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.

Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson's disease

“…Aminochrome also induces protein degradation dysfunction by: i) impairing the proteasomal system in different in vitro models (Xiong et al, 2014; Zafar et al, 2006; Zhou and Lim, 2009); ii) inhibiting autophagy by preventing the adequate formation of microtubules required for the fusion of autophagic vacuoles and lysosomes in rat and human cell lines (Huenchuguala et al, 2014; Muñoz et al, 2012a; Paris et al, 2010); iii) inducing lysosomal dysfunction in human cell lines by affecting lysosomal acidification required for protein degradation inside these organelles (Huenchuguala et al, 2014). Aminochrome can induce oxidative stress when it is one-electron reduced by flavoenzymes to leukoaminochrome- o -semiquinone radical, which is extremely reactive with oxygen and auto-oxidizes immediately to aminochrome generating a redox cycling between aminochrome and leukoaminochrome- o -semiquinone radical, depleting both NADH or NADPH and oxygen, the latter being reduced to superoxide radical with consequent formation of hydroxyl radical, as demonstrated in rat cell lines experiments (Arriagada et al, 2004).…”

“…Interestingly, the products of DA oxidation (DA- o -quinone, aminochrome and 5,6-indolequinone) are directly involved in mitochondria dysfunction, protein degradation alteration, α-synuclein aggregation to neurotoxic oligomers and oxidative stress in dopaminergic neurons (Fig. 1, 2), suggesting that these o -quinones can play an important role in the degenerative process, resulting in the loss of SN dopaminergic neurons containing NM (Aguirre et al, 2012; Arriagada et al, 2004; Bisaglia et al, 2007; Dibenedetto et al, 2013; Hauser and Hastings, 2013; Huenchuguala et al, 2014; LaVoie et al, 2005; Martinez-Vicente et al, 2008; Muñoz et al, 2012a, 2015; Norris et al, 2005; Paris et al, 2010; Van Laar et al, 2009; Whitehead et al, 2001; Xiong et al, 2014; Xu et al, 1998; Zafar et al, 2006; Zhou and Lim, 2009). …”

Silica nanoparticles induce autophagy dysfunction via lysosomal impairment and inhibition of autophagosome degradation in hepatocytes

“…17,30 For instance, lysosome damage induced by aminochrome resulted in autophagy disorder, 31 and HBV X protein could inhibit autophagic degradation through destroying the nature of lysosomes. 32 The current investigations on SiNPs and autophagy are mostly focused on autophagy detection; however, the mechanisms of autophagosome accumulation and whether the autophagic flux was blocked remain unclear.…”

Molecular and Neurochemical Mechanisms Dopamine Oxidation To O-Quinones in Parkinson’s Disease Pathogenesis

“…This conjugate was found to be stable under oxidative conditions in the presence of hydrogen peroxide, superoxide radicals and oxygen [63]. The GSH conjugation of aminochrome has been proposed to be a protective reaction because it prevents aminochrome-induced cell death in an astrocyte model cell line [59].…”

“…However, aminochrome has been reported to inactivate the proteasome [108,109]. Aminochrome forms adducts with α-and β-tubulin preventing the formation of microtubules required for autophagosome fusion with lysosomes and induces lysosome dysfunction by increasing its internal pH [58,59,110]. (iv) Aminochrome induces oxidative stress via hydroxyl radical formation (Fig.…”