Efeito do meio condicionado de glioma sobre a diferenciação de macrófagos : envolvimento da sinalização purinérgica

Gliomas são os tumores mais comuns e devastadores do sistema nervoso central. A presença de um ambiente inflamatório, incluindo macrófagos associados ao tumor (TAM), auxiliam a proliferação tumoral e estão associados a um pior prognóstico em pacientes com essa neoplasia. A interrupção da sinalização purinérgica também tem sido implicada na progressão do câncer. O ATP pode atuar como uma molécula de sinalização de dano celular, enquanto a adenosina, o produto da degradação do ATP, serve como um mecanismo de feedback negativo para limitar a inflamação. A comunicação de nucleotídeos é controlada pela ação de ectonucleotidases, tais como ENTPDase e ecto-5'- nucleotidase/CD73. Com isso, investigamos se a exposição de macrófagos ao meio condicionado de GL261 (GL-CM) modulam o fenótipo de macrófagos, bem como as atividades das ectonucleotidases. O envolvimento de receptores purinérgicos na secreção de citocinas por macrófagos também foi investigado. Os macrófagos expostos a GL-CM apresentam uma diminuição da atividade da iNOS, baixos níveis de secreção de citocinas pró-inflamatórias TNF-α e IL-12 e alto nível de secreção da citocina anti-inflamatória IL-10. A expressão de genes como FIZZ1 e Ym1 foi aumentada. A hidrólise de ATP, ADP e AMP foi diminuída em macrófagos estimulados com GL-CM. A citometria de fluxo e Real-Time PCR mostraram uma significativa mudança na expressão de proteínas para a ENTPDase 2 e 3 e houve alteração do nível de mRNA para a ENTPDase 1 e ecto-5'-NT/CD73. Houve também um aumento da secreção das citocinas IL-6, IL-10 e MCP-1 por macrófagos expostos a GL-CM. Este efeito foi revertido pela suramina, indicando a participação de ATP na secreção dessas citocinas. Em conclusão, os macrófagos expostos ao meio condicionado de gliomas foram modulados a um fenótipo análogo ao M2 (TAM), que foram acompanhadas por uma diminuição na hidrólise de nucleotídeos. Portanto, nossos resultados fornecem evidências para o envolvimento da hidrólise extracelular de nucleotídeos no processo inflamatório associado à progressão tumoral.Gliomas are the most common and devastating type of primary brain tumors. The presence of an inflammatory environment, including tumor-associated macrophages (TAM), promote tumor proliferation and are associated with a poor prognosis in patients with this malignancy. Disruption of purinergic signalling also has been implicated in cancer progression. ATP can act as danger signaling whereas adenosine, the ATP breakdown product, serves as a negative feedback mechanism to limit inflammation. The nucleotide communication is controlled by the action of ectonucleotidases, such as ENTPDase and ecto-5′-nucleotidase/CD73. Herewith we investigate if the exposure of macrophage cells to conditioned medium of GL261 (GL-CM) cell cultures modulate macrophage differentiation as well as the ectonucleotidase activities. The involvement of purine receptors in the secretion of cytokines by macrophages was also investigated. Macrophages exposed to GL-CM exhibits decreased iNOS activity, release low levels of the pro-inflammatory cytokine TNF-α and IL-12 and high levels of the anti-inflammatory cytokine IL-10. The expression of genes FIZZ1 and Ym1 was increased too. The hydrolysis of ATP, ADP and AMP was decreased in macrophages stimulated with GL-CM. Flow cytometry and Real Time-PCR analysis showed a significant changed in NTPDase 2 and 3 protein and mRNA expression of NTPDase 1 and ecto-5´- NT/CD73, respectively. There was also an increase in IL-6, IL-10 and MCP-1 secretion by macrophages exposed to GL-CM. This effect was reversed by suramin, indicating the participation of ATP in the secretion of these cytokines. In conclusion, macrophages exposed to glioma conditioned medium showed a polarized to M2-like (TAM) phenotype, which were accompanied by a decrease in the nucleotide hydrolysis, providing further evidence for the involvement of the extracellular nucleotide hydrolysis in the inflammatory process associated to tumor progression

Perfil hemoglobínico de amostras do Rio Grande do Sul - Brasil

02

Ectonucleotidases in Tumor Cells and Tumor-Associated Immune Cells: An Overview

Increasing evidence points out that genetic alteration does not guarantee the development of a tumor and indicates that complex interactions of tumor cells with the microenvironment are fundamental to tumorigenesis. Among the pathological alterations that give tumor cells invasive potential, disruption of inflammatory response and the purinergic signaling are emerging as an important component of cancer progression. Nucleotide/nucleoside receptor-mediated cell communication is orchestrated by ectonucleotidases, which efficiently hydrolyze ATP, ADP, and AMP to adenosine. ATP can act as danger signaling whereas adenosine, acts as a negative feedback mechanism to limit inflammation. Many tumors exhibit alterations in ATP-metabolizing enzymes, which may contribute to the pathological events observed in solid cancer. In this paper, the main changes occurring in the expression and activity of ectonucleotidases in tumor cells as well as in tumor-associated immune cells are discussed. Furthermore, we focus on the understanding of the purinergic signaling primarily as exemplified by research done by the group on gliomas

Extracellular mycobacterial DnaK polarizes macrophages to the M2-like phenotype

Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-c and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses

Differential macrophage activation alters the expression profile of NTPDase and Ecto-5´-nucleotidase

Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-59-nucleotidase/CD73 (ecto-59-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6–8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-59-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-59-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set

Envolvimento do sistema purinérgico, da enzima ciclooxigenase 2 e sistema imune no desenvolvimento e progressão de glioblastoma multiforme e novas alternativas terapêuticas para esse tipo tumoral

Glioblastoma multiforme é o tumor maligno mais comum do sistema nervoso central em adultos e a sobrevida média é de apenas 12 a 15 meses após o diagnóstico. Por isso, é extremamente importante desenvolver tratamentos mais eficazes e específicos contra essa neoplasia. A presença do sistema imune, incluindo macrófagos associados ao tumor, promove a proliferação tumoral e está associada com um pior prognóstico em pacientes com essa doença maligna. A sinalização purinérgica e o receptor purinérgico P2X7, um canal iônico, têm sido implicados na progressão de diferentes tipos de tumores tanto in vitro como in vivo. A ciclooxigenase 2 (COX-2) desempenha um papel importante na regulação da proliferação celular, diferenciação e na tumorigênese. O ácido ursólico é um triterpeno pentacíclico encontrado em uma variedade de plantas e exibe diversas atividades biológicas e farmacológicas. O objetivo dessa tese é verificar a participação do sistema purinérgico, sistema imune e COX-2 no desenvolvimento e progressão do glioblastoma multiforme, e também investigar os efeitos citotóxicos do ácido ursólico. Primeiramente, verificamos que macrófagos expostos ao meio condicionado de glioma (GL-CM) foram modulados para um fenótipo do tipo M2 e houve um aumento da liberação de IL-10, IL-6 e MCP-1. Esses efeitos foram diminuídos na presença de antagonistas dos receptores P2X7 e A2A. Portanto, os resultados apresentados contribuem para o melhor entendimento da interação entre inflamação e câncer e demonstram que os receptores purinérgicos são importantes para a progressão do glioma. Após, analisamos o papel do receptor P2X7 na proliferação de células de glioma. Surpreendentemente, in vitro, não se observou nenhuma diferença no crescimento das células quando houve a transfecção com o P2X7. Entretanto, in vivo, essas células geraram tumores maiores quando comparado com o controle. Os nossos resultados demonstram que, como em outros tipos de cânceres, o P2X7 tem um papel importante no desenvolvimento e progressão tumoral. Uma vez verificado o importante papel do receptor P2X7 nos macrófagos associados ao tumor e nas células de glioma, investigamos se esse receptor poderia interagir com a enzima COX-2 em células de glioma. Porém, não houve diferença na expressão do P2X7 ou da COX-2 tanto in vitro como in vivo. E também não houve nenhum efeito adicional entre o antagonista de P2X7 e o inibidor seletivo de COX-2. Esse trabalho fornece evidências de que não há relação entre o P2X7 e COX-2 em células de glioma. Em conclusão, todos esses resultados reforçam a hipótese do envolvimento da sinalização purinérgica na progressão do glioblastoma multiforme e tornam o P2X7 como um interessante alvo terapêutico. Finalmente, também investigamos a possível atividade anticâncer do ácido ursólico contra as células de glioma. Essa molécula foi capaz de diminuir o número de células e induziu parada no ciclo celular. In vivo, o ácido ursólico reduziu ligeiramente o tamanho do tumor, mas não alterou as características malignas. Em conclusão, o ácido ursólico pode ser um potencial candidato como adjuvante para o tratamento do glioblastoma multiforme. Em conjunto, todos os resultados apresentados nessa tese indicam possíveis novas abordagens terapêuticas no tratamento e novos conhecimentos em relação a esse maligno câncer cerebral.Glioblastoma multiforme is the most common malignant tumor of central nervous system in adults and the median survival is only 12 to 15 months after diagnosis. Therefore, it is extremely important to develop more effective and specific treatments. The presence of an inflammatory environment, including tumor-associated macrophages, promotes tumor proliferation and is associated with a poor prognosis in patients with this malignancy. Disruption of purinergic signaling has also been implicated in cancer progression. P2X7R is an ion channel receptor, whose participation in tumor progression has been demonstrated in in vitro and in vivo studies. Cyclooxygenase 2 (COX-2) plays an important role in regulating cell proliferation, differentiation, and tumorigenesis. Ursolic acid is a pentacyclic triterpenoid found in a variety of plants that exhibits several biological and pharmacological activities. The aim of this study is to verify the participation of the purinergic system, immune system and COX-2 in the glioblastoma multiforme development and progression, and also to investigate the anti-proliferative effects of ursolic acid. We first verified that macrophages exposed to glioma conditioned medium (GL-CM) were modulated to an M2-like phenotype and there was an increased IL-10, IL-6 and MCP-1 secretion and these effects were diminished by P2X7 and A2A receptors antagonists. Therefore, the results presented contribute to advancing in the field of cancer-related inflammation and point specific purinergic receptors as targets for glioma progression. After that, we analyzed the role of P2X7 receptor in glioma cell proliferation. Surprisingly, in vitro, no difference in cell growth was observed when the cells were transfected with P2X7R but in vivo these cells generated larger tumors when compared to the control. Our data demonstrate that, as in other type of cancers, P2X7R has an important role in sustaining the development of glioma. Once verified the important role of P2X7 receptor in tumorassociated macrophages and glioma cells, we verified whether this receptor could interact with the COX-2 enzyme in glioma cells. No differences in mRNA expression of P2X7R or COX-2 were verified both in vitro and in vivo experiments. And any additional effect with selective P2X7R antagonist and COX-2 inhibitor were observed in in vitro and in vivo experiments. This work provides evidence that there is no relationship between the P2X7R and COX-2 in glioma. In conclusion, all these results reinforce the hypothesis of purinergic signaling involvement in glioma progression and point to P2X7R as an interesting target for glioma treatment. Finally, we also investigated the potential anticancer activity of ursolic acid against to glioma cells. Ursolic acid decreased the cell number and induced an arrest in the cell cycle in glioma cells. In vivo, ursolic acid slightly reduced the glioma tumor size but did not alter the malignant features. In conclusion, the ursolic acid may be a potential candidate as adjuvant for glioblastoma therapy. Taken together, the results presented herein indicate new adjuvant treatment approaches and new knowledge regarding to this deadliest brain tumor

Perfil hemoglobínico de amostras do Rio Grande do Sul - Brasil

02