Chitosan-Coated Liposome Formulations for Encapsulation of Ciprofloxacin and Etoposide.

Cancer and bacterial infections rank among the most significant global health threats. accounting for roughly 25 million fatalities each year. This statistic underscores the urgent necessity for developing novel drugs, enhancing current treatments, and implementing systems that boost their bioavailability to achieve superior therapeutic outcomes. Liposomes have been recognised as effective carriers; nonetheless, they encounter issues with long-term stability and structural integrity, which limit their pharmaceutical applicability. Chitosomes (chitosan-coated liposomes) are generally a good alternative to solve these issues. This research aims to demonstrate the effective individual encapsulation of ciprofloxacin (antibacterial, hydrophilic) and etoposide (anticancer, hydrophobic), within chitosomes to create more effective drug delivery systems (oral administration for ciprofloxacin, parenteral administration for etoposide). Thus, liposomes and chitosomes were prepared using the thin-film hydration technique and were characterised through ATR-FTIR, Dynamic Light Scattering (DLS), zeta potential, and release profiling. In both cases, the application of chitosomes enhanced long-term stability in size and surface charge. Chitosome-encapsulated ciprofloxacin formulations exhibited a slower and sustained release profile, while the combined effect of etoposide and chitosan showed heightened efficacy against the glioblastoma cell line U373. Therefore, coating liposomes with chitosan improved the encapsulation system's properties, resulting in a promising method for drug delivery.

Metabolomics Analysis Revealed Significant Metabolic Changes in Brain Cancer Cells Treated with Paclitaxel and/or Etoposide.

Cancer of the central nervous system (CNS) is ranked as the 19th most prevalent form of the disease in 2020. This study aims to identify candidate biomarkers and metabolic pathways affected by paclitaxel and etoposide, which serve as potential treatments for glioblastoma, and are linked to the pathogenesis of glioblastoma. We utilized an untargeted metabolomics approach using the highly sensitive ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) for identification. In this study, 92 and 94 metabolites in U87 and U373 cell lines were profiled, respectively. The produced metabolites were then analyzed utilizing t-tests, volcano plots, and enrichment analysis modules. Our analysis revealed distinct metabolites to be significantly dysregulated (nutriacholic acid, L-phenylalanine, L-arginine, guanosine, ADP, hypoxanthine, and guanine), and to a lesser extent, mevalonic acid in paclitaxel and/or etoposide treated cells. Furthermore, both urea and citric acid cycles, and metabolism of polyamines and amino acids (aspartate, arginine, and proline) were significantly enriched. These findings can be used to create a map that can be utilized to assess the antitumor effect of paclitaxel and/or etoposide within the studied cancer cells.

Thallium Induces Antiproliferative and Cytotoxic Activity in Glioblastoma C6 and U373 Cell Cultures via Apoptosis and Changes in Cell Cycle.

Thallium (Tl+) is a heavy metal that causes toxicity in several organs, including the brain. Its cytotoxic profile, combined with its affinity for tumor cells when used as a radioligand for labeling these cells, suggests its potential use as antitumor therapy. In this study, glioblastoma cell lines C6 (from rat) and U373 (from human) were exposed to increased concentrations of thallium(I) acetate (5, 10, 50, 100, or 200 µM) and several toxic endpoints were evaluated, including loss of confluence and morphological changes, loss of cell viability, changes in cell cycle, and apoptosis. Tl+ was detected in cells exposed to thallium(I) acetate, demonstrating efficient uptake mechanism. Confluence in both cell lines decreased in a concentration-dependent manner (50-200 µM), while morphological changes (cell shrinkage and decreased cell volume) were more evident at exposures to higher Tl+ concentrations. For both parameters, the effects of Tl+ were more prominent in C6 cells compared to U373 cells. The same trend was observed for cell viability, with Tl+ affecting this parameter in C6 cells at low concentrations, whereas U373 cells showed greater resistance, with significant changes observed only at the higher concentrations. C6 and U373 cells treated with Tl+ also showed morphological characteristics corresponding to apoptosis. The cytotoxic effects of Tl+ were also assessed in neural and astrocytic primary cultures from the whole rat brain. Primary neural and astrocytic cultures were less sensitive than C6 and U373 cells, showing changes in cell viability at 50 and 100 µM concentrations, respectively. Cell cycle in both brain tumor cell lines was altered by Tl+ in G1/G2 and S phases. In addition, when combined with temozolamide (500 µM), Tl+ elicited cell cycle alterations, increasing SubG1 population. Combined, our novel results characterize and validate the cytotoxic and antiproliferative effects of Tl+ in glioblastoma cells.

Sulforaphane Causes Cell Cycle Arrest and Apoptosis in Human Glioblastoma U87MG and U373MG Cell Lines under Hypoxic Conditions.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. The median survival rate from diagnosis ranges from 15 to 17 months because the tumor is resistant to most therapeutic strategies. GBM exhibits microvascular hyperplasia and pronounced necrosis triggered by hypoxia. Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has already demonstrated the ability to inhibit cell proliferation, by provoking cell cycle arrest, and leading to apoptosis in many cell lines. In this study, we investigated the antineoplastic effects of SFN [20-80 µM for 48 h] in GBM cells under normoxic and hypoxic conditions. Cell viability assays, flow cytometry, and Western blot results revealed that SFN could induce apoptosis of GBM cells in a dose-dependent manner, under both conditions. In particular, SFN significantly induced caspase 3/7 activation and DNA fragmentation. Moreover, our results demonstrated that SFN suppressed GBM cells proliferation by arresting the cell cycle at the S-phase, also under hypoxic condition, and that these effects may be due in part to its ability to induce oxidative stress by reducing glutathione levels and to increase the phosphorylation of extracellular signal-regulated kinases (ERKs). Overall, we hypothesized that SFN treatment might serve as a potential therapeutic strategy, alone or in combination, against GBM.

Quercetin-loaded platelets as a potential targeted therapy for glioblastoma multiforme cell line U373-MG.

Over the globe, the incidence of glioblastoma multiforme (GM) is very low, that is, 1-4 cases per 100,000, but it is fatal and cancer grows very fast inside the brain tissues, namely astrocytes and oligodendrocytes. Because of the rapid growth, it is difficult to halt the dissemination of tumor in adjacent tissues. Although temozolomide (TMZ) is a currently approved standard of care, it develops resistance over the period. Therefore, there is a need to develop a novel drug delivery system. In this work, authors have developed platelets as drug delivery carriers-loaded with quercetin (QCT) for targeting GM. The effect of QCT and QCT-platelet was assessed on the U373-MG cell line. Natural human platelets were used as carriers for drug loading and drug delivery. Platelets possess an open canalicular system that allows the uptake of drug molecules in the platelet cytoplasm. The study showed that the maximum encapsulation efficiency of QCT-platelet was 93.96 ± 0.12% and the maximum drug release in 24 h was 76.26 ± 0.13% in-vitro at pH 5.5 that mimics the tumor microenvironment. In this work, there is a three-fold enhancement of solubility of QCT. The cytotoxic activity of QCT-platelets was studied in the U373-MG human astrocytoma glioblastoma cell line and the cell viability was 14.52 ± 1.53% after 48 h. Thus, platelets were proved as good carriers for therapeutic moieties and can be effectively used to target the glioblastoma tumor in the near future.

Rutin-Loaded Solid Lipid Nanoparticles: Characterization and In Vitro Evaluation.

This study was aimed at preparing and characterizing solid lipid nanoparticles loading rutin (RT-SLNs) for the treatment of oxidative stress-induced diseases. Phospholipon 80H® as a solid lipid and Polysorbate 80 as surfactant were used for the SLNs preparation, using the solvent emulsification/diffusion method. We obtained spherical RT-SLNs with low sizes, ranging from 40 to 60 nm (hydrodynamic radius) for the SLNs prepared starting from 2% and 5% (w/w) theoretical amount. All prepared formulations showed negative zeta-potential values. RT was efficiently encapsulated within SLNs, obtaining high encapsulation efficiency and drug content percentages, particularly for SLNs prepared with a 5% theoretical amount of RT. In vitro release profiles and analysis of the obtained data applying different kinetic models revealed Fickian diffusion as the main mechanism of RT release from the SLNs. The morphology of RT-SLNs was characterized by scanning electron microscopy (SEM), whereas the interactions between RT and the lipid matrix were investigated by Raman spectroscopy, evidencing spectral modifications of characteristic bands of RT due to the establishment of new interactions. Finally, antioxidant activity assay on human glioblastoma astrocytoma (U373) culture cells showed a dose-dependent activity for RT-SLNs, particularly at the highest assayed dose (50 µM), whereas the free drug showed the lesser activity.

Enhanced brain targeting efficiency using 5-FU (fluorouracil) lipid-drug conjugated nanoparticles in brain cancer therapy.

The present investigation was aimed to synthesize, optimize, and characterize lipid/drug conjugate nanoparticles for delivering 5-fluorouracil (5-FU) to treat brain cancer. The Box-Behnken design was used to optimize the formulation, evaluate the particle size, entrapment efficiency, morphology, in vitro drug release study, and stability profiles. The in vitro performance was executed using cell line studies. The in vivo performance was carried out for pharmacokinetic studies, sterility test, biodistribution studies, and distribution lipid-drug conjugated (LDC) nanoparticles in the brain. Particle size, zeta potential, entrapment efficiency, and morphology of the optimized formulation demonstrated desirable results. In vitro release pattern showed initial fast release, followed by sustained release up to 48 h. Cytotoxic effects of blank stearic acid nanoparticles, LDC nanoparticles, and 5-FU solution on human glioma cell lines U373 MG cell showed more cytotoxicity by LDC-NPs compared to others. The values reported for LDC (AUC = 19.37 ± 0.09 µg/mL h and VD 2.4 ± 0.24 mL) and pure drug (AUC = 8.37 ± 0.04 µg/mL h and VD = 5.24 ± 0.29 mL) indicate higher concentrations of LDC in systemic circulation, while pure 5-FU was found to be largely available in tissue rather than blood circulation. The t1/2 for LDC represents an approximate rise by ninefold, while MRT (12.10 ± 0.44 h) denotes 12-fold rise than pure 5-FU indicating the prolonged circulation of LDC. Free 5-FU concentration in the brain was maximum (5.24 ± 0.01 µg/g) after 3 h, while for the optimized formulation of LDC it was twofold greater estimated as 11.52 ± 0.32 µg/g. In conclusion, the efficiency of 5-FU to treat the brain is increased when it is formulated with LDC nanoparticles.

Physicochemical Characterization and Antioxidant Activity Evaluation of Idebenone/Hydroxypropyl-ß-Cyclodextrin Inclusion Complex †.

Idebenone (IDE) is an antioxidant drug active at the level of the central nervous system (CNS), whose poor water solubility limits its clinical application. An IDE/2-hydroxypropyl-ß-cyclodextrin (IDE/HP-ß-CD) inclusion complex was investigated by combining experimental methods and theoretical approaches. Furthermore, biological in vitro/ex vivo assays were performed. Phase solubility studies showed an AL type diagram, suggesting the presence of a 1:1 complex with high solubility. Scanning electron microscopy (SEM) allowed us to detect the morphological changes upon complexation. The intermolecular interactions stabilizing the inclusion complex were experimentally characterized by exploring the complementarity of Fourier-transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR) with mid-infrared light, Fourier-transform near-infrared (FT-NIR) spectroscopy, and Raman spectroscopy. From the temperature evolution of the O-H stretching band of the complex, the average enthalpy ΔHHB of the hydrogen bond scheme upon inclusion was obtained. Two-dimensional (2D) rotating frame Overhauser effect spectroscopy (ROESY) analysis and computational studies involving molecular modeling and molecular dynamics (MD) simulation demonstrated the inclusion of the quinone ring of IDE inside the CD ring. In vitro/ex vivo studies evidenced that complexation produces a protective effect of IDE against the H2O2-induced damage on human glioblastoma astrocytoma (U373) cells and increases IDE permeation through the excised bovine nasal mucosa.

Protective Effects of Crude Plant Extracts against Aminochrome-induced toxicity in Human Astrocytoma Cells: Implications for Parkinson's Disease.

BACKGROUND/AIMS: Aminochrome, an endogenous compound formed during dopamine oxidation can induce neurotoxicity under certain aberrant conditions and induce Parkinson-like syndrome. Glutathione transferase M2 (GSTM2) activity of astrocytes by catalysing the conjugation of aminochrome with glutathione, can offer protection against aminochrome toxicity. Some medicinal toxicity through this plants may exert protective effect against aminochrome mechanism. METHODS: In the present study, extracts from plants native to Cameroon, such as Alchornea laxiflora (leaves), Dacryodes edulis (barks), Annona muricata (seeds), Annona senegalensis (barks) were evaluated for their protection against aminochrome-induced toxicity in human glioblastoma/ astrocytoma U373MG wild type and U373MGsiGT6 cells in which GSTM2 expression was 74% silenced. The cells were pre-incubated with the plant extracts for 2 hr before addition of aminochrome (75 µM) and measurement of cell death/viability by flow cytometry after 24 hr incubation. RESULTS: The extract of A. laxiflora (1 µg/ml), D. edulis (25 µg/ml), A. muricata (25 µg/ml) and A. senegalensis (25µg/ml) significantly decreased aminochrome-induced toxicity in U373siGST6 and U373MG cells. However, only A. laxiflora and A. muricata significantly increased the mitochondria membrane potential in U373siGST6 cells following aminochrome treatment. CONCLUSION: The results indicate that extracts of some Cameroon plants can provide protection against aminochrome-induced toxicity and mitochondria dysfunction in human glioblastoma/astrocytoma cells. Although further identification of active components of these extracts is needed, potential usefulness of these compounds in Parkinson's disease may be suggested.

Gene Silencing of TGFßRII Can Inhibit Glioblastoma Cell Growth

Objective: Glioblastoma (GBM) is the most malignant and aggressive type of glioma, associated with a high rate of mortality. The transforming growth factor-ß receptor II (TGFß RII) is involved in glioma initiation and progression. On the other hand, TGFß RII silencing is critical to the inhibition of GBM. Therefore, we aimed to determine the effects of specific TGFß RII siRNA on the survival of U-373MG cells. Methods: TGFß RII siRNA was transfected, and qRT-PCR was performed to examine TGFß RII mRNA expression. Cell survival was determined using colorimetric MTT assay, and platelet-derived growth factor-BB (PDGF-BB) level was measured in the culture supernatant using ELISA assay. Result: Our findings indicated that specific siRNAs could dose-dependently suppress TGFß RII mRNA expression after 48 hours. In addition, treatment with TGFß RII siRNA significantly reduced tumor cell survival and decreased the amount of PDGF-BB protein in the cell culture supernatant. Conclusion: Our results suggest that TGFß RII silencing can be a promising complementary treatment for glioma.

AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner.

AIM: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. MATERIALS AND METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. RESULTS: In U373 cells, A-769662 (100 µM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 µM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.

Hydrogen Peroxide-Induced Oxidative Stress Activates Proteasomal Trypsin-Like Activity in Human U373 Glioma Cells.

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal, a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. The major proteolytic system for modified protein degradation is the ubiquitin-proteasome pathway. However, our previous studies using U937 human leukemic cells showed that 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, U373 human glioma cells were cultured in the presence of hydrogen peroxide (H2O2) to investigate the relationships of proteasome and/or cathepsin G activities and H2O2-induced GAPDH degradation. Treatment of cells with H2O2 for 5 h in culture decreased GAPDH activity as well as its protein concentration in a concentration-dependent manner. Two proteasomal activities (peptidylglutamyl-peptide hydrolase and chymotrypsin-like hydrolase activities) and cathepsin G activity were decreased by H2O2 treatment in a concentration-dependent manner, but proteasomal trypsin-like hydrolase activity increased with cell exposure to high H2O2 concentrations. Among the protease inhibitors examined here, H2O2-induced activation of trypsin-like activity and GAPDH degradation were inhibited by the proteasome inhibitor lactacystin. Furthermore, H2O2-induced activation of trypsin-like activity was also inhibited by another proteasome inhibitor MG-132. These results suggested that proteasomal trypsin-like activity played an important role in eliminating oxidatively modified GAPDH formed in these cells during H2O2 exposure.

Intracellular GSH Alterations and Its Relationship to Level of Resistance following Exposure to Cisplatin in Cancer Cells.

One of the major complications in cancer chemotherapy with cisplatin as one of the important medicines in treatment regimens of different cancers is the development of resistance. One of the most described cellular defense mechanisms involved in resistance is glutathione (GSH), thus in this study, the effects of cisplatin on the total intracellular GSH level (GSHi) in some sensitive and resistant variants of human cell lines (hepatocarcinoma HepG2, skin A375, cisplatin sensitive glioblastoma U373MG and cisplatin resistant glioblastoma U373MGCP, cisplatin sensitive ovary A2780S and cisplatin resistant A2780CP cells) were studied. MTT assay was performed to measure cytotoxicity of cisplatin (33.3 µM for 1 hour). Following cisplatin exposure, GSHi (per million cells) was evaluated using a photometrical assay up to 90 minutes. Our results indicate that there are significant differences between GSHi content of A2780CP and U373MGCP cells compared to other cell lines. Moreover, IC50 of cisplatin in different cells seems to have a relation with mean of GSH level in 90 minutes (GSH (mean)90). As a conclusion, it seems that resistance to cisplatin in different cell lines is more related with the diverse patterns of GSHi variations following cisplatin exposure than its original level, and/or its cellular increase or decrease. It is also suggested that GSH (mean)90 may be used as a factor for the prediction of cellular resistance to cisplatin.

Synthesis and preliminary evaluation of a new (99m)tc labeled substance p analogue as a potential tumor imaging agent.

Neurokinin 1 receptors (NK1R) are overexpressed on several types of important human cancer cells. Substance P (SP) is the most specific endogenous ligand known for NK1Rs. Accordingly,a new SP analogue was synthesized and evaluated for detection of NK1R positive tumors.[6-hydrazinopyridine-3-carboxylic acid (HYNIC)-Tyr(8)-Met(O)(11)-SP] was synthesized and radiolabeled with (99m)Tc using ethylenediamine-N,N'-diacetic acid (EDDA)and Tricine as coligands. Common physicochemical properties of radioconjugate were studied and in-vitro cell line biological tests were accomplished to determine the receptor mediated characteristics. In-vivo biodistribution in normal and tumor bearingnude mice was also assessed. The cold peptide was prepared in high purity (>99%) and radiolabeled with (99m)Tc at high specific activities (84-112GBq/µmol) with an acceptable labeling yield (>95%). The radioconjugate was stable in-vitro in the presence of human serum and showed 44% protein binding to human serumalbumin. In-vitro cell line studies on U373MG cells showed an acceptable uptake up to 4.91 ± 0.22% with the ratio of 60.21 ± 1.19% for its specific fraction and increasing specific internalization during 4 h. Receptor binding assays on U373MG cells indicated a mean Kd of 2.46 ± 0.43 nM and Bmax of 128925 ± 8145 sites/cell. In-vivo investigations determined the specific tumor uptake in 3.36 percent of injected dose per gram (%ID/g) for U373MG cells and noticeable accumulations of activity in the intestines and lung. Predominant renal excretion pathway was demonstrated. Therefore, this new radiolabeled peptide could be a promising radiotracer for detection of NK1R positive primary or secondary tumors.

Desferrioxamine, an iron chelator, inhibits CXCL10 expression induced by polyinosinic-polycytidylic acid in U373MG human astrocytoma cells.

Although iron is essential in physiological processes, accumulation of iron in central nervous system is associated with various neurological diseases including Alzheimer's disease and Parkinson's disease. Innate immune reactions are involved in the pathogenesis of those diseases, but roles of iron in innate immunity are not known well. In the present study, pretreatment of U373MG human astrocytoma cells with an iron chelator desferrioxamine (DFX) inhibited the expression of CXCL10 induced by a Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly IC). Induction of interferon-ß (IFN-ß) was not affected, but phosphorylation of signal transducer and transcription 1 (STAT1) was decreased by DFX. We have previously reported that various IFN-stimulated genes (ISGs) are involved in CXCL10 induction by poly IC. Pretreatment with DFX also decreased the expression of these ISGs. Pretreatment of cells with FeSO4 counteracted inhibitory effects of DFX on ISG56, retinoic acid-inducible gene-I (RIG-I), CXCL10 and phosphorylation of STAT1. These results suggest that iron may positively regulate STAT1 phosphorylation and following signaling to express ISG56, RIG-I and CXCL10 in U373MG cells treated with poly IC. Iron may contribute to innate immune and inflammatory reactions elicited by the TLR3 signaling in astrocytes, and may play an important role in neuroinflammatory diseases.

U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells.

It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line.

ISG54 and ISG56 are induced by TLR3 signaling in U373MG human astrocytoma cells: possible involvement in CXCL10 expression.

Toll-like receptor (TLR) 3 is a pattern recognition receptor that recognizes double-stranded RNA (dsRNA). TLR3 signaling in astrocytes leads to the expression of interferon-ß (IFN-ß), and IFN-ß regulates immune and inflammatory reactions by inducing IFN-stimulated genes (ISGs). We demonstrated in the present study that polyinosinic-polycytidylic acid (poly IC), an authentic dsRNA, up-regulated the expression of ISG54 and ISG56 in U373MG human astrocytoma cells. This reaction was confirmed to be mediated via the TLR3/IFN-ß pathway. We also found that ISG56 positively regulates the expression of ISG54, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). In addition, positive feedback loops were found between ISG54 and ISG56, and also between ISG54 and RIG-I. RNA interference experiments revealed that all of ISG54, ISG56, RIG-I and MDA5 were involved in the poly IC-induced expression of a chemokine CXCL10. These results suggest that ISG54 and ISG56 are involved in the induction of CXCL10 in TLR3/IFN-ß signaling at least partly by co-operating with RIG-I and MDA5. ISG54 and ISG56 may contribute to immune and inflammatory reactions elicited by the TLR3/IFN-ß signaling pathway in astrocytes, and may play an important role both in antiviral immunity and in neuroinflammatory diseases.

MDA5 and ISG56 mediate CXCL10 expression induced by toll-like receptor 4 activation in U373MG human astrocytoma cells.

Toll-like receptor (TLR) 4 is a pattern recognition receptor, and recognizes not only bacterial lipopolysaccharide (LPS) but also endogenous danger-associated molecular patterns released from dying or injured cells. It has been reported that TLR4 signaling in astrocytes plays an important role in various neurological diseases. However, details of TLR4 signaling in astrocytes are not fully elucidated. In the present study, we demonstrated that TLR4 signaling, induced by LPS, increases the expression of melanoma differentiation-associated gene 5 (MDA5) and interferon (IFN)-stimulated gene 56 (ISG56) in U373MG human astrocytoma cells. We also found that nuclear factor-κB, p38 mitogen-activated protein kinase and IFN-ß are involved in the expression of MDA5 and ISG56 induced by LPS. RNA interference experiments revealed that MDA5 and ISG56 positively regulate the LPS-induced expression of a chemokine CXCL10, but not CCL2. In addition, it was suggested that MDA5 and ISG56 constitute a positive feedback loop. These results suggest that MDA5 and ISG56 may contribute not only to physiological inflammatory reactions but also to the pathogenesis of various neurological diseases elicited by TLR4 in astrocytes, at least in part, by regulating the expression of CXCL10.

Neuroprotective properties of Spanish red wine and its isolated polyphenols on astrocytes.

The neuroprotective effect of Merlot red wine and its isolated polyphenols was evaluated in an oxidative stress model induced by Fenton reaction and hydrogen peroxide in the human astrocytoma U373 MG cell line. Compared with cells treated only with oxidative stress inductors, the pre-incubation with Merlot red wine for 24h caused a significant increase in cell viability for all concentrations assayed. The most abundant polyphenols found in Merlot red wine were the flavonoids catechin (37.8mg/l), epicatechin (52.3mg/l), quercetin (5.89mg/l) and procyanidins (15.2mg/l), the hydroxybenzoic acid gallic acid (16.7mg/l), and the phenolic alcohol tyrosol (31.4mg/l). The potential protective role of these polyphenols when isolated was then assessed in treated Fenton reaction U373 MG cells. Polyphenols decreased reactive oxygen species generation and increased the activity and the protein expression of the antioxidant enzymes catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase. Of the polyphenols, quercetin and procyanidins showed the highest neuroprotective effect.