Ana Maria Oliveira Battastini

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats.

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.

Indomethacin-loaded nanocapsules treatment reduces in vivo glioblastoma growth in a rat glioma model

Multimodal combinations of target agents with radiation and chemotherapy may enhance cancer treatment efficacy; however, despite these treatments, gliomas recur early due to their highly proliferative, infiltrative and invasive behaviors. Nanoparticles of biodegradable polymers for anticancer drug delivery have attracted intensive interest in recent years since they may provide a sustained, controlled and targeted delivery. In the present study, we investigated the effect of indomethacin-loaded nanocapsules in an experimental glioma model. The rats treated with indomethacin-loaded nanocapsules demonstrated a significant reduction in tumor size and half of these animals presented just cells with characteristics of a residual tumor, as shown by immunostaining for nestin. Pathological analyses showed that the treated gliomas presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. An important finding of the present study is that indomethacin carried by polymeric nanocapsules achieved higher intracerebral drug concentrations than those of indomethacin in solution. Furthermore, indomethacin achieved a greater concentration in the hemisphere where the glioma was implanted, compared with the contralateral healthy hemisphere. Indomethacin-loaded nanocapsule treatment did not cause characteristics of toxicity and increased the survival of animals. Thus, our results show that polymeric nanocapsules are able to increase the intratumoral bioavailability of indomethacin and reduce the growth of implanted gliomas. Data suggest that indomethacin-loaded nanocapsules could offer new and potentially highly effective strategies for the treatment of malignant gliomas.

ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) in human blood platelets

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca(2+) dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2 μg/d ouabain, 1.0 μM levamisole, 10 μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100 μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes

Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.

NFκB inhibitors induce cell death in glioblastomas

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.

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.

The curry spice curcumin selectively inhibits cancer cells growth in vitro and in preclinical model of glioblastoma.

Previous studies suggested that curcumin is a potential agent against glioblastomas (GBMs). However, the in vivo efficacy of curcumin in gliomas remains not established. In this work, we examined the mechanisms underlying apoptosis, selectivity, efficacy and safety of curcumin from in vitro (U138MG, U87, U373 and C6 cell lines) and in vivo (C6 implants) models of GBM. In vitro, curcumin markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of GBM growth. Curcumin effects occurred irrespective of the p53 and PTEN mutational status of the cells. Interestingly, curcumin did not affect viability of primary astrocytes, suggesting that curcumin selectivity targeted transformed cells. In U138MG and C6 cells, curcumin decreased the constitutive activation of PI3K/Akt and NFkappaB survival pathways, down-regulated the antiapoptotic NFkappaB-regulated protein bcl-xl and induced mitochondrial dysfunction as a prelude to apoptosis. Cells developed an early G2/M cell cycle arrest followed by sub-G1 apoptosis and apoptotic bodies formation. Caspase-3 activation occurred in the p53-normal cell type C6, but not in the p53-mutant U138MG. Besides its apoptotic effect, curcumin also synergized with the chemotherapeutics cisplatin and doxorubicin to enhance GBM cells death. In C6-implanted rats, intraperitoneal curcumin (50 mg kg(-1) d(-1)) decreased brain tumors in 9/11 (81.8%) animals against 0/11 (0%) in the vehicle-treated group. Importantly, no evidence of tissue (transaminases, creatinine and alkaline phosphatase), metabolic (cholesterol and glucose), oxidative or hematological toxicity was observed. In summary, data presented here suggest curcumin as a potential agent for therapy of GBMs.

Differential Macrophage Activation Alters the Expression Profile of NTPDase and Ecto-5′-Nucleotidase

Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5′-nucleotidase/CD73 (ecto-5′-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6–8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5′-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5′-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.

Curcumin-loaded lipid-core nanocapsules as a strategy to improve pharmacological efficacy of curcumin in glioma treatment

In this study, we developed curcumin-loaded lipid-core nanocapsules (C-LNCs) in an attempt to improve the antiglioma activity of this polyphenol. C-LNC showed nanotechnological properties such as nanometric mean size (196 nm), 100% encapsulation efficiency, polydispersity index below 0.1, and negative zeta potential. The in vitro release assays demonstrated a controlled release of curcumin from lipid-core nanocapsules. In C6 and U251MG gliomas, C-LNC promoted a biphasic delivery of curcumin: the first peak occurred early in the treatment (1-3h), whereas the onset of the second phase occurred after 48 h. In C6 cells, the cytotoxicity of C-LNC was comparable to non-encapsulated curcumin only after 96 h, whereas C-LNCs were more cytotoxic than non-encapsulated curcumin after 24h of incubation in U251MG. Induction of G2/M arrest and autophagy were observed in C-LNC as well as in free-curcumin treatments. In rats bearing C6 gliomas, C-LNC (1.5mg/kg/day, i.p.) decreased the tumor size and malignance and prolonged animal survival when compared to same dose of non-encapsulated drug. In addition, serum markers of tissue toxicity and histological parameters were not altered. Considered overall, the data suggest that the nanoencapsulation of curcumin in LNC is an important strategy to improve its pharmacological efficacy in the treatment of gliomas.

Amyloid‐β neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK‐3β, tau and neuroinflammation

Abstract:  Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid‐β (Aβ) peptide in brain regions that are important for memory and cognition. The buildup of Aβ aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Aβ‐induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 μm of Aβ25–35 in the absence or in the presence of melatonin (25, 50, or 100 μm). In addition, the authors have investigated the involvement of GSK‐3β, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Aβ‐induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Aβ25–35. In addition, melatonin significantly reduced the activation of GSK‐3β, the phosphorylation of tau protein, the glial activation and the Aβ‐induced increase of TNF‐α and IL‐6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Aβ‐induced phosphorylation of tau protein, and preventing GSK‐3β activation and neuroinflammation.