IDO, COX and iNOS have an important role in the proliferation of Neospora caninum in neuron/glia co-cultures.

Central nervous system (CNS) is the main site for encystment of Neospora caninum in different animal species. In this tissue, glial cells (astrocytes and microglia) modulate responses to aggression in order to preserve homeostasis and neuronal function. Previous data showed that when primary cultures of glial cells are infected with N. caninum, they develop gliosis and the immune response is characterized by the release of TNF and IL-10, followed by the control of parasite proliferation. In order to elucidate this control, three enzymatic systems involved in parasite-versus-host interactions were observed on a model of neuron/glia co/cultures obtained from rat brains. Indoleamine 2,3-dioxygenase (IDO), induced nitric oxide synthase (iNOS) responsible for the catabolism of tryptophan and arginine, respectively, and cycloxigenase (COX) were studied comparing their modulation by respective inhibitors with the number of tachyzoites or the immune response measured by the release of IL-10 and TNF. Cells were treated with the inhibitors of iNOS (1.5 mM L-NAME), IDO (1 mM 1-methyl tryptophan), COX-1 (1 µM indomethacin) and COX-2 (1 µM nimesulide) before infection with tachyzoites of N. caninum (1:1 cell: parasite). After 72 h of infection, immunocytochemistry showed astrogliosis and a significant increase in the number and length of neurites, compared with uninfected co-cultures, while an increase of IL-10 and TNF was verified. N. caninum did not change iNOS activity, but the inhibition of the basal levels of this enzyme stimulated parasite proliferation. Additionally, a significant increase of about 40% was verified in the IDO activity, whose inhibition caused 1.2-fold increase in parasitic growth. For COX-2 activity, infection of cultures stimulated a significant increase in release of PGE2 and its inhibition by nimesulide allowed the parasitic growth. These data indicate that iNOS, IDO and COX-2 control the proliferation of N. caninum in this in vitro model. On the other hand, the release of IL-10 by glia besides modulating the inflammation also allow the continuity of parasitism.

Antiproliferative, proapoptotic and morphogenic effects of the flavonoid rutin on human glioblastoma cells.

In this study, we investigated the effects of the flavonoid rutin (3,3',4',5,7-pentahydroxyflavone-3-rutinoside) on glioma cells, using the highly proliferative human cell line GL-15 as a model. We observed that rutin (50-100µM) reduced proliferation and viability of GL-15 cells, leading to decreased levels of ERK1/2 phosphorylation (P-ERK1/2) and accumulation of cells in the G2 phase of the cell cycle. On the other hand, 87.4% of GL-15 cells exposed to 100µM rutin entered apoptosis, as revealed by flow cytometry after AnnexinV/PI staining. Nuclear condensation and DNA fragmentation were also observed, further confirming that apoptosis had occurred. Moreover, the remaining cells that were treated with 50µM rutin presented a morphological pattern of astroglial differentiation in culture, characterised by a condensed cell body and thin processes with overexpression of GFAP. Because of its capacity to induce differentiation and apoptosis in cultured human glioblastoma cells, rutin could be considered as a potential candidate for malignant gliomas treatment.

Catechol cytotoxicity in vitro: induction of glioblastoma cell death by apoptosis.

The exposure to benzene is a public health problem. Although the most well-known effect of benzene is hematopoietic toxicity, there is little information about the benzene and its metabolites effects on the central nervous system (CNS). This study examined the toxic effects of 1,2-dihydroxybenzene (catechol), a benzene metabolite, to human glioblastoma GL-15 cells. GL-15 cell cultures were used as a model to provide more information about the toxic effects of aromatic compounds to the CNS. Catechol induced time- and concentration-dependent cytotoxic effects. Morphological changes, such as the retraction of the cytoplasm and chromatin clumping, were seen in cells exposed to 200 microM catechol for 48 hours. In cells exposed to 600 microM catechol for 48 hours, 78.0% of them presented condensed nuclei, and the Comet assay showed DNA damage. The percentage of cells labeled with annexin V (apoptotic cells) was greater in the group exposed to catechol (20.7%) than in control cells (0.4%). Exposure to catechol at concentrations greater than 100 microM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 microM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.