Marcia Rosangela Wink

In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model

BackgroundATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model.MethodsTo deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family.ResultsC6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group.ConclusionThese results indicate that the participation of extracellular ATP and the ecto-nucleotidases may be associated with the development of this type of brain tumor in an in vivo glioma model.

STUDIES ON THE ANCHORAGE OF ATP DIPHOSPHOHYDROLASE IN SYNAPTIC PLASMA MEMBRANES FROM RAT BRAIN

ATP diphosphohydrolases are described as ecto-enzymes in several tissues. In the present study, synaptic plasma membrane (SPM) was exposed to a series of agents used to distinguish between peripheral (hydrophilic), G-PI-anchored and transmembrane-polypeptide-anchored membrane proteins. These procedures included: (a) nondetergent extraction, (b) Triton X-114 phase partitioning, (c) phosphatidylinositol-specific phospholipase C (PI-PLC) extraction and (d) protease incubation. In cases (a), (c) and (d) the SPM was incubated with different agents and the ATPase-ADPase activities and the protein concentration was determined in the original sample, in the pellet and in the supernatant obtained after 100,000 g centrifugation. In procedure (b), the SPM was solubilized in 1% triton X-114 and submitted to phase separation onto a sucrose cushion. The aqueous and detergent rich phases obtained by this treatment were assayed for ATPase-ADPase activities and protein determination. The results obtained suggest an intrinsic behaviour for ATP diphosphohydrolase since none of the nondetergent treatments was efficient in removing the enzyme from SPM. Moreover, ATPase and ADPase activities were recovered predominantly (> 50%) in the detergent-rich phase obtained by Triton X-114 partitioning. The enzyme was not released by PI-PLC or proteases. These results indicate that the enzyme is not a GPI-anchored protein, but is probably deeply anchored on the plasma membrane in agreement with the amino acid sequence of the enzyme recently published.

CD73 Downregulation Decreases In Vitro and In Vivo Glioblastoma Growth

AbstractGlioblastoma is the worst and most common primary brain tumor. Here, we demonstrated the role of CD73, an enzyme responsible for adenosine (ADO) production, in glioblastoma progression. ADO increased glioma cell viability via A1 receptor sensitization. CD73 downregulation decreased glioma cell migration and invasion by reducing metalloproteinase-2 and vimentin expression and reduced cell proliferation by 40%, which was related to necrosis and sub-G1 phase blockage of cell cycle. Those effects also involved the stimulation of Akt/NF-kB pathways. Additionally, CD73 knockdown or enzyme inhibition potentiated temozolomide cytotoxic effect on glioma cells by decreasing the IC50 value and sensitizing cells to a non-cytotoxic drug concentration. CD73 inhibition also decreased in vivo rat glioblastoma progression. Delivery of siRNA-CD73 or APCP reduced tumor size by 45 and 40%, respectively, when compared with control. This effect was followed by a parallel 95% reduction of ADO levels in cerebrospinal fluid, indicating the role of extracellular ADO in in vivo glioma growth. Treatment did not induce systemic damage or mortality. Altogether, we conclude that CD73 is an interesting target for glioblastoma treatment and its inhibition may provide new opportunities to improve the treatment of brain tumors. Graphical Abstractᅟ

The Role of Ectonucleotidases in Glioma Cell Proliferation

Glioma invasion is a multifactorial process consisting of numerous genetic and physiological alterations, which affect glioma cell interactions with neurons, glia, and vascular cells. Purinergic signaling is emerging as an important component to give invasive potential to glioma cells. Specific purinergic receptor subtypes have been implicated in a variety of biological effects, including proliferation, differentiation, trophic actions and immune/inflammatory responses. Signaling events induced by extracellular nucleotides are controlled by the action of ectonucleotidases. These enzymes operate in concert for the complete nucleotide hydrolysis to nucleoside and represent a powerful manner to control the effects mediated by extracellular purines. It was demonstrated that glioma cell lines have altered extracellular ATP, ADP and AMP catabolism, presenting low rates of extracellular ATP hydrolysis and high rates of extracellular AMP hydrolysis when compared to astrocytes. Therefore, the ATP released by tumor adjacent cells, often damaged by growing tumors or due to ongoing inflammation together with the low glioma ability to hydrolyze extracellular ATP could result in powerful purinergic receptor activation, which in turn modulates glioma cell proliferation and neuronal toxicity. In addition, the high expression and activity of ecto-5ʹ-NT/CD73 in glioma cells and the extracellular adenosine generation could also be involved in the immunosupression process, angiogenesis and glioma invasion. These alterations could have important consequences in the activation of purinergic receptors and modulate events related to glioma advance. Although more studies are necessary, the ectonucleotidases may be considered as new molecular markers of gliomas and future target for pharmacological or gene therapy.