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. 2006 Sep 23:6:226.
doi: 10.1186/1471-2407-6-226.

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

Affiliations

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

Fernanda B Morrone et al. BMC Cancer. .

Abstract

Background: ATP 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.

Methods: To 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.

Results: C6 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.

Conclusion: These 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.

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Figures

Figure 1
Figure 1
Comparison of NTPDase family expression in rat cortical astrocytes and rat C6 gliomas by quantitative real-time RT-PCR analysis. The expression of NTPDase members in astrocytes and C6 gliomas was quantitatively analyzed by real-time PCR as described in material and methods. C6: rat glioma cell line. Astr: cortical rat astrocytes. The results are presented as a ratio of cDNA enzymes/GAPDH.
Figure 2
Figure 2
Tumor size of implanted gliomas. Tumor size was measured 20 d after implantation of C6 cells in the different groups. (A) Rats were treated with 10% DMSO (vehicle control) or temozolomide in 10% DMSO (n = 5). (B) C6 cells were co-injected with: denaturated apyrase (C apyrase) or 2 U of apyrase (apyrase group) (n = 6). Data are means ± SEM. **p < 0.01; *p < 0.05 for comparison versus control, as determined by ANOVA, followed by Tukey-Krammer test.
Figure 3
Figure 3
The sections of implanted rat glioma were stained with haematoxylin and eosin (H&E). Histological characteristics that define glioblastoma multiform as seen in rats implanted with gliomas (a,c) and in rats co-injected with apyrase (b,d). Necrosis (N) and microvascular proliferation (V), giant cell formation and nuclear pleomorphism (arrow). Scale bars = 100 μm (a,b); 20 μm (c,d).
Figure 4
Figure 4
Immunohistochemical stainings of gliomas. Glioma cell proliferation was assessed by immunostaining for Ki67 positive glioma cell nuclei (arrows) in rats implanted with gliomas (a) and in rats co-injected with apyrase (b). The sections were immunostained for VEGF, in rats implanted with gliomas (c) and in rats co-injected with apyrase (d). Scale bars = 20 μm (a,b); 100 μm (c,d).

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