Human Astrocytoma Research Articles

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells.

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells.

Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress.

Abstract 4038: Role of the Rho-GTPase CDC42 in glioma migration

Abstract Introduction: Malignant astrocytoma is a highly proliferative and invasive neoplasm that infiltrates diffusely into regions of normal brain rendering total surgical extirpation impossible and effective local radiation therapy difficult. Conventional chemotherapy frequently fails because most agents administered systemically will not cross the blood-brain barrier. Despite recent reports which have shown a 2-month survival advantage when adjuvant temozolomide chemotherapy is used for glioblastoma multiforme, death ensues in most cases within 2 years of diagnosis. For truly significant advances to be made in the treatment of patients with malignant astrocytoma, we must develop a greater understanding of the basic biology of these neoplasms. PURPOSE: The aim of this study is to examine a key pathway involving the Rho-GTPase family of proteins that regulates glioma cell migration from the tumor mass into normal brain tissue. One of the Rho-GTPase family proteins, CDC42 is the major regulator of cell polarity. It plays a crucial role in controlling the direction of cell migration by determining the cell membrane localization of lamellipodia, and by moving the microtubule-organizing center and Golgi apparatus in front of the nucleus and towards the leading edge of the cell. However, there have been few reports on the role of CDC42 in human astrocytomas. METHOD: In order to examine the role of CDC42 in glioblastoma, we generated stable doxycycline (DOX) inducible cell lines expressing wild type (WT) -, constitutively active (CA) -, and dominant negative (DN) -CDC42. Cell viability, migration, and invasion assays were performed using the cell lines. We also used orthotopic xenograft mouse models and evaluated the survival by Kaplan-Meier analysis. RESULTS: Administration of DOX induced the expression of CA-CDC42 and demonstrated significantly increased migration and invasion properties compared to WT and DN-CDC42. Furthermore, CA-CDC42 bearing mice with DOX treatment had significantly decreased survival compared to WT and DN with DOX. CONCLUSION: Our results demonstrated that CA-CDC42 enhances the astrocytoma invasion and migration. Furthermore, CA-CDC42 increased the size of implanted tumor in the murine brain leading to early mortality. We concluded that CA-CDC42 plays an important role for astrocytoma malignancy. Citation Format: Hidehiro Okura, Brian J. Golbourn, Amanda J. Luck, Christian A. Smith, James T. Rutka. Role of the Rho-GTPase CDC42 in glioma migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4038. doi:10.1158/1538-7445.AM2015-4038

Abstract 1560: FGFR4 is expressed in the tumor and stromal compartments of human gliomas of all grades and histologies

Abstract BACKGROUND: Fibroblast growth factor receptor (FGFR) 4 is reportedly expressed in several cancer types, and is a high-affinity transmembrane receptor for FGF2 which is expressed human astrocytomas. However, the expression of FGFR4 across gliomas of different grades and histologies has not been extensively investigated. METHODS: We performed immunohistochemistry (IHC) of 178 grade II-IV human gliomas from our tumor bank, including 117 GBMs and 61 lower grade gliomas of astrocytic and oligodendroglial lineage. The samples were scored (0-2) separately for staining intensity and fraction of area that was stained and whether staining was predominantly cytoplasmic or nuclear. This scoring was performed separately for tissue from tumor center and periphery, as well as in tumor areas around pseudopalisading (PP) and large necroses. We also performed western blot analysis (n = 22) and real-time qPCR (n = 37). On one tumor sample we performed FISH for EGFR (red)/Centromere chromosome 7 (green), combined with immunofluorescence (IF) for FGFR4. RESULTS: I) FGFR4 is uniformly expressed in human gliomas, although at a varying degree, as confirmed by IHC, western blotting and RT-qPCR. II) FGFR4 expression was detected both in the nuclei and cytoplasm. III) Both a homogenous and a granular staining pattern was observed the cytoplasm, suggesting involvement of different cell types, whereas FISH demonstrated expression in the tumor and stromal cell compartments. IV) Tumors of grade II and III show a statistically significant higher expression than tumors of grade IV as shown by RT-qPCR. V) There is no difference in expression by IHC between tumors of grade II, III and IV. Citation Format: Christiane H. Gjerde, Ercan Mutlu, Lina Leiss, Bjarne Winther Kristensen, Mohummad Aminur Rahman, Per Øyvind Enger. FGFR4 is expressed in the tumor and stromal compartments of human gliomas of all grades and histologies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1560. doi:10.1158/1538-7445.AM2015-1560

Abstract 3047: Mitochondrial DNA copy variation and TFAM expression in astrocytoma

Abstract Mitochondrial dysfunction plays a role in determining the phenotype through bioenergetic depletion and increased production of ROS in several diseases, including cancer. We have previously demonstrated a relevant reduction of mitochondrial DNA (mtDNA) copy number in astrocytoma of different grades of malignancy, predominantly in grade IV-glioblastoma (GBM). We observed a stepwise increase of TFAM in parallel to the increase of malignancy, and TFAM expression was higher in GBM patients with overall survival longer than 24 months than less than 12 months. TFAM is codified in the nucleus and transported into mitochondria, where is essential for mtDNA replication, repair and protection by forming nucleoid complexes. To further understand the role of TFAM in astrocytoma progression we performed TFAM immunohistochestry in human astrocytoma samples, confocal analysis in GBM cell lines, in vitro functional assays after silencing TFAM by siRNA in two GBM cell lines (U87MG, A172), and high throughput RNA-Seq in Illumina platform for network analysis by Metacore. TFAM protein was detected in cytoplasm in granular pattern of low grade astrocytomas, and also in nuclei of malignant atsrocytomas. Such localization was also confirmed by confocal analysis in two GBM cell lines. TFAM-siRNA transfection decreased TFAM expression level >80% in D2, which persisted up to D7 with evident decrease of mtDNA copy number after 4 days, and maintained until 7 days. Interestingly, a decrease in migration but not in proliferation was observed in both cell lines after TFAM knockdown. Comparison of RNA-Seq of TFAM-siRNA to NTC- siRNA, with 20 million of reads in each experiment performed in duplicate, resulted in 1000 genes differentially expressed and among them 315 were detected in both cell lines. These genes were in pathways related to cell adhesion (integrin-mediated: p = 5.79e-5, histamine H1 receptor signaling in the interruption of cell barrier integrity: p = 3.98e-5, chemokines-mediated: p = 1.22e-5) and ECM remodeling (p = 9.17e-5), supporting the results of in vitro migration assay. 82 genes were downregulated together with TFAM in both cell lines. These genes were in pathways related to immune response IL15 signaling via JAK-STAT cascade involving MyD88-independent toll-like receptor signaling pathway involving ADAM17 and STAT2 (p = 1.89e-5), regulation of cytoskeleton proteins (p = 1.65e-2), hypoxia-induced EMT in cancer and fibrosis (p = 3.37e-2), polyamine and arginine metabolism (p = 2.73e-2, p = 4.65e-2) and as expected, regulation of apoptotic process (p = 9.50e-21). The role of TFAM in cytoskeleton and ECM remodeling may worth additional studies. Also, TFAM in extracellular space, as released by necrotic cells, may potentiate mitochondrial N-formyl peptide-induced secretion of chemokines and activate inflammatory responses, which may open another strategy to further understand TFAM role in tumorigeniesis. Citation Format: Suely K. Marie, Roseli Silva, Antonio Lerario, Miyuki Uno, Sueli Mieko Oba-Shinjo. Mitochondrial DNA copy variation and TFAM expression in astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2015-3047

MiR-25 promotes glioblastoma cell proliferation and invasion by directly targeting NEFL.

Glioblastoma multiforme (GBM) is the most malignant and common brain tumor; it is aggressive growth pattern means that GBM patients face a poor prognosis even when receiving the best available treatment modalities. In recent years, an increasing number of reports suggest that the discovery of microRNAs (miRNAs) might provide a novel therapeutic target for human cancers, including GBM. One miRNA in particular, microRNA-25 (miR-25), is overexpressed in several cancers, wherein accumulating evidence indicates that it functions as an oncogene. However, the function of miR-25 in GBM has not been totally elucidated. In this study, we demonstrated that miR-25 was significantly up-regulated in astrocytoma tissues and glioblastoma cell lines. In vitro studies further demonstrated that overexpressed miR-25 was able to promote, while its antisense oligos inhibited cell proliferation and invasion in U251 cells. Moreover, we identified neurofilament light polypeptide (NEFL) as a novel target molecule of miR-25. Also of note was the fact that NEFL was down-regulated with increased levels of miR-25 expression in human astrocytoma clinical specimens. In addition, via the mTOR signaling pathway, NEFL-siRNA could significantly attenuate the inhibitory effects of knockdown miR-25 on the proliferation and invasion of U251 cells. Overall, our results showed an important role for miR-25 in regulating NEFL expression in GBM, and suggest that miR-25 could be a potential target for GBM treatment.

Superoxide anion radical ([formula omitted]) degrades methylmercury to inorganic mercury in human astrocytoma cell line (CCF-STTG1)

Methylmercury (MeHg) is a global pollutant that is affecting the health of millions of people worldwide. However, the mechanism of MeHg toxicity still remains somewhat elusive and there is no treatment. It has been known for some time that MeHg can be progressively converted to inorganic mercury (iHg) in various tissues including the brain. Recent work has suggested that cleavage of the carbon–metal bond in MeHg in a biological environment is facilitated by reactive oxygen species (ROS). However, the oxyradical species that actually mediates this process has not been identified. Here, we provide evidence that superoxide anion radical (O2-) can convert MeHg to iHg. The calculated second-order rate constant for the degradation of 1μM MeHg by O2- generated by xanthine/xanthine oxidase was calculated to be 2×105M−1s−1. We were also able to show that this bioconversion can proceed in intact CCF-STTG1 human astrocytoma cells exposed to paraquat (PQ), a O2- generating viologen. Notably, exposure of cells to increasing amounts of PQ led to a dose dependent increase in both MeHg and iHg. Indeed, a 24h exposure to 500μM PQ induced a ∼13-fold and ∼18-fold increase in intracellular MeHg and iHg respectively. These effects were inhibited by superoxide dismutase mimetic MnTBAP. In addition, we also observed that a 24h exposure to a biologically relevant concentration of MeHg (1μM) did not induce cell death, oxidative stress, or even changes in cellular O2- and H2O2. However, co-exposure to PQ enhanced MeHg toxicity which was associated with a robust increase in cell death and oxidative stress. Collectively our results show that O2- can bioconvert MeHg to iHg in vitro and in intact cells exposed to conditions that simulate high intracellular O2- production. In addition, we show for the first time that O2- mediated degradation of MeHg to iHg enhances the toxicity of MeHg by facilitating an accumulation of both MeHg and iHg in the intracellular environment.

MicroRNA-182 inhibits the proliferation and migration of glioma cells through the induction of neuritin expression.

Astrocytomas are the most common type of glial tumors and carry a poor prognosis. However, the pathogenesis of astrocytomas remains to be elucidated. Neuritin, a novel member of the neurotrophic factors family, has been shown to be associated with tumor malignancy, via the regulation of apoptosis and proliferation. In the present study, microRNA-182 (miR-182) was cloned and transfected into the U251 human astrocytoma cell line, in order to investigate its regulatory effects on the proliferation and migration of these cells, as well as its association with the expression of neuritin. The results showed that miR-182 specifically targets the gene encoding neuritin, NRN1, as demonstrated by a reduction in the protein and mRNA levels of NRN1. In addition, overexpression of miR-182 affected cell cycle regulation and cell migration capacity in vitro, which may have been associated with the promotion of apoptosis by this molecule. In conclusion, endogenous miR-182 may be involved in the pathogenesis of astrocytoma, which is associated with the miR-182-regulated gene, NRN1.

Suppression of methylmercury-induced IL-6 and MCP-1 expressions by N-acetylcysteine in U-87MG human astrocytoma cells.

The aim of this study was to clarify the involvement of oxidative stress in methylmercury (MeHg)-induced pro-inflammatory cytokine expressions and the suppressive effects of N-acetylcysteine (NAC) in MeHg-induced cytokine expression. Using U-87-MG human astrocytoma cell line, interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 expressions induced by 4 μM MeHg were measured at mRNA and protein levels. Hydrogen peroxide (H2O2) and superoxide anion (O2(-)) were quantified by flow-cytometry analysis. To examine the suppressive effects of NAC on the cytokine expressions among different timing of NAC treatment, cells were treated with 0.5 or 5mM NAC before, simultaneously, or after MeHg administration. MeHg exposure at 4 μM, a non-cytotoxic concentration, significantly induced MCP-1 and IL-6 expressions at both mRNA and protein levels. A significant increase of H2O2 production but not O2(-) was observed. MeHg-induced expression of MCP-1 and IL-6 mRNA was reduced by 10-20% in the presence of 5mM NAC (co-treatment experiment) compared to cells treated with MeHg only. Pre-treatment of cells with 0.5 or 5mM NAC at 0.5 or 1h and its subsequent washout before MeHg addition suppressed MCP-1 and IL-6 cytokine expressions. Post-treatment of cells with NAC after MeHg addition also suppressed the cytokine induction, but the magnitude of suppression was evidently lower than in co-treated cells even though the H2O2 generation was almost completely suppressed by NAC. NAC may effectively suppress the MeHg-induced cytokine production through both, inhibition of reactive oxygen species as well as extracellular chelation of MeHg in astrocytes.

The ubiquitin proteasome system plays a role in venezuelan equine encephalitis virus infection.

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.

Neurotrophic Activity of Cultured Cell Line U87 is Up-Regulated by Proline-Rich Polypeptide Complex and Its Constituent Nonapeptide.

Neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor, as well as cytokines, for example, interleukin-6 (IL-6) play an important role in neuroprotection and in the control of the central nervous system (CNS) function. Reduced expression of neurotrophic factors can lead to dysregulation of neuron function and neuronal death. There is also evidence for mutual interactions between neurotrophins and IL-6. Therefore, the up-regulating the level of neuroprotective substances is one of the key manners to control the nervous system development and function. It can be a promising aim in the therapy of neurodegenerative disease in which the decreased level of neurotrophins is observed. In our recent studies, the role of proline-rich polypeptide complex (PRP) and its nonapeptide fragment (NP) in the regulation of neurotrophic activity in cultured astrocytes was shown. PRP and NP stimulate human astrocytoma cell line U87 to release the significant amounts of NGF to the extracellular space both in its precursor and mature form. We also provide the evidence that in NP-treated cells, the level of βNGF mRNA was increased. NP-treated cells used in this study produced also increasing amounts of IL-6. This finding indicates that PRP and its nonapeptide fragment NP up-regulate neurotrophic activity of U87 cell line by increase of NGF synthesis and its release into the extracellular space. It was also shown that NP-dependent increased production of IL-6 can enhance the NGF activity.

HOTAIR is a therapeutic target in glioblastoma.

HOTAIR is a negative prognostic factor and is overexpressed in multiple human cancers including glioblastoma multiform (GBM). Survival analysis of Chinese Glioma Genome Atlas (CGGA) patient data indicated that high HOTAIR expression was associated with poor outcome in GBM patients. NLK (Nemo-like kinase), a negative regulator of the β-catenin pathway, was negatively correlated with HOTAIR expression. When the β-catenin pathway was inhibited, GBM cells became susceptible to cell cycle arrest and inhibition of invasion. Introduction of the HOTAIR 5' domain in human glioma-derived astrocytoma induced β-catenin. An intracranial animal model was used to confirm that HOTAIR depletion inhibited GBM cell migration/invasion. In the orthotopic model, HOTAIR was required for GBM formation in vivo. In summary, HOTAIR is a potential therapeutic target in GBM.

MicroRNA-144 suppresses tumorigenesis and tumor progression of astrocytoma by targeting EZH2.

Our previous study demonstrated that enhancer of zeste homolog 2 (EZH2) overexpression may be associated with aggressive tumor progression and poor prognosis in human astrocytoma. The aim of this study was to investigate the underlying mechanisms of EZH2 on astrocytoma tumorigenesis. An online program miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was used to predict possible microRNAs (miRNAs) that might target EZH2 messenger RNA (mRNA). Then the functions of the miRNA-EZH2 mRNA axis in astrocytoma cell proliferation, invasion, and migration were also assessed. We further evaluated the clinical value of the miRNA-EZH2 mRNA axis in astrocytomas. As a result, we identified EZH2 as a target gene of miR-144. In addition, forced expression of miR-144 suppressed astrocytoma cell proliferation, invasion, and migration by down-regulating EZH2. Moreover, miR-144 down-regulation and EZH2 mRNA up-regulation were both significantly associated with advanced World Health Organization grades and low Karnofsky performance status score of astrocytoma patients. Importantly, survival analysis identified the combined expression of miR-144 and EZH2 (miR-144/EZH2) as an independent prognostic factor for overall survival in astrocytoma patients. In conclusion, miR-144 may function as a tumor suppressor by regulating EZH2 expression, and miR-144/EZH2 expression may be a highly sensitive marker for the prognosis in astrocytoma patients.

Expression of CD150 in tumors of the central nervous system: identification of a novel isoform.

CD150 (IPO3/SLAM) belongs to the SLAM family of receptors and serves as a major entry receptor for measles virus. CD150 is expressed on normal and malignant cells of the immune system. However, little is known about its expression outside the hematopoietic system, especially tumors of the central nervous system (CNS). Although CD150 was not found in different regions of normal brain tissues, our immunohistochemical study revealed its expression in 77.6% of human CNS tumors, including glioblastoma, anaplastic astrocytoma, diffuse astrocytoma, ependymoma, and others. CD150 was detected in the cytoplasm, but not on the cell surface of glioma cell lines, and it was colocalized with the endoplasmic reticulum and Golgi complex markers. In addition to the full length mRNA of the mCD150 splice isoform, in glioma cells we found a highly expressed novel CD150 transcript (nCD150), containing an 83 bp insert. The insert is derived from a previously unrecognized exon designated Cyt-new, which is located 510 bp downstream of the transmembrane region exon, and is a specific feature of primate SLAMF1. Both mCD150 and nCD150 cDNA variants did not contain any mutations and had the leader sequence. The nCD150 transcript was also detected in normal and malignant B lymphocytes, primary T cells, dendritic cells and macrophages; however, in glioma cells nCD150 was found to be the predominant CD150 isoform. Similarly to mCD150, cell surface expression of nCD150 allows wild type measles virus entry to the cell. Our data indicate that CD150 expression in CNS tumors can be considered a new diagnostic marker and potential target for novel therapeutic approaches.

Direct immunomodulatory influence of IFN-β on human astrocytoma cells

Astrocytes actively play a pivotal role in inflammatory disease intensity of central nervous system especially multiple sclerosis (MS). Although IFN-β is a selective therapy for MS but the role of IFN-β in stimulating the astrocytes to produce cytokines is not clearly revealed. Therefore, it is encouraging to assess the modulatory role of IFN-β on astrocytes of brain tissue. The aim of our study was to analyze the molecular mechanisms of recombinant IFN-β 1a directly affecting IL-10, iNOS, MMP-9 and TIMP-1 expression in central nervous system for the first time. In this way, in vitro procedures conducted by human astrocytoma A172 and 1321N1 cell lines as a model system. The total RNA from A172 and 1321N1 cells treated with IFN-β and LPS/IFN-γ/IFN-β and untreated cells were extracted and evaluated for IL-10, iNOS, MMP-9 and TIMP-1 expression by real-time RT-PCR. We found a significant dose-dependent increase in IL-10 gene expression in A172 and 1321N1 cells treated with IFN-β or LPS/IFN-γ/IFN-β. Moreover, a significant decrease was observed in iNOS expression suggesting a similar mechanism of action for both cells. Eventually there were no significant changes concerning the modulation of the MMP-9 and TIMP-1 in response to IFN-β treatment. In part, the immunomodulatory effect of IFN-β may be due to increase of IL-10 and suppression of iNOS expression in astrocytes of brain tissue.

NADP+-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification

An NADP+-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33µM, kcat 405min−1), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic–δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

Effect of new hybrids based on 5,16-pregnadiene scaffold linked to an anti-inflammatory drug on the growth of a human astrocytoma cell line (U373)

In spite of the fact that anaplastic astrocytoma is an uncommon disease, very often the pathology of this disease is associated with lethal effects due to the late diagnosis and unspecific treatments. This paper reports the synthesis and the biological effect on the growth of U373 cell line (human anaplastic astrocytoma) of new hybrid compounds based on 5,16-pregnadiene scaffold linked to an anti-inflammatory drug (6a–e). Moreover, we also determined the cell growth effect of five non-steroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, indomethacin and sulindac) as well as the free steroidal alcohol 5. The results from this study indicated that sulindac as well as compound 5 decreased the number of U373 cells at different concentrations. However, when an anti-inflammatory drug was bound to the steroidal structure (5), the resulting compounds (6a–e) showed an enhanced biological effect with exception of hybrid 6c. Furthermore, derivative 6e (sulindac hybrid) did not allow cell growth during six days of experiment at a concentration of 10 μM. The overall data indicated that these molecules showed an anti-proliferative activity on anaplastic astrocytoma cell line.

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-β (IFN-β) 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.

Inhibition of KIF14 Suppresses Tumor Cell Growth and Promotes Apoptosis in Human Glioblastoma

Background/Aims: The mitotic kinesin superfamily protein KIF14 is essential for cytokinesis and chromosome segregation, and increased KIF14 expression is related to a variety of human cancers. However, the role of KIF14 in the development and malignant progression of astrocytomas and the underlying mechanisms remain unclear. The present study examined the relation between KIF14 and the pathogenesis of malignant astrocytoma. Methods and Results: The role of KIF14 in astrocytoma development and progression was investigated by analyzing KIF14 expression using SYBR Green quantitative real-time RT-PCR, western blotting and immunohistochemistry in human astrocytoma and normal brain tissues. KIF14 expression was higher in astrocytoma samples, and was positively correlated with pathological grade and proliferative activity indicated by Ki-67 staining. SiRNA knockdown of KIF14 inhibited tumor growth in vitro and in vivo, attenuated anchorage-independent growth, and induced G2/M phase arrest, cytokinesis failure and apoptosis in glioblastoma cell lines in association with decreased AKT phosphorylation and activity. Conclusions: The upregulation of KIF14 in astrocytoma is associated with disease severity, and suppression of KIF14 inhibits cell proliferation and induces apoptosis through a mechanism involving the inactivation of AKT signaling, suggesting that KIF14 plays an important role in astrocytoma tumorigenesis and could be a promising molecular target for anticancer therapy.

Identification of Stably Expressed lncRNAs as Valid Endogenous Controls for Profiling of Human Glioma

Background: Recent research indicates that long non-coding RNAs (lncRNA) represent a new family of RNAs that is of fundamental importance for controlling transcription and translation. Thereby, there is increasing evidence that lncRNAs are also important in tumourigenesis. Thereby valid expression profiling using quantitative PCR requires suitable, stably expressed normalisers to achieve reliable and reproducible data. However, no systematic analysis of suitable references in lncRNA studies in human glioma has been performed yet.Methods: In this study, we investigated 90 lncRNAs in 30 tissue specimen for the expression stability in human diffuse astrocytoma (WHO-Grade II), anaplastic astrocytoma (WHO-Grade III) and glioblastoma (WHO-Grade IV) both alone as well as in comparison with normal white matter. Our identification procedure included a rigorous bioinformatical selection process that resulted in the inclusion of only highly abundant, equally expressed lncRNAs for further analysis. Additionally, lncRNAs were classified according to their stability value using the NormFinder algorithm.Results: We identified 24 appropriate normalisers suitable for studies in diffuse astrocytoma, 22 for studies in anaplastic astrocytoma and 12 for studies in glioblastoma. Comparing all three glioma entities 7 lncRNAs showed stable expression levels. Addition of normal brain tissue resulted in only 4 suitable lncRNAs.Conclusions: Our findings indicate that 4 lncRNAs (HOXA6as, H19 upstream conserved 1 and 2, Zfhx2as and BC200) are suitable as normalisers in glioma and normal brain. These lncRNAs may thus be regarded as universal references being applicable for the accurate normalisation of lncRNA expression profiling in various glioma (WHO-Grades II-IV) alone and in combination with brain tissue. This enables to perform valid longitudinal studies, e.g. of glioma before and after malignisation to identify changes of lncRNA expressions probably driving malignant transformation.