Perfil antioxidante e efeito dos extratos, frações e das antraquinonas crisofanol, emodina e fisciona de Rhamnus sphaerosperma var. pubescens sobre a viabilidade celular e estresse oxidativo em células tumorais de colo uterino e carcinoma oral
Data
2016-11-25
Autores
Moreira, Thais Fernanda
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os produtos naturais apresentam imensa diversidade estrutural, característica útil para alcançar alvos moleculares específicos importantes para a terapia de diversas doenças, contribuindo assim para a ampliação do arsenal farmacológico. Como observado para o gênero Rhamnus, a espécie vegetal Rhamnus sphaerosperma var. pubescens, nativa no Brasil, pode apresentar um grande potencial químico e farmacológico; popularmente é conhecida como “fruto-de-pombo”, com poucos estudos relatando suas propriedades. Tem composição rica em compostos antraquinônicos, onde já foram identificados crisofanol, fisciona e emodina. Os objetivos deste estudo foram verificar o potencial antioxidante via estudo da capacidade de captura sobre espécies reativas de oxigênio e nitrogênio, assim como a capacidade de indução de morte celular dos extratos, frações e antraquinonas isoladas de Rhamnus sphaerosperma var. pubescens (Rhamnaceae) em células tumorais de colo uterino com e sem o genoma do vírus HPV (SiHa e C33-A, respectivamente), e carcinoma oral de células escamosas (HSC-3). A antraquinona emodina apresentou a melhor capacidade de captura sobre as espécies NO• e O2 •- em relação as demais antraquinonas e extratos brutos. No ensaio de captura sobre o HOCl/OCl- todas as amostras foram efetivas, sendo os extratos brutos com melhor atividade, e dentre as antraquinonas, o crisofanol apresentou maior efetividade. Nenhuma amostra demonstrou capacidade na captura do H2O2. As antraquinonas emodina e fisciona demonstraram efeito citotóxico sobre as linhagens celulares SiHa, C33-A, HSC-3 e HaCaT, com morte celular mista (apoptose e necrose), a qual ocorre de maneira independente de caspases, constatada nos ensaios citomorfológico de exclusão por fluorocromos, anexina-V e avaliação da atividade de capsase-3. O crisofanol não apresentou citotoxicidade. Emodina, fisciona e o extrato bruto promoveram aumento do estresse oxidativo intracelular, uma vez que foi observado aumento de espécies reativas ao ácido tiobarbitúrico (TBARS) e aumento de danos ao DNA como observado nos ensaios de micronúcleo e cometa. As amostras foram capazes de promover supressão da via PI3K/AKT, diminuindo os níveis de fosforilação de AKT: a emodina apresenta melhor atividade e ação apenas sobre as linhagens tumorais SiHa, C33-A e HSC-3; fisciona e extrato bruto apresentaram este efeito sobre as linhagens HSC-3 (tumoral) e HaCaT (não tumoral). Estes dados sugerem que o mecanismo de indução de morte promovido pelas antraquinonas emodina e fisciona, assim como pelo extrato bruto de Rhamnus sphaerosperma var. pubescens, pode ser mediado pelo aumento do estresse oxidativo intracelular, danos ao DNA e também supressão da via PI3K/AKT, importante para a sobrevivência celular.
Natural products have immense structural diversity, useful feature to achieve specific molecular targets in cancer treatment, and thus contribute to the expansion of the therapeutic arsenal. As notorious for the Rhamnus genus, plant species Rhamnus sphaerosperma var. pubescens, native in Brazil may present a great chemical and pharmacological potential; It is popularly known as the Fruto-de-pombo, with few studies reporting its properties. This species has rich composition with anthraquinones, which have been identified: chrysophanol, physcion and emodin. The objectives of this study were to verify the capture capability of reactive species of oxygen and nitrogen, as well as cell death inducing ability of the extracts, fractions and isolated anthraquinones of Rhamnus sphaerosperma var. pubescens (Rhamnaceae) in cervical tumor cells with and without the genome of HPV virus (SiHa and C33-A, respectively), and oral squamous cell carcinoma (HSC-3). The anthraquinone emodin showed the best capture capability of the species NO• and O2 •- compared with other anthraquinones and crude extracts. In the scavenge assay of HOCl / OCl- all samples were effective, and the crude extracts showed better activity and chrysophanol showed better effectiveness among anthraquinones. No samples demonstrated ability in capturing the H2O2. The anthraquinones emodin and physcion demonstrated cytotoxic effect on the cell lines SiHa, C33-A, HSC-3 and HaCaT with mixed cell death (apoptosis and necrosis), independent of caspases pathway, detected in cytomorphological test with fluorochromes exclusion, annexin-V and evaluation of capsase-3 activity. The chrysophanol was not cytotoxic in these assays. Emodin, physcion and the crude extract promoted increased intracellular oxidative stress, verified by thiobarbituric acid reactive species assay (TBARS), and an increase of DNA damage as observed in the micronucleus test. Further, these samples were capable of promoting the supression of PI3K/AKT, reducing levels of activation (phosphorylation) of AKT. In this parameter, emodin showed activity only on the tumor cell lines (SiHa, C33-A and HSC-3), on the other hand, physcion and crude extract showed this effect on HSC-3 and HaCaT cell lines. Therefore, the death induction mechanism promoted by emodin, physcion and crude extract of this plant species should be mediated by an increase of the intracellular oxidative stress, DNA damage, and suppression of the PI3K/AKT important for cell survival.
Natural products have immense structural diversity, useful feature to achieve specific molecular targets in cancer treatment, and thus contribute to the expansion of the therapeutic arsenal. As notorious for the Rhamnus genus, plant species Rhamnus sphaerosperma var. pubescens, native in Brazil may present a great chemical and pharmacological potential; It is popularly known as the Fruto-de-pombo, with few studies reporting its properties. This species has rich composition with anthraquinones, which have been identified: chrysophanol, physcion and emodin. The objectives of this study were to verify the capture capability of reactive species of oxygen and nitrogen, as well as cell death inducing ability of the extracts, fractions and isolated anthraquinones of Rhamnus sphaerosperma var. pubescens (Rhamnaceae) in cervical tumor cells with and without the genome of HPV virus (SiHa and C33-A, respectively), and oral squamous cell carcinoma (HSC-3). The anthraquinone emodin showed the best capture capability of the species NO• and O2 •- compared with other anthraquinones and crude extracts. In the scavenge assay of HOCl / OCl- all samples were effective, and the crude extracts showed better activity and chrysophanol showed better effectiveness among anthraquinones. No samples demonstrated ability in capturing the H2O2. The anthraquinones emodin and physcion demonstrated cytotoxic effect on the cell lines SiHa, C33-A, HSC-3 and HaCaT with mixed cell death (apoptosis and necrosis), independent of caspases pathway, detected in cytomorphological test with fluorochromes exclusion, annexin-V and evaluation of capsase-3 activity. The chrysophanol was not cytotoxic in these assays. Emodin, physcion and the crude extract promoted increased intracellular oxidative stress, verified by thiobarbituric acid reactive species assay (TBARS), and an increase of DNA damage as observed in the micronucleus test. Further, these samples were capable of promoting the supression of PI3K/AKT, reducing levels of activation (phosphorylation) of AKT. In this parameter, emodin showed activity only on the tumor cell lines (SiHa, C33-A and HSC-3), on the other hand, physcion and crude extract showed this effect on HSC-3 and HaCaT cell lines. Therefore, the death induction mechanism promoted by emodin, physcion and crude extract of this plant species should be mediated by an increase of the intracellular oxidative stress, DNA damage, and suppression of the PI3K/AKT important for cell survival.
Descrição
Palavras-chave
Rhamnus sphaerosperma var. pubescens., Emodina., Fisciona., Crisofanol., Atividade citotóxica., Mutagenicidade., Estresse oxidativo.