title: Both GLS silencing and GLS2 overexpression synergize with oxidative stress against proliferation of glioma cells.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Molecular Biology and Biochemistry, Faculty of Sciences, Campus de Teatinos, University of Málaga, 29071, Málaga, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Martín-Rufián
authors.forename: Renata
authors.initials: R
authors.lastname: Nascimento-Gomes
authors.forename: Ana
authors.initials: A
authors.lastname: Higuero
authors.forename: Amanda R
authors.initials: AR
authors.lastname: Crisma
authors.forename: José A
authors.initials: JA
authors.lastname: Campos-Sandoval
authors.forename: María C
authors.initials: MC
authors.lastname: Gómez-García
authors.forename: Carolina
authors.initials: C
authors.lastname: Cardona
authors.forename: Tzuling
authors.initials: T
authors.lastname: Cheng
authors.forename: Carolina
authors.initials: C
authors.lastname: Lobo
authors.forename: Juan A
authors.initials: JA
authors.lastname: Segura
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Alonso
authors.forename: Monika
authors.initials: M
authors.lastname: Szeliga
authors.forename: Jan
authors.initials: J
authors.lastname: Albrecht
authors.forename: Rui
authors.initials: R
authors.lastname: Curi
authors.forename: Javier
authors.initials: J
authors.lastname: Márquez
authors.forename: Alison
authors.initials: A
authors.lastname: Colquhoun
authors.forename: Ralph J
authors.initials: RJ
authors.lastname: Deberardinis
authors.forename: José M
authors.initials: JM
authors.lastname: Matés
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D001932:Brain Neoplasms / Q000201:enzymology* / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D020868:Gene Silencing / Q000187:drug effects; D005910:Glioma / Q000201:enzymology* / Q000473:pathology*; D005972:Glutaminase / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D010087:Oxides / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D000975:Antioxidants; D001152:Arsenicals; D051023:BH3 Interacting Domain Death Agonist Protein; D010087:Oxides; D016271:Proto-Oncogene Proteins c-myc; D017382:Reactive Oxygen Species; C579480:GLS protein, human; C586504:GLS2 protein, human; D005972:Glutaminase; D005978:Glutathione; D000077237:Arsenic Trioxide