Effects of gallic acid on expressions of MMP-2 and MMP-9 through the pathway of p38/JNK in C6 glioma cells.

In our study, the effects of gallic acid (GA), a natural therapeutic agent, on oxidative stress biomarkers and MMP-2 and MMP-9 expressions via the p38/JNK pathway in C6 glioma cells were investigated. The toxicity of GA was determined by the WST-1 method. JNK, p38 and MMP-2/-9 mRNA expressions in the cell line were detected by RT-qPCR. JNK/SAPK, Grap-2/p38 and MMP-2/-9 protein levels were analyzed by using ELISA methods. Biochemical markers were analyzed. GA reduced the cell viability of C6 glioma cells after 24, 48 and 72h of treatment. The expression levels of MMP-2 and MMP-9 mRNA decreased in C6 glioma cells treated with 150µg/ml GA for 24 and 48h compared to the control cells. Unlike SOD activity, GA treatment significantly increased PCO and MDA levels in the cells treated with 150µg/ml GA for 24 and 48h compared to the non-treated cells. According to our results, GA inhibited the proliferation of C6 glioma cells. Also, it reduced MMP-2 and MMP-9 expressions and increase oxidative stress. Therefore, GA may have preventive effects on gliomas progression and/or invasion.

Quercetin-loaded nanoparticles enhance cytotoxicity and antioxidant activity on C6 glioma cells.

Quercetin (Qu) is a natural flavonoid present in many commonly consumed food items. The dietary phytochemical quercetin prevents tumor proliferation and is a potent therapeutic cancer agent. The purpose of this study was to synthesize and characterize quercetin-loaded poly(lactic-co-glycolic acid) nanoparticles (Qu1NP, Qu2NP, and Qu3NP) with different size and encapsulation properties and to evaluate their in vitro activity on C6 glioma cells. Nanoparticles were synthesized by single emulsion solvent evaporation method. Then, particle size, zeta potential, polydispersity index and encapsulation efficiency of nanoparticles were determined. Particle size of Qu1NP, Qu2NP, and Qu3NPs were determined as 215.2 ± 6.2, 282.3 ± 7.9, and 584.5 ± 15.2 nm respectively. Treating C6 glioma cells with all nanoparticle formulations effectively inhibited the cell proliferation. Qu1NPs were showed the lowest IC50 value in 48 h with 29.9 µg/ml and achieved higher cellular uptake among other nanoparticles and Qu. Additionally, 48-h treatment with Qu1NPs significantly decreased MDA level (14.90 nmol/µg protein) on C6 glioma cells which is related to reduced oxidative stress in cells. Findings of this study revealed that quercetin's cellular uptake and anti-oxidant activity is improved by small-sized Qu1NPs in C6 glioma cells.

Cytotoxic and apoptotic effects of ethanolic propolis extract on C6 glioma cells.

Propolis is a natural resinous substance obtained from beehives, and emerging evidence supports that it has antitumor, antiinflammatory, antioxidant, and antimicrobial activities. The aim of the study is to examine the cytotoxic, antioxidant, and apoptotic features of ethanolic propolis extract (PE) on C6 glioma cells. The cells were treated with ethanolic PE at various concentrations for 24 hours, after which the total antioxidant status (TAS) and total oxidant status; malondialdehyde, protein carbonyl, 8-hydroxy-2'-deoxyguanosine, and glutathione (GSH) levels; Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity; and apoptotic markers were measured. Ethanolic PE at 100, 250, and 500 µg/mL concentrations showed optimal activity on C6 glioma cells. TAS and GSH levels were significantly increased in C6 glioma cells treated with 100 and 500 µg/mL PE compared to control cells (P < .05). Similarly, the activity of Cu/Zn-SOD was higher in C6 glioma cells treated with 250 or 500 µg/mL ethanolic PE compared to control cells (P < .05), as was the caspase-3 mRNA expression level. The highest levels of caspase-8 and -9 expression were in C6 glioma cells treated with 500 µg/mL PE. Collectively, our results indicate that ethanolic PE has cytotoxic and apoptotic effects on C6 glioma cells. Furthermore, it may provide a protective role in the antioxidant defense system. PE shows potential for development as a natural antioxidant and apoptotic agent for the treatment of brain tumors.

Investigation of the biochemical and apoptotic changes in breast cancer cells treated with leaf extract from tea (Camellia sinensis L.) grown with added boric acid.

Tea obtained from the leaves of Camellia sinensis L., a medicinal plant, is a widely popular beverage. Deficiency in boron, a micronutrient for C. sinensis, affects the growth as well as the quality of tea. The aim of this study was to explore whether boric acid at various concentrations added to soil improves the quality of C. sinensis and also whether it changes the apoptotic, anti-proliferative, and anti-oxidative effects of C. sinensis leaf extract on breast cancer (MCF-7) cells. C. sinensis was grown in Rize-Turkey. Boric acid at concentrations of 100 (group B), 300 (group C), and 500 (group D) mg/m2 in sodium tetraborate buffer was administered as a single dose to the soil; group A (no boric acid) was the control. Boron, glutathione (GSH), malondialdehyde and protein carbonyl levels in the C. sinensis leaves were measured. C. sinensis leaf extracts at different concentrations was applied to MCF-7 cells for 24 and 48h. Cytotoxicity, proliferation, and apoptosis were examined. The highest TUNEL+ cell percentage was in MCF-7 cells treated with D group leaf extract compared to the control group (p<0.001 at concentrations of 2.3, 2.6 and 3mg/mL). Moreover, the GSH level increased in the MCF-7 cells under the same conditions (p<0.001 for each concentration). Leaf extracts from C. sinensis grown in soil with boric acid have more anti-proliferative, apoptotic and anti-oxidative effects on the MCF 7 cells.

Kruppel-Like Transcription Factor-4 Gene Expression and DNA Methylation Status in Type 2 Diabetes and Diabetic Nephropathy Patients.

BACKGROUND/AIM: Diabetic nephropathy (DN) is one of the most serious microvascular complications in diabetic patients. The kruppel-like transcription factor-4 (KLF-4) affects the expression of genes involved in the pathogenesis of DN. The present study aims to identify the KLF-4 expression and DNA methylation (DNAMe) status in patients with type-2 diabetes (T2D) and DN and to reveal the contribution of the KLF-4 to the development of DN. MATERIAL AND METHODS: The cohort study was performed with blood samples from 120 individuals; T2D group (n = 40), DN group (n = 40) and control group (n = 40). The expression level of the KLF-4 gene was analyzed using the real-time polymerase chain reaction (qRT-PCR) and the methylation profile detected using the methylation-specific PCR (MS-PCR) technique. RESULTS: According to our findings, KLF-4 mRNA expression in the T2D group was 1.60 fold lower than in the control group (p = 0.001). In the DN group, the expression of KLF-4 mRNA was 2.92-fold less than that of the T2D group (p = 0.001). There was no significant alteration in the DNAMe status among the groups. CONCLUSION: Our findings showed that regardless of the DNAMe status, KLF-4 gene expression may play a role in the development of T2D and DN. This suggests that the KLF-4 gene may be the target gene in understanding the mechanism of nephropathy, which is the most important complication of diabetes, and planning nephropathy-related treatments, but the data should be supported with more studies.

Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells.

The aim of this study was to evaluate anti-cancer properties of hesperetin (Hsp) and hesperetin-loaded poly(lactic-co-glycolic acid) nanoparticles (HspNPs) for glioblastoma treatment. Nanoparticles prepared by single emulsion method had a size of less than 300 nm with 70.7 ± 3.9% reaction yield and 26.4 ± 1.1% Hsp loading capacity. Treatment of C6 glioma cells with HspNPs for 24 and 48 h resulted in dose- and time-dependent decrease in cell viability, with approximate IC50 of 28 and 21 µg/mL, respectively (p = .036 for 24 h, p = .025 for 48 h). The percentage of PCNA positive cells decreased to 20% and 10%, respectively, for Hsp- and HspNP-treated cells at concentration of 100 µg/mL. Treatment with increasing concentrations of HspNPs (25, 50, 75 and 100 µg/mL) resulted in 9.1-, 7-, 12.5- and 12.7-fold in increase in apoptotic cell number. Optimum doses of Hsp and HspNPs were found to increase oxidative damage in C6 glioma cells. MDA levels, an indicator of lipid peroxidation, were found to be significantly elevated at 75 and 100 µg/mL exposure concentration of HspNPs with (p = .002) and (p = .018), respectively for 48-h treatment. The results obtained with this study showed biocompatible polymeric nanoparticle systems has great advantages to enhance anti-cancer activity and poor solubility of therapeutic agents. Overall our findings suggest that Hsp-loaded PLGA nanoparticle systems showed significant anti-cancer activity and HspNPs could be used as promising novel anti-cancer agent.

The antioxidant and antiapoptotic effect of boric acid on hepatoxicity in chronic alcohol-fed rats.

The harmful use of alcohol is a worldwide problem involving all ages. This study aims to investigate chronic alcohol exposure related hepatotoxicity on the rat liver and possible hepatoprotective effects of boric acid. Rats were separated into 4 different groups: control, ethanol, ethanol+boric acid, and boric acid. We measured (i) malondialdehyde (MDA), total sialic acid (TSA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels, which are known to be the markers of alcohol damage; and also (ii) caspase-3, tumor necrosis factor-alpha (TNF-α), and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) as the markers of apoptosis. In the ethanol group, MDA, TSA, and TNF-α levels increased whereas SOD and CAT levels decreased compared with the control group. Ethanol+boric acid group MDA, TSA, caspase-3, and TNF-α levels decreased whereas SOD and CAT levels increased compared with the ethanol group. Using histopathological evaluation of light microscope images, immunohistochemical caspase-3 and TNF-α activity in the ethanol+boric acid group were shown to be decreased compared with that in the ethanol group. Our results revealed that ethanol is capable of triggering oxidative stress and apoptosis in the rat liver. We propose that boric acid is an effective compound in protecting the rat liver against ethanol.

Screening of antimicrobial activity and cytotoxic effects of two Cladonia species.

The present study explores the antimicrobial activity and cytotoxic effects in culture assays of two fruticose soil lichens, Cladonia rangiformis Hoffm. and Cladonia convoluta (Lamkey) Cout., to contribute to possible pharmacological uses of lichens. In vitro antimicrobial activities of methanol and chloroform extracts against two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), two Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), and the yeast Candida albicans were examined using the paper disc method and through determination of minimal inhibitory concentrations (MICs). The data showed the presence of antibiotic substances in the chloroform and the methanol extracts of the lichen species. The chloroform extracts exhibited more significant antimicrobial activity than the methanol extracts. However, a higher antifungal activity was noted in the methanol extract of C. rangiformis. The maximum antimicrobial activity was recorded for the chloroform extract of C. convoluta against E. coli. The cytotoxic effects of the lichen extracts on human breast cancer MCF-7 cells were evaluated by the trypan blue assay yielding IC50 values of ca. 173 and 167 microg/ml for the extracts from C. rangiformis and C. convoluta, respectively.

Estradiol induces JNK-dependent apoptosis in glioblastoma cells.

Estrogens exert multiple regulatory actions on cellular events in a variety of tissues including the brain. In the present study, the signaling mechanisms of the concentration-dependent effects of 17-ß-estradiol (estradiol) on glioblastoma cells were investigated. Cell viability was evaluated by the trypan blue exclusion assay. Cell growth and kinase activities were evaluated by immunocytochemistry and Western blotting. The results showed that high concentrations of estradiol inhibit growth and induce apoptosis in C6 rat glioma and T98G human glioblastoma cells. The blockade of the c-jun NH(2)-terminal kinase (JNK) signaling pathway prevented these effects of estradiol, indicating the critical role of the JNK/c-jun signaling cascade in glioblastoma cell growth inhibition and cell death in response to high concentrations of estradiol. Collectively, these findings highlight the potential of new discoveries in sensitizing estrogen-sensitive tumors to chemotherapeutic drugs, and may lead to the development of new JNK-based effective therapies.

Common variants in the ATP-binding cassette transporter 1 gene with decreased HDL-cholesterol levels and coronary artery disease.

BACKGROUND: Our aim was to determine whether the common variants within the coding sequence of ABCA1 gene affects low plasma high-density lipoprotein cholesterol (HDL-C) levels in Turkish patients with coronary artery disease (CAD). The study group was composed of 552 CAD patients, of which 251 had HDL-C levels < or =40 mg/dL, and 301 had HDL-C levels >40 mg/dL. METHODS: PCR-RFLP was used to determine the A2589G and G3456C DNA polymorphisms of the ABCA1 gene. The study group was analyzed for potential clinical predictors of low HDL-C. RESULTS: The GG variant of the ABCA1 gene A2589G polymorphism was found in 3.6% patients within the HDL-C < or =40 mg/dL group and in 4% of HDL-C levels >40 mg/dL group. Frequency distributions of the A2589G genotypes were not found to differ significantly among groups. The CC genotype of the G3456C polymorphism was found in 6.8% of HDL-C < or =40 mg/dL group and in 11.6% individuals of the HDL-C levels >40 mg/dL group. Frequency distributions of the G3456G genotypes were not significantly different among groups. The A2589G genotypes were not found to be effective over the analyzed lipid parameters. Among G3456C genotypes, in CAD patients with HDL-C < or =40 mg/dL the low-density lipoprotein (LDL-C) levels were elevated, whereas HDL-C levels decreased in CC genotype carriers compared to GG and GC. CONCLUSIONS: No significant association was found between cardiovascular endpoints and ABCA1 gene A2589G and G3456C genotypes in this study population.

Simvastatin induces apoptosis in human breast cancer cells: p53 and estrogen receptor independent pathway requiring signalling through JNK.

The effect of simvastatin, a widely used statin for the treatment of hypercholesterolemia, was investigated in the estrogen receptor (ER)-positive MCF-7, and the ER-negative MDA-MB 231 human breast cancer cell lines. Simvastatin induced cell cycle arrest and apoptosis in both cells. These effects of simvastatin were not altered by 17-beta-estradiol treatment. MCF-7 cells express wild-type tumor suppressor protein p53, whereas MDA-MB 231 cells carry a p53 mutation. However, no alteration in the level or localisation of p53 was observed with simvastatin treatment in either cell line. On the other hand, simvastatin strongly stimulated phosphorylation of c-jun which was completely abolished by the c-jun NH2-terminal kinase (JNK) inhibitor SP600125, which also significantly reduced the antiproliferative and apoptotic effects of simvastatin in these cells. In conclusion, we describe here that simvastatin induces apoptosis via involvement of JNK in breast cancer cells independent of their ER or p53 expression status. These findings indicate a great potential for statins for the treatment of cancers resistant to currently used drugs, and target the JNK signalling pathway for a novel approach of breast cancer treatment.

Ginkgo biloba extract regulates differentially the cell death induced by hydrogen peroxide and simvastatin.

Several human diseases have been associated with the overproduction of reactive oxygen species (ROS) and subsequently various antioxidants emerged as potential therapeutic agents that scavenge ROS. As an oxidative stress model of human disease, we used hydrogen peroxide (H2O2) to study effect of ROS on C6 glioma cells as a surrogate for astrocytes. H2O2 induced dose- and time-dependent apoptotic cell death which was preceded by growth arrest, and transiently activated the signalling proteins ATF-2, ERK1/2 and AKT in C6 glioma cells. While several antioxidants failed to block H2O2-induced apoptosis of these cells, Ginkgo biloba extract (EGb) totally prevented the cell death and growth inhibition induced by H2O2. Interestingly, EGb did not prevent the activation of ATF-2, ERK1/2 and AKT induced by H2O2 excluding the role of these factors in the pro-apoptotic effect of H2O2. We have previously shown that the lipid-lowering drug, simvastatin, causes apoptotic cell death in C6 glioma cells [Koyuturk M, Ersoz M, Altiok N. Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase. Neurosci Lett 2004;370(2-3):212-7]. However, in parallel experiments with H2O2, EGb was unable to prevent cell death induced by simvastatin suggesting the involvement of separate signalling pathways between H2O2 and simvastatin. Thus, EGb and other plant flavonoids might have potential as protective agents against apoptosis through scavenging ROS upon cerebral or myocardial diseases associated with free radical generation.

Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase.

The lipid-lowering drugs, statins, induce apoptosis in a variety of tumor cells. Here we investigated the apoptotic effect of the lipophilic statin, simvastatin, in C6 glioma cells and the underlying effects on intracellular signal transduction. Simvastatin inhibited cell proliferation totally after 20h of treatment as shown by the decrease in proliferating cell nuclear antigen expression in the nucleus. Subsequently, simvastatin caused apoptotic cell death by shrinkage of cytoplasm and condensation of chromatin, and DNA fragmentation. The features of apoptosis were visible only after 48 h of treatment, possibly reflecting a requirement for cell commitment to growth arrest. In immunocytochemical and immunoblotting experiments we have shown that simvastatin markedly increased the phosphorylation of ATF-2 and c-jun in the nucleus of the C6 glioma cells at early time points which was preserved even 24 h after treatment. In contrast, activities of protein kinases Erk1/2 and AKT in the cell survival pathway remained unchanged throughout the treatment. Selective inhibitor of JNK, but not p38 kinase, reduced simvastatin-induced cell death and ATF-2 and c-jun phosphorylation suggesting that JNK-dependent activation of ATF-2 and c-jun may play an important role in simvastatin-induced proliferation inhibition and apoptosis in C6 glioma cells. These observations suggest that statins may have clinical significance in the prevention of glial tumors beyond their cholesterol-lowering effect and JNK may be a rational target for sensitizing glioma cells to chemotherapeutic agents.