Envolvimento da sinalização Notch e de metaloproteases em co-culturas de células endoteliais e células leucêmicas
Data
2021-08-10
Autores
Souza, Emanoelle Fabossi de [UNIFESP]
Orientadores
Justo, Giselle Zenker [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A leucemia mieloide crônica (LMC) é uma doença mieloproliferativa, caracterizada pela translocação BCR-ABL, que gera uma tirosina quinase constitutivamente ativa, a qual estimula a proliferação e sobrevivência das células de LMC. A resistência à quimioterapia é um dos principais aspectos da falha dos tratamentos no câncer, estando relacionada frequentemente à superexpressão dos transportadores ABCB1, incluindo a glicoproteína P (Pgp). Além disso, interações entre as células leucêmicas e as células endoteliais vizinhas no microambiente da medula óssea são importantes para o desenvolvimento da doença e suscetibilidade à quimioterapia. Estudos sugerem uma associação entre a via Notch e o fenótipo MDR. Em adição, a via Notch estaria envolvida no estímulo da função de células endoteliais em co-culturas com células leucêmicas e este processo envolve a ativação de metaloproteases (MMP-2 e MMP-9). Portanto, neste trabalho investigou-se diferenças na sinalização Notch e na expressão/atividade de metaloproteases devido ao fenótipo de resistência a múltiplas drogas (MDR) em sistemas de co-cultura entre células de LMC (linhagens K562 e sua variante superexpressando Pgp, Lucena 1) e células endoteliais (linhagem ECV-304). Os resultados mostraram que as células Lucena 1 apresentam níveis superiores de Notch1 e 2, bem como do domínio interacelular ativo de Notch1 (NICD1), em relação à K562. Uma alta expressão de Hes1 foi observada em ambas linhages, sugerindo ativação de Notch nas células MDR+. As culturas de células de LMC com meio condicionado das células endoteliais levaram a um aumento na expressão de NICD1 e Hes1 nas células Lucena 1, sugerindo ativação desta via também nestas condições. Por outro lado, houve redução significativa de Notch2 e c-Myc em ambas linhagens. A interação célula-célula de maneira contato-dependente estimulou a maior expressão de Notch 2 e de c-Myc nas células com fenótipo MDR, enquanto que uma redução significativa de Notch1 nas células Lucena 1 e de NICD1 em ambas células foi observado. O meio condicionado das células de LMC reduziu a expressão de Notch1 e NICD1 nas células endoteliais e os efeitos sobre c-Myc e Hes1 corroboraram esses achados. Não se observou diferença na atividade gelatinolítica de MMP-2 e MMP-9 entre as linhagens de LMC. Os efeitos da co-cultura sobre as gelatinases foram mais proeminentes nas células K562, aumentando a expressão de MMP-9 na presença do meio condicionado das células endoteliais e de MMP-2 na co-cultura em contato direto. A comunicação célula-célula, por meio de mediadores solúveis e também contato físico, levou ao estímulo da atividade de MMPs nos sobrenadantes das culturas em todas as condições experimentais. Em conjunto os resultados sugerem uma associação entre o fenótipo MDR e a sinalização Notch nas células de LMC, que pode ser influenciada pelo cross-talk entre as células leucêmicas e as células endoteliais, sendo acompanhada da modulação de MMP-2 e MMP-9.
Chronic myeloid leukemia (CML) is a myeloproliferative disease, characterized by BCR-ABL translocation, which generates a constitutively active tyrosine kinase, which stimulates proliferation and survival of CML cells. Resistance to chemotherapy is one of the most relevant aspects of treatment failure in cancer, which is frequently associated with the overexpression of ABCB1 transporters, including the P glycoprotein (Pgp). In addition, interactions between leukemic cells and neighboring endothelial cells within the bone marrow microenvironment are important for disease development and susceptibility to chemotherapy. Studies also suggests an association between the Notch pathway and the MDR phenotype. In addition, the Notch sinaling seems to be involved in stimulating the function of endothelial cells in co-cultures with leukemic cells and this process involves the activation of metalloproteases (MMP-2 and MMP-9). Therefore, this work investigated differences in the Notch signaling and in the expression/activity of metalloproteases due to the multiple drug resistance (MDR) phenotype in co-culture systems of CML cells (K562 and its variant overexpressing Pgp, Lucena 1, cell lines) and endothelial cells (ECV-304 cell line). The results showed that Lucena 1 cells have higher levels of Notch1 and 2, as well as the active interacellular domain of Notch1 (NICD1), compared to K562. This was followed by a high expression of Hes1 was observed in both cell lines, suggesting activation of Notch in MDR+ cells. Cultures of CML cells with conditioned medium of endothelial cells led to an increase in the expression of NICD1 and Hes1 in Lucena 1 cells, suggesting activation of this pathway also in these condition. On the other hand, there was a significant reduction in Notch2 and c-Myc levels in both strains. The cell-cell interaction in a contact-dependent manner stimulated a greater expression of Notch 2 and c-Myc in MDR cells, while a significant reduction of Notch1 in Lucena 1 cells and NICD1 in both cells was observed. The conditioned medium of the CML cells reduced the expression of Notch1 and NICD1 in the endothelial cells and the effects on c-Myc and Hes1 corroborated this findings. There was no difference in the gelatinolytic activity of MMP-2 and MMP-9 between both CML cell lines. The effects of co-culture on gelatinases were more prominent in K562 cells, increasing the expression of MMP-9 in the presence of the conditioned medium of the endothelial cells, and of MMP-2 in direct contact co-culture. Cell-cell communication, through soluble mediators and also physical contact, led to the stimulation of MMP activity in culture supernatants at all experimental conditions. Together, the results suggest an association between the MDR phenotype and Notch signaling in CML cells, which can be influenced by the cross-talk between leukemic and endothelial cells, being accompanied by the modulation of MMP-2 and MMP-9.
Chronic myeloid leukemia (CML) is a myeloproliferative disease, characterized by BCR-ABL translocation, which generates a constitutively active tyrosine kinase, which stimulates proliferation and survival of CML cells. Resistance to chemotherapy is one of the most relevant aspects of treatment failure in cancer, which is frequently associated with the overexpression of ABCB1 transporters, including the P glycoprotein (Pgp). In addition, interactions between leukemic cells and neighboring endothelial cells within the bone marrow microenvironment are important for disease development and susceptibility to chemotherapy. Studies also suggests an association between the Notch pathway and the MDR phenotype. In addition, the Notch sinaling seems to be involved in stimulating the function of endothelial cells in co-cultures with leukemic cells and this process involves the activation of metalloproteases (MMP-2 and MMP-9). Therefore, this work investigated differences in the Notch signaling and in the expression/activity of metalloproteases due to the multiple drug resistance (MDR) phenotype in co-culture systems of CML cells (K562 and its variant overexpressing Pgp, Lucena 1, cell lines) and endothelial cells (ECV-304 cell line). The results showed that Lucena 1 cells have higher levels of Notch1 and 2, as well as the active interacellular domain of Notch1 (NICD1), compared to K562. This was followed by a high expression of Hes1 was observed in both cell lines, suggesting activation of Notch in MDR+ cells. Cultures of CML cells with conditioned medium of endothelial cells led to an increase in the expression of NICD1 and Hes1 in Lucena 1 cells, suggesting activation of this pathway also in these condition. On the other hand, there was a significant reduction in Notch2 and c-Myc levels in both strains. The cell-cell interaction in a contact-dependent manner stimulated a greater expression of Notch 2 and c-Myc in MDR cells, while a significant reduction of Notch1 in Lucena 1 cells and NICD1 in both cells was observed. The conditioned medium of the CML cells reduced the expression of Notch1 and NICD1 in the endothelial cells and the effects on c-Myc and Hes1 corroborated this findings. There was no difference in the gelatinolytic activity of MMP-2 and MMP-9 between both CML cell lines. The effects of co-culture on gelatinases were more prominent in K562 cells, increasing the expression of MMP-9 in the presence of the conditioned medium of the endothelial cells, and of MMP-2 in direct contact co-culture. Cell-cell communication, through soluble mediators and also physical contact, led to the stimulation of MMP activity in culture supernatants at all experimental conditions. Together, the results suggest an association between the MDR phenotype and Notch signaling in CML cells, which can be influenced by the cross-talk between leukemic and endothelial cells, being accompanied by the modulation of MMP-2 and MMP-9.
Descrição
Citação
SOUZA, E. F. Envolvimento da sinalização Notch e de metaloproteases em co-culturas de células endoteliais e células leucêmicas. São Paulo, 2021. 114 f. Dissertação (Mestrado em Ciências Biológicas (Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2021