Caracterização do sistema purinérgico e seu envolvimento na progressão de meduloblastomas

Meduloblastoma é um tipo de tumor neuroepitelial que acomete principalmente o cerebelo de crianças com idade média de aproximadamente nove anos. Estes tumores são identificados como a principal malignidade cerebral entre crianças e são classificados pela Organização Mundial de Saúde como tumores de alto grau. Inúmeras alterações gênicas, cromossômicas e em vias de sinalização têm sido utilizadas como ferramentas para a classificação de tumores. Alterações evidenciadas no sistema purinérgico, o qual se baseia no estudo das funções desencadeadas pelo ATP e outros nucleotídeos e nucleosídeos no meio extracelular têm sido identificadas por favorecerem a progressão de malignidades. Tais funções são mediadas pela ativação de um amplo grupo de receptores purinérgicos denominados P2, e reguladas por uma família de enzimas denominadas ectonucleotidases. Nosso grupo de pesquisa tem publicado resultados que apontam para a íntima relação entre o sistema purinérgico e a progressão de gliomas. No intuito de dar contunuidade a estes estudos em outros tumores do sistema nervoso central, o objetivo principal desta tese de doutorado foi caracterizar a expressão e funcionalidade do sistema purinérgico em diferentes linhagens de meduloblastoma humano fazendo uso de técnicas tanto in vitro quanto in vivo. Observamos que as linhagens Daoy, ONS76 e D283 apresntaram a expressão e funcionalidade dos receptores purinérgicos P2 de forma bastante significativa. Dentre os receptores expressos, enfatizamos os estudos sobre o P2X₇ o qual, quando estimulado por concentrações farmacológicas de ATP desencadeou morte celular na linhagem Daoy de MB humano, confirmando o potencial deste receptor como um eficiente alvo de terapias contra esse tipo de câncer. Na sequencia, avaliamos a família das ectonucleotidases, onde observamos que assim como em gliomas, os meduloblastomas apresentam uma baixa capacidade de hidrólise do ATP no meio extracelular e uma alta atividade AMPásica, atribuída a elevada expressão de enzima ecto-5'NT/CD73 pelas linhagens Daoy e ONS76. Já a linhagem D283 apresentou ausência da atividade e expressão desta enzima. Sabendo que a ecto-5'NT/CD73 tem sido descrita por favorecer a progressão de tumores, superexpressamos essa enzima na linhagem D283 e avaliamos o crescimento dos tumores formados após o implante in vivo. Observamos que, para os meduloblastomas, a presença da enzima promoveu uma significativa redução no tamanho dos tumores implantados quando comparado com o grupo controle. Esses resultados nos levam a sugerir que a expressão da ecto-5'NT/CD73 por células de meduloblastoma pode ser considerada um marcador de prognóstico assim como ser utilizada como um possível alvo terapêutico no tratamento desta malignidade. Por fim, de uma forma geral, mostramos o funcionamento do sistema purinérgico em mais um tipo de tumor e que a sua modulação pode servir, de diferentes formas, como ferramenta para o controle da progressão de meduloblastomas.Medulloblastoma is a kind of neuroepithelial tumor that mainly affects the cerebellum of children with median age of nine years old. These tumor are identified as the principal brains malignancy in children and are classified by the World Health Organization as tumors of high malignancy. Numerous genetic mutations as well chromosomal and signaling pathways alterations have been used as tools to classify these tumors. Changes in the purinergic system, wich is based on the study of functions triggering by ATP and others nucleotides and nucleosides into the extracellular medium, have been described to favoring the malignancies progression. These functions are mediated by stimulation of a broad group of purinergic receptors named P2 and regulated by enzimes family called ectonucleotidases. Our research group has published results that indicate the relationship between the purinergic system and the glioma progression. In view of the continuation of these studies in other tumors of the central nervous system, the main objective of this thesis was to characterize the expression and function of the purinergic system in different human medulloblastoma cell lines making use of in vitro and in vivo techniques. We observe that Daoy, ONS76 and D283 showed the pronounced expression and function of P2 purinergic receptors. Among the expressed receptors we highlight the studies about the P2X₇. When stimulated by pharmacological ATP concentrations it triggered cell death in Daoy cell line, confirming the potencial of this receptor as a target for effective cancer therapies. Considering the necessity for the modulation of this system, we avaluated at the sequence the ectonucleotidases family. We observed that the medulloblastoma cell lines presented reduced ATP hydrolysis in the extracellular medium and presented a prominent AMP hydrolysis wich was attributed to ecto-5'NT/CD73 expression presented by Daoy and ONS76 cell lines. D283 cell line did not shown expression or activity attributed to this enzyme. Knowing that ecto-5'NT/CD73 has been described to favor the tumor progression, we performed the overexpression of this enzyme in the D283 cell line and its functionality was evaluated in an in vivo xenograph tumor model. The overexpression of this enzyme caused a significant reduction in the implanted tumors growth when compared to the control group. These results lead us to suggest that the expression of ecto-5'NT/CD73 by medulloblastoma might be considered as a prognostic marker and can be planned to be used as a therapeutic target in the treatment to this malignancy. Overall, we show the purinergic system functionality in the another type of tumor and that its modulation might be suggested as a tool to control of medulloblastoma progression

Efeito de diferentes componentes da matriz extracelular sobre a ecto-5'-nucleotidase, proliferação, adesão e migração celular na linhagem de células de glioma humano U138-MG

Glioblastoma multiforme é a forma mais comum e agressiva de tumor cerebral que apresenta um severo crescimento e um comportamento altamente invasivo. Linhagens de células de glioma em cultura apresentam alta atividade da enzima ecto-5’-nucleotidase que metaboliza AMP em adenosina. Em adição, ela também interage com componentes da matriz extracelular como molécula adesiva. Neste trabalho, nós avaliamos o efeito de diferentes componentes da matriz extracelular sobre a atividade da ecto-5’-nucleotidase, proliferação, adesão e migração celular na linhagem de células de glioma humano U138-MG. Os resultados obtidos mostraram uma inibição da atividade enzimática da ecto-5’- nucleotidase quando tratada com laminina sozinha e com fibronectina ou laminina em co-tratamento com dextran sulfato. O dextran sulfato mostrou reduzir a proliferação em 37%. O mesmo efeito foi observado para os co-tratamentos entre dextran/laminina (29%) e dextran/colágeno (28%). A presença de adenosina diminuiu a adesão celular em torno de 40% e o APCP aumentou a adesão em 75%. Laminina inibiu a adesão celular, já a condroitina sulfato aumentou em 70%. As células U138 apresentaram uma redução da adesão e migração celular quando tratadas apenas com dextran e também no co-tratamento deste com adenosina e APCP. Diante dos resultados podemos sugerir a modulação da atividade da ecto5’-nucleotidase e assim uma modulação da produção de adenosina por moléculas da matriz extracelular, afetando assim eventos celulares envolvidos no comportamento invasivo destas células tumorais.Glioblastoma multiforme is the most aggressive form of brain tumor that shows a severe growth and highly invasiveness behavior. Glioma cell lines in culture present a high activity of the ecto-5’-nucleotidase (ecto-5’-NT/CD73) which metabolizes AMP to adenosine. In addition, ecto-5’-NT/CD73 also has contact with extracellular matrix components like adhesive molecule. In the present paper, we evaluate the effect of distinct extracellular matrix components on the ecto-5’- NT/CD73 activity, proliferation, adhesion and migration in U138-MG human glioma cell line. The results showed an inhibition of enzymatic activity of ecto-5’NT/CD73 in the presence of laminin, fibronectin plus dextran sulfate and laminin plus dextran sulfate. In the proliferation assay it was observed that dextran sulfate reduced the proliferation in 37% and in association with collagen type I or laminin, the proliferation was reduced too (28% and 29%, respectively). The presence of adenosina decreased of adhesion at about 40% and when treated with APCP increased in 75%. Laminin inhibited the cell adhesion and chondroitin sulfate increased in 70%. U138 cells presented reduction of adhesion and cell migration in the presence of dextran sulfate alone and in the presence of adenosine and APCP. Taken together these results suggest the modulation of ecto-5’-NT/CD73 activity and production of adenosin by extracellular matrix molecules, affecting cell events involved in the invasiveness behavior this tumor cells

Involvement of ecto-5-nucleotidase/CD73 in U138MG glioma cell adhesion

Glioblastoma multiform is the most common and aggressive type of brain tumor. The overexpression of ecto-5′-nucleotidase/CD73 (ecto-5′-NT/CD73), an adhesion molecule and the main enzymatic source of extracellular adenosine, has been reported in tumor cells, and it is emerging as a component of glioma progression. Here, we evaluated the involvement of ecto-5′-NT/CD73 in cell adhesion through its interaction with different components of the extracellular matrix in the human U138MG glioma cell line. The results indicated that adenosine induced an increase in glioma cell adhesion. The treatment of glioma cells with adenosine receptor antagonists, APCP (α,β-methylene ADP) and dipyridamole prevented the adenosine effect, indicating the participation of extracellular and intracellular signaling pathways in cell adhesion mediated by adenosine. The ECM protein laminin (lam) and chondroitin sulfate (ChS) modulated the ecto-5′-NT/CD73 activity and glioma adhesion in a parallel manner, suggesting the involvement of purinergic signaling in the effects mediated by the extracellular matrix. Taken together, these results suggest that ecto-5′-NT/CD73, an important producer of extracellular adenosine, may modulate glioma cell adhesion and tumor cell–extracellular matrix interactions

Bradykinin promotes neuron-generating division of neural progenitor cells through ERK activation

During brain development, cells proliferate, migrate and differentiate in highly accurate patterns. In this context, published results indicate that bradykinin functions in neural fate determination, favoring neurogenesis and migration. However, mechanisms underlying bradykinin function are yet to be explored. Our findings indicate a previously unidentified role for bradykinin action in inducing neurongenerating division in vitro and in vivo, given that bradykinin lengthened the G1-phase of the neural progenitor cells (NPC) cycle and increased TIS21 (also known as PC3 and BTG2) expression in hippocampus from newborn mice. This role, triggered by activation of the kinin-B2 receptor, was conditioned by ERK1/2 activation. Moreover, immunohistochemistry analysis of hippocampal dentate gyrus showed that the percentage of Ki67+ cells markedly increased in bradykinin-treated mice, and ERK1/2 inhibition affected this neurogenic response. The progress of neurogenesis depended on sustained ERK phosphorylation and resulted in ERK1/2 translocation to the nucleus in NPCs and PC12 cells, changing expression of genes such as Hes1 and Ngn2 (also known as Neurog2). In agreement with the function of ERK in integrating signaling pathways, effects of bradykinin in stimulating neurogenesis were reversed following removal of protein kinase C (PKC)-mediated sustained phosphorylation

Ecto-5’-nucleotidase overexpression reduces tumor growth in a xenograph medulloblastoma model

Background Ecto-5’-nucleotidase/CD73 (ecto-5’-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5’-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children. Materials and Methods The effects of ecto-5’-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified. Results The human MB cell line D283, transfected with ecto-5’-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5’-NT. Conclusion This work suggests that ecto-5’-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5’-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy

The role of ecto-5´-nucleotidase/CD73 in glioma cell line proliferation

Malignant gliomas are the most common and devastating primary tumors in the brain and, despite treatment, patients with these tumors have a poor prognosis. The participation of ecto-5′-NT/CD73 per se as a proliferative factor, being involved in the control of cell growth, differentiation, invasion, migration and metastasis processes has been previously proposed. In the present study, we evaluated the activity and functions of ecto-5′-NT/CD73 during the proliferation process of rat C6 and human U138MG glioma cell lines. Increasing confluences and culture times led to an increase in ecto-5′-NT/CD73 activity in both C6 and U138MG glioma cells. RT-PCR analysis and flow cytometry analysis showed a significant increase in ecto-5′-NT/CD73 mRNA and protein levels, respectively, comparing confluent with sub-confluent cultures in human U138MG glioma cells. Ecto-5′-nucleotidase/CD73 may regulate the extracellular adenosine 5′-monophosphate (AMP) and adenosine levels. Treatment with 1 μM APCP, a competitive ecto-5′-NT/CD73 inhibitor, caused a significant reduction of 30% in glioma cell proliferation. In addition, 100 μM adenosine increases cell proliferation by 36%, and the treatment with adenosine plus NBTI and dipyridamole, produced an additional and significant increase of on cell proliferation. The inhibitory effect on cell proliferation caused by APCP was reverted by co-treatment with NBTI and dipyridamole. AMP (1 mM and 3 mM) decreased U138MG glioma cell proliferation by 29% and 42%, respectively. Taken together, these results suggest the participation of ecto-5′-NT/CD73 in cell proliferation and that this process is dependent upon the enzyme’s production of adenosine, a proliferative factor, and removal of AMP, a toxic molecule for gliomas