Influência do tratamento com melatonina sobre mecanismos celulares de adaptação redox em células eritroleucêmicas K562
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Data
2021-04-28
Autores
Torres, Flaviene Felix
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Universidade Estadual Paulista (Unesp)
Resumo
A homeostase redox já foi descrita como “o meio áureo da vida saudável” e sua regulação pode ocorrer através do controle de atividade enzimática ou em nível transcricional. Importantes fatores de transcrição relacionados a esta homeostase são FoxO3 e Nrf2, ambos influenciados pela ação da melatonina (MEL). Esta indolamina e seus metabólitos constituem uma família química particularmente eficiente, com grande capacidade de combate ao estresse oxidativo, podendo atuar de forma direta contra os mesmos ou indireta, atuando na indução da expressão de enzimas antioxidantes. Um tipo celular constantemente exposto a insultos oxidativos é o eritrócito, dessa forma, o presente estudo utilizou a linhagem eritroleucêmica K562 induzidas a diferenciação em células eritróides. Assim, a presente proposta teve como objetivo investigar o possível efeito modulador da MEL sobre as vias de sinalização redox PI3K/AKT/FoxO3 e Keap1/Nrf2/ARE, em cultivo de células eritroleucêmicas K562 submetidas a indução de estresse oxidativo, favorecendo a manutenção da homeostase redox celular. Para tanto, avaliou-se a viabilidade, bem como os níveis de transcritos dos genes de importantes enzimas antioxidantes, utilizando células K562 em diferentes períodos de diferenciação eritroide como modelo experimental. Assim, o estudo contou com três amostras pseudoreplicadas acompanhadas durante cinco dias de acordo com os períodos de incubação, sendo as avaliações realizadas: antes do início (D0), no início de diferenciação (D2) e final da diferenciação (D4). Além disso, cada amostra foi dividida nos seguintes grupos: células eritroides sem indução de estresse oxidativo e tratamento antioxidante (Referência); células sob indução de estresse com peróxido de hidrogênio (100 µM H2O2); células tratadas com 1 nM (C1) e com 1 mM (C2) de MEL; por fim, dois conjuntos de células tratados com as mesmas concentrações de MEL associados com a indução do estresse (C1+ H2O2 e C2+ H2O2, respectivamente). Dentre os principais resultados, observou-se o reestabelecimento de níveis fisiológicos de Nrf2 em ambas concentrações de MEL, sob indução de estresse com peróxido, quando comparado ao grupo sob a ação apenas do agente estressor, sugerindo um efeito citoprotetor. A menor concentração (C1) relacionou-se com a indução da expressão de genes antioxidantes por intermédio da via NRF2-ARE, quando comparados com suas respectivas referências. Este padrão foi observado no dia 0 para os genes da CAT, SOD1, PRXD1 e 6; No dia 2, foi constatado para CAT, PRDX1 e 6. Em relação ao dia 4, o mesmo padrão foi observado para SOD1, PRDX1 e 6, sugerindo, inclusive, um efeito protetor da MEL em relação a esta primeira peroxirredoxina, uma vez que os tratamentos utilizando MEL ocasionaram uma expressão semelhante aos níveis fisiológicos. Quanto a C2, esta relacionou-se com a indução da expressão de FOXO3, quando associada ou não a indução de estresse oxidativo; sendo que, no D4, C2 + H2O2 mostrou-se relacionada a uma redução de viabilidade celular possivelmente relacionada a ativação de vias apoptóticas. Dessa forma, os efeitos da administração de MEL nestas células apresentaram um padrão período e dose-dependentes contra o estresse oxidativo induzidos com H2O2, com ação direta sobre a detoxificação do agente estressor e indireta, visto que a via FoxO3 apresentou papel sugestivo de indução de vias apoptóticas, enquanto o fator de transcrição predominantemente responsável pela manutenção da homeostase redox nas células K562 foi o Nrf2. Dessa forma, o presente trabalho reforça a importância dessa indolamina na regulação da homeostase celular, sendo uma alternativa terapêutica promissora para doenças que apresentam um dano oxidativo exacerbado, devido ao seu potente efeito redutor.
Redox homeostasis has already been described as “the golden mean of healthy living” and its regulation can occur through the control of enzymatic activity or at the transcriptional level. Important transcription factors related to this homeostasis are FoxO3 and Nrf2, both influenced by the action of melatonin (MEL). This indolamine and its metabolites constitute a particularly efficient chemical family, with a great capacity to fight oxidative stress, which can act directly against them or indirectly, acting on the induction of the expression of antioxidant enzymes. A cell type constantly exposed to oxidative insults is the erythrocyte, so the present study used the erythroleukemic lineage K562 induced to differentiate into erythroid cells. Thus, this proposal aimed to investigate the possible modulating effect of MEL on the redox signaling pathways PI3K/AKT/FoxO3 and Keap1/Nrf2/ARE, in cultivation of K562 erythroleukemic cells subjected to oxidative stress induction, favoring the maintenance of cellular redox homeostasis. For this, the viability, as well as the transcript levels of genes of important antioxidant enzymes, were evaluated, using K562 cells in different periods of erythroid differentiation as an experimental model. Thus, the study had three pseudo-replicated samples followed for five days according to the incubation periods, the evaluations was carried out: before the start (D0), at the beginning of differentiation (D2) and end of differentiation (D4). In addition, each sample was divided into the following groups: erythroid cells without oxidative stress and antioxidant treatment (Reference); cells under stress induction by hydrogen peroxide (100 µM H2O2); cells treated with 1 nM (C1) and 1 mM (C2) MEL; finally, two sets of cells treated with the same concentrations of MEL associated with stress induction (C1 + H2O2 and C2 + H2O2, respectively). Among the main results, it was observed the restoration of physiological levels of Nrf2 in both concentrations of MEL, under stress induction with peroxide, when compared to the group under the action of only the stressor, suggesting a cytoprotective effect. The lowest concentration (C1) was related to the induction of the expression of antioxidant genes through the Nrf2-ARE pathway, when compared with their respective references. This pattern was observed on day 0 for the CAT, SOD1, PRXD1 and 6 genes; On day 2, it was found for CAT, PRDX1 and 6. In relation to day 4, the same pattern was observed for SOD1, PRDX1 and 6, even suggesting a protective effect of MEL in relation to this first peroxiredoxin, since the treatments using MEL caused an expression similar to the physiological levels. As for C2, it was related to the induction of FOXO3 expression, when associated or not with the induction of oxidative stress; and on D4, C2 + H2O2 was shown to be related to a reduction in cell viability possibly related to activation of apoptotic pathways. Thus, the effects of MEL administration in these cells showed a period and dose-dependent pattern against oxidative stress induced with H2O2, with direct action on the detoxification of the stressor and indirect, since the FoxO3 pathway had a suggestive role in inducing apoptotic pathways, while the transcription factor predominantly responsible for maintaining redox homeostasis in K562 cells was Nrf2. Thus, the present work reinforces the importance of this indolamine in the regulation of cellular homeostasis, being a promising therapeutic alternative for diseases that present an exacerbated oxidative damage, due to its potent reducing effect.
Redox homeostasis has already been described as “the golden mean of healthy living” and its regulation can occur through the control of enzymatic activity or at the transcriptional level. Important transcription factors related to this homeostasis are FoxO3 and Nrf2, both influenced by the action of melatonin (MEL). This indolamine and its metabolites constitute a particularly efficient chemical family, with a great capacity to fight oxidative stress, which can act directly against them or indirectly, acting on the induction of the expression of antioxidant enzymes. A cell type constantly exposed to oxidative insults is the erythrocyte, so the present study used the erythroleukemic lineage K562 induced to differentiate into erythroid cells. Thus, this proposal aimed to investigate the possible modulating effect of MEL on the redox signaling pathways PI3K/AKT/FoxO3 and Keap1/Nrf2/ARE, in cultivation of K562 erythroleukemic cells subjected to oxidative stress induction, favoring the maintenance of cellular redox homeostasis. For this, the viability, as well as the transcript levels of genes of important antioxidant enzymes, were evaluated, using K562 cells in different periods of erythroid differentiation as an experimental model. Thus, the study had three pseudo-replicated samples followed for five days according to the incubation periods, the evaluations was carried out: before the start (D0), at the beginning of differentiation (D2) and end of differentiation (D4). In addition, each sample was divided into the following groups: erythroid cells without oxidative stress and antioxidant treatment (Reference); cells under stress induction by hydrogen peroxide (100 µM H2O2); cells treated with 1 nM (C1) and 1 mM (C2) MEL; finally, two sets of cells treated with the same concentrations of MEL associated with stress induction (C1 + H2O2 and C2 + H2O2, respectively). Among the main results, it was observed the restoration of physiological levels of Nrf2 in both concentrations of MEL, under stress induction with peroxide, when compared to the group under the action of only the stressor, suggesting a cytoprotective effect. The lowest concentration (C1) was related to the induction of the expression of antioxidant genes through the Nrf2-ARE pathway, when compared with their respective references. This pattern was observed on day 0 for the CAT, SOD1, PRXD1 and 6 genes; On day 2, it was found for CAT, PRDX1 and 6. In relation to day 4, the same pattern was observed for SOD1, PRDX1 and 6, even suggesting a protective effect of MEL in relation to this first peroxiredoxin, since the treatments using MEL caused an expression similar to the physiological levels. As for C2, it was related to the induction of FOXO3 expression, when associated or not with the induction of oxidative stress; and on D4, C2 + H2O2 was shown to be related to a reduction in cell viability possibly related to activation of apoptotic pathways. Thus, the effects of MEL administration in these cells showed a period and dose-dependent pattern against oxidative stress induced with H2O2, with direct action on the detoxification of the stressor and indirect, since the FoxO3 pathway had a suggestive role in inducing apoptotic pathways, while the transcription factor predominantly responsible for maintaining redox homeostasis in K562 cells was Nrf2. Thus, the present work reinforces the importance of this indolamine in the regulation of cellular homeostasis, being a promising therapeutic alternative for diseases that present an exacerbated oxidative damage, due to its potent reducing effect.
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Palavras-chave
Melatonina, Estresse oxidativo, Antioxidantes, Estresse antioxidante, Melatonin, Oxidative stress, Antioxidants, Antioxidant stress