Avaliação da atividade apoptótica de substância pura isolada de Cryptocarya mandioccanna em células de carcinoma cervical imortalizadas pelo papilomavirus humano (HPV)

Abstract

Diversos estudos buscam identificar compostos com atividade seletiva para celulas tumorais e que possuam mecanismo de acao para desencadear a apoptose. Dentre as substancias isoladas de Cryptocarya sp, algumas estirilpironas, como a goniotalamina, apresentam atividade antiproliferativa e apoptogenica em diferentes linhagens celulares. No presente estudo, foram avaliadas as atividades citotoxica e pro-apoptotica da estirilpirona (criptomoscatona D2) isolada de Cryptocarya mandioccana, em linhagens celulares de carcinoma cervical humano infectada por HPV (HeLa e SiHa), nao infectada (C33A) e fibroblasto pulmonar humano transformado pelo SV-40 (MRC-5). A atividade citotoxica foi avaliada pelo ensaio do MTT e a apoptose foi avaliada, respectivamente, pelos ensaios de anexina V e a expressao de bak/bcl-2, por citometria de fluxo. Para o ensaio do MTT, as celulas foram tratadas com estirilpirona (criptomoscatona D2) nas concentracoes de 15, 30, 60 e 90-ÊM por 6, 24 e 48 horas e por 6 horas com periodo de recuperacao de 24, 48 e 72 horas pos tratamento. Para os ensaios de apoptose, as celulas foram tratadas por 6 horas e periodo de recuperacao de 24, 48 e 72 horas. O tratamento com a estirilpirona (criptomoscatona D2) ocasionou elevada citotoxicidade dose-resposta e tempo-resposta em HeLa, SiHa, C33A e MRC-5. Embora nao haja diferenca estatisticamente significativa de citotoxicidade entre as linhagens, aparentemente a citotoxicidade foi maior em HeLa e C33A (tratamento de 24 e 48 horas) que em MRC-5 e SiHa. Ainda, no periodo de recuperacao, HeLa e SiHa aparentemente restabelecem sua capacidade proliferativa, que e diretamente proporcional ao tempo de recuperacao, enquanto o mesmo comportamento nao e observado em C33A. Ao avaliar a expressao de duas proteinas da via intrinseca de apoptose (bcl-2 e bak), nao foi observada modulacao dessa expressao entre as linhagens celulares, nas diferentes tempos de recuperação pos-tratamento.

Several attempts have been made to identify chemical compounds with selective cytotoxicity against cancer cells and apoptosis trigger activity. Among the substances isolated from Cryptocarya sp, some styrylpyrones, such as goniothalamine, demonstrate antiproliferative and apoptotic activity in abroad human cell lines. In the present study, we evaluated the antiproliferative and apoptotic activities of the styrylpyrone (cryptomoschatone D2) isolated from Cryptocarya mandioccana in HPV-infected (HeLa and SiHa) and non-infected (C33A) human cervical carcinoma cell lines, and in human lung's fibroblast immortalized with SV-40 (MRC-5). The antiproliferative activity was evaluated by the MTT assay and the apoptotic activity was investigated by measuring the expression levels of annexin V and bak/bcl-2 by flow cytometry. In the MTT assay, cells were treated with styrylpyrone (cryptomoschatone D2) at a 15, 30, 60 or 90ìM concentration for 6, 24 or 48 hours as well as for 6 hours followed by a recovery posttreatment period of 24, 48 or 72 hours. In the apoptotic assays, cells were treated for 6 hours followed by a recovery posttreatment period of 24, 48 or 72 hours. High cytotoxicity (dose-response and time-response) was observed in HeLa, SiHa, C33A and MRC-5 cell lines. Although the styrylpyrone cytotoxicity was not significantly different among the cell lines tested, the citotoxicity was apparently higher in HeLa and C33A than MRC-5 and SiHa in the case of treatments for 24 or 48 hours. Moreover, HeLa and SiHa were able to recover their prolifetative status, which were directly proportional to the posttreatment recovery time. On the other hand, C33A did not demonstrate a similar posttreatment recovery. Despite the posttreatment recovery time, the expression of the apoptotic proteins bcl-2 and bak seems not to be modulated by the treatment.