title: Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction.
abstract: U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A 1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A 1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Sandro
authors.initials: S
authors.lastname: Huenchuguala
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Patricia
authors.initials: P
authors.lastname: Muñoz
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Patricio
authors.initials: P
authors.lastname: Zavala
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Mónica
authors.initials: M
authors.lastname: Villa
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Carlos
authors.initials: C
authors.lastname: Cuevas
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Ulises
authors.initials: U
authors.lastname: Ahumada
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Graumann
authors.affiliation: Laboratory of Medicine; Clinical Research Center-Novum; Karolinska Institutet; Sweden; Department of Medical Biochemistry; School of Medicine; Faculty of Medical Sciences; University of Sulaimani; Ministry of Higher Education and Research; Kurdistan Regional Government; Iraq.
authors.forename: Beston F
authors.initials: BF
authors.lastname: Nore
authors.affiliation: Department of Biology and Environmental sciences; University of Valparaiso; Valparaiso, Chile.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Couve
authors.affiliation: Department of Neurochemistry; Stockholm University; Stockholm, Sweden.
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile; Department of Basic Sciences; Santo Tomas University; Viña del Mar, Chile.
authors.forename: Irmgard
authors.initials: I
authors.lastname: Paris
authors.affiliation: Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
authors.forename: Juan
authors.initials: J
authors.lastname: Segura-Aguilar
mesh_terms: D001253:Astrocytes / Q000378:metabolism*; D001343:Autophagy / Q000502:physiology*; D002460:Cell Line; D005909:Glioblastoma / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D045563:Indolequinones / Q000633:toxicity*; D008247:Lysosomes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D020011:Protective Agents / Q000378:metabolism
chemical_list: D045563:Indolequinones; D020011:Protective Agents; C091584:aminochrome 1; D005982:Glutathione Transferase; C412602:glutathione S-transferase Mu 2
keywords: Parkinson disease; aminochrome; astrocytes; autophagy; dopamine; glutathione transferase; lysosome dysfunction; siRNA