Flavonoids suppress human glioblastoma cell growth by inhibiting cell metabolism, migration, and by regulating extracellular matrix proteins and metalloproteinases expression.

The malignant gliomas are very common primary brain tumors with poor prognosis, which require more effective therapies than the current used, such as with chemotherapy drugs. In this work, we investigated the effects of several polyhydroxylated flavonoids namely, rutin, quercetin (F7), apigenin (F32), chrysin (F11), kaempferol (F12), and 3',4'-dihydroxyflavone (F2) in human GL-15 glioblastoma cells. We observed that all flavonoids decreased the number of viable cells and the mitochondrial metabolism. Furthermore, they damaged mitochondria and rough endoplasmic reticulum, inducing apoptosis. Flavonoids also induced a delay in cell migration, related to a reduction in filopodia-like structures on the cell surface, reduction on metalloproteinase (MMP-2) expression and activity, as well as an increase in intra- and extracellular expression of fibronectin, and intracellular expression of laminin. Morphological changes were also evident in adherent cells characterized by the presence of a condensed cell body with thin and long cellular processes, expressing glial fibrillary acidic protein (GFAP). Therefore, these flavonoids should be tested as potential antitumor agents in vitro and in vivo in other malignant glioma models.

Flavonoids inhibit angiogenic cytokine production by human glioma cells.

VEGF and TGF-ß1 are cytokines that stimulate tissue invasion and angiogenesis. These factors are considered as molecular targets for the therapy of glioblastoma. Bevacizumab, a recombinant humanized monoclonal antibody developed against VEGF, inhibits endothelial cell proliferation and vessel formation. Flavonoids obtained from Dimorphandra mollis and Croton betulaster have been described as proliferation inhibitors of a human glioblastoma derived cell line. VEGF and TGF-ß1 levels were dosed by ELISA in a GL-15 cell line treated with bevacizumab and also with the flavonoids rutin, 5-hydroxy-7,4'-dimethoxyflavone, casticin, apigenin and penduletin. Rutin reduced the VEGF and TGF-ß1 levels after 24 h but not after 72 h. The other flavonoids significantly reduced TGF-ß1 production. Bevacizumab reduced only the VEGF levels in the supernatant from GL-15 cultures. These results suggest that the flavonoids studied, and commonly used in popular medicine, present an interesting subject of study due to their potential effect as angiogenic factor inhibitors.

The flavonoid rutin but not the alkaloid arborinine induces apoptosis in a B-cell hybridoma cell line.

The effects of arborinine, an alkaloid extracted from Erthela bahiensis and of rutin, a flavonoid obtained from Dimorphandra mollis (Benth.), Brazilian medicinal plants, on the viability and function of a murine B-cell hybridoma as a tumor model were investigated. The flavonoid rutin at 50 microM induced an increase in the number of apoptotic cells of one- to fivefold and reductions in cellular proliferation and monoclonal antibody production. Less but still significant necrosis was also induced by rutin under the same experimental conditions. On the other hand, the alkaloid arborinine exerted no significant effects on the studied parameters.

Cytotoxicity effect of alkaloidal extract from Prosopis juliflora Sw. D.C. (Algaroba) pods on glial cells

Prosopis juliflora is largely used for feeding cattle and humans. Neurological signals have been reported in cattle due to intoxication with this plant. In this study, an alkaloidal fraction (AF) obtained from P. juliflora pods was tested on astrocyte primary cultures. Astrocytes display physiological functions essential to development, homeostasis and detoxification in the central nervous system (CNS). These cells are known for their role on energetic support and immune response in the CNS. Concentrations between 0.03 to 30 µg/ml AF were assayed for 24 - 72 h. The mitochondrial activity, assayed by MTT test, showed cytotoxicity at 30 µg/ml AF after 24 h. At concentrations ranging between 0.3 - 3 µg/ ml, the AF induced an increase on mitochondrial activity, indicating cell reactivity. lmmunocytochemistry assay for GFAP cytoskeletal protein, revealed alterations on cell morphology after treatment with 0.3 - 3 µg/ ml AF for 72 h. This result corroborates with western blot analysis when ceUs treated with 0.3 - 3 I-µg/ml AF for 72 h showed GFAP upregulation. The vimentin expression was not significantly altered in all tested concentrations. These results suggest that alkaloids induce astrocyte reactivity and might be involved in the neurotoxic effects induced by P. juliflora consumption.

A Prosopis juliflora é amplamente utilizada na alimentação humana e de várias espécies animais, especialmente bovinos. Quadros de intoxicação por esta planta, nesta espécie, têm sido relatados, principalmente quando a mesma é oferecida como única fonte alimentar, desencadeando uma doença de sintomatologia nervosa. Neste estudo, objetivou-se avaliar os efeitos in vitro da fração de alcalóides totais (F A) extraída das vagens da Prosopis juliflora utilizando cultura primária de astrócitos obtidos do córtex cerebral de ratos como modelo de estudo. A avaliação da atividade mitocondrial pelo teste do MTT demonstrou a citotoxicidade em 30 µg/ ml da FA após 24 h. As concentrações de 0,3 e 3 µg/ ml da FA induziram um aumento da atividade mitocondrial, indicando reatividade celular. Testes imunocitoquimicos para a GFAP, principal proteína de citoesqueleto de astrócitos, revelaram alterações morfológicas nas células após tratamento com 0,3 e 3 µg/ml da FA por 72 h. Tais resultados são consoantes à análise desta proteína por westernblot, quando as culturas foram tratadas com 0,3 e 3 µg/ml da FA por 72 h, demonstrando interferências na regulação da expressão da GFAP. A expressão de vimentina não foi significativamente alterada em nenhuma das concentrações testadas. Estes resultados sugerem que os alcalóides da P.juliflora induzem a reatividade astrocitária, o que pode estar envolvido nos efeitos neurotóxicos providos pelo consumo desta planta.

Contribution of gap junctional communication between tumor cells and astroglia to the invasion of the brain parenchyma by human glioblastomas.

BACKGROUND: Gliomas are "intraparenchymally metastatic" tumors, invading the brain in a non-destructive way that suggests cooperation between glioma cells and their environment. Recent studies using an engineered rodent C6 tumor cell line have pointed to mechanisms of invasion that involved gap junctional communication (GJC), with connexin 43 as a substrate. We explored whether this concept may have clinical relevance by analyzing the participation of GJC in human glioblastoma invasion. RESULTS: Three complementary in vitro assays were used: (i) seeding on collagen IV, to analyze homocellular interactions between tumor cells (ii) co-cultures with astrocytes, to study glioblastoma/astrocytes relationships and (iii) implantation into organotypic brain slice cultures, that mimic the three-dimensional parenchymal environment. Carbenoxolone, a potent blocker of GJC, inhibited cell migration in the two latter models. It paradoxically increased it in the first one. These results showed that homocellular interaction between tumor cells supports intercellular adhesion, whereas heterocellular glioblastoma/astrocytes interactions through functional GJC conversely support tumor cell migration. As demonstrated for the rodent cell line, connexin 43 may be responsible for this heterocellular functional coupling. Its levels of expression, high in astrocytes, correlated positively with invasiveness in biopsied tumors. CONCLUSIONS: our results underscore the potential clinical relevance of the concept put forward by other authors based on experiments with a rodent cell line, that glioblastoma cells use astrocytes as a substrate for their migration by subverting communication through connexin 43-dependent gap junctions.

P-glycoprotein (ABCB1) but not multidrug resistance-associated protein 1 (ABCC1) is induced by doxorubicin in primary cultures of rat astrocytes.

At least two drug efflux pumps involved in multidrug resistance, P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (Mrp1), are expressed in rat astrocyte primary cultures. The aim of this study was to compare the expression of P-gp and Mrp1 in primary cultures exposed to 50 or 500 ng/mL doxorubicin (DOX). Among the two P-gp genes expressed in rodents, mdr1a and mdr1b, a time- and dose-dependent increase in mdr1b mRNA levels was revealed by northern blot analysis. This up-regulation was inhibited by actinomycin D and occurred as early as 2 h after exposure to 50 or 500 ng/mL DOX, whereas mdr1a and mrp1 transcripts were not modified by the DOX exposure. In addition, DOX also strongly enhanced, in a time- and dose-dependent manner, P-gp but not Mrp1 expression. Moreover, DOX raised the cellular efflux of vincristine, a substrate for both P-gp and Mrp1. This efflux was inhibited by the P-gp modulators PSC833 and GW918, but not by the Mrp1 modulator MK571. On the other hand, a 24-h exposure to 500 ng/mL DOX, but not 50 ng/mL DOX, induced apoptosis in primary cultures of rat astrocytes. Fumonisin B1, a ceramide synthase inhibitor, reduced DOX-induced apoptosis, suggesting that de novo synthesis of the ceramide regulatory pathway might be involved in DOX-induced apoptosis. Moreover, western blot analysis showed that fumonisin B1 was not able to decrease the overexpression of P-gp induced by DOX. Our results provide evidence that DOX up-regulates a functional P-gp in primary cultures of rat astrocytes and might cause astrocyte apoptosis via the ceramide pathway.

Efeitos in vitro do ácido retinóico em células de glioblastoma / Effects in vitro of the retinoic acid in glioblastoma cells

Os gliomas malignos são tumores muito infiltrantes, cujas células proliferam rapidamente, e apresentam um prognóstico muito reservado. Neste estudo, investigamos o efeito em potencial do ácido retinóico (AR) sobre a linhagem de células de glioblastoma multiforme humano GL-15. A exposição a uma única dose de AR (1-10 mM) inibiu a proliferação celular, induziu uma diferenciação transitória e, finalmente, conduziu estas células à apoptose. Observamos que as células GL-15 expressam os isotipos dos RARs a, b e g, e que as isoformas RARa1/2, RARb2 e RARg2 são induzidas pelo AR. Estes resultados sugerem que a relação entre a expressão das diferentes isoformas de RARs pode ser um elemento fundamental para a indução seja de uma diferenciação completa, seja de apoptose das células de glioblastoma, e que o uso de ligantes específicos a cada isotipo de receptor pode vir a ser um elemento importante para terapias futuras de gliomas.

Role of laminin bioavailability in the astroglial permissivity for neuritic outgrowth

The mechanisms involved in the failure of an adult brain to regenerate post-lesion remain poorly understood. The reactive gliosis which occurs after an injury to the CNS and leads to the glial scar has been considered as one of the major impediments to neurite outgrowth and axonal regeneration. A glial scar consists mainly of reactive, hypertrophic astrocytes. These reactive cells acquire new properties, leading to A non-permissive support for neurons. Astrogial reactivity is mainly characteriized by a high overexpression of the major component of the gliofilaments, the glial fibrillary acidic protein (GFAP). This GFAP overexpression is related to the astroglial morphological response to injury. We hypothesized that modulation of GFAP synthesis, reversing the hypertrophic phenotype, might also reverse the blockage of neuritic outgrowth observed after a lesion. In this article, we review findings of our group, confirming our hypothesis in a model of lesioned neuron-astrocyte cocultures. We demonstrate that permissivity for neuritic outgrowth is related to phenotypic changes induced in reactive astrocytes transfected by antisense GFAP-mRNA. We also found that this permissivity was related to a neuron-regulated extracellular laminin bioavailability

Molecular and functional MDR1-Pgp and MRPs expression in human glioblastoma multiforme cell lines.

The aim of our study was to investigate the functional expression of P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) in 2 distinct glioma cells (GL15 and 8MG) from patients with glioblastoma multiforme. MDR1 gene and Pgp expression was not detected in either cell line by RT-PCR and Western blotting, respectively. In contrast, MRP1 was detected at both mRNA and protein level in both cell lines, with a higher expression in the 8MG cells that occur predominantly at the cell membrane. Three other MRPs (MRP3, MRP4 and MRP5) were detected by RT-PCR in both cell lines, whereas MRP2 was not expressed. In addition, MRP3 protein was also detected by immunocytochemistry in both GL15 and 8MG cell lines. Indomethacin and probenecid, 2 modulators of MRPs activity, increased the accumulation of vincristine and etoposide, 2 substrates of MRPs, by both cell lines. These modulators also decreased the efflux of vincristine from both cell lines with a more pronounced effect in 8MG cells. In conclusion, our results show functional expression of MRPs leading to a decrease in the intracellular vincristine and etoposide concentrations in human glioblastoma cell lines. Furthermore, our results that exhibit protein expression of MRP1 and MRP3 and gene expression of MRP4 and MRP5 in these 2 glioblastoma cell lines suggest new mechanisms that could lead to a MDR phenotype of tumour cells in patients with glioblastoma multiforme.

Role of laminin bioavailability in the astroglial permissivity for neuritic outgrowth.

The mechanisms involved in the failure of an adult brain to regenerate post-lesion remain poorly understood. The reactive gliosis which occurs after an injury to the CNS and leads to the glial scar has been considered as one of the major impediments to neurite outgrowth and axonal regeneration. A glial scar consists mainly of reactive, hypertrophic astrocytes. These reactive cells acquire new properties, leading to A non-permissive support for neurons. Astrogial reactivity is mainly characteriized by a high overexpression of the major component of the gliofilaments, the glial fibrillary acidic protein (GFAP). This GFAP overexpression is related to the astroglial morphological response to injury. We hypothesized that modulation of GFAP synthesis, reversing the hypertrophic phenotype, might also reverse the blockage of neuritic outgrowth observed after a lesion. In this article, we review findings of our group, confirming our hypothesis in a model of lesioned neuron-astrocyte cocultures. We demonstrate that permissivity for neuritic outgrowth is related to phenotypic changes induced in reactive astrocytes transfected by antisense GFAP-mRNA. We also found that this permissivity was related to a neuron-regulated extracellular laminin bioavailability.

Astroglial permissivity for neuritic outgrowth in neuron-astrocyte cocultures depends on regulation of laminin bioavailability.

The molecular determinants underlying the failure of axons to regenerate in the CNS after injury were studied in an in vitro model of astrogliosis and neuronal coculture. Mechanically lesioned neuron-astrocyte mouse cortical cocultures were treated with antisense glial fibrillary acidic protein (GFAP)-mRNA in order to inhibit the formation of gliofilaments that occurs in response to injury. This inhibition relieves the blockage of neuron migration and neuritic outgrowth observed after lesion, and migrating neurons reappeared, supported by a laminin-labeled extracellular network (permissive conditions). We then questioned the relationship between this permissivity and laminin production. Follow-up studies on the concentration of laminin indicated that, after antisense treatment, the laminin level was increased in the cocultures and was under the control of astrocyte-neuron interactions. The addition of exogenous laminin favored neuronal migration and neurite outgrowth, whereas neutralizing laminin bioavailability with antibodies recognizing the astroglial laminin resulted in an inhibition of both neuronal access to the lesion site and neurite outgrowth, suggesting an active role for laminin in the permissive process. This permissive process could be associated with modulation of extracellular matrix (ECM) molecule degradation by proteinases. Among the latter, matrix metalloproteinases (MMPs) are involved in the breakdown of the ECM component. Our investigation showed a net decrease of the matrix metalloproteinase MMP-2 expression and activity and an increase of its endogenous inhibitor TIMP-2 expression. Both proteins associated with permissivity should be involved in the laminin stabilization and cell-matrix interactions. High levels of laminin and laminin bioavailability, consequent to a reduction in astrogliosis, may be important permissive elements for neuronal migration and neurite outgrowth postlesion.