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Background/aim Glioblastoma multiforme is the most malignant type of glioma. Alpha-bisabolol is an essential oil reported as a potent cell death agent. In the present work, we evaluated the effect of alpha-bisabolol on ecto-5'-nucleotidase/CD73, the most well-characterized enzymatic source of adenosine, present in lipid rafts. Materials and methods Glioma cells were treated with alpha-bisabolol and, in some experiments, pre-treated with an A3 antagonist. MTT assay (viability), malachite green method (ecto-5'-nucleotidase/CD73 activity) and quantitative polymerase chain reaction (qPCR) (A3 mRNA) were carried out. Results Alpha-bisabolol led to a decrease in C6 and U138-MG glioma cells viability, accompanied by an increase in ecto-5'-NT/CD73 activity. Pre-treatment with an A3 antagonist reverted the effect of α-bisabolol with an increase of mRNA expression of this receptor. Conclusion Our data indicated the participation of ecto-5'-nucleotidase/CD73 and A3 receptor in the anti-proliferative effect of α-bisabolol on glioma cells.

Glioblastoma multiforme is a devastating cerebral tumor with an exceedingly poor prognosis. Methotrexate (MTX) is a folic acid analogue that inhibits DNA synthesis by binding to dihydrofolate reductase. Biodegradable nanoparticles are emerging as a promising system for drug delivery to specific tissues. The aims of the current study were pharmacological improvement and preclinical evaluation of MTX-loaded lipid-core nanocapsules (MTX-LNCs) in a glioblastoma model. Cell viability was assessed using the MTT assay, and the cell cycle was characterized by flow cytometry analysis of propidium iodide staining. Apoptosis was measured using an AnnexinV kit and by examining active caspase-3 immunocontent. In vivo glioma implantation was performed in rats, followed by measurement of the tumor size and tumoral apoptosis, BCL-2 immunohistochemistry and analyses of toxicological parameters. MTX-LNCs with increased encapsulation efficiency were successfully prepared. Our in vitro results showed a decrease in glioma cell viability after MTX-LNC treatment that was preceded by cell cycle arrest, leading the cells to undergo apoptotic death, as indicated by AnnexinV staining and increased active caspase-3 protein levels. In the in vivo glioma model, we observed a decrease in the tumor size and an increase in apoptosis in the tumor microenvironment (based on the AnnexinV assay and BCL-2 measurement). MTX-LNC treatment decreased the leukocyte number but altered neither toxicological tissue marker expression nor metabolic parameters. The present results reveal that MTX-LNCs represented an efficient formulation in a preclinical model of glioma and are a potential candidate for clinical trials.

Glioblastoma multiforme (GBM) is a deadly cancer characterized by a pro-tumoral immune response. T-regulatory (Treg) lymphocytes suppress effector immune cells through cytokine secretion and the adenosinergic system. Ecto-5'-nucleotidase/CD73 plays a crucial role in Treg-mediated immunosuppression in the GBM microenvironment (GME). Methotrexate (MTX) is an immunosuppressive drug that can increase the extracellular concentration of adenosine. In this manuscript, C6 GBM cells were treated with 1.0 μM MTX, and ecto-5'-nucleotidase/CD73 expression and extracellular AMP metabolism were analyzed in vitro. For in vivo studies, rats with implanted GBM were treated for 10 days with MTX-loaded lipid-core nanocapsules (MTX-LNCs, 1 mg/kg/day). The activity of ectonucleotidase and the expression of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 were measured. The frequencies of T lymphocytes (CD3(+)CD4(+), CD3(+)CD8(+), and CD4(+)CD25(high)CD39(+)) were quantified. In vitro, treatment with MTX increased CD73 expression and activity in C6 cells, which is in agreement with higher levels of extracellular adenosine. In vivo, MTX-LNC treatment increased CD39 expression on CD3(+)CD8(+) lymphocytes. In addition, MTX-LNC treatment up-regulated CD73 expression in tissue isolated from GBM, a finding that is in agreement with the higher activity of this enzyme. More specifically, the treatment increased CD73 expression on CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes. Treatment with MTX-LNCs decreased the frequencies of T-cytotoxic, T-helper, and Treg lymphocytes in the GME. Although more studies are necessary to better understand the complex cross-talk mediated by supra-physiological concentrations of adenosine in the GME, these studies demonstrate that MTX treatment increases CD73 enzyme expression and AMP hydrolysis, leading to an increase in adenosine production and immunosuppressive capability.

Glioblastoma multiforme is the most malignant type of glioma. Ecto-5'-nucleotidase (ecto-5'NT), a glioma-overexpressed enzyme can induce a protective effect on tumor cells. Monastrol, a kinesin spindle protein-specific inhibitor, is reported to be an interesting prototype for cancer therapy. We describe the effect of LaSOM 63, a monastrol derivative, on ecto-5'NT activity and on glioma cell survival.Glioma cells were treated with LaSOM 63 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue assay (viability), flow cytometry (cell cycle/cell death) and malachite green method for ecto-5'NT activity were carried out.Treatment with LaSOM 63 reduces glioma cell viability and cell growth. In contrast to monastrol, LaSOM 63 did not cause glioma cell-cycle arrest, but inhibited ecto-5'NT enzyme activity. Furthermore, this compound induces apoptotic death of C6 and U138 glioma cells.LaSOM 63 may be useful for in vivo experiments on the treatment of GBM.

Despite recent progress in glioblastoma treatment, prognosis is still poor. Monastrol is a kinesin spindle protein (KSP) inhibitor and anticancer effects for this molecule have been reported. Here we describe the effect of LaSOM 65, a monastrol derivated compound, against glioma cell lines.Cell counting, viability assay, lactate dehydrogenase (LDH) activity, cell-cycle analysis, immunofluorescence and organotypic hippocampal slice cultures were performed.LaSOM 65 reduced cell number and cell viability of gliomas cells, but did not cause arrest in the cell cycle at the G2/M phase. Measurement of LDH activity showed that LaSOM 65 induces necrosis after 48 h of treatment.LaSOM 65 appears to a be promising new molecule to treat glioblastoma since it promotes a decrease of cell growth and cell viability of glioma cells in vitro and does not induces the neurotoxic characteristics of the anti-mitotic drugs currently used.