Normal Astrocyte Cells Research Articles

Long noncoding RNA HOXD-AS2 regulates cell cycle to promote glioma progression.

Now, numerous exciting findings have been found that long noncoding RNAs (lncRNAs) play a vital role in cancer malignant progression. However, their potential involvement in glioma is not well understood. Here, we performed a high-throughput microarray to detect the lncRNA expression profiles between glioma cell lines and normal astrocyte cell lines. HOXD-AS2 was increased in glioma cells and it was associated with glioma grade and poor prognosis. Loss of HOXD-AS2 can inhibit glioma cell growth by inducing cell-cycle G1 arrest in vitro. The proliferation of glioma was inhibited followed by knocking down the expression of HOXD-AS2 not only in subcutaneous injection model but also in orthotopic implantation model. These findings indicate that HOXD-AS2 promotes the glioma progression and may serve as a potential target for glioma diagnosis and therapy.

MiR-1254 inhibits proliferation, migration and invasion of human brain tumour cell lines

Purpose: To investigate the expression of miR-1254 in 5 astrocytoma cell lines, and the mechanism involved.Methods: Total RNA was isolated by RNeasy RNA isolation kit while cDNA was prepared by RevertAid cDNA synthesis kit. The transcripts were analysed by real-time polymerase chain reaction (RT-PCR). Transfection of miR-1254 was carried out using FuGENE HD (Promega). Apoptosis was determined by DAPI, acridine orange (AO)/ethidium bromide (EB) and annexin V/PI double staining. Cell migration and invasion were investigated by wound healing and Martigel invasion assays, respectively. Protein expression was measured by western blotting.Results: Expression of miR-1254 was significantly down-regulated in the astrocytoma cell lines when compared to normal astrocyte cells (p < 0.05). Ectopic expression of miR-1254 in astrocytoma SW 1088 cells inhibited cell proliferation via initiation of apoptosis and cell cycle arrest. Over-expression of miR- 1254 also led to significant decrease in cell migration and invasion of SW 1088 astrocytoma cells (p < 0.05).Conclusion: The results show that the expression of miR-1254 is down-regulated in astrocytoma cell lines, but over-expression of miR-1254 inhibits proliferation of the cell lines via cell cycle arrest and apoptosis. Thus, miR-1254 has promising potential for use in the treatment of brain tumour.Keywords: Brain tumour, Astrocytoma, miR-1254, Apoptosis, Cell migration

The Effects of Synthesized Rhenium Acetylsalicylate Compounds on Human Astrocytoma Cell Lines.

PurposeBecause of the scarcity of suitable brain cancer drugs, researchers are frantically trying to discover novel and highly potent drugs free of side effects and drug-resistance. Rhenium compounds are known to be nontoxic and exhibit no drug resistance. For that reason, we have developed a series of novel rhenium acetylsalicylato (RAC or ASP) complexes to test their cytotoxicity on brain cancer cells. Also we have attempted to explore the DNAbinding properties of these compounds because many drugs either directly or indirectly bind to DNA.MethodsWe have treated the RAC series compounds on human astrocytoma brain cancer cell lines and rat normal brain astrocyte cells and determined the efficacy of these complexes through in vitro cytotoxicity assay. We carried out the DNA-binding study through UV titrations of a RAC compound with DNA. Also we attempted to determine the planarity of the polypyridyl ligands of the RAC series compounds using DFT calculations.ResultsRAC6 is more potent than any other RAC series compounds on HTB-12 human astrocytoma cancer cells as well as on Glioblastoma Multiforme D54 cell lines. In fact, The IC-50 value of RAC6 on HTB-12 cancer cells is approximately 2 μM. As expected, the RAC series compounds were not active on normal cells. The DFT calculations on the RAC series compounds were done and suggest that the polypyridyl ligands in the complexes are planar. The UV-titrations of RAC9 with DNA were carried out. It suggests that RAC9 and possibly all RAC series compounds bind to minor grooves of the DNA.ConclusionBecause of the very low activity of RAC6 on normal cells and low lC50 value of on astrocytoma (HTB-12) cell lines, it is possible that RAC6 and its derivatives may potentially find application in the treatment of brain cancers. The DFT calculations and UV titrations suggest that RAC series compounds either bind to DNA intercalatively or minor grooves of the DNA or both. However, it is highly premature to make any definite statement in the absence of other techniques.

Curcumin-lipoic acid conjugate as a promising anticancer agent on the surface of gold‑iron oxide nanocomposites: A pH-sensitive targeted drug delivery system for brain cancer theranostics

Brain tumor is a lethal, fast growing cancer and a difficult case for treatment. Receptor-mediated endocytosis has been recognized as one of the most effective methods for drug delivery to brain tissue by overcoming obstacles associated with conventional therapeutics. In this work, a targeted theranostic drug delivery system (DDS) was prepared based on gold‑iron oxide nanocomposites (Fe3O4@Au NCs). Lipoic acid-curcumin (LA-CUR) was synthesized and introduced as a novel anticancer drug, and glutathione (GSH) was exploited as the targeting ligand. Both LA-CUR and GSH were easily attached to Fe3O4@Au NCs via Au-S interaction. As a negatively charged nanocarrier, the prepared DDS showed relatively less protein adsorption. Accordingly, hemocompatibility assays (complement, platelet, and leucocyte activation) revealed its hemocompatible virtue, especially in respect of free LA-CUR. GSH functionalization led to 2-fold increase of cellular uptake in GSH receptor-positive astrocyte cells which could primarily indicate the probable ability of the DDS to bypass BBB. Cytotoxicity and apoptosis assays together showed the noticeably enhanced cytotoxicity of LA-CUR against cancerous U87MG cells (IC50=2.69μg/ml) in comparison with curcumin (IC50=21.31μg/ml); moreover, the DDS demonstrated relatively higher cytotoxicity against cancerous U87MG cells than normal astrocyte cells which was in accordance with pH sensitive mechanism of LA-CUR release. Besides, the results of in vitro magnetic resonance imaging (MRI) (relaxation rate (r2)=80.73 (s−1·mM−1)) primarily revealed that the DDS can be applied as a negative MRI contrast agent. In sum, the prepared DDS appeared to be a promising candidate for brain cancer treatment and a favorable MRI contrast agent.

Long non-coding RNA MEG3 inhibits cell growth of gliomas by targeting miR-93 and inactivating PI3K/AKT pathway

Gliomas are the most common cancers in the brain, accompanied with high morbility, occurrence, disability and mortality. Long non-coding RNAs (lncRNAs) have been proposed as promoter or inhibitor in many cancer processes. Previous findings have indicated that lncRNA-maternally expressed gene 3 (MEG3) is involved in tumorigenesis of several cancers, including glioma. However, the underlying mechanism of MEG3 in glioma remains elusive. In our study, MEG3 was found downregulated in glioma tissues compared with normal brain tissues. Downregulated expression of MEG3 was also detected in two human glioma cell lines (U-251, M059J) compared with normal astrocyte cells. MEG3 was then overexpressed by ligating to a lentiviral vector. Overexpressed MEG3 inhibited the proliferation of U-251 cells, and restrained the expression of proliferation marker proteins Ki67 and proliferating cell nuclear antigen (PCNA). However, cell apoptosis rate of U-251 cells and the expression of apoptosis marker proteins (caspase-3 and caspase-9) were elevated by MEG3. Furthermore, miR-93 was predicted a direct target of lncRNA-MEG3 by bioinformatics analysis. Overexpressed MEG3 counteracted the role of miR-93 in facilitating proliferation and inhibiting apoptosis in U-251 cells. Moreover, MEG3 restained the activation of phosphatidylinositol3 kinase/protein kinaseB (PI3K/AKT) pathway by reducing cytomembrane translocation of AKT. Finally, the invivo experiment revealed that MEG3 strongly reduced tumor growth, tumor volume and the expression of Ki67 and PCNA. lncRNA-MEG3 also inhibited the level of miR-93 and the expression of PI3K/AKT pathway related proteins invivo. Taken together, our research indicated a MEG3-miR-93-PI3K-AKT pathway in regulating the growth of glioma, providing a promising therapy for glioma.

Knockdown of Ras-Related Protein 25 (Rab25) Inhibits the In Vitro Cytotoxicity and In Vivo Antitumor Activity of Human Glioblastoma Multiforme Cells.

Ras-related protein 25 (Rab25) is a member of the Rab family, and it has been reported to play an important role in tumorigenesis. However, its direct involvement in human glioblastoma multiforme (GBM) is still unclear. The aim of the current study was to investigate the potential role of Rab25 in the growth, proliferation, invasion, and migration of human GBM. Our results showed that Rab25 expression was significantly higher in human GBM cell lines compared with a normal astrocyte cell line. In vitro functional studies revealed that knockdown of Rab25 reduced cell proliferation and inhibited invasion and migration of GBM cells. In vivo experiments showed that knockdown of Rab25 attenuated the tumor growth in nude mice. Finally, knockdown of Rab25 significantly inhibited the phosphorylation levels of PI3K and AKT in GBM cells. Taken together, these findings indicate that Rab25 may act as a tumor promoter in human GBM and that approaches to target Rab25 may provide a novel strategy to treat this disease.

Exosomal microRNAs in Tumoral U87 MG versus Normal Astrocyte Cells

Exosomal microRNAs in Tumoral U87 MG versus Normal Astrocyte Cells

Discovery of potent and selective cytotoxic activity of new quinazoline-ureas against TMZ-resistant glioblastoma multiforme (GBM)

Herein, we report new quinazoline-urea based compounds with potent cytotoxic activities against TMZ-resistant glioblastoma multiforme (GBM) cells. Low micromolar IC50 values were exhibited over a panel of three primary GBM patient-derived cell cultures belonging to proneural (GBM-1), mesenchymal (GBM-2), and classical (GBM-3) subtypes. Eight compounds showed excellent selectivity indices for GBM cells comparing to a normal astrocyte cell line. In JC-1 assay, analogues 11, 12, 20, 22, and 24 exerted promising rates of mPTP opening induction towards proneural GBM subtype. Compounds 11, 20, and 24 bound to the translocator protein 18 kDa (TSPO) in submicromolar range using [3H] PK-11195 binding affinity assay. A homology model was built and docked models of 11, 12, 20, 22 and 24 were generated for describing their plausible binding modes in TSPO. In 3D clonogenic assay, compound 20 manifested potent tumoricidal effects on TMZ-resistant GBM cells even at submicromolar concentrations. In addition, CYP450 and hERG assays presented a safe toxicity profile of 20. Taken as a whole, this report presents compound 20 as a potent, selective and safe GBM cytotoxic agent which constitutes a promising direction against TMZ-resistant GBM.

PTBP1 induces ADAR1 p110 isoform expression through IRES-like dependent translation control and influences cell proliferation in gliomas.

Internal ribosomal entry site (IRES)-mediated translation initiation is constitutively activated during stress conditions such as tumorigenesis and hypoxia. The RNA editing enzyme ADAR1 plays an important role in physiology and pathology. Initially, we found that the ADAR1 p150 or p110 transcript levels were decreased in glioma cells compared with normal astrocyte cells. In contrast, protein levels of ADAR1 p110 were significantly upregulated in glioma tissues and cells. This expression pattern indicated translationally controlled regulation. We identified an 885-nt sequence that was located between AUG1 and AUG2 within the ADAR1 mRNA that exhibited IRES-like activity. Furthermore, we confirmed that the translational mode of ADAR1 p110 was mediated by PTBP1 in glioma cells. The protein levels of PTBP1 and ADAR1 were cooperatively expressed in glioma tissues and cells. Knocking down ADAR1 p110 significantly decreased cell proliferation in three types of glioma cells (T98G, U87MG and A172). The removal of a minimal IRES-like sequence in a p150-overexpression construct could effectively abolish p110 induction and resulted in the slight suppression of cell proliferation compared with ADAR1-p150 overexpression in siPTBP1-treated T98G cells. In summary, our study revealed a mechanism whereby ADAR1 p110 can be activated by PTBP1 through an IRES-like element in glioma cells, and ADAR1 is essential for the maintenance of gliomagenesis.

Epigenetic approaches in glioblastoma multiforme and their implication in screening and diagnosis.

Epigenetic modifications have been reported in a number of non-germ-line tumor types. Epigenetic modifications to the genome, especially DNA methylation and histone modifications, affect gene expression causing increased risk for cancers and other diseases. We have summarized information about DNA methylation percentages in Glioblastoma multiforme (GBM) line HTB-12, alveolar cell carcinoma, and acute lymphocytic leukemia samples and determined H3 (K27) methyltransferase activity in GBM and leukemia cells and made comparisons to H3 (K27) methyltransferase activity in normal astrocyte, lung, and lymphocyte cells. GBM and alveolar cell carcinoma gDNA possessed lower gDNA methylation percentages compared to normal cells. Methyl-sensitive cut counting analysis (MSCC) showed fold decreases in GBM CpG methylation sites for genes PBK, KIF23, COL6A3, and LOX. There was no significant difference in CpG DNA methylation, but less histone methyltransferase activity in acute lymphocytic leukemia compared to normal cells. GBM possessed increased histone methyltransferase activity compared to normal samples. Challenges in the field in diagnosis and prognosis for cancer risk especially with regard to the results of this work are discussed.

Investigation into the potential for hypoxic interior of neoplasms to enhance HSPA expression in glioma

Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia. This study investigated the level of HSPA gene expression in human cell lines exposed to hypoxic conditions. Three human glioma cell lines were selected for this study, each representing different types of glioma (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in three glioma cell lines were approximately sixfold higher than in a normal astrocyte cell line. The expression of HSPA was induced in normal cell lines immediately after exposure to hypoxia with 33% of cells exhibiting expression. However, the effects of hypoxia on gene expression were marginal in glioma cells, due to the already increased levels of HSPA with both pre- and post-hypoxia samples showing expression in approximately 90% of cells. These results show that whilst the stress caused by both cancer and hypoxia induce HSPA expression the underlying imprint of tumourgenesis leads to sustained expression.

Reliability of HSP70 (HSPA) expression as a prognostic marker in glioma.

Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia, with production also being linked to tumourigenesis. HSPA mRNA transcripts and proteins were examined in three human glioma cell lines, representing astrocytoma, oligodendroglioma and glioblastoma, plus 18 clinical brain tissue samples. GAPDH was used as a control gene throughout these studies and exhibited a consistent level of expression in a normal astrocyte cell line, tumourous cell lines and tissue samples. In contrast, the average HSPA mRNA copy numbers detected in glioblastoma tissue were between 1.8- and 8.8-fold higher than in lower grade glioma and control tissue, respectively, which is suggestive of a grade-related transcription profile. Similar patterns of grade-related expression were also observed in glioma cell lines. This study indicates for the first time that HSPA expression in glioma cells may possibly be grade related, and hence could have potential as a prognostic marker.

EDL-360: A Potential Novel Antiglioma Agent

Glioma is a brain tumor that arises from glial cells or glial progenitor cells, and represents 80% of malignant brain tumor incidence in the United States. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor malignancy with fewer than 8% of patients with GBM surviving for more than 3 years. Over the past 10 years, despite improvement in diagnosis and therapies for cancer, the survival rate for high-grade glioma patients remains dismal. The main focus of our research is to identify potent novel antiglioma small molecules. We previously showed that EDL-360, a tetrahydroisoquinoline (THIQ) analog, as being highly cytotoxic to human glioma cell cultures. Here we show that EDL-360 significantly induced apoptosis in human glioma cell lines (U87 and LN18). However, in normal astrocytic cells, EDL-360 induced a modest G0/G1 cell cycle arrest but did not induce apoptosis. In an attempt to enhance EDL-360 induced cell death, we tested simultaneous treatment with EDL-360 and embelin (an inhibitor of the anti-apoptotic protein, XIAP). We found that, glioma cells had significant lower viability when EDL-360 and embelin were used in combination when compared to EDL-360 alone. We also used combination treatment of EDL-360 with decylubiquinone (dUb), a caspase-9 inhibitor, and found that the combination treatment induced a significant cell death when compared to treatment with EDL-360 alone. This is the first report that suggests that dUb has anticancer activity, and perhaps acts as a XIAP inhibitor. Finally, our in vivo data showed that EDL-360 treatment induced a partial regression in glioma tumorigenesis and induced cell death in the treated tumors as shown by H&E staining. Taken together these data suggests that EDL-360 has a potential therapeutic application for treating glioma, especially when combined with XIAP inhibitors.

14-3-3β regulates the proliferation of glioma cells through the GSK3β/β-catenin signaling pathway

We previously demonstrated that 14-3-3β is overexpressed in astrocytomas; however, the underlying mechanisms are poorly understood. Based on the reported multiple functions of 14-3-3β, we hypothesized that it interacts with glycogen synthase kinase 3 β (GSK3β), which regulates β-catenin-mediated oncogene expression and contributes to tumorigenesis and astrocytoma progression. To test these hypotheses, we used 14-3-3β overexpression vectors and small interfering RNA (siRNA) transfection in the human normal astrocyte cell line SVGp12 and the glioma cell line U87, respectively. The results showed that overexpression of 14-3-3β promoted the proliferation of SVGp12 cells, while knockdown of 14-3-3β inhibited the proliferation of U87 cells as analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assays. In Flag-tagged 14-3-3β-overexpressing cells, GSK3β was co-immunoprecipitated with 14-3-3β using a Flag antibody. Knockdown of β-catenin by siRNA blocked cell proliferation induced by overexpression of 14-3-3β. Furthermore, overexpression of 14-3-3β suppressed the phosphorylation of β-catenin leading to its accumulation and nuclear translocation as revealed by western blot analysis. In addition, β-catenin nuclear translocation induced by overexpression of 14-3-3β activated the transcription of oncogenes including c-myc and cyclin D1. Collectively, these results revealed that 14-3-3β regulates the proliferation of astrocytes and glioma cells through the GSK3β/β-catenin signaling pathway. The delineated mechanism of 14-3-3β may be responsible for the tumorigenesis and progression of human astrocytomas. Thus, new therapeutic strategies or drugs aimed at 14-3-3β may have potential for the treatment of human astrocytomas.

Abstract 4523: Targeted delivery to tumor cells by using tunable nano-delivery system with chlorotoxin.

Abstract The aim of this research is to develop the Tunable Nano-Delivery System (TNDS) for specific targeting of the tumor cells using Chlorotoxin (CTX). We present preliminary findings for the TNDS localized drug delivery system comprising of non-ionic surfactant vesicles (niosomes) with CTX embedded in a biodegradable and temperature sensitive chitosan network. The system demonstrates a new approach in the treatment of cancers, through the controlled and targeted delivery to tumor cells (the release time and dosage can be accurately controlled) while sparing normal cells. CTX is a scorpion-derived peptide that binds specifically to neuroectodermal tumors, e.g., glioma, but not to normal tissues. Previously, we have shown that there is a specific interaction between chitosan and Mucin1 (MUC1) receptors over expressed in epithelial ovarian carcinoma resulting in higher chitosan accumulation on the cancer cell surface than on normal ovarian epithelial cells. The current research addresses the incorporation of CTX embedded in Chitosan network to enhance specific targeting, which already benefits from the intrinsic interaction of MUC1 with Chitosan. Our current studies incorporating CTX in chitsoan resulting in TNDS-CTX complex are preformed in cell-free system. First, the release rates and release kinetics of TNDS-CTX are being measured in cell-free system using High Performance Liquid Chromatography (HPLC) and Attenuated Total Reflectance- Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. Results are being compared to our established TNDS delivery system to better determine CTX specific chemical binding sites along with the sites already determined to be specific for MUC1. Next, we will be testing TNDS-CTX in vitro in glioma, epithelial ovarian carcinoma, and normal ovarian epithelial and astrocyte cells. The level of fluorescence of normal cells and tumor cells exposed to the TNDS-CTX-fluorescently labeled are evaluated in in vitro by confocal microscopy. The studies in cell-free system showed that the release rates could be finely controlled depending on the specific design of the TNDS-CTX e.g., ratio of niosomes to chitosan and amount of crosslinker for the chitosan network. We are currently evaluating TNDS-CTX containing benchmark chemotherapeutics such as Paclitaxel, and it is expected that the presence of CTX will enhance cellular uptake of chemotherapeutics when compared to the noisome-chitosan delivery system without CTX. Based on our preliminary data, it is anticipated that CTX will perform as a targeting ligand improving the TNDS-CTX's anti-tumor efficacy owing to enhanced tumor targeting and its tunable and localized delivery. Citation Format: Rana Falahat, Eva Christabel Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Targeted delivery to tumor cells by using tunable nano-delivery system with chlorotoxin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2013-4523

MeCP2 modulates gene expression pathways in astrocytes

BackgroundMutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.MethodsTo identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.ResultsA total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.ConclusionsThe first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.

Identification of the Transmembrane Glucose Regulated Protein 78 as a Biomarker for the Brain Cancer Glioblastoma Multiforme by Gene Expression and Proteomic Studies.

The prognosis of patients with Glioblastoma Multiforme (GBM), the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation and chemotherapy. Genetic heterogeneity of GBM warrants extensive studies to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. In the present study, we report a novel proteomic approach by using two-dimensional difference gel electrophoresis (2D-DIGE) followed by spot picking and analysis of proteins/peptides by Mass Spectrometry. We report Glucose Regulated Protein 78 (GRP78) as a differentially expressed protein in the GBM cell line compared to human normal Astrocyte cells.In addition to proteomic studies, we performed microarray analysis which further confirmed up regulation of GRP78 in GBM cells compared to human normal Astrocyte cells.GRP78 has long been recognized as a molecular chaperone in the endoplasmic reticulum (ER) and can be induced by the ER stress response. Besides its location in the ER, GRP78 has been found in cell plasma membrane, cytoplasm, mitochondria, nucleus and other cellular secretions. GRP78 is implicated in tumor cell proliferation, apoptosis resistance, immune escape, metastasis and angiogenesis, and its elevated expression usually correlates with a variety of tumor micro environmental stresses, including hypoxia, glucose deprivation, lactic acidosis and inflammatory response. GRP78 protein acts as a centrally located sensor of stress, which senses and facilitates the adaptation to the tumor microenvironment.Our findings showed differential expression of this gene in brain cancer GBM and thus confirm similarities in findings in existing transcriptional and translational studies. Thus, these findings could be of further importance for diagnostic, therapeutic and prognostic approaches for dealing with this highly malignant cancer.

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.

Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

BackgroundHOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.ResultsIn this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).ConclusionsOur results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

Induction of Cyclooxygenase-2 Expression by Interleukin-1.BETA. in Human Glioma Cell Line, U87MG

Cyclooxygenase-2 (COX-2) is up-regulated in most high-grade gliomas, and high COX-2 expression is associated with aggressive character and poor prognosis. However, the effect of COX-2 in human glioma cell lines is not well known. This study examined the effect of several stimuli, including interleukin-1beta (IL-1beta) and carcinogens, on COX-2 induction in normal astrocyte cells and human glioma cell lines U87MG, A172, and T98G. IL-1beta-induced COX-2 expression strongly at both protein and messenger ribonucleic acid levels in only the U87MG cells of the glioma cell lines. Furthermore, carcinogen induced COX-2 expression. Similar findings were also observed in normal human astrocyte cells. The U87MG glioma cell line is a good model for COX-2 induction in glioma cell lines.