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Autophagy. 2014 Apr;10(4):618-30. doi: 10.4161/auto.27720. Epub 2014 Jan 14.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction.

Author information

1
Molecular and Clinical Pharmacology; ICBM-Instituto de Ciencias Biomédicas; Faculty of Medicine; University of Chile; Santiago, Chile.
2
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.
3
Department of Biology and Environmental sciences; University of Valparaiso; Valparaiso, Chile.
4
Department of Neurochemistry; Stockholm University; Stockholm, Sweden.
5
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.

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.

KEYWORDS:

Parkinson disease; aminochrome; astrocytes; autophagy; dopamine; glutathione transferase; lysosome dysfunction; siRNA

PMID:
24434817
PMCID:
PMC4091149
DOI:
10.4161/auto.27720
[Indexed for MEDLINE]
Free PMC Article

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