Autor(es): Aranha, Elenn Suzany Pereira
Data: 2015
Origem: Oasisbr
Assunto(s): E. cuspidifolia; E. tapacumensis; Citotoxicidade; Agentes anticancer??genos; HCT 116; Genotoxicidade; E. cuspidifolia; E. tapacumensis; Citotoxicity; Genotoxicity; CI??NCIAS DA SA??DE: FARM??CIA
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CNPq - Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico
Cancer is one of the most causes of death in the world. The research of natural products is strategy to develop anticancer agents. The aim this research was to identify chemical components, to investigate the anticancer potential in vitro and genotoxicity of Eugenia genus??s essential oil. For this, were used the cytotoxicity assay of Alamar blue and the hemolytic potential in erythrocytes of mice, as initial screening for selection of samples with cytotoxic potential, from the determination of the samples IC50. After the samples selection, we attemped to evaluate anticancer effect of the essential oil and its genotoxicity. Nine essential oils were tested, in a single concentration (50 ??g/mL) which only two- Eugenia cuspidifolia (1) e Eugenia tapacumensis (3)- were selected for later tests. The IC50, in 72 hours, obtained to these samples varied between 4,69 to 24,35 ??g/mL and 6,22 to 26,17 ??g/mL, respectively, among tumor cell lines. For non-tumor cell line, in 72 hours, the values for the IC50 were 25,51 ??g/mL to E. cuspidifolia and 36,12 ??g/mL to E. tapacumensis besides they didn??t cause hemolysis to mice erythrocytes. The species E. cuspidifolia and E. tapacumensis were more active to colorectal line (HCT 116) and ovary (ES-2), and the HCT 116 line was chosen to be used for later tests, because it??s one of the most frequent kind of cancer in diagnostics and in deaths worldwide at a world level . The chemical constitution of E. cuspidifolia and E. tapacumensis essential oils was investigated through gas chromatography. A total 26 constituents were identified for both samples. The majority components were caryophyllene oxide, ??-copaene, hepoxid of humulene II and cis-nerolidol. The essential oils in concentrations 7,5, 15 and 30 ??g/mL, were tested in the clonogenic assay and they significantly reduced the number of colonies (p<0,05), in the 7,5 and 15 ??g/mL concentrations, when compared with DMSO (0,2%). In the wound-healing assay, 24 and 48 hours, the essential oils reduced the migration in vitro (p<0,05) only on the concentration 30 ??g/mL, at 48h. The inhibitor potential of metalloproteinase MMP-2 and MMP-9 was evaluated through of zymography method. In the results, the better inhibition effect was from the E. tapacumensis essential oil, that reduced the enzymatic activity (p<0,05) in the concentrations 15 and 30 ??g/mL, in both treatments times (24 and 48h). Genotoxicity was evaluated by comet assay, using two versions, alkaline pH, which detects any damage caused to DNA, and neutral pH, specific to double-stranded breaks in DNA. Through damage index analisis, the results in alkaline pH assay were similar to the neutral pH. Only E. tapacumensis (30 ??g/mL) caused damage DNA (p<0,05) in the alkaline version. At the neutral pH version, all concentrations tested (p<0,05) caused DNA damage. Thus, is concluded that E. cuspidifolia and E. tapacumensis essential oils are cytotoxic in tumor cell lines, and they have anticancer potencial in HCT 116 cell line acting as cytotoxic and cytostatic depending on the time and the tested concentration. Only E. tapacumensis is genotoxic, in non-tumor cell line, however more specific studies are needed to determine whether this genotoxicity is reversible or if the mechanism of cytotoxic action of essential oil is related to the cell DNA damage.
O c??ncer ?? uma das maiores causas de morte no mundo. A pesquisa de produtos naturais constitui uma estrat??gia para o desenvolvimento de agentes anticancer??genos. Objetivou-se nessa pesquisa identificar os componentes qu??micos, investigar o potencial antic??ncer in vitro e a genotoxicidade de ??leos essenciais do g??nero Eugenia. Para isso, foram utilizados os ensaios de citotoxicidade do alamar blue e o potencial hemol??tico em eritr??citos de camundongos, como triagem inicial para a sele????o das amostras com potencial citot??xico, a partir da determina????o da CI50 das amostras. Posteriormente a sele????o das amostras, buscouse avaliar o efeito antic??ncer dos ??leos essenciais e a sua genotoxicidade. Foram testados nove ??leos essenciais, em concentra????o ??nica (50 ??g/mL) dos quais apenas dois - Eugenia cuspidifolia (1) e Eugenia tapacumensis (3)- foram selecionados para os testes posteriores. A CI50, em 72 horas, obtida para essas duas amostras variou entre 4,69 a 24,35 ??g/mL e 6,22 a 26,17 ??g/mL, respectivamente, entre as linhagens tumorais. Para a linhagem n??o tumoral, em 72 horas, os valores de CI50 foram de 25,51??g/mL para E. cuspidifolia (1) e 36,12 ??g/mL para E. tapacumensis (3), al??m de n??o causarem hem??lise a eritr??citos de camundongos. As esp??cies E. cuspidifolia (1) e E. tapacumensis (3) foram mais ativas para as linhagens de colorretal (HCT 116) e ov??rio (ES-2), e escolheu-se para os testes posteriores utilizar a linhagem HCT 116, por ser um dos tipos de c??ncer mais frequentes em diagn??sticos e tamb??m em mortes a n??vel mundial. A constitui????o qu??mica dos ??leos essenciais de E. cuspidifolia (1) e E. tapacumensis (3) foi investigada atrav??s de cromatografia gasosa. Foram identificados um total de 26 constituintes para as duas amostras. Os componentes majorit??rios foram ??xido de cariofileno, ??-copaeno, hep??xido de humuleno II e cis-nerolidol. Os ??leos nas concentra????es de 7,5, 15 e 30 ??g/mL, foram testados no ensaio clonog??nico e reduziram significativamente o n??mero de col??nias (p<0,05) na concentra????o de 7,5 e 15 ??g/mL, quando comparado ao controle DMSO (0,2%). No ensaio de motilidade celular, nos tempos de 24 e 48 horas, os ??leos essencias reduziram a migra????o (p<0,05) somente na concentra????o de 30 ??g/mL, no tempo de 48h. O potencial inibidor de metaloproteinases MMP-9 e MMP-2 foi avaliado atrav??s de m??todo zimogr??fico. Nos resultados obtidos, o melhor efeito inibit??rio foi do ??leo essencial de E. tapacumensis (3), o qual reduziu a atividade enzim??tica (p<0,05) nas concentra????es de 15 e 30 ??g/mL, nos dois tempos de tratamento (24 e 48h). A genotoxicidade foi avaliada pelo ensaio do cometa, usando duas vers??es, a de pH alcalino, o qual detecta qualquer dano causado ao DNA, e a de pH neutro, espec??fico para quebras da fita dupla de DNA. Atrav??s da an??lise do ??ndice de dano, os resultados no ensaio em pH alcalino foram semelhantes ao do pH neutro. Somente E. tapacumensis (3) (30 ??g/mL) causou dano ao DNA (p<0,05) na vers??o alcalina. Na vers??o em pH neutro todas as concentra????es causaram dano (p<0,05). Assim, conclui-se que os ??leos essenciais de E. cuspidifolia (1) e E. tapacumensis (3) s??o citot??xicos nas linhagens tumorais, tem potencial antic??ncer na linhagem HCT116 agindo como citot??xico e citost??tico dependendo do tempo e da concentra????o testada e somente a E. tapacumensis (3) ?? genot??xica, em c??lulas n??o neopl??sicas, , entretanto estudos mais espec??ficos precisam determinar se essa genotoxicidade ?? revers??vel ou se o mecanismo de a????o citot??xica do ??leo est?? relacionada a les??o do DNA das c??lulas.
