ABSTRACT
Vimentin is a cytoeskeletal intermediate filament protein commonly observed in mesenchymal cells; however, it can also be found in malignant epithelial cells. It is demonstrated in several carcinomas, such as those of the cervix, breast and bladder, in which it is widely used as a marker of the epithelial to mesenchymal transition that takes place during embryogenesis and metastasis. Vimentin is associated with tumors that show a high degree of invasiveness, being detected in invasion front cells. Its expression seems to be influenced by the tumor microenvironment. The aim of this study was to evaluate vimentin expression in head and neck squamous cell carcinoma (HNSCC) cell lines, and to investigate the contribution of the microenvironment to its expression. HNSCC cell lines (HN6, HN30 and HN31) and an immortalized nontumorigenic cell line (HaCaT) were submitted to a three-dimensional assay with Matrigel. Cytoplasmatic staining of the HN6 cell line cultured without Matrigel and of the HN30 and HN31 cell lines cultured with Matrigel was demonstrated through immunohistochemistry. Western Blotting revealed a significant decrease in vimentin expression for the HN6 cell line and a significant increase for the HN30 and HN31 cell lines cultured with Matrigel. The results suggest that vimentin can be expressed in HNSCC cells and its presence is influenced by the microenvironment of a tumor.
Subject(s)
Humans , Carcinoma, Squamous Cell/metabolism , Collagen/pharmacology , Head and Neck Neoplasms/metabolism , Laminin/pharmacology , Neoplasm Proteins/metabolism , Proteoglycans/pharmacology , Vimentin/metabolism , Blotting, Western , Cell Line, Tumor , Carcinoma, Squamous Cell/pathology , Drug Combinations , Extracellular Matrix , Head and Neck Neoplasms/pathology , Immunohistochemistry , Neoplasm Proteins/analysis , Vimentin/analysisABSTRACT
O Celecoxib, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, mas o seu papel no carcinoma epidermóide de boca (CEB) não é totalmente compreendido. Sabe-se que diversas alterações genéticas estão associadas à patogênese do CEB, a neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. É sabido que Akt pode ativar o fator de transcrição NF-kB, o qual tem importante participação na fisiologia normal e no câncer. A proteína COX-2, descrita inicialmente em processos inflamatórios, está associada com a oncogênese e recentemente tem sido associada com a via de sinalização do Akt e com o NF-kB. Portanto, o objetivo deste estudo foi analisar o efeito do Celecoxib sobre linhagens celulares de carcinoma epidermóide de boca e verificar a localização intracelular e a expressão das proteínas pAkt, NF-kB e COX-2 em linhagens celulares de carcinoma epiermóide de boca após o tratamento com o Celecoxib.Através da técnica de imunofluorescência, foram analisados os padrões de expressão das proteínas pAkt, NFkB e COX-2 em quatro linhagens celulares de carcinoma epidermóide bucal submetidas ao tratamento com Celecoxib, cuja a dose e o tempo foram obtidos a partir de ensaios de viabilidade celular. Também se realizou ensaio de apoptose celular. Como controle utilizou-se células não tratadas com o medicamento. O Celecoxib na dose de 30 M por 24 horas causa apoptose.Na técnica de western blot, somente a linhagem SCC15 apresentou uma diminuição significativa para a COX-2. Entretanto, para p-Akt e NF-kB nenhuma alteração na expressão foi observada entre os grupos controle e tratado.Na imunofluorescência, houve alteração no padrão de expressão das proteínas pAkt, NF-kB e COX-2, quando se comparou os grupos contrele e tratado. Portanto, o Celecoxib pode ser um eficaz agente terapêutico, uma vez que demonstrou grande eficácia na inibição da proliferação celular de linhagens celulares de CEB.
Celecoxib, nonsteroidal anti-inflammatory COX-2 selective inhibitor, has proven to be an important anticancer agent. However its role in oral squamous cell carcinoma (OSCC) is not entirely understood. This is the most common malignancy of head and neck regionand it is known that various genetic alterations are associated with its pathogenesis. Some of these changes affect proteins belonging to the Akt signaling pathway, involved in different cellular processes. It is known that Akt can activate the transcription factor NF-kB, which has important role in normal physiology and cancer.The COX-2 proteinwas firstly described in the inflammatory processes, is associated with oncogenesis and has recently been related with the Akt signaling pathwayand with NF-kB. Therefore, the aim of this study was to analyze the effect of Celecoxib on squamous cell carcinoma cell lines and to determine pAkt, NF-kB and COX-2 intracellular localization and levels of expressionin this cell lines after treated with Celecoxib. By immunofluorescence, we analyzed the pAkt, NF-kB and COX-2 expression patterns in four oral squamous cell carcinoma cell lines treated with Celecoxib, which the dose and time were obtained from cell viability assays. Cellular apoptosis assay was also performed. As control the cells were not treated with this drug. Celecoxibcauses apoptosisin the dose of 30 M for 24 hours. In the western blottechnique, only the SCC15 cell line shows a significant decrease for COX-2. However, for p-Akt and NF-kB no change in expression was observed between control and treated groups. On immunofluorescence, there were changes in the pAkt, NFkB and COX-2protein expression pattern when the control group was compared with treated group. Therefore, the Celecoxib can be an effective therapeutic agent, since it has shown great efficacy in thecelular proliferation inhibition of the OSCC cell lines.