic modulators. Genistein (GN), a soy flavonoid, and Epigallocatechin-3-gallate (EGCG), a major green tea component, were associated with epigenetic effects and previously identified as potential HbF inductors without cytotoxic effects in K562 cells. Objective: Evaluate the epigenetic modulation by EGCG and GN in their potential HbF induction. Material and Methods: Total RNA from cells of the K562 lineage previously exposed for 72 or 96 hours to GN and EGCG in 3 concentrations (100, 250, and 500 ƞg/mL) and to HU at 25 μg/mL was quantified and converted to cDNA. In turn, this one was used to evaluate their transcriptional effects in epigenetic modulators (DNMT1, DNMT3A, DNMT3B e class I HDAC) by qRT-PCR using specific primers. Results: EGCG mostly affects transcription of HDAC2 and HDAC8 at both exposure times, while GN influences the genetic expression levels of methylation at 72 hours (especially DNMT1) and deacetylation at both times (HDAC1, HDAC3, and HDAC8). The transcriptional analysis of both reveals a non-monotonic dose-response (NMDR). HU appears to have divergent effects, with an impact on methylation transcription at both exposure times (DNMT3A) and deacetylation at 96 hours (HDAC1 and HDAC8). Conclusion: Succinctly, our results support the hypothesis that EGCG and GN might act as epigenetic modulators in relation to their potential HbF induction and are might be future candidates as new therapeutic agents in hemoglobinopathies.
Description
Mestrado em Tecnologias Clínico-Laboratoriais
Keywords
Epigalocatequina-3-galato Genisteína Hemoglobina fetal Moduladores epigenéticos Hidroxiureia Epigalocatechin-3-galate Epigenetic modulator Fetal hemoglobin Genistein Hemoglobinopaties Hydroxyurea
Citation
Santos RS. Uso de epigalocatequina-3-galato e genisteína como potenciais indutores epigenéticos da hemoglobina fetal [dissertation]. Lisboa: Escola Superior de Tecnologia da Saúde de Lisboa/Instituto Politécnico de Lisboa; 2021.
Publisher
Instituto Politécnico de Lisboa, Escola Superior de Tecnologia da Saúde de Lisboa