Análises celulares e moleculares das lignanas extraídas da Piper cubeba em linhagens tumorigênicas de cabeça e pescoço
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Data
2019-02-28
Autores
Gusson, Juliana Prado
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Universidade Estadual Paulista (Unesp)
Resumo
O carcinoma espinocelular de cabeça e pescoço com acometimento de sítios anatômicos do trato aerodigestivo superior representa o sexto tipo mais comum de câncer no mundo. Algumas plantas vêm sendo utilizadas no tratamento do câncer e uma delas tem despertado interesse científico, a Piper cubeba. Com relação à importância da atividade antitumoral das lignanas extraídas da Piper cubeba, incluindo seus elementos químicos, foi proposto o presente trabalho que teve como objetivo geral avaliar o potencial efeito citotóxico e genotóxico de lignanas nas células sobre a morfologia, proliferação e migração celular, citotoxicidade, genotoxicidade, apoptose e necrose celular, e expressão gênica, observando como o fitoterápico age e como essas alterações participam do processo tumorigênico. Para isso foram utilizadas duas linhagens de células tumorigênicas e uma de células normais, após o tratamento com as lignanas cubebina, dihidrocubebina, etilcubebina, extrato total, hinoquinina e metilcubebina, todas extraídas das sementes de Piper cubeba em três concentrações (10 µg/mL, 50 µg/mL e 100 µg/mL), por 4, 24, 48 e 72 horas. A observação da morfologia foi realizada em microscópio invertido, o índice de proliferação pela curva de crescimento, a viabilidade celular foi determinada pelo método colorimétrico, MTS (CC50), migração celular pelo método de transwell, genotoxicidade pelo ensaio cometa, avaliação da apoptose por citômetro de fluxo e expressão gênica pela técnica de PCR quantitativo. A estatística foi realizada por análise de variância para comparações múltiplas (ANOVA), seguido do ajuste de Bonferroni, pelo software GraphPad Prism 5.0. Os resultados evidenciaram que as diferentes lignanas não alteram a morfologia celular, mas diminuem a proliferação e migração das células, apresentam pouca citotoxicidade, e efeitos genotóxicos, provavelmente por indução de apoptose tardia e necrose celular, e ainda alteram a expressão de genes envolvidos com o processo inflamatório. Dessa maneira, as lignanas da Piper cubeba atuam diretamente nas células tumorigênicas, seja pelas vias apoptóticas e/ou inflamatórias e abrem novas possibilidades de estudos futuros para terapias associadas no câncer de cabeça e pescoço.
Head and neck Squamous cell carcinoma with involvement of anatomical sites of the upper aerodigestive tract represents the sixth most common type of cancer in the world. Some plants have been used in the treatment of cancer and one of them has aroused scientific interest, Piper cubeba. Regarding the importance of the antitumor activity of the lignans extracted from Piper cubeba, including its chemical elements, the present work was proposed whose general objective was to evaluate the potential cytotoxic and genotoxic effects of lignans in cells on cell morphology, proliferation and migration, cytotoxicity, genotoxicity, apoptosis and cell necrosis and gene expression, observing how the phytotherapic acts and how these alterations participate in the tumorigenic process. After the treatment with the lignans cubebin, dihydrocubebin, ethylcubebin, total extract, hinokinin and metilcubebin, all of them were extracted from Piper cubeba seeds at three concentrations (10, 50 and 100μg/ml) for 4, 24, 48 and 72 hours. The morphology was determined by inverted microscopy, proliferation index by growth curve, cell viability was determined by the colorimetric method, MTS (IC50), cell migration by transwell method, genotoxicity by comet assay, flow cytometric apoptosis evaluation and gene expression by quantitative PCR technique. Statistical analysis was performed by analysis of variance for multiple comparisons (ANOVA), followed by Bonferroni adjustment by GraphPad Prism 5.0 software. The results showed that the different lignans do not alter the cellular morphology, but decrease cell proliferation and migration, have little cytotoxicity, and genotoxic effects, probably due to the induction of late apoptosis and cellular necrosis, and also alter the expression of genes involved with inflammatory process. Thus, Piper cubeba lignans act directly on tumorigenic cells, either through apoptotic and/or inflammatory pathways and open up new possibilities for future studies for associated therapies in head and neck cancer.
Head and neck Squamous cell carcinoma with involvement of anatomical sites of the upper aerodigestive tract represents the sixth most common type of cancer in the world. Some plants have been used in the treatment of cancer and one of them has aroused scientific interest, Piper cubeba. Regarding the importance of the antitumor activity of the lignans extracted from Piper cubeba, including its chemical elements, the present work was proposed whose general objective was to evaluate the potential cytotoxic and genotoxic effects of lignans in cells on cell morphology, proliferation and migration, cytotoxicity, genotoxicity, apoptosis and cell necrosis and gene expression, observing how the phytotherapic acts and how these alterations participate in the tumorigenic process. After the treatment with the lignans cubebin, dihydrocubebin, ethylcubebin, total extract, hinokinin and metilcubebin, all of them were extracted from Piper cubeba seeds at three concentrations (10, 50 and 100μg/ml) for 4, 24, 48 and 72 hours. The morphology was determined by inverted microscopy, proliferation index by growth curve, cell viability was determined by the colorimetric method, MTS (IC50), cell migration by transwell method, genotoxicity by comet assay, flow cytometric apoptosis evaluation and gene expression by quantitative PCR technique. Statistical analysis was performed by analysis of variance for multiple comparisons (ANOVA), followed by Bonferroni adjustment by GraphPad Prism 5.0 software. The results showed that the different lignans do not alter the cellular morphology, but decrease cell proliferation and migration, have little cytotoxicity, and genotoxic effects, probably due to the induction of late apoptosis and cellular necrosis, and also alter the expression of genes involved with inflammatory process. Thus, Piper cubeba lignans act directly on tumorigenic cells, either through apoptotic and/or inflammatory pathways and open up new possibilities for future studies for associated therapies in head and neck cancer.
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Câncer, Potencial antitumorigênico, Fitoterápico, Processo inflamatório, Antitumorigenic potential, Phytotherapeutic, Inflammatory process