Potencial apoptótico do cobre: estudo in vitro: expressão das caspases 3, 8 e 9, do AIF e da proteína p53
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2014
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A apoptose define-se como um processo de morte celular programada, controlada geneticamente, que possui um papel essencial no desenvolvimento e na manutenção da homeostasia nos organismos, sendo fundamental na eliminação de células ectópicas, danificadas, mutantes e infetadas. A ativação da apoptose pode ser desencadeada através de duas vias: a via extrínseca ou citoplasmática, onde os recetores de membrana funcionam como ponto de iniciação do processo apoptótico, e a intrínseca ou mitocondrial, na qual a mitocôndria tem o papel principal. Após vários estímulos, pode-se verificar também, a indução da via apoptótica independente das caspases, mediada pelo fator de indução de apoptose (AIF- Apoptosis Inducing Factor).
O cobre é um metal essencial para os organismos, sendo necessário para garantir a função celular. Este elemento funciona como um cofator catalítico para diversas enzimas metabólicas. Contudo, a exposição de células e tecidos a quantidades excessivas de cobre pode provocar danos em diferentes mecanismos celulares, levando à perda da integridade da célula e, consequentemente, à apoptose.
A morte celular induzida pelo cobre foi detetada in vivo e in vitro, contudo a via de sinalização subjacente ainda não se encontra bem caracterizada, pois ainda não se sabe se esta ocorre como resultado da ação direta do metal ou de efeitos indiretos. Assim, no presente trabalho, pretendeu-se identificar a influência de diferentes concentrações de cobre (IC50 e 1/8 do IC50) na indução de apoptose, e explorar, através de dois diferentes modelos celulares in vitro, Caco-2 e Hep-G2, as potenciais vias de sinalização ativadas.
Os resultados obtidos demonstraram que, em ambos os modelos celulares, o cobre altera a expressão das caspases 3, 8 e 9, do AIF e da proteína p53. Na linha celular Caco-2, a concentração mais alta de cobre parece ativar a via intrínseca precocemente (6 horas de exposição). Relativamente à menor concentração de cobre, não se observaram diferenças significativas na expressão das caspases contudo, a via independente das caspases é ativada às 6 horas de exposição. Às 24 horas de exposição o aumento da p53 parece promover um aumento da caspase 8. Nas células Hep-G2, em ambas as concentrações de cobre, o aumento da expressão das caspases 9 e 8, concordante com o aumento da caspase 3, sugere a ativação de apoptose por via intrínseca e extrínseca. A análise da expressão do AIF indica, também, o envolvimento da via independente das caspases, na indução de apoptose pela menor concentração de cobre.
Em conclusão, no presente trabalho, demonstrou-se que em células epiteliais do intestino e células hepáticas humanas, o cobre induz a ativação da apoptose, através de vias dependentes e independentes de caspases. Os resultados sugerem também que o mecanismo pelo qual ocorre a ativação da apoptose está dependente da concentração e do tempo de exposição ao cobre, bem como do tipo celular.
Apoptosis is defined as a process of programmed cell death, genetically controlled, that plays an essential role in the development and maintenance of organisms homeostasis, being instrumental in the elimination of ectopic, damaged, mutants or infected cells. Apoptosis activation can be initiated through two distinct pathways: extrinsic or citoplasmatic, where membrane receptors act as a point of initiation of the apoptotic process, and intrinsic or mitochondrial, in which mitochondria have the lead. Upon several stimuli, there is also, induction of caspase independent apoptotic pathways, mediated by apoptosis inducing factor (AIF). Copper is an essential metal for organisms, being necessary to ensure cell function. This element serves as a catalytic cofactor for numerous metabolic enzymes. However, exposure of cells and tissues to excessive amounts of copper may lead to cell protein mechanisms damage, leading to the loss of cell integrity and, therefore, apoptosis. Cell death induced by copper was detected in vivo and in vitro, although the underlying signaling pathway has not yet been well characterized, it is still not known whether this occurs as a result of direct action of the metal or of indirect effects. In the present work we intended to identify the influence of different copper concentrations (IC50 and 1/8 of IC50) in the induction of apoptosis, and explore the potential signaling pathways using two different cell models in vitro, Caco-2 and Hep-G2. The results showed that in both cell lines, copper alters caspase 3, 8 and 9, AIF and p53 expression. In Caco-2 cell line, the highest copper concentration appears to activate an early intrinsic pathway (6h of exposure). For the lower copper concentration, no significant differences were observed in the expression of both caspases however, caspase independent pathways is activated at 6h of exposure. At 24h of exposure p53 upregulation appears to promote an increase of caspase 8. In Hep-G2 cells, in both copper concentrations exposure, caspases 9 and 8 expression, confirmed by caspase 3 expression, suggest apoptosis activation through intrinsic and extrinsic pathway. Analysis of AIF expression also indicates the involvement of the caspase independent pathway in apoptosis induction by the lower copper concentration. In conclusion, in this study, it was demonstrated that in intestine epithelial cells and liver cells, copper induces the activation of apoptosis through caspase dependent and independent pathways. The results also suggest that apoptosis activation mechanism is dependent on concentration and time of exposure to copper, as well as the cell type.
Apoptosis is defined as a process of programmed cell death, genetically controlled, that plays an essential role in the development and maintenance of organisms homeostasis, being instrumental in the elimination of ectopic, damaged, mutants or infected cells. Apoptosis activation can be initiated through two distinct pathways: extrinsic or citoplasmatic, where membrane receptors act as a point of initiation of the apoptotic process, and intrinsic or mitochondrial, in which mitochondria have the lead. Upon several stimuli, there is also, induction of caspase independent apoptotic pathways, mediated by apoptosis inducing factor (AIF). Copper is an essential metal for organisms, being necessary to ensure cell function. This element serves as a catalytic cofactor for numerous metabolic enzymes. However, exposure of cells and tissues to excessive amounts of copper may lead to cell protein mechanisms damage, leading to the loss of cell integrity and, therefore, apoptosis. Cell death induced by copper was detected in vivo and in vitro, although the underlying signaling pathway has not yet been well characterized, it is still not known whether this occurs as a result of direct action of the metal or of indirect effects. In the present work we intended to identify the influence of different copper concentrations (IC50 and 1/8 of IC50) in the induction of apoptosis, and explore the potential signaling pathways using two different cell models in vitro, Caco-2 and Hep-G2. The results showed that in both cell lines, copper alters caspase 3, 8 and 9, AIF and p53 expression. In Caco-2 cell line, the highest copper concentration appears to activate an early intrinsic pathway (6h of exposure). For the lower copper concentration, no significant differences were observed in the expression of both caspases however, caspase independent pathways is activated at 6h of exposure. At 24h of exposure p53 upregulation appears to promote an increase of caspase 8. In Hep-G2 cells, in both copper concentrations exposure, caspases 9 and 8 expression, confirmed by caspase 3 expression, suggest apoptosis activation through intrinsic and extrinsic pathway. Analysis of AIF expression also indicates the involvement of the caspase independent pathway in apoptosis induction by the lower copper concentration. In conclusion, in this study, it was demonstrated that in intestine epithelial cells and liver cells, copper induces the activation of apoptosis through caspase dependent and independent pathways. The results also suggest that apoptosis activation mechanism is dependent on concentration and time of exposure to copper, as well as the cell type.
Descrição
Dissertação de Mestrado em Biologia Clinica Laboratorial
Palavras-chave
Apoptose , Cobre , In vitro , Caspases