Estudo da ação do peptídeo ANXA1Ac2-26 e da interação entre células endoteliais e carcinogênicas de cabeça e pescoço.
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Data
2019-03-01
Autores
Picão, Thaís Bravo
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Universidade Estadual Paulista (Unesp)
Resumo
O carcinoma epidermoide de cabeça e pescoço (CECP) representa o sexto câncer mais comum em todo o mundo, sendo o câncer na cavidade oral o mais comum entre as regiões anatômicas de abrangência do CECP. Há evidências de que o desenvolvimento e a progressão do câncer dependem não somente de suas características genéticas, mas também de interações de suas células com as células do microambiente tumoral. A carcinogênese também está relacionada com angiogênese e processos inflamatórios, que podem ser controlados pela ação de mediadores anti-inflamatórios, e entre esses, destacamos a proteína anti-inflamatória anexina A1 (ANXA1). Mas os mecanismos pelos quais a ANXA1 atua na tumorigênese e a sua relação com a interação entre células endoteliais e tumorigênicas não são bem conhecidos e, considerando seu papel na inflamação, compreendem um alvo importante para estudo. Desse modo, o presente trabalho teve como objetivo, analisar a interação de células endoteliais e tumorigênicas juntamente com a ação do peptídeo sintético Ac2-26 da proteína ANXA1(ANXA1Ac2-26) em linhagem de carcinoma de cabeça e pescoço, verificando como o peptídeo atua junto ao meio condicionado de células endoteliais e/ou de células tumorais, elucidando como os fatores excretados por ambas as células e o peptídeo atuam no processo tumorigênico. Para isso, 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 (IC50), a migração celular pelo método bicamada transwell, genotoxicidade pelo ensaio Cometa, avaliação de apoptose e necrose por citômetro de fluxo, padrãode liberação de citocinas/quimiocinas por ELISA e expressão gênica pela técnica de PCR quantitativa. 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 mostraram que o efeito do meio condicionado por células tumorais (Hep-2) em células endoteliais (HUVEC) e vice-versa, tratadas com o peptídeo ANXA1Ac2-26, não modifica a morfologia e a migração celular, mas altera a proliferação; não é citotóxico, porém é genotóxico; induz ao processo de apoptose tardia, modifica a expressão das citocinas/quimiocinas IL-8, RANTES e TNF-α e modula a expressão de genes envolvidos com o processo inflamatório. Assim, mostramos que a interação entre células endoteliais e tumorigênicas, juntamente com a ação do ANXA1Ac2-26 nas células endoteliais e/ou tumorais provavelmente participam do processo tumorigênico por mecanismos relacionados às vias anti-proliferativas, apoptóticas e/ou inflamatórias e abrem novas possibilidades de estudos para alternativas terapêuticas no câncer de cabeça e pescoço.
Head and Neck Squamous Cells Carcinoma (HNSCC) is the sixth most common cancer in the world, being cancer in the oral cavity the most common among the anatomical regions of HNSCC. There is evidence that the development and progression of cancer depends not only on its genetic characteristics, but also because of the interactions of cells, cancer and tumor microenvironment cells. Carcinogenesis is also related to angiogenesis and inflammatory processes which can be controlled by the action of anti-inflammatory mediators, among which we highlight the anti-inflammatory protein annexin A1 (ANXA1). But the mechanisms that ANXA1 acts in tumorigenesis and its relation to the interaction between endothelial and tumorigenic cells are not well known and considering their role in inflammation, comprise an important target for study. Therefore, the present study aimed to analyze the interaction of endothelial and tumorigenic cells with the action of the synthetic peptide Ac2-26 of the ANXA1 protein (ANXA1Ac2-26) in head and neck carcinoma cell line, verifying how the peptide acts with the conditioned medium of endothelial cells and/or tumor cells, elucidating how the factors excreted by both cells and the peptide acts in the tumorigenic process. For this, the morphology observation was performed under inverted microscope, proliferation index by growth curve, cell viability was determined by the colorimetric method MTS (IC50), cell migration by transwell bilayer method, genotoxicity by the Comet assay, apoptosis and necrosis by flow cytometry, cytokine/chemokine release pattern by ELISA and gene expression by the quantitative PCR technique. The statistic was performed by Analysis of Variance for multiple comparisons, followed by Bonferroni adjustment, made it with GraphPad Prism 5.0 software. The results showed that the effect of tumor cell conditioned medium (Hep-2) on endothelial cells (HUVEC) and the other way, treated with the peptide ANXA1Ac2-26, does not modify cell morphology and migration, but alters proliferation; is not cytotoxic, but is genotoxic; induces the late apoptosis process, modifies the expression of cytokines/chemokines IL-8, RANTES and TNF- and modulates the expression of genes involved in the inflammatory process. Thus, we show that the interaction of the tumor microenvironment with the action of ANXA1Ac2-26 on endothelial and/or tumor cells probably participates in the tumorigenic process by mechanisms related to antiproliferative, apoptotic and/or inflammatory pathways and open new possibilities for studies to therapeutic alternatives in head and neck cancer.
Head and Neck Squamous Cells Carcinoma (HNSCC) is the sixth most common cancer in the world, being cancer in the oral cavity the most common among the anatomical regions of HNSCC. There is evidence that the development and progression of cancer depends not only on its genetic characteristics, but also because of the interactions of cells, cancer and tumor microenvironment cells. Carcinogenesis is also related to angiogenesis and inflammatory processes which can be controlled by the action of anti-inflammatory mediators, among which we highlight the anti-inflammatory protein annexin A1 (ANXA1). But the mechanisms that ANXA1 acts in tumorigenesis and its relation to the interaction between endothelial and tumorigenic cells are not well known and considering their role in inflammation, comprise an important target for study. Therefore, the present study aimed to analyze the interaction of endothelial and tumorigenic cells with the action of the synthetic peptide Ac2-26 of the ANXA1 protein (ANXA1Ac2-26) in head and neck carcinoma cell line, verifying how the peptide acts with the conditioned medium of endothelial cells and/or tumor cells, elucidating how the factors excreted by both cells and the peptide acts in the tumorigenic process. For this, the morphology observation was performed under inverted microscope, proliferation index by growth curve, cell viability was determined by the colorimetric method MTS (IC50), cell migration by transwell bilayer method, genotoxicity by the Comet assay, apoptosis and necrosis by flow cytometry, cytokine/chemokine release pattern by ELISA and gene expression by the quantitative PCR technique. The statistic was performed by Analysis of Variance for multiple comparisons, followed by Bonferroni adjustment, made it with GraphPad Prism 5.0 software. The results showed that the effect of tumor cell conditioned medium (Hep-2) on endothelial cells (HUVEC) and the other way, treated with the peptide ANXA1Ac2-26, does not modify cell morphology and migration, but alters proliferation; is not cytotoxic, but is genotoxic; induces the late apoptosis process, modifies the expression of cytokines/chemokines IL-8, RANTES and TNF- and modulates the expression of genes involved in the inflammatory process. Thus, we show that the interaction of the tumor microenvironment with the action of ANXA1Ac2-26 on endothelial and/or tumor cells probably participates in the tumorigenic process by mechanisms related to antiproliferative, apoptotic and/or inflammatory pathways and open new possibilities for studies to therapeutic alternatives in head and neck cancer.
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Palavras-chave
Cultura de células, Estroma, Anexina A1, ELISA, Expressão gênica, Cell culture, Annexin A1, Genic expression