Avaliação do procesamento proteolítico da citocina TGF beta em secretomas de melanoma
Arquivos
Data
2022-07-28
Autores
Barcick, Uilla 
Orientadores
Zelanis, André
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O câncer é o termo utilizado que abrange um conjunto de mais de 100 doenças que
possuem em comum o crescimento desordenado e descontrolado de células
anormais. Dentre esse tipo de doença encontra-se o melanoma, que é um tipo de
câncer de pele que acomete os melanócitos, e é tido como o mais grave dentre os
câncer de pele por sua alta capacidade de progredir para metástase. Num estudo
recente realizado pelo nosso grupo acerca do processamento proteolítico no
secretoma de um modelo pareado de melanoma murino, composto pelas linhagens
Melan- a (melanócito normal) e Tm1 (o fenótipo tumoral correspondente) foi possível
identificar, exclusivamente no fenótipo tumoral, eventos de processamento proteolítico
em proteínas de importante papel no desenvolvimento tumoral. Dentre os sítios de
clivagem observados, identificamos um peptídeo correspondente ao processamento
da proteína precursora do TGFβ-3 em uma porção imediatamente anterior ao sítio de
clivagem canônico para esta citocina. Do ponto de vista funcional, este achado sugere
uma ativação não-canônica e autócrina de TGFβ-3 por melanócitos transformados.
Para identificar formas processadas da citocina TGFβ em secretomas de melanócitos
tumorais, foi realizado um ensaio de Western Blotting em que foi possível perceber a
presença dessa citocina em uma banda com massa adicional exclusivamente nos
secretomas das linhagens tumorais. Com o intuito de associar esse achado com vias
biológicas foram realizados ensaios de migração celular. Esse ensaio resultou numa
diferença nos tempos de migração de uma linhagem endotelial ao sofrer estímulos
dos secretomas de melanócitos e de melanoma. Por fim, foi feito uma lista de
proteases que poderiam ser as responsáveis pela clivagem do TGF-3 no sítio não
canônico. Porém, para confirmarmos o processamento não canônico e identificarmos
o mecanismo envolvido nesse evento mais ensaios devem ser realizados.
Cancer is the term used to cover a set of more than 100 diseases that have in common the disordered and uncontrolled growth of abnormal cells. Among this type of disease lies melanoma, which is a type of skin cancer that affects melanocytes, and is considered the most serious among skin cancers due to its high capacity to progress to metastasis. In a recent study, performed by our group, on proteolytic processing in the secretome of a paired model of murine melanoma, composed of Melan-a (normal melanocyte) and Tm1 (the corresponding tumor phenotype) lineages, it was possible to identify, exclusively in the tumor phenotype, proteolytic processing events in proteins important role in tumor development. Among the cleavage sites observed, we identified a peptide corresponding to the processing of the TGFβ-3 precursor protein in a portion immediately anterior to the canonical cleavage site for this cytokine. From a functional point of view, this finding suggests a non-canonical and autocrine activation of TGFβ 3 by transformed melanocytes. To identify processed forms of the cytokine TGFβ in secretomes of tumor melanocytes, a Western Blotting assay was performed. It was possible to perceive the presence of this cytokine in a band with additional mass exclusively in the secretomes of the tumor cell lines. In order to associate this finding with biological pathways, cell migration assays were performed. This assay resulted in a difference in the migration times of an endothelial cell line when undergoing stimuli from melanocyte and melanoma secretomes. Finally, a list of proteases that could be responsible for the cleavage of TGF-3 at the non-canonical site was made. In order to confirm the non-canonical processing and identify the mechanism involved in this event, further tests should be performed.
Cancer is the term used to cover a set of more than 100 diseases that have in common the disordered and uncontrolled growth of abnormal cells. Among this type of disease lies melanoma, which is a type of skin cancer that affects melanocytes, and is considered the most serious among skin cancers due to its high capacity to progress to metastasis. In a recent study, performed by our group, on proteolytic processing in the secretome of a paired model of murine melanoma, composed of Melan-a (normal melanocyte) and Tm1 (the corresponding tumor phenotype) lineages, it was possible to identify, exclusively in the tumor phenotype, proteolytic processing events in proteins important role in tumor development. Among the cleavage sites observed, we identified a peptide corresponding to the processing of the TGFβ-3 precursor protein in a portion immediately anterior to the canonical cleavage site for this cytokine. From a functional point of view, this finding suggests a non-canonical and autocrine activation of TGFβ 3 by transformed melanocytes. To identify processed forms of the cytokine TGFβ in secretomes of tumor melanocytes, a Western Blotting assay was performed. It was possible to perceive the presence of this cytokine in a band with additional mass exclusively in the secretomes of the tumor cell lines. In order to associate this finding with biological pathways, cell migration assays were performed. This assay resulted in a difference in the migration times of an endothelial cell line when undergoing stimuli from melanocyte and melanoma secretomes. Finally, a list of proteases that could be responsible for the cleavage of TGF-3 at the non-canonical site was made. In order to confirm the non-canonical processing and identify the mechanism involved in this event, further tests should be performed.