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

Impact of elevated nitrate on sulfate-reducing bacteria: A comparative study of Desulfovibrio vulgaris

Sulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70 mM NaNO{sub 3} but not by 70 mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level

Demographic and anthropometrical analysis and genotype distribution of chronic hepatitis C patients treated in public and private reference centers in Brazil

Hepatitis C virus (HCV) infection is a serious public health problem, since 80% to 85% of HCV carriers develop a persistent infection that can progress into liver cirrhosis and hepatocarcinoma. Considering that the response of hepatitis C patients to combination therapy with interferon and ribavirin depends on HCV characteristics as well as on host features, we made a retrospective analysis of demographic and anthropometrical data and HCV genotype distribution of chronic hepatitis C patients treated in public and private reference centers in Brazil. The medical records of 4,996 patients were reviewed, 81% from public and 19% from private institutions. Patients' median age was 46 years, and there was a higher prevalence of male (62%) and white patients (80%). The analysis of HCV-infecting strains showed a predominance of genotype 1 (64%) over genotypes 2 and 3. The patients' mean weight was 70.6 kg, and 65% of the patients weighed less than 77kg. Overweight and obesity were observed in 37.8% and 13.6% of the patients, respectively. Since a body weight of 75 kg or less has been considered an independent factor that significantly increases the odds of achieving a sustained virological response, the Brazilian population seems to have a more favorable body weight profile to achieve a sustained response than the American and European populations. The finding that 65% of chronic hepatitis C patients have a body weight of 77 kg or less may have a positive pharmacoeconomic impact on the treatment of genotype 1 HCV patients with weight-based doses of peginterferon

Methionine- and choline-deficient diet induces hepatic changes characteristic of non-alcoholic steatohepatitis

CONTEXT: Non-alcoholic steatohepatitis is a disease with a high incidence, difficult diagnosis, and as yet no effective treatment. So, the use of experimental models for non-alcoholic steatohepatitis induction and the study of its routes of development have been studied. OBJECTIVES: This study was designed to develop an experimental model of non-alcoholic steatohepatitis based on a methionine- and choline-deficient diet that is manufactured in Brazil so as to evaluate the liver alterations resulting from the disorder. METHODS: Thirty male C57BL6 mice divided in two groups (n = 15) were used: the experimental group fed a methionine- and choline-deficient diet manufactured by Brazilian company PragSoluções®, and the control group fed a normal diet, for a period of 2 weeks. The animals were then killed by exsanguination to sample blood for systemic biochemical analyses, and subsequently submitted to laparotomy with total hepatectomy and preparation of the material for histological analysis. The statistical analysis was done using the Student's t-test for independent samples, with significance level of 5%. RESULTS: The mice that received the methionine- and choline-deficient diet showed weight loss and significant increase in hepatic damage enzymes, as well as decreased systemic levels of glycemia, triglycerides, total cholesterol, HDL and VLDL. The diagnosis of non-alcoholic steatohepatitis was performed in 100% of the mice that were fed the methionine- and choline-deficient diet. All non-alcoholic steatohepatitis animals showed some degree of macrovesicular steatosis, ballooning, and inflammatory process. None of the animals which were fed the control diet presented histological alterations. All non-alcoholic steatohepatitis animals showed significantly increased lipoperoxidation and antioxidant enzyme GSH activity. CONCLUSION: The low cost and easily accessible methionine- and choline-deficient diet explored in this study is highly effective in inducing steatosis and steatohepatitis in animal model, alterations that are similar to those observed in human livers

The vitamin E reduces liver lipoperoxidation and fibrosis in a model of nonalcoholic steatohepatitis A vitamina E reduz a lipoperoxidação hepática e a fibrose em modelo experimental de esteatohepatite não-alcoólica

CONTEXT: No effective treatment is available for nonalcoholic steatohepatitis in nowadays. OBJECTIVES: To develop a model of nonalcoholic steatohepatitis induced by a methionine and choline deficient diet, as well as to evaluate the role of metformin, vitamin E and simvastatin in the nonalcoholic steatohepatitis progression. METHODS: The study analyzed prospectively 50 Wistar rats for a 90-day period and divided them into five groups of 10 rats. One group was given standard rat diet and the others received the methionine and choline deficient diet. Among the four groups that received this diet, one received saline 0,9% and the others received metformin, vitamin E or simvastatin. After the study period, the animals were sacrificed and their blood was collected for biochemical analysis. The livers were removed for lipoperoxidation analysis and for the histological examinations. RESULTS: The methionine and choline deficient diet was able to induce steatosis in 100% of the animals and nonalcoholic steatohepatitis in 27 (69.2%). The alanine aminotransferase levels were significantly higher in the simvastatin group. The aspartate aminotransferase levels were also higher in the simvastatin group, but were statistically significant only in relation to the standard diet group. When lipoperoxidation values were compared, the groups that received standard rat diet and methionine and choline deficient with vitamin E presented significantly lower rates than the others. The presence of fibrosis was significantly smaller in the group receiving vitamin E. CONCLUSIONS: The diet used was able to induce steatosis and nonalcoholic steatohepatitis. Besides vitamin E showed to reduce the liver oxidative stress, as well as the fibrosis development<br>CONTEXTO: Ainda não há um tratamento comprovadamente eficaz para a esteatohepatite não-alcoólica. OBJETIVO: Desenvolver um modelo experimental de esteatohepatite não-alcoólica induzida por dieta deficiente em metionina e colina, bem como avaliar o papel do metformina, da vitamina E e da sinvastatina na evolução da esteatohepatite não-alcoólica. MÉTODOS: Foram estudados prospectivamente 50 ratos da linhagem Wistar, por um período de 90 dias, sendo os mesmos divididos em cinco grupos de 10 ratos. Um grupo recebeu ração convencional e os demais a dieta deficiente em metionina e colina. Dentre os quatro grupos que receberam a dieta, para um foi administrado soro fisiológico e para os demais o metformina, a vitamina E e a sinvastatina. Após o período de estudo os animais foram mortos, sendo colhido sangue para análise bioquímica e removido o fígado para análise da lipoperoxidação através do teste de substâncias reativas ao ácido tiobarbitúrico e para a realização dos exames histológicos. RESULTADOS: A dieta deficiente em metionina e colina foi capaz de induzir esteatose em 100% dos animais enquanto que esteatohepatite não-alcoólica foi diagnosticada em 27 (69,2%). Os níveis da ALT foram significativamente maiores no grupo da sinvastatina. Os níveis de AST também foram maiores no grupo da sinvastatina, mas apenas foram estatisticamente significantes em relação ao grupo da ração. Quando se compararam os valores de lipoperoxidação, os grupos da ração convencional e da vitamina E apresentaram índices significativamente menores que os demais. A presença de fibrose foi significativamente menor no grupo que recebeu a vitamina E. CONCLUSÕES: A dieta utilizada foi capaz de induzir esteatose e esteatohepatite não-alcoólica, sendo que a vitamina E demonstrou reduzir o estresse oxidativo hepático, bem como o desenvolvimento de fibros