glutaminase and Brain-Neoplasms

Glutamine (Gln) and glutamate (Glu) play pivotal roles in the malignant phenotype of brain tumors via multiple mechanisms. Glutaminase (GA, EC 3.5.1.2) metabolizes Gln to Glu and ammonia. Human GA isoforms are encoded by two genes: GLS gene codes for kidney-type isoforms, KGA and GAC, whereas GLS2 codes for liver-type isoforms, GAB and LGA. The expression pattern of both genes in different neoplastic cell lines and tissues implicated that the kidney-type isoforms are associated with cell proliferation, while the liver-type isoforms dominate in, and contribute to the phenotype of quiescent cells. GLS gene has been demonstrated to be regulated by oncogene c-Myc, whereas GLS2 gene was identified as a target gene of p53 tumor suppressor. In glioblastomas (GBM, WHO grade IV), the most aggressive brain tumors, high levels of GLS and only traces or lack of GLS2 transcripts were found. Ectopic overexpression of GLS2 in human glioblastoma T98G cells decreased their proliferation and migration and sensitized them to the alkylating agents often used in the chemotherapy of gliomas. GLS silencing reduced proliferation of glioblastoma T98G cells and strengthen the antiproliferative effect evoked by previous GLS2 overexpression.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Glutaminase; Humans; Isoenzymes; Phenotype

Glutaminase is expressed in most mammalian tissues and cancer cells, but recent studies are now revealing a considerably degree of complexity in its pattern of expression and functional regulation. Novel transcript variants of the mammalian glutaminase Gls2 gene have been recently found and characterized in brain. Co-expression of different isoforms in the same cell type would allow cells to fine-tune their Gln/Glu levels under a wide range of metabolic states. Moreover, the discovery of protein interacting partners and novel subcellular localizations, for example nucleocytoplasmic in neurons and astrocytes, strongly suggest non-neurotransmission roles for Gls2 isoforms associated with transcriptional regulation and cellular differentiation. Of note, Gls isoforms have been considered as an important trophic factor for neuronal differentiation and postnatal development of brain regions. On the other hand, glutaminases are taking center stage in tumor biology as new therapeutic targets to inhibit metabolic reprogramming of cancer cells. Interestingly, glutaminase isoenzymes play seemingly opposing roles in cancer cell growth and proliferation; this issue will be also succinctly discussed with special emphasis on brain tumors.

Topics: Animals; Astrocytes; Brain; Brain Neoplasms; Glutaminase; Humans; Isoenzymes; Neurons

Cellular energy metabolism is one of the main processes affected during the transition from normal to cancer cells, and it is a crucial determinant of cell proliferation or cell death. As a support for rapid proliferation, cancer cells choose to use glycolysis even in the presence of oxygen (Warburg effect) to fuel macromolecules for the synthesis of nucleotides, fatty acids, and amino acids for the accelerated mitosis, rather than fuel the tricarboxylic acid cycle and oxidative phosphorylation. Mitochondria biogenesis is also reprogrammed in cancer cells, and the destiny of those cells is determined by the balance between energy and macromolecule supplies, and the efficiency of buffering of the cumulative radical oxygen species. In glioblastoma, the most frequent and malignant adult brain tumor, a metabolic shift toward aerobic glycolysis is observed, with regulation by well known genes as integrants of oncogenic pathways such as phosphoinositide 3-kinase/protein kinase, MYC, and hypoxia regulated gene as hypoxia induced factor 1. The expression profile of a set of genes coding for glycolysis and the tricarboxylic acid cycle in glioblastoma cases confirms this metabolic switch. An understanding of how the main metabolic pathways are modified by cancer cells and the interactions between oncogenes and tumor suppressor genes with these pathways may enlighten new strategies in cancer therapy. In the present review, the main metabolic pathways are compared in normal and cancer cells, and key regulations by the main oncogenes and tumor suppressor genes are discussed. Potential therapeutic targets of the cancer energetic metabolism are enumerated, highlighting the astrocytomas, the most common brain cancer.

Topics: Brain Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Citric Acid Cycle; Glutaminase; Glutamine; Glycolysis; Humans; Oncogenes; Pentose Phosphate Pathway; Stem Cells

Circular RNA CREB-binding protein (circ-CREBBP) has been reported to involve in the tumorigenesis of glioma. However, the role and underlying molecular mechanism of circ-CREBBP in glioma glutamine catabolism remain unclear. The expression of circ-CREBBP, microRNA (miR)-375 and glutaminase (GLS) was detected using quantitative real-time polymerase chain reaction and western blot. The 3‑(4, 5‑dimethylthiazol‑2‑y1)‑2, 5‑diphenyl tetrazolium bromide (MTT), colony formation, flow cytometry and transwell assays were used to determine the effects of them on glioma cell malignant biological behaviors in vitro. Glutamine metabolism was analyzed using assay kits. Murine xenograft model was established to investigate the role of circ-CREBBP in vivo. The binding interactions between miR-375 and circ-CREBBP or GLS were confirmed by the dual-luciferase reporter assay. Circ-CREBBP was highly expressed in glioma tissues and cells, and high expression of circ-CREBBP predicted poor prognosis. Circ-CREBBP knockdown suppressed cell proliferation, migration, invasion and glutamine metabolism while expedited cell apoptosis in glioma in vitro, as well as impeded tumor growth in vivo. Circ-CREBBP directly targeted miR-375, which was demonstrated to restrain glioma cell growth, motility and glutamine metabolism. Moreover, miR-375 inhibition reverted the anticancer effects of circ-CREBBP knockdown on glioma cells. GLS was a target of miR-375, GLS silencing or the treatment of GLS inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) impaired glioma cell malignant phenotypes and glutamine metabolism. Importantly, GLS up-regulation weakened the tumor-suppressive functions of miR-375 on glioma cells. Mechanistically, circ-CREBBP indirectly regulated GLS expression through sponging miR-375. In all, circ-CREBBP expedited glioma tumorigenesis and glutamine metabolism through miR-375/GLS axis, suggesting a promising target for combined glioma therapy.

Topics: Brain Neoplasms; Carcinogenesis; Cell Proliferation; CREB-Binding Protein; Female; Gene Expression Regulation, Neoplastic; Glioma; Glutaminase; Glutamine; Humans; Male; MicroRNAs; Middle Aged; RNA, Circular; Survival Rate

Glioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system in adults. The discovery of novel anti-GBM agents based on the isocitrate dehydrogenase (IDH) mutant phenotypes and classifications have attracted comprehensive attention.. Diterpenoids are a class of naturally occurring 20-carbon isoprenoid compounds, and have previously been shown to possess high cytotoxicity for a variety of human tumours in many scientific reports. In the present study, 31 cassane diterpenoids of four types, namely, butanolide lactone cassane diterpenoids (I) (1-10), tricyclic cassane diterpenoids (II) (11-15), polyoxybutanolide lactone cassane diterpenoids (III) (16-23), and fused furan ring cassane diterpenoids (IV) (24-31), were tested for their anti-glioblastoma activity and mechanism underlying based on IDH1 mutant phenotypes of primary GBM cell cultures and human oligodendroglioma (HOG) cell lines.. We confirmed that tricyclic-type (II) and compound 13 (Caesalpin A, CSA) showed the best anti-neoplastic potencies in IDH1 mutant glioma cells compared with the other types and compounds. Furthermore, the structure-relationship analysis indicated that the carbonyl group at C-12 and an α, β-unsaturated ketone unit fundamentally contributed to enhancing the anti-glioma activity. Studies investigating the mechanism demonstrated that CSA induced oxidative stress via causing glutathione reduction and NOS activation by negatively regulating glutaminase (GLS), which proved to be highly dependent on IDH mutant type glioblastoma. Finally, GLS overexpression reversed the CSA-induced anti-glioma effects in vitro and in vivo, which indicated that the reduction of GLS contributed to the CSA-induced proliferation inhibition and apoptosis in HOG-IDH1-mu cells.. Therefore, the present results demonstrated that compared with other diterpenoids, tricyclic-type diterpenoids could be a targeted drug candidate for the treatment of secondary IDH1 mutant type glioblastoma through negatively regulating GLS.

Topics: Apoptosis; Brain Neoplasms; Cell Line, Tumor; Diterpenes; Glioblastoma; Glutaminase; Humans; Isocitrate Dehydrogenase; Mutation; Oxidative Stress

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. SIGNIFICANCE: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.

Topics: Amino Acids; Astrocytes; Benzeneacetamides; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Citric Acid Cycle; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Glioblastoma; Glutamic Acid; Glutaminase; Humans; Neoplastic Stem Cells; Signal Transduction; Thiadiazoles

Topics: Adolescent; Biomarkers, Tumor; Brain Neoplasms; Child; China; Computational Biology; Databases, Genetic; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Glioblastoma; Glutaminase; Humans; Male; NADPH Oxidases; Prognosis; Protein Interaction Maps; Transcriptome

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1

Topics: 4-Aminobutyrate Transaminase; Animals; Brain Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Glutamate Dehydrogenase; Glutamic Acid; Glutaminase; Humans; Isocitrate Dehydrogenase; Lactic Acid; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Neoplasm Invasiveness; Stress, Physiological; Succinate-Semialdehyde Dehydrogenase; Transcriptome; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Phosphate-activated glutaminase (GA), a ubiquitous glutamine-metabolizing enzyme, is encoded by two genes, GLS and GLS2. In mammalian cancers, GLS isoforms are perceived as molecules promoting cell proliferation and invasion, whereas the role of GLS2 isoforms seems to be more complex and cell type-specific. Previous studies have shown abundance of GLS and lack of GLS2 transcripts in T98G human glioblastoma (GBM) cell line and patient-derived GBM. Transfection with GAB sequence, the whole GLS2 cDNA transcript, suppressed malignant phenotype of T98G cells. Microarray analysis revealed upregulation of GATA3, the product of which has been implicated in suppressing growth of some peripheral cancers. In this study we confirmed a significant upregulation of GATA3 expression in the transfected cells both at mRNA and protein level. Considerable expression of GATA3 was also observed in GBM tissues (previously shown as not expressing GLS2), while only traces or no GATA3 was detected in (GLS2-expressing) non-tumorigenic brain samples. In conclusion, while mechanistic relation between GAB and GATA3 expression is evident following in vitro manipulation of GBM cell line, it does not appear to be an intrinsic property of GBM nor non-tumorigenic brain tissue.

Topics: Adult; Aged; Brain; Brain Neoplasms; Cell Line, Tumor; Female; GATA3 Transcription Factor; Glioblastoma; Glutaminase; Humans; Male; Middle Aged

Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is now a widely-used modality for glioblastoma (GBM) treatment. However, intratumoral heterogeneity of fluorescence intensity may reflect different onco-metabolic programs. Here, we investigated the metabolic mechanism underlying the heterogeneity of 5-ALA fluorescence in GBM. Using an in-house developed fluorescence quantification system for tumor tissues, we collected 3 types of GBM tissues on the basis of their fluorescence intensity, which was characterized as strong, weak, and none. Expression profiling by RNA-sequencing revealed 77 genes with a proportional relationship and 509 genes with an inverse relationship between gene expression and fluorescence intensity. Functional analysis and in vitro experiments confirmed glutaminase 2 (GLS2) as a key gene associated with the fluorescence heterogeneity. Subsequent metabolite profiling discovered that insufficient NADPH due to GLS2 underexpression was responsible for the delayed metabolism of 5-ALA and accumulation of protoporphyrin IX (PpIX) in the high fluorescence area. The expression level of GLS2 and related NADPH production capacity is associated with the regional heterogeneity of 5-ALA fluorescence in GBM.

Topics: Aminolevulinic Acid; Brain Neoplasms; Cell Line, Tumor; Fluorescence; Fluorescent Dyes; Gene Expression Profiling; Glioblastoma; Glutaminase; Humans; Levulinic Acids; NADP; Prospective Studies; Protoporphyrins; Surgery, Computer-Assisted

Glioblastoma is the most aggressive manifestation of malignant gliomas and considered to be among the deadliest forms of human cancers. MicroRNAs are found to tightly regulate diverse biological processes and considered to play important roles in cancer etiology. In this study, we found that microRNA-153 was significantly downregulated in glioblastoma tissues compared to matched non-tumor tissues and in glioblastoma cell lines. To investigate the potential function of microRNA-153 in glioblastoma, we transfected glioblastoma cell line U87MG as well as U373MG with synthetic microRNA-153 oligos and observed decreased cell proliferation and increased apoptosis. We further found that microRNA-153 restrained glutamine utilization and glutamate generation. Bioinformatics analysis revealed that glutaminase, which catalyzed the formation of glutamate from glutamine, is the potential target of microRNA-153. Indeed, microRNA-153 cannot further reduce glutamine utilization when glutaminase was knocked down. Overexpression of glutaminase abrogates the effect of microRNA-153 on glutamine utilization. Furthermore, the relative expression of microRNA-153 and glutaminase in glioblastoma versus matched non-tumor tissues showed a reverse correlation, further indicating that microRNA-153 may negatively regulate glutaminase in vivo. These results demonstrate an unexpected role of microRNA-153 in regulating glutamine metabolism and strengthen the role of microRNA-153 as a therapeutic target in glioblastoma.

Topics: Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Glioblastoma; Glutaminase; Glutamine; Humans; Immunohistochemistry; In Situ Hybridization; MicroRNAs

Human phosphate-activated glutaminase (GA) is encoded by two genes: GLS and GLS2. Glioblastomas (GB) usually lack GLS2 transcripts, and their reintroduction inhibits GB growth. The GLS2 gene in peripheral tumors may be i) methylation- controlled and ii) a target of tumor suppressor p53 often mutated in gliomas. Here we assessed the relation of GLS2 downregulation in GB to its methylation and TP53 status. DNA demethylation with 5-aza-2'-deoxycytidine restored GLS2 mRNA and protein content in human GB cell lines with both mutated (T98G) and wild-type (U87MG) p53 and reduced the methylation of CpG1 (promoter region island), and CpG2 (first intron island) in both cell lines. In cell lines and clinical GB samples alike, methylated CpG islands were detected both in the GLS2 promoter (as reported earlier) and in the first intron of this gene. CpG methylation of either island was absent in GLS2-expressing non-tumoros brain tissues. Screening for mutation in the exons 5-8 of TP53 revealed a point mutation in only one out of seven GB examined. In conclusion, aberrant methylation of CpG islands, appear to contribute to silencing of GLS2 in GB by a mechanism bypassing TP53 mutations. © 2015 Wiley Periodicals, Inc.

Topics: Brain; Brain Neoplasms; Cell Line, Tumor; CpG Islands; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Genes, p53; Glioblastoma; Glutaminase; Humans; Point Mutation; Promoter Regions, Genetic; Tumor Suppressor Protein p53

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

Topics: Brain Neoplasms; Cell Line, Tumor; Cyclic S-Oxides; Glioblastoma; Glutamic Acid; Glutaminase; Homeostasis; Humans; Metabolome; Receptors, Notch; Thiadiazoles

By histological, morphological criteria, and malignancy, brain tumors are classified by WHO into grades I (most benign) to IV (highly malignant), and gliomas are the most frequently occurring class throughout the grades. Similar to peripheral tumors, the growth of glia-derived tumor cells largely depends on glutamine (Gln), which is vividly taken up by the cells, using mostly ASCT2 and SN1 as Gln carriers. Tumor growth-promoting effects of Gln are associated with its phosphate-activated glutaminase (GA) (specifically KGA)-mediated degradation to glutamate (Glu) and/or with its entry to the energy- and intermediate metabolite-generating pathways related to the tricarboxylic acid cycle. However, a subclass of liver-type GA are absent in glioma cells, a circumstance which allows phenotype manipulations upon their transfection to the cells. Gln-derived Glu plays a major role in promoting tumor proliferation and invasion. Glu is relatively inefficiently recycled to Gln and readily leaves the cells by exchange with the extracellular pool of the glutathione (GSH) precursor Cys mediated by xc- transporter. This results in (a) cell invasion-fostering interaction of Glu with ionotropic Glu receptors in the surrounding tissue, (b) intracellular accumulation of GSH which increases tumor resistance to radio- and chemotherapy.

Topics: Brain Neoplasms; Cell Proliferation; Glioma; Glutamic Acid; Glutaminase; Glutamine; Humans; Neoplasm Invasiveness

Gliosarcoma cell line K308 was established from a primary tumor specimen removed from a 51-year-old male Han Chinese patient. Besides the typical characteristics of gliosarcoma cells, K308 cells express abundant glutaminase and can release large amount of glutamate. K308 exhibited cell-density-dependent expression of neuronal precursor markers, particularly nestin. At low density, the majority of K308 cells were nestin negative (approximately 70%) and nestin levels remained homogenous within each single-cell-derived colony when K308 proliferated. After reaching confluence, however, the majority of K308 cells turned nestin positive. These confluent K308 cells were also Sox2 positive and could form tumor spheres even in serum-containing media.

Topics: Animals; Biomarkers; Brain Neoplasms; Cell Count; Cell Line, Tumor; Gliosarcoma; Glutamic Acid; Glutaminase; Humans; Karyotype; Male; Mice, Inbred BALB C; Middle Aged; Nestin; Neurons; SOXB1 Transcription Factors; Xenograft Model Antitumor Assays

The mechanistic target of rapamycin (mTOR) is hyperactivated in many types of cancer, rendering it a compelling drug target; however, the impact of mTOR inhibition on metabolic reprogramming in cancer is incompletely understood. Here, by integrating metabolic and functional studies in glioblastoma multiforme (GBM) cell lines, preclinical models, and clinical samples, we demonstrate that the compensatory upregulation of glutamine metabolism promotes resistance to mTOR kinase inhibitors. Metabolomic studies in GBM cells revealed that glutaminase (GLS) and glutamate levels are elevated following mTOR kinase inhibitor treatment. Moreover, these mTOR inhibitor-dependent metabolic alterations were confirmed in a GBM xenograft model. Expression of GLS following mTOR inhibitor treatment promoted GBM survival in an α-ketoglutarate-dependent (αKG-dependent) manner. Combined genetic and/or pharmacological inhibition of mTOR kinase and GLS resulted in massive synergistic tumor cell death and growth inhibition in tumor-bearing mice. These results highlight a critical role for compensatory glutamine metabolism in promoting mTOR inhibitor resistance and suggest that rational combination therapy has the potential to suppress resistance.

Topics: Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzophenanthridines; Brain Neoplasms; Cell Line, Tumor; Citric Acid Cycle; Drug Resistance, Neoplasm; Drug Synergism; Energy Metabolism; Gas Chromatography-Mass Spectrometry; Glioblastoma; Glutamic Acid; Glutaminase; Glutamine; Glycolysis; Humans; Indoles; Ketoglutaric Acids; Magnetic Resonance Spectroscopy; Male; Metabolome; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Targeted Therapy; Neoplasm Proteins; Protein Kinase Inhibitors; Purines; RNA, Small Interfering; Rotarod Performance Test; Signal Transduction; Temporal Lobe; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Topics: Animals; Brain Neoplasms; Cell Survival; Gene Expression Regulation, Neoplastic; Genomics; Glioblastoma; Glutaminase; Homeostasis; Humans; Isoenzymes; Kidney; Liver; Mice; Phosphatidylinositol 3-Kinases; Proteomics; Signal Transduction; TOR Serine-Threonine Kinases

Mitochondrial glutaminase (GA) plays an essential role in cancer cell metabolism, contributing to biosynthesis, bioenergetics, and redox balance. Humans contain several GA isozymes encoded by the GLS and GLS2 genes, but the specific roles of each in cancer metabolism are still unclear. In this study, glioma SFxL and LN229 cells with silenced isoenzyme glutaminase KGA (encoded by GLS) showed lower survival ratios and a reduced GSH-dependent antioxidant capacity. These GLS-silenced cells also demonstrated induction of apoptosis indicated by enhanced annexin V binding capacity and caspase 3 activity. GLS silencing was associated with decreased mitochondrial membrane potential (ΔΨm) (JC-1 dye test), indicating that apoptosis was mediated by mitochondrial dysfunction. Similar observations were made in T98 glioma cells overexpressing glutaminase isoenzyme GAB, encoded by GLS2, though some characteristics (GSH/GSSG ratio) were different in the differently treated cell lines. Thus, control of GA isoenzyme expression may prove to be a key tool to alter both metabolic and oxidative stress in cancer therapy. Interestingly, reactive oxygen species (ROS) generation by treatment with oxidizing agents: arsenic trioxide or hydrogen peroxide, synergizes with either KGA silencing or GAB overexpression to suppress malignant properties of glioma cells, including the reduction of cellular motility. Of note, negative modulation of GLS isoforms or GAB overexpression evoked lower c-myc and bcl-2 expression, as well as higher pro-apoptotic bid expression. Combination of modulation of GA expression and treatment with oxidizing agents may become a therapeutic strategy for intractable cancers and provides a multi-angle evaluation system for anti-glioma pre-clinical investigations.. Silencing GLS or overexpressing GLS2 induces growth inhibition in glioma cell lines. Inhibition is synergistically enhanced after arsenic trioxide (ATO) or H2O2 treatment. Glutatione levels decrease in GLS-silenced cells but augment if GLS2 is overexpressed. ROS synergistically inhibit cell migration by GLS silencing or GLS2 overexpression. c-myc, bid, and bcl-2 mediate apoptosis resulting from GLS silencing or GLS2 overexpression.

Topics: Antioxidants; Apoptosis; Arsenic Trioxide; Arsenicals; BH3 Interacting Domain Death Agonist Protein; Brain Neoplasms; Cell Movement; Cell Proliferation; Cell Survival; Flow Cytometry; Gene Silencing; Glioma; Glutaminase; Glutathione; Humans; Mitochondria; Oxidative Stress; Oxides; Proto-Oncogene Proteins c-myc; Reactive Oxygen Species; Staining and Labeling

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

Topics: Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Glioma; Glutaminase; Glutamine; Glutarates; HEK293 Cells; Humans; Isocitrate Dehydrogenase; Ketoglutaric Acids; Metabolome; Mutation; Temozolomide

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.

Topics: Animals; Brain Neoplasms; Glioblastoma; Gluconeogenesis; Glucose; Glutamate-Ammonia Ligase; Glutamic Acid; Glutaminase; Glutamine; Glycolysis; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Neoplasm Transplantation; Neostriatum; Oxidation-Reduction; Phenotype; Pyruvate Carboxylase; Statistics, Nonparametric; Tumor Cells, Cultured

A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.

Topics: Astrocytes; Brain; Brain Neoplasms; Cell Line, Tumor; Cells, Cultured; Glutaminase; Humans; Immunohistochemistry; Mitochondria; Mitochondrial Membranes; Neuroblastoma

Topics: Ammonia; Animals; Brain Neoplasms; Glioma; Glutamates; Glutaminase; Glutamine; Mice; Neoplasms, Experimental; Neuroglia; Rabbits