Investigação da ecto-5’nucleotidase/cd73 como alvo terapêutico em gliomas

Abstract

Gliomas are tumors that have genetic and phenotypic characteristics of glial cells. The ecto5'-nucleotidase/CD73 (5’-NT/CD73) enzyme is considered the limiting step of the purinergic system responsible for the hydrolysis of AMP to adenosine and shows increased expression in solid tumors. Studies silencing the gene expression using RNA interference tool (siRNA) have been proposed as new therapeutic alternative. Therefore, the aim of this study was to evaluate the effect of inhibitors of the enzymatic activity and expression of 5'-NT/CD73 as a therapeutic target in gliomas. To evaluate the effect of inhibitor AMPCP (adenosine 5' -( α , β - methylene) diphosphate ) on cell proliferation was used cell counting method and AMP hydrolysis in C6 cells was evaluated by malachite green method. To perform the silencing of gene expression, two new sequences of RNA interference for 5' - NT/CD73 were designed and nanoemulsions (NE) were used as carriers. Cytotoxicity of NE in primary astrocyte cultures and C6 glioma cell line was determined by MTT 3(4, 5-dimethyl)-2,5diphenyl tetrazolium bromide) method. To characterize the physicochemical properties of the complex formed by NE and siRNA-CD73 parameters of particle size, zeta potential and polydispersity index using the Zetasizer apparatus and images of NE-siRNA complexes were captured and analyzed by transmission electron microscopy (TEM). The efficiency of silencing complexes promoted by transfection of NE-siRNA in C6 glioma was determined by evaluating the AMPase activity and expression analysis of 5'- NT/CD73 by immunocytochemistry and flow cytometry using specific antibody. The assessment of the antiproliferative effect of the complexes NE-siRNA in C6 glioma was performed by MTT. The results of this work are consistent with the hypothesis that AMPCP reduced cell proliferation of C6 glioma which was followed by the inhibition of AMP hydrolysis. The sequences of siRNA were effective in promoting 5'NT/CD73 silencing and NE formulation showed good complexing ability with the oligonucleotide, with low toxicity in normal cells and selectivity for tumor cells. It was observed that the NE-CD73-siRNA complex was effective to silence the enzyme and reduce AMPase activity and viability of C6 glioma. It is important to note that the nanostructured system (NE) was effective both in complex with oligonucleotides as them for delivery into target cells. However, there is a need for further studies to understand the effect of treatment of NE-CD73-siRNA complex on tumor growth in vivo. Finally, our data indicate for the first time that the potential of siRNA-loaded nanoemulsion CD73 as a therapeutic alternative for the treatment of GBM. Nevertheless there is a need for further studies to understand the effect of treatment of complex NE-CD73-siRNA on tumor growth in vivo. Despite the need for further studies to understand the effect of treatment of complex NE- CD73 - siRNA on tumor growth in vivo