Análise do perfil de expressão de genes relacionados à resistência a múltiplas drogas em cultura tridimensional de células de melanoma cutâneo humano

• Autor(a) principal: Faria, Letícia Cristine de
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/10156
• topic: Cultura celular tridimensional; Esferóides; Melanoma cutâneo; Resistência a múltiplas drogas; Transportadores ABC; ABC transportes; Cutaneous melanoma; Resistance to multiple drugs; Three-dimensional culture; Spheroids; BIOQUIMICA::BIOLOGIA MOLECULAR

Cutaneous melanoma is a type of skin cancer being considered aggressive due to its rapid evolution, high predisposition to metastasis and the low effectiveness of current treatments in advanced stages. Poor prognosis results mainly from the intrinsic resistance to chemotherapy with the ability to confer resistance to multiple drugs (MDR). Among the several mechanisms related to the MDR phenotype, the best characterized involves expression of the superfamily of ATPdependent ABC transporters, identified as facilitators of drug efflux. In this context, it becomes important to search for models that make it possible to understand the behavior of the cell and the tumor microenvironment. Three-dimensional culture models have been proposed by mimicking the tumor microenvironment through cell-cell and extracellular matrix-cell interaction. Therefore, this study analyzed the expression profile of the ABCB1, ABCB5 and ABCB8 transporters in human cutaneous melanoma cells in a three-dimensional culture model through the formation of spheroids. Cutaneous melanoma cells from the A-375 and SKMEL-37 lineage were cultured in a monolayer and later were used for spheroid formation using 3 different methods: 1) agarose-coated plate culture; 2) culture in hanging drop; 3) culture in plate with non-adherent surface. Expression analysis of the ABCB1, ABCB5 and ABCB8 genes was performed by RT-PCR and visualization of the amplicons was done by agarose gel electrophoresis. Real-time RT-PCR reactions were performed for the quantification of the expression of these genes using the CT method. Data analysis was performed through One Way ANOVA, followed by Tukey post-test (p≤0.05). Our results demonstrated that the three-dimensional cell culture model in plate with the non-adherent surface was the most efficient, simple to perform and high efficiency technique for spheroid formation. Through RT-PCR we observed that the ABCB5 gene was not expressed in the cell lines studied and this data was confirmed in real time RT-PCR. The ABCB1 and ABCB8 genes were expressed on the spheroids formed by cells A-375 and SKMEL-37. In these two cell lines we observed that there is a significant increase in the expression of the ABCB1 and ABCB8 genes at time 07 in relation to the control. These findings show that the three-dimensional culture model through spheroid formation provided expression analysis of the ABCB1, ABCB5 and ABCB8 transporters, which are associated with drug resistance activity in human cutaneous melanoma.