glutaminase and Glioma

Inhibiting cancer metabolism via glutaminase (GLS) is a promising strategy to disrupt tumor progression. However, the mechanism regarding GLS acetylation remains largely unknown.. Mitochondrial protein isolation and glutaminase activity assay were used to examine GLS activity. RT-qPCR, western blot, sphere-formation, ALDH activity, and tumor-initiating assays were performed to evaluate the alteration of cell stemness. Co-IP and rescuing experiments were conducted to explore the underlying mechanisms.. In this study, we demonstrated that GLS acetylation is a vital post-translational modification that inhibits GLS activity in glioma. We identified GLS as deacetylated by HDAC4, a class II deacetylase. GLS acetylation stimulated the interaction between GLS and SIRT5, thereby promoting GLS ubiquitination and inhibiting GLS activity. Furthermore, GLS overexpression suppressed the stemness of glioma cells, which was rescued by the deacetylation of GLS.. Our findings reveal a novel mechanism of GLS regulation by acetylation and ubiquitination that participate in glioma stemness.>.

Topics: Cell Line, Tumor; Glioma; Glutaminase; Histone Deacetylases; Humans; Repressor Proteins

To compare tissue levels of the regulatory enzymes related to the Krebs cycle between low, and high-grade supratentorial gliomas.. Forty patients who underwent surgery for supratentorial gliomas (19 with low-grade and 21 with high-grade gliomas) were evaluated. The regulatory enzymes directly involved in the Krebs cycle, namely pyruvate dehydrogenase, citrate synthase, ?-ketoglutarate dehydrogenase, and isocitrate dehydrogenase, and two enzymes that indirectly regulate the Krebs cycle, namely glutamate dehydrogenase and glutaminase, were quantitatively studied in tumor tissues using ELISA. The results were compared between the two groups.. The levels of all enzymes were higher in the high-grade glioma group but only pyruvate dehydrogenase, citrate synthase, and isocitrate dehydrogenase levels showed statistical significance. Moreover, all enzymes showed higher tissue levels in grade- II compared to grade-I gliomas, but only two enzymes, glutamate dehydrogenase and glutaminase, reached significantly higher levels. In the high-grade glioma group, all enzymes again showed higher tissue levels in grade-IV gliomas than in grade-III gliomas, but none showed statistical significance.. Regulatory enzymes of the Krebs cycle are increased in high-grade gliomas compared to low-grade gliomas. Glutaminolysis enzymes, namely glutamate dehydrogenase and glutaminase, which are required for resupplying the Krebs cycle, are also increased in order to meet the high energy demand in high-grade gliomas.

Topics: Citrate (si)-Synthase; Citric Acid Cycle; Glioma; Glutamate Dehydrogenase; Glutaminase; Humans; Isocitrate Dehydrogenase; Pyruvates

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

Topics: Adenosine Deaminase; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Ferroptosis; Glioma; Glutaminase; Humans; RNA-Binding Proteins; RNA, Long Noncoding; Temozolomide

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

LncRNA HOX transcript antisense intergenic RNA (HOTAIR) has been shown to play prominent roles in tumorigenesis. However, its precise molecular mechanism in glioma has not been completely elucidated. In this study, we found that HOTAIR was aberrantly up-regulated in glioma tissues and was negatively correlated with miR-126-5p expression. Next, we determined that HOTAIR promote glioma progression by sponging miR-126-5p. Subsequently, glutaminase (GLS) was confirmed to be a direct target of miR-126-5p using bioinformatics software and a luciferase reporter assay. Moreover, HOTAIR could modulate GLS expression by functioning as a competing endogenous RNA (ceRNA) for miR-126-5p. Taken together, our study clarified that the HOTAIR/miR-126/GLS pathway is involved in glioma progression and may potentially serve as a target for glioma therapy.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Disease Progression; Gene Expression Regulation, Neoplastic; Glioma; Glutaminase; Humans; Male; Mice; Mice, Nude; MicroRNAs; RNA, Long Noncoding; Up-Regulation

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

Resisting cell death, reprogrammed metabolism and immune escape are fundamental traits of hard-to-treat cancers. Therapeutic improvement can be expected by designing drugs targeting all three aspects. 5'-Triphosphate RNA (ppp-RNA), a specific ligand of the pattern recognition receptor retinoic acid-inducible gene I (RIG-I), has been shown to trigger intrinsic apoptosis of malignant cells and to activate antitumor immune responses via type I interferons (IFNs). In our study, we designed a ppp-modified siRNA specifically silencing glutaminase (ppp-GLS), a key enzyme of glutaminolysis that is indispensable for many cancer types. Bifunctional ppp-GLS induced more prominent antitumor responses than RNA molecules that contained either the RIG-I ligand motif or GLS silencing capability alone. The cytopathic effect was constrained to tumor cells as nonmalignant cells were not affected. We then analyzed the mechanisms leading to the profound antitumor efficacy. First, ppp-GLS effectively induced intrinsic proapoptotic signaling. In addition, GLS silencing sensitized malignant cells to RIG-I-induced apoptosis. Moreover, disturbed glutaminolysis by GLS silencing contributed to enhanced cytotoxicity. Finally, RIG-I activation blocked autophagic degradation leading to dysfunctional mitochondria and reactive oxygen species (ROS) generation, whereas GLS silencing severely impaired ROS scavenging systems, leading to a vicious circle of ROS-mediated cytotoxicity. Taken together, ppp-GLS combines cell death induction, immune activation and glutaminase inhibition in a single molecule and has high therapeutic efficacy against cancer cells.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; DEAD Box Protein 58; DEAD-box RNA Helicases; Enzyme Activation; Female; Glioma; Glutaminase; HeLa Cells; Humans; Lung Neoplasms; Mitochondria; Neoplasms; Pancreatic Neoplasms; Reactive Oxygen Species; Receptors, Immunologic; RNA Interference; RNA, Small Interfering; Uterine Cervical Neoplasms

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

Mutation at the R132 residue of isocitrate dehydrogenase 1 (IDH1), frequently found in gliomas and acute myelogenous leukemia, creates a neoenzyme that produces 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). We sought to therapeutically exploit this neoreaction in mutant IDH1 cells that require α-KG derived from glutamine. Glutamine is converted to glutamate by glutaminase and further metabolized to α-KG. Therefore, we inhibited glutaminase with siRNA or the small molecule inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and found slowed growth of glioblastoma cells expressing mutant IDH1 compared with those expressing wild-type IDH1. Growth suppression of mutant IDH1 cells by BPTES was rescued by adding exogenous α-KG. BPTES inhibited glutaminase activity, lowered glutamate and α-KG levels, and increased glycolytic intermediates while leaving total 2-HG levels unaffected. The ability to selectively slow growth in cells with IDH1 mutations by inhibiting glutaminase suggests a unique reprogramming of intermediary metabolism and a potential therapeutic strategy.

Topics: Blotting, Western; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Glioma; Glutamic Acid; Glutaminase; Glutarates; Humans; Isocitrate Dehydrogenase; Ketoglutaric Acids; Mutation; RNA Interference; Sulfides; Thiadiazoles; Time Factors

Liver-type glutaminase (LGA) is a glutaminase isoform that has been implicated in transcription modulation. LGA mRNA is absent from postoperative samples of primary gliomas and is low in cultured astrocytes. In this study, stable transfection of T98G cells with a vector carrying human LGA sequence increased the expression of LGA mRNA and protein, and the ability of the cells to degrade glutamine (Gln), as manifested by a three-fold reduction of their steady-state Gln content and a 2.5-fold increase of their glutamate (Glu) content. The transfected cells (TLGA cells) showed a 40% decrease of cell survival as assessed by colony formation, well correlated with significant reduction of mitochondrial activity as demonstrated with MTT test. Also, a 45% reduction of cell migration and a 47% decrease of proliferation index (Ki67 immunostaining) were found as compared with sham-transfected cells. Microarray analysis, which included over 47,000 transcripts, revealed a significantly altered expression of 85 genes in TLGA, but not in sham-transfected or control cells (P < 0.005). Microarray data were confirmed with real-time PCR analysis for eight genes potentially relevant to malignancy: S100A16, CAPN2, FNDC3B, DYNC1LI1, TIMP4, MGMT, ADM, and TIMP1. Of these changes, decreased expression of S100A16 and MGMT can be best reconciled with the current views on the role of their protein products in glioma malignancy. Malignancy-reducing effect of newly inserted LGA mRNA in glioblastoma cells can be reconciled with a hypothesis that absence of such a modulatory mechanism in glia-derived tumors deprived of LGA mRNA may facilitate some aspects of their progression.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; DNA, Complementary; Gene Expression Regulation, Neoplastic; Glioma; Glutamic Acid; Glutaminase; Glutamine; Humans; Isoenzymes; Microarray Analysis; Mitochondria; Polymerase Chain Reaction; RNA, Messenger; Transfection

In the central nervous system (CNS), liver-type glutaminase (LGA) shows a unique nuclear localization suggesting its role in the regulation of transcription rather than in the cellular glutamine metabolism. RT-PCR analysis of RNA derived from postoperative tissue samples revealed the absence or only traces of LGA mRNA in all (9) cases of malignant gliomas (astrocytoma anaplasticum, AA, WHO grade III; glioblastoma multiforme, WHO grade IV) examined. The RNA was strongly expressed in the non-neoplastic tissue derived from the same patients (6 cases), and in most of the brain metastases from different organs (5 out of 7 cases). By contrast, the mRNAs coding for the kidney-type glutaminase (KGA) and its less ubiquitous isoform GAC, which catalyze degradation of the cytoplasmic pool of Gln, were expressed in all the tissues examined. The lack of LGA may be thus considered as a useful negative diagnostic marker of highly malignant gliomas in situ.

Topics: Blotting, Northern; Blotting, Southern; DNA, Complementary; Glioma; Glutaminase; Humans; Liver; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric

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