Overexpression of NTPDase2 in gliomas promotes systemic inflammation and pulmonary injury.

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.

Selective NTPDase2 expression modulates in vivo rat glioma growth.

The ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are a family of ectoenzymes that hydrolyze extracellular nucleotides, thereby modulating purinergic signaling. Gliomas have low expression of all E-NTPDases, particularly NTPDase2, when compared to astrocytes in culture. Nucleotides induce glioma proliferation and ATP, although potentially neurotoxic, does not evoke cytotoxic action on the majority of glioma cultures. We have previously shown that the co-injection of apyrase with gliomas decreases glioma progression. Here, we tested whether selective re-establishment of NTPDase2 expression would affect glioma growth. NTPDase2 overexpression in C6 glioma cells had no effect on in vitro proliferation but dramatically increased tumor growth and malignant characteristics in vivo. Additionally, a sizable platelet sequestration in the tumor area and an increase in CD31 or platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor and OX-42 immunostaining were observed in C6-Enhanced Yellow Fluorescent Protein (EYFP)/NTPDase2-derived gliomas when compared to controls. Treatment with clopidogrel, a P2Y(12) antagonist with anti-platelet properties, decreased these parameters to control levels. These data suggest that the ADP derived from NTPDase2 activity stimulates platelet migration to the tumor area and that NTPDase2, by regulating angiogenesis and inflammation, seems to play an important role in tumor progression. In conclusion, our results point to the involvement of purinergic signaling in glioma progression.

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

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.

Effect of nicotine and cigarette smoke on an experimental model of intraepithelial lesions and pancreatic adenocarcinoma induced by 7,12-dimethylbenzanthracene in mice.

OBJECTIVES: To evaluate the effects of nicotine and cigarette smoke exposure on mice submitted to 7,12-dimethylbenzanthracene (DMBA) model of pancreatic carcinogenesis. METHODS: One hundred fourteen male mice were divided into the DMBA-n and DMBA-s groups: the DMBA-n group was given 2 mg/kg per dose of nicotine ([3-(1-methyl-2-pyrrolidinyl)pyridine]) subcutaneously for 45 days, and the DMBA-s group was exposed to 100 mg/m of cigarette smoke. At day 16, 1 mg of DMBA crystals was implanted in the pancreatic head of both groups. Euthanasia was performed in all mice 30 days after the surgery. The specimens were evaluated according to the following criteria: normal ducts, reactive hyperplasia, pancreatic intraepithelial neoplasm 3 (PanIN-3), and carcinoma. For statistical analysis, DMBA-exclusive ([DMBA-e] historical control group) was included. RESULTS: The frequency of PanIN in the 3 groups was almost the same when considering the higher-grade lesions: DMBA-e (16 [66.7%]), DMBA-s (20 [66.7%]), and DMBA-n (12 [44.4%]). Pancreatic adenocarcinoma has a higher frequency in the DMBA-n group (14 [51.9%]) than in the DMBA-e (4 [16.7%]) and DMBA-s (4, 13.3%) groups. The DMBA-s group has the highest score of PanIN-3 (40%). The differences among the groups were statistically significant (P = 0.05, Fisher exact test). CONCLUSIONS: Nicotine but not cigarette smoke promotes pancreatic DMBA carcinogenesis in mice. Pancreatic adenocarcinomas and PanINs have the same phenotypic appearance as those that occur in humans.

[Ovine abortion associated with toxoplasmosis: serological, anatomo-pathological and immunohistochemistry characterization]. / Aborto em ovinos associado à toxoplasmose: caracterização sorológica, anátomo-patológica e imunoistoquímica.

Ovine toxoplasmosis is a major cause of abortion and placentitis. Lambs with congenital infection are born with encephalitis. Abortions occurred between May and August 2006 in an estate in Três Palmeiras, southern Brazil. This study aimed to assess the frequency of abortion associated with toxoplasmosis among ewes in southern Brazil using indirect hemagglutination and pathoanatomical and immunohistochemical analyses. Blood samples were collected from nine animals for indirect hemagglutination. One fetus underwent necropsy and histopathological and immunohistochemical analyses. Three samples, one of which belonged to the necropsied animals mother, were positive on the serological test. The necropsy revealed pale foci in the liver, lungs with a marbled appearance, heart with severe pallor and brain and cerebellar congestion. Fragments of all organs were collected, fixed in 10% buffered formalin, processed, and stained with hematoxylin-eosin. Microscopically, there were cysts and tachyzoites in the brain in areas of malacia with microgliosis and lymphoplasmacytic infiltration, suggesting toxoplasmic encephalitis. There was also interstitial pneumonia, centrolobular necrosis with structures compatible with tachyzoites, focal lymphocytic myocarditis and acute tubular nephrosis. The immunohistochemical test was positive for Toxoplasma gondii. The results allowed diagnosing the ovine toxoplasmosis.

Effect of temozolomide treatment on the adenine nucleotide hydrolysis in blood serum of rats with implanted gliomas

Objective: Adenine nucleotides and adenosine have many important functions in the physiological and pathological conditions. The measurement of these nucleotides in serum may be an auxiliary tool in the identification of cellular damage in many pathological conditions. The aim of this study is to examine the effect of chemotherapy treatment on nucleotide hydrolysis in the serum of rats following glioma implantation. Methods: C6 glioma cells were injected in the right striatum of 60 day-old Wistar rats, and 20 days after the induction of gliomas, blood serum samples were prepared for measurement of ATP and AMP hydrolysis. Results: The pathological analysis showed that the malignant gliomas induced by C6 injection and treated with temozolomide exhibited a reduction in malignant characteristics. The results demonstrated that the rats that underwent temozolomide treatment had a significant decrease (p<0.05) in blood serum hydrolysis of ATP and AMP when compared with the glioma group. None of the animals included in this study presented significant alterations in the activities of the serum enzymes alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. Conclusion: The decrease in the enzymatic hydrolysis of the ATP and AMP is probably related to the diminished malignant characteristics caused by temozolomide treatment on the gliomas in vivo.