Abstract
Hydrogen peroxide (H2O2) induces the hypertrophy in cultured H9c2 cardiomyocytes and cell death in glutathione (GSH)-depleted H9c2 cells. In the present study, we observed that pretreatment with a serine protease inhibitor, N-a-tosyl-L-lysine chloromethyl ketone (TLCK), significantly prevented the H2O2-induced cell damages in GSH-depleted H9c2 cells in a concentration-dependent manner. The phase contrast microscopy revealed that although the exposure of the GSH-depleted H9c2 cells to H2O2 resulted in a globular shape of the cells, TLCK prevented the occurrence of H2O2-induced morphological changes. TLCK also inhibited the generation of reactive oxygen species in the cells after addition of H2O2, suggesting that the antioxidant action of TLCK is involved in the protection against the cell damages by H2O2. Application of TLCK after ~30 min of exposure to H2O2 could significantly protect the cells from cell damages. The other serine protease inhibitors that were tested could not prevent the cell damages in GSH- depleted H9c2 cells. Pretreatment with an inhibitor of nuclear factor-kB translocation into the nucleus and a proteasome inhibitor did not prevent the cell damages in GSH-depleted H9c2 cells. An inhibitor of p53 significantly prevented the cell damages in GSH-depleted H9c2 cells. These results suggest that antioxidative action of TLCK prevents the death of GSH-depleted H9c2 cardiomyocytes induced by H2O2.
Highlights
Studies on the reactive oxygen species (ROS), such as superoxide, H2O2, and the hydroxyl radical, have indicated their involvement in pathogenesis of several diseases
Regarding the dose-related cytotoxic effect of H2O2, an increase in the concentration of H2O2 resulted in an increase in the damages of the cells; tosyl-L-lysine chloromethyl ketone (TLCK) significantly prevented H2O2-induced cell damages caused by ~250 μM H2O2 (Figure 1(b))
TLCK, which is known as an inhibitor of serine proteases, significantly protected GSHdepleted H9c2 cardiomyocytes from oxidative damages (Figure 1) and morphological changes (Figure 2) induced by H2O2 up to 6 h of test, but did not from cell death for 24 h, suggesting that TLCK prevented the oxidative damages and delayed the induction of cell death
Summary
Studies on the reactive oxygen species (ROS), such as superoxide, H2O2, and the hydroxyl radical, have indicated their involvement in pathogenesis of several diseases. In GSH-depleted H9c2 cells, hypertrophy did not occur after treatment with H2O2 at any concentrations, but the viability of the cells decreased markedly in comparison with that of normal GSH-containing cells [4]. These findings suggest that the GSH-depleted cells are vulnerable to oxidative stress. It has been reported that GSH functions as an important cellular defense system against oxidative stress and that GSH participates in the detoxification reactions of free radicals and peroxides, either directly or indirectly as a coenzyme of antioxidant enzymes, such as glutathione peroxidases [5,6]
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