Mecanismos de Cardiotoxicidade da Doxorrubicina em Ratos Wistar e Potencial Cardioprotetor da Alda-1
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Data
2019-02-01
Autores
Souza, Leonardo da Cunha Menezes
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Universidade Estadual Paulista (Unesp)
Resumo
A cardiotoxicidade induzida pela doxorrubicina (DOX), antraciclina isolada da actinobacteria Streptomyces peucetius e amplamente utilizada na terapia antineoplásica, corresponde a um dos mais importantes eventos patofisiológicos que limitam sua aplicação clínica. No entanto, não são completamente conhecidos todos os mecanismos envolvidos nessa toxicidade, o que diminui as possiblidades de intervenção e a redução dos efeitos colaterais para os pacientes sob tratamento. Uma das hipósteses é que os aldeídos gerados pela ação da DOX atuam sobre membranas mitocondriais, alterando o estado redox e formando adutos com proteínas, os quais prejudicam o correto funcionamento da organela. Atividades deletérias da DOX sobre outros componentes celulares, como, por exemplo, os ácidos ribonucleicos, são, também, possíveis mecanismos de toxicidade do antineoplásico. Várias estratégias têm sido utilizadas para minimizar os efeitos adversos da DOX. Uma delas, é a busca por compostos que possam proteger as células da ação citotóxica. Nesse sentido, a Alda-1, pertencente ao grupo das chaperonas e agonista da enzima aldeído desidrogenase mitocondrial (ALDH2), vem sendo testada com o objetivo de reduzir os efeitos adversos dos metabólitos e radicais gerados pelo antineoplásico. Para investigar outros possíveis mecanismos de ação da DOX e o efeito cardiprotetor da Alda-1, este estudo foi delineado utilizando duas abordagens distintas: experimentos in vivo, com ratos Wistar machos submetidos a tratamentos agudos e crônicos com o antineoplásico, e, in vitro, em fibroblastos de camundongos e cíbridos com heteroplasmia mitocondrial para o gene ND5 (codifica subunidade ND5 do Complexo I mitocondrial). Foram avaliados perfis de expressão gênica (genes das vias de beta oxidação de ácidos graxos; Bax, Bcl-2,C1QBP e ALDH2) e do microRNA miR-34a (regulador da expressão da ALDH2), e o processo de lipoperoxidação em cardiomiócitos de ratos tratados com a DOX isoladamente ou em combinação com a Alda-1. In vitro, foram investigados os efeitos dos dois compostos sobre a viabilidade celular, sobre a estrutura mitocondrial e sobre o perfil das espécies moleculares da cardiolipina. Além disso, foi avaliado se os níveis de heteroplasmia podiam interferir na ação da DOX. Os resultados confirmaram a ação da DOX sobre o perfil lipídico, aumentando os níveis de triglicerídeos e do VLDL. O tratamento simultâneo com a Alda-1 reverteu tal efeito. As análises de expressão gênica revelaram a hiperexpressão de Fabp4, Slc27a2, Bcl-2 e C1QBP nos cardiomiócitos dos animais tratados com a DOX (tramento agudo). Novamente, a administração associada da Alda-1 foi capaz de reverter as alterações na expressão dos genes afetados. In vitro, a DOX promoveu alterações na heteroplasmia mitocondrial em favor do tipo mutante, a diminuição na viabilidade celular e alterações morfológicas nas mitocondriais. Esses eventos foram reduzidos pela ação da Alda-1. Diante de todos esses achados, pode-se concluir que, além dos mecanismos de ação já conhecidos, a DOX também atua modulando genes envolvidos no transporte de ácidos graxos e estresse oxidativo mitocondrial, bem como alterando a heteroplasmia do mtDNA. Por outro lado, os resultados confirmaram o efeito protetor da Alda-1 contra os efeitos tóxicos da DOX, mostrando sua ação redutora da hiperlipidemia e expressão gênica.
The cardiotoxicity induced by doxorubicin (DOX), anthracycline isolated from the actinobacteria Streptomyces peucetius, and widely used as an antineoplastic drug, is one of the most important pathophysiological events that limit its clinical application. However, all the mechanisms involved in this toxicity are not fully understood. One hypothesis is that the aldehydes generated by DOX act on mitochondrial membranes, modifying the redox state and proting adducts with proteins. DOX activities on other cellular components, such as ribonucleic acids, are also possible mechanisms of toxicity. Several strategies have been used to reduce the DOX adverse effects. One of them is the identification of compounds that can protect cells against cytotoxic. Alda-1, which belongs to a group of chaperones and is an agonist of the mitochondrial aldehyde dehydrogenase (ALDH2), has been tested to reduce the adverse effects of metabolites and radicals generated by DOX. To investigate other possible DOX mechanisms of action, and the cardioprotective activity of Alda-1, this study was designed using two different approaches: in vivo, with male Wistar rats submitted to acute and chronic treatments; and, in vitro, in mice fibroblasts and cybrids with ND5 (gene that encodes the mitochondrial Complex I subunit) mitochondrial gene heteroplasmy. The expression profiling of genes related to beta oxidation pathways, Bax, Bcl-2, C1QBP, ALDH2 and miR34a microRNA (ALDH2 expression regulator), and the lipoperoxidation process were investigated in cardiomyocytes of rats treated with DOX, or with DOX plus Alda-1. In vitro, the effects of the two compounds on cell viability, mitochondrial structure and cardiolipin molecular species profile, were investigated. In addition, it was assessed whether heteroplasmy levels could interfere on DOX toxic activity. The results confirmed the DOX effect on lipid profile, increasing triglycerides and VLDL concentration. Simultaneous treatment with Alda-1 decreased such effect. Gene expression analyses revealed the overexpression of Fabp4, Slc27a2, Bcl-2 and C1QBP in cardiomyocytes of rats treated with DOX (acute treatment). Again, the Alda-1 simultaneous administration was able to modulate the changes on gene expressions. In vitro, DOX promoted changes in mitochondrial heteroplasmy towards a mutant genotype, decreased cell viability and morphological changes in the mitochondrias. These events were reduced by the action of Alda-1. In conclusion, besides the known mechanisms of action, DOX was able to modulate genes involved in the fatty acids transportation and in oxidative stress pathways. Furthermore, DOX promoted changes in mitochondrial heteroplasmy. On the other hand, the results confirmed the protective effect of Alda-1 against the DOX toxic activity through its ability to reduce hyperlipidemia and gene expression.
The cardiotoxicity induced by doxorubicin (DOX), anthracycline isolated from the actinobacteria Streptomyces peucetius, and widely used as an antineoplastic drug, is one of the most important pathophysiological events that limit its clinical application. However, all the mechanisms involved in this toxicity are not fully understood. One hypothesis is that the aldehydes generated by DOX act on mitochondrial membranes, modifying the redox state and proting adducts with proteins. DOX activities on other cellular components, such as ribonucleic acids, are also possible mechanisms of toxicity. Several strategies have been used to reduce the DOX adverse effects. One of them is the identification of compounds that can protect cells against cytotoxic. Alda-1, which belongs to a group of chaperones and is an agonist of the mitochondrial aldehyde dehydrogenase (ALDH2), has been tested to reduce the adverse effects of metabolites and radicals generated by DOX. To investigate other possible DOX mechanisms of action, and the cardioprotective activity of Alda-1, this study was designed using two different approaches: in vivo, with male Wistar rats submitted to acute and chronic treatments; and, in vitro, in mice fibroblasts and cybrids with ND5 (gene that encodes the mitochondrial Complex I subunit) mitochondrial gene heteroplasmy. The expression profiling of genes related to beta oxidation pathways, Bax, Bcl-2, C1QBP, ALDH2 and miR34a microRNA (ALDH2 expression regulator), and the lipoperoxidation process were investigated in cardiomyocytes of rats treated with DOX, or with DOX plus Alda-1. In vitro, the effects of the two compounds on cell viability, mitochondrial structure and cardiolipin molecular species profile, were investigated. In addition, it was assessed whether heteroplasmy levels could interfere on DOX toxic activity. The results confirmed the DOX effect on lipid profile, increasing triglycerides and VLDL concentration. Simultaneous treatment with Alda-1 decreased such effect. Gene expression analyses revealed the overexpression of Fabp4, Slc27a2, Bcl-2 and C1QBP in cardiomyocytes of rats treated with DOX (acute treatment). Again, the Alda-1 simultaneous administration was able to modulate the changes on gene expressions. In vitro, DOX promoted changes in mitochondrial heteroplasmy towards a mutant genotype, decreased cell viability and morphological changes in the mitochondrias. These events were reduced by the action of Alda-1. In conclusion, besides the known mechanisms of action, DOX was able to modulate genes involved in the fatty acids transportation and in oxidative stress pathways. Furthermore, DOX promoted changes in mitochondrial heteroplasmy. On the other hand, the results confirmed the protective effect of Alda-1 against the DOX toxic activity through its ability to reduce hyperlipidemia and gene expression.
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Palavras-chave
Alda-1, alterações mitocondriais, cardioproteção, cardiotoxicidade, doxorrubicina, transcriptoma, mitochondrial alterations, cardioprotection, cardiotoxicity, doxorubicin, transcriptome