Avaliação in vitro e in vivo da transição epitélio-mesenquimal e seus efeitos em diferentes modelos de melanoma
| Ano de defesa: | 2022 |
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| Autor(a) principal: | |
| Orientador(a): | |
| Banca de defesa: | |
| Tipo de documento: | Tese |
| Tipo de acesso: | Acesso aberto |
| Idioma: | por |
| Instituição de defesa: |
Universidade Federal de Minas Gerais
Brasil ICB - DEPARTAMENTO DE PATOLOGIA Programa de Pós-Graduação em Patologia UFMG |
| Programa de Pós-Graduação: |
Não Informado pela instituição
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| Departamento: |
Não Informado pela instituição
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| País: |
Não Informado pela instituição
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| Palavras-chave em Português: | |
| Link de acesso: | http://hdl.handle.net/1843/77972 https://orcid.org/0000-0003-4696-7738 |
Resumo: | Melanoma is the malignant neoplasm originating from melanocytes, being the most aggressive and with the highest mortality rate among skin neoplasms. Melanocyte homeostasis is controlled by the interaction with keratinocytes mediated by E-cadherin expression. Epithelial-mesenchymal transition (EMT), a process associated with progression in epithelial neoplasms, is characterized by the change from an epithelial to mesenchymal phenotype, with a reduction in E-cadherin and an increase in N-cadherin expression. In melanomas, the occurrence and role of EMT, although observed with some similarities, remains under discussion due to the non-epithelial embryological origin of melanocytes and the high histological and molecular heterogeneity of melanomas. The regulatory mechanisms of EMT are widely studied, due to its important role in neoplastic progression, and it has been suggested that metformin has the potential to inhibit its occurrence. Thus, in this work we investigated the effects of metformin on EMT in human melanoma A-375 and murine melanoma B16-F10 cell lines as well as the relationship between EMT-associated markers and the formation of metastases in an in vivo experimental model of melanoma in C57BL/6 mice. For the in vitro experiments, the cell lines were cultivated, treated with 0mM (control), 0.5mM or 5mM metformin, and cell viability was evaluated by the MTT assay, gene expression of EMT markers and cell differentiation by RT-qPCR , assessment of invasive and migratory potential by matrigel and wound healing assays. To evaluate lung colonization, B16-F10 cells cultured and treated in vitro were inoculated via the caudal vein into C57BL/6 mice that were followed for 20 days. The animals were euthanized and the metastatic foci in the lung were evaluated, as well as the immunohistochemical expression of E-cadherin, N-cadherin, ZEB1, ZEB2, Twist, Vimentin, Nanog, Sox10 and CDC47 in the areas of lung metastases. In in vitro assays, treatment with metformin did not impact cell viability at 24 hours of treatment, but at 48 hours, treatment with 5mM significantly reduced cell metabolism in both cell lines in relation to the respective controls. The gene expression of CDH1, CDH2, Snail, ZEB1, ZEB2, OCT3/4, Nanog and miR200-c did not show significant changes with the concentrations used in any of the studied cell lines, since the migratory potential of both cell lines was reduced against the treatment with the two concentrations of metformin. In the in vivo model, metastatic formations were observed in only 10% (1/10) of the animals that received cells treated with 5mM of metformin, while in those that received cells without previous treatment or treated with only 0.5mM, 100% of the animals showed development of lung metastases. In addition, the formations observed in the group that received cells treated with 5mM metformin were smaller than those observed in the other groups. A reduction in the protein expression of N-cadherin, vimentin, CDC47, ZEB2 and Sox10 was also observed in the metastases of cells previously treated with 5mM. The results indicate that proteins involved in TEM apparently play an important role in the migration and development of metastases in experimental models of melanoma, as evidenced by pharmacological inhibition with metformin. |