Quercetin promotes glioma growth in a rat model.

We have previously demonstrated that quercetin (Quer), a polyphenol widely found in vegetables, decreased glioma cell growth in vitro. Here, we asked whether this compound could affect glioma growth in an in vivo rat glioma model. We found that daily intraperitoneal Quer (50 mg/kg) injections lead to a concentration of 0.15 µg of Quer per gram of brain tissue, which increased the tumor volume in a time dependent manner. We observed a small reduction in lymphocytic infiltration, a marker of good prognosis in gliomas that was accompanied by a small reduction in cell viability of peripheral T-cells. Moreover, after Quer treatment neither body weight alteration nor liver pathology markers were detected. Although in vitro studies and massive literature reports point to the antitumoral properties of Quer, the present results indicate that great caution has to be taken in the design of clinical trials and the indiscriminate use of this polyphenol as dietary supplement.

Resveratrol abrogates the temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the temozolomide-induced senescence in glioma cells.

BACKGROUND: Temozolomide (TMZ) is the most widely used drug to treat glioblastoma (GBM), which is the most common and aggressive primary tumor of the Central Nervous System and one of the hardest challenges in oncotherapy. TMZ is an alkylating agent that induces autophagy, apoptosis and senescence in GBM cells. However, therapy with TMZ increases survival after diagnosis only from 12 to 14.4 months, making the development of combined therapies to treat GBM fundamental. One candidate for GBM therapy is Resveratrol (Rsv), which has additive toxicity with TMZ in several glioma cells in vitro and in vivo. However, the mechanism of Rsv and TMZ additive toxicity, which is the aim of the present work, is not clear, especially concerning cell cycle dynamics and long term effects. METHODS: Glioma cell lines were treated with Rsv and TMZ, alone or in combinations, and the induction and the role of autophagy, apoptosis, cell cycle dynamics, protein expression and phosphorylation status were measured. We further evaluated the long term senescence induction and clonogenic capacity. RESULTS: As expected, temozolomide caused a G2 cell cycle arrest and extensive DNA damage response. Rsv did not reduced this response, even increasing pATM, pChk2 and gammaH2Ax levels, but abrogated the temozolomide-induced G2 arrest, increasing levels of cyclin B and pRb(S807/811) and reducing levels of pWee1(S642) and pCdk1(Y15). This suggests a cellular state of forced passage through G2 checkpoint despite large DNA damage, a scenario that may produce mitotic catastrophe. Indeed, the proportion of cells with high nuclear irregularity increased from 6 to 26% in 48 h after cotreatment. At a long term, a reduction in clonogenic capacity was observed, accompanied by a large induction of senescence. CONCLUSION: The presence of Rsv forces cells treated with TMZ through mitosis leading to mitotic catastrophe and senescence, reducing the clonogenic capacity of glioma cells and increasing the chronic effects of temozolomide.

Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells.

Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

Protective profile of oxcarbazepine against oxygen-glucose deprivation in organotypic hippocampal slice culture could involve PI3K cell signaling pathway.

OBJECTIVE: Brain ischemia results in cellular degeneration and loss of brain function. Oxcarbazepine (OXC), one of the newer antiepileptic drugs, has been demonstrating its efficacy on wide spectrum neurological disorders. In this paper, we investigated the neuroprotective profile of OXC in an in vitro model of ischemia, which consists in the exposure of organotypic hippocampal slice cultures to oxygen and glucose deprivation. METHODS: OXC (30 microM) was added to the medium before and/or during and/or after the oxygen and glucose deprivation induction. Cell death was quantified by propidium iodide uptake measurement. Immunoblotting was used to detect the phosphorylation of Akt. RESULTS: Our results showed a decrease in propidium iodide incorporation when OXC was added before oxygen and glucose deprivation, suggesting a neuroprotective effect. This effect was prevented when cultures were previously treated with LY294002, an inhibitor of phosphoinositide-3-kinase (PI3K) pathway. We also analysed the effect of OXC on Akt phosphorylation. Immunoblotting revealed that OXC did not induce any change in phosphorylation/activation of Akt. DISCUSSION: Our results reinforce the neuroprotective effect of OXC and add some evidence that its mechanism may involve the PI3K pathway, suggesting that such effect could be upstream Akt. This indicates that with respect to OXC neuroprotective, Akt may not play a crucial role in determining cell survival.

Resveratrol and quercetin cooperate to induce senescence-like growth arrest in C6 rat glioma cells.

Glioma is the most frequent and malignant primary human brain tumor with dismal prognosis despite multimodal therapy. Resveratrol and quercetin, two structurally related and naturally occurring polyphenols, are proposed to have anticancer effects. We report here that resveratrol and quercetin decreased the cell number in four glioma cell lines but not in rat astrocytes. Low doses of resveratrol (10 microM) or quercetin (25 microM) separately had no effect on apoptosis induction, but had a strong effect on caspase 3/7 activation when administered together. Western blot analyses showed that resveratrol (10 microM) and quercetin (25 microM) caused a reduction in phosphorylation of Akt, but this reduction was not sufficient by itself to mediate the effects of these polyphenols. Most important, resveratrol and quercetin chronically administered presented a strong synergism in inducing senescence-like growth arrest. These results suggest that the combination of polyphenols can potentialize their antitumoral activity, thereby reducing the therapeutic concentration needed for glioma treatment.

Beta-amyloid peptide toxicity in organotypic hippocampal slice culture involves Akt/PKB, GSK-3beta, and PTEN.

In the present study we investigated the toxicity induced by exposing organotypic slice culture to beta-amyloid peptide 25-35 (25microM) for 1, 3, 6, 12, 24 and 48h. To elucidate a mechanism involved in its toxicity, we studied the PI3-K cell signaling pathway, particularly Akt/PKB, GSK-3beta, and PTEN proteins. Cell death was quantified by propidium iodide uptake and proteins were analyzed by immunoblotting. Our results showed a significant cell death after 48h of beta-amyloid 25-35 peptide exposition. The exposition of cultures to beta-amyloid peptide resulted in an increase in the phosphorylation state of Akt and GSK-3beta proteins after 6h, followed by a decrease of the phosphorylation state of these proteins after 12h of exposition. However, after 24h of peptide treatment, the phosphorylation of GSK-3beta presented a new increase while the phosphorylation of Akt remained down. The immunocontent of the PTEN protein, an indirect Akt phosphatase, increased after 24 and 48h of beta-amyloid exposition. These results suggest an involvement of Akt dephosphorylation/inactivation in the toxicity induced by the beta-amyloid 25-35 peptide in organotypic slice hippocampal culture, probably induced by increasing PTEN immunocontent. Taken together, our results provide more information about the molecular mechanisms involved on beta-amyloid peptide toxicity.

Homocysteine increases neuronal damage in hippocampal slices receiving oxygen and glucose deprivation.

Homocystinuria is an inherited metabolic disorder caused by severe deficiency of cystationine beta-synthase activity, resulting in the tissue accumulation of homocysteine (Hcy). Affected patients usually present many signs and symptoms such as seizures, mental retardation, atherosclerosis and stroke. The aim of this study is to evaluate in vivo and in vitro effects of Hcy using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, an in vitro model of hypoxic-ischemic events. Neural cell injury was quantified by the measurement of lactate dehydrogenase (LDH) released from damaged cells into the extracellular fluid. The results showed that both in vivo and in vitro Hcy increased the LDH released to de incubation medium, suggesting an increase of tissue damage caused by OGD. This fact can be related with the high incidence of stroke in homocystinuric patients.

Antiproliferative effect of quercetin in the human U138MG glioma cell line.

Recent epidemiological and dietary intervention studies in animals and humans have suggested that diet-derived flavonoids, in particular quercetin, may play a beneficial role by preventing or inhibiting tumorigenesis. The aim of this study was to evaluate whether quercetin may act differently on cancer and normal neuronal tissue. In order to investigate this, the U138MG human glioma cell line and hippocampal organotypic cultures were used. The study showed that quercetin induced in glioma cell cultures results in (a) a decrease in cell proliferation and viability, (b) necrotic and apoptotic cell death, (c) arrest in the G2 checkpoint of the cell cycle, and (d) a decrease of the mitotic index. Furthermore, we demonstrated that while quercetin promotes cancer regression it was able to protect the hippocampal organotypic cultures from ischemic damage. To sum up, our results suggest that quercetin induced growth inhibition and cell death in the U138MG human glioma cell line, while exerting a cytoprotective effect in normal cell cultures.

Protective effect of resveratrol against oxygen-glucose deprivation in organotypic hippocampal slice cultures: Involvement of PI3-K pathway.

Here we investigated the neuroprotective effect of resveratrol in an in vitro model of ischemia. We used organotypic hippocampal cultures exposed to oxygen-glucose deprivation (OGD). In OGD-vehicle exposed cultures, about 46% of the hippocampus was labeled with PI, indicating a robust percentage of cell death. When cultures were treated with resveratrol 10, 25 and 50 microM, the cell death was reduced to 22, 20 and 13% respectively. To elucidate a possible mechanism by which resveratrol exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase (PI3-k) pathway using LY294002 (5 microM) and mitogen-activated protein kinase (MAPK) using PD98059 (20 microM). The resveratrol (50 microM) neuroprotection was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that resveratrol 50 microM induced the phosphorylation/activation of Akt and extracellular signal-regulated kinase-1 and -2 (ERK1/2) and the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that PI3-k/Akt pathway are involved in the neuroprotective effect of resveratrol.

The effects of estradiol on estrogen receptor and glutamate transporter expression in organotypic hippocampal cultures exposed to oxygen--glucose deprivation.

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) alpha and beta and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17beta-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERalpha protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERbeta was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERbeta and, consequently, of the genes influenced by this receptor.

Hyperhomocysteinemia increases damage on brain slices exposed to in vitro model of oxygen and glucose deprivation: prevention by folic acid.

In the present study we evaluate the effects of homocysteine on cellular damage using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation), an in vitro model of hypoxic-ischemic events. For chronic treatment, we induced elevated levels of homocysteine in blood (500 microM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by subcutaneous injections of homocysteine (0.3-0.6 micromol/g of body weight), twice a day with 8 h intervals, from the 6 th to the 28 th postpartum day and controls received saline. Rats were sacrificed 1, 3 or 12 h after the last injection. For acute treatment, 29-day-old rats received one single injection of homocysteine (0.6 micromol homocysteine/g body weight) or saline and were sacrificed 1h later. In another set of experiments rats were pretreated with Vitamins E (40 mg/kg) and C (100 mg/kg) or folic acid (5 mg/kg) during 1 week; 12 h after the last administration they received a single injection of homocysteine or saline and were sacrificed 1 h later. Results showed that both chronic (1 h after homocysteine administration) and acute hyperhomocysteinemia increased the cellular damage measured by LDH released to de incubation medium, suggesting an increase of tissue damage caused by OGD. Pretreatment with folic acid completely prevented the damage caused by acute hyperhomocysteinemia, whereas Vitamin E just partially prevented such effect. These findings may be relevant to explain, at least in part, the higher susceptibility of hyperhomocysteinemic patients to be susceptible to ischemic events and point to a possible preventive treatment.

Neuroprotection and protein damage prevention by estradiol replacement in rat hippocampal slices exposed to oxygen-glucose deprivation.

Here we investigated the effects of estradiol replacement in ovariectomized female rats using hippocampal slices exposed to oxygen-glucose deprivation (OGD). OGD induced lactate dehydrogenase (LDH) release to the incubation medium, what was assumed as a parameter of cellular death. In the estradiol-treated group the LDH release was markedly decreased by 23% as compared to the vehicle-treated group. In attempt to study a possible mechanism by which estradiol acts, we investigated some parameters of oxidative stress. In both vehicle-treated and estradiol-treated groups, OGD significantly increased the free radical production by 34% and 16%, respectively, although no significant differences on total antioxidant capacity were observed. Interestingly, estradiol replacement prevented the significant reduction in tryptophan and tyrosine contents caused by OGD observed in vehicle-treated animals. Our results show that estradiol replacement in ovariectomized female rats decreases cellular susceptibility to an ischemic-like injury and suggest a role for the hormone on protein damage prevention.

Estradiol protects against oxygen and glucose deprivation in rat hippocampal organotypic cultures and activates Akt and inactivates GSK-3beta.

Here we investigated the neuroprotective effect of 17beta-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17beta-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17beta-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17beta-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3beta. Our results show a clear neuroprotective effect of 17beta-estradiol and suggest that this effect could involve PI3-K pathway.