Fernanda Bueno Morrone

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.

Increased resistance of glioma cell lines to extracellular ATP cytotoxicity

Glioblastomas are the most common form of primary tumors of the central nervous system (CNS) and despite treatment, patients with these tumors have a very poor prognosis. ATP and other nucleotides and nucleosides are very important signaling molecule in physiological and pathological conditions in the CNS. ATP is degraded very slowly by gliomas when compared to astrocytes, potentially resulting in the accumulation of extracellular ATP around gliomas. Cell lysis caused by excitotoxic death or by tumor resection may liberate intracellular ATP, a known mitotic factor for glioma cells. The aim of this study is to examine the effects on cytotoxicity induced by extracellular ATP in U138-MG human glioma cell line and C6 rat glioma cell line compared to hippocampal organotypic cell cultures. The cytotoxicity of ATP (0.1, 0.5, 5 mM) was measured using propidium iodide and LDH assays. Caspases assay was performed to identify apoptotic cell death. Results showed that the glioma cells present resistance to death induced by ATP when compared with a normal tissue. High ATP concentrations (5 mM) induced cell death after 24 h in organotypic cell cultures but not in glioma cell lines. Our data indicate that ATP released in these situations can induce cell death of the normal tissue surrounding the tumor, potentially opening space to the fast growth and invasion of the tumor.