Abstract
Purpose: :
Glutaredoxin 2 (Grx2) is an isozyme of thioltransferase (TTase or Grx1) present in the mitochondria but its function is not well understood. The purpose of this study was to evaluate the anti-apoptotic function, and to examine if protecting complex I enzyme in the mitochondrial electron transport system is part of the anti-apoptotic mechanism.
Methods: :
Human lens epithelial B3 cells (HLE-B3) were transfected with either a Grx2-containing plasmid or an empty vector (control). Normal HLE-B3 cells and transfected cells were incubated in MEM with or without 200 µM H2O2 for 24 h. Grx2 activity was assayed by spectrophotometric method in the mitochondrial fraction. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. Protein expression levels for Bax, Bcl-2, cytochrome C and caspase 3 activation were determined by Western Blot. The enzymatic activity of complex I was assayed by spectrophotometric method. The protective effect of Grx2 was further examined using siRNA to knock down Grx2 in HLE-B3 cells.
Results: :
Grx2 activity in transfected cells was significantly increased as compared to normal cells. Grx2 overexpression protected HLE-B3 cells from H2O2-induced cell viability loss and apoptosis. These cells also exhibited high level of anti-apoptotic protein Bcl-2 and inhibited the classical apoptotic changes in cytochrome C release and caspase 3 activation induced by H2O2. In addition, upregulation of Grx2 also protected mitochondria from H2O2-induced loss in complex I activity. In contrast, silencing of Grx2 expression using short-interference RNA in HLE-B3 cells enhanced H2O2-induced cellular damage.
Conclusions: :
Grx2 can protect human lens epithelial cells against H2O2 induced apoptosis. The mechanism of this protection is likely associated with its ability to protect complex I in the electron transport chain and preserve the mitochondrial membrane integrity.
Keywords: enzymes/enzyme inhibitors • apoptosis/cell death • oxidation/oxidative or free radical damage