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Doctoral Thesis
DOI
10.11606/T.42.2009.tde-02042009-170820
Document
Author
Full name
Leticia Nogueira da Gama de Souza
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Jaeger, Ruy Gastaldoni (President)
Carvalho, Hernandes Faustino de
Chaves, Maria Luiza Morais Barreto de
Zanata, Silvio Marques
Zorn, Telma Maria Tenorio
Title in Portuguese
Peptídeo C16 derivado da laminina regula migração, invasão e secreção de protease em linhagem celular derivada de carcinoma adenóide cístico humano através de integrinas e das vias de sinalização AKT e ERK.
Keywords in Portuguese
Carcinoma adenóide cístico
Integrinas
Laminina
Matriz extracelular
Metaloproteinase da matriz
Neoplasia das glândulas salivares
Abstract in Portuguese
Avaliamos a capacidade de indução de migração, invasão e secreção de protease pelo peptídeo derivado da laminina, C16 (KAFDITYVRLKF, cadeia g1) em linhagem celular (CAC2) de carcinoma adenóide cístico humano. Laminina g1 foi imunolocalizada no carcinoma adenóide in vivo e in vitro. Ensaio de ferida, em câmara bipartite e em vídeo microscopia (time-lapse) mostraram que C16 estimula migração em células CAC2. C16 também estimulou invasão em ensaio com câmaras bipartites cobertas com Matrigel. Invasão depende de atividade de protease. Zimografia mostrou que C16 aumentou secreção de MMPs 2 e 9. Diferentes vias de sinalização podem estar relacionadas com os efeitos de C16. Immunoblot revelou que C16 aumentou a fosforilação de AKT e ERK. Para o estudo de possíveis receptores do peptídeo, preparações de membrana foram passadas em colunas de afinidade com C16 acoplado. Banda de 40kDa foi eluída e analisada por espectrometria de massa (LC-MS/MS) que identificou a cadeia a1 do colágeno. O fragmento de colágeno eluído poderia ser parte de um complexo protéico envolvendo C16. Integrinas são receptores de colágeno e candidatas a fazerem parte desse complexo. Células CAC2 expressaram as integrinas av, a5, b3 and b1. Silenciamento dessas integrinas promoveu redução da migração e secreção de protease induzidas por C16. Sugerimos que C16 estimularia migração, invasão e secreção de protease em células de carcinoma adenóide cístico através de integrinas a5b1 e avb3. O sinal gerado por C16 seria transduzido pelas vias AKT e ERK1/2.
Title in English
Laminin peptide C16 regulates migration, invasion and protease activity of adenoid cystic carcinoma cells through integrins, AKT and ERK.
Keywords in English
Adenoid cystic carcinoma
Extracelular matrix
Integrin
Laminin
Matrix metalloproteinases
Salivary gland neoplasia
Abstract in English
We studied induction of migration, invasion and protease activity by laminin-derived peptide C16 (KAFDITYVRLKF, g1 chain) in a cell line (CAC2) from adenoid cystic carcinoma. Laminin g1 was immunolocalized in adenoid cystic carcinoma in vivo and in vitro. C16 increased migratory activity of CAC2 cells, as shown by monolayer wound assay, Transwell migration assay and time-lapse video microscopy. This peptide also stimulated cell invasion in Transwell chambers coated with Matrigel. Invasion depends on protease activity. Zymograms showed that C16 increased secretion of MMPs 2 and 9. Different signaling pathways could be related to C16 regulation in CAC2 cells. Immunoblot showed that C16 increased phosphorylation of both AKT and ERK compared to controls. To study putative receptors of this peptide we used affinity chromatography. Membrane preparations were run through C16-affinity columns. A 40kDa band was eluted and analyzed by mass spectrometry (LC-MS/MS) identifying a collagen a1 chain. The collagen fragment eluted could be part of a protein complex involving C16. This protein complex may include integrins, which are collagen receptors. CAC2 cells exhibited av, a5, b3 and b1 integrins. siRNA knockdown of these integrins inhibited both C16-induced migration and protease activity. We propose that C16 increases migration, invasion and protease activity of a human salivary gland adenoid cystic carcinoma cell line through a5b1 and avb3 integrins. The signal generated by C16 is transduced by AKT and ERK1/2 signaling pathways.
 
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Anexo_1.pdf (3.59 Mbytes)
Anexo_2.pdf (2.68 Mbytes)
Publishing Date
2009-04-07
 
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