Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-buthionine (S, R) sulfoximine (BSO), TLCK, N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK), pifithrin-a, and catalase were obtained from Sigma Chemical Co., St. Louis, MO. SN50 and MG-132 were obtained from BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA. Phenylmethanesulfonyl fluoride (PMSF) and H₂O₂ were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan. 4- (2-aminoethyl)-benzensulfonyl fluoride hydrochloride (AEBSF) was obtained from Roche Diagnostics GmbH, Mannheim, Germany. Leupeptin was obtained from Peptide Institute, Inc., Osaka, Japan. 2’,7’-dichlorofluorescin diacetate (DCFH₂-DA) was purchased from CALBIOCHEM, San Diego, CA. The 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay kit was obtained from Promega Co., Madison, WI. All the other chemicals used in this study were of the highest grade available from commercial suppliers.

Rat cardiomyocyte H9c2 cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in DMEM containing 10% (v/v) heatinactivated FBS. The cells were incubated in an atmosphere of 5% CO₂ and 95% air at 37°C. The details of the standard treatment of H9c2 cells are as follows: the cells were seeded onto 96-well plates at a concentration of 1 x 10⁴ cells/well for performing cell survival assays. After overnight preincubation with 0.1 mM BSO, the cells were incubated with different concentrations of H₂O₂ for various time intervals. TLCK and TPCK were usually added to the cell suspension concurrently with BSO unless otherwise noted, and the other inhibitors were added 30 min (leupeptin, PMSF, SN50) [15] or 1 h (AEBSF, MG-132, pifithrin-a) prior to the addition of H₂O₂ [15-17]. The details regarding the intracellular GSH levels in H9c2 cells pretreated with or without BSO have been described in our previous report [4].

Cell damages were determined using an MTT assay according to the manufacturer’s instructions. Briefly, after the incubation with H₂O₂, DMEM containing 10% FBS and a dye solution were added to each of the 96 wells and the assay plates were incubated for 4 h at 37°C. A solubilization/stop solution was added for overnight incubation. The absorbances at 540 nm and 690 nm were recorded using a microplate reader (Labsystems Multiskan Bichromatic, Helsinki, Finland). Other culture conditions were same as those mentioned earlier. Cell morphology was investigated using a phase contrast microscope (Nikon Diaphoto-TMD, Tokyo, Japan).

Intracellular ROS levels were assessed using DCFH₂- DA [18,19]. Briefly, DCFH₂-DA was preincubated with cells in culture medium at a final concentration of 10 μM for 15 min. Following incubation with H₂O₂ for 15 min, the cells were trypsinized, collected and washed with PBS by centrifugation at 300 ´ g for 5 min. The cell pellets were resuspended with PBS (1 ml) and analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA). DCF fluorescence was measured by fluorescence channel 1 of the flow cytometer [20,21]. Antioxidant effect of TLCK on lipid peroxidation was examined in rat liver microsomes incubated with ascorbic acid and FeCl₃ [22]. Briefly, microsomes (0.5 mg protein/mL) prepared from male Wistar strain rats were incubated with ascorbic acid (0.5 mM) and FeCl₃ (10 μM) in 50 mM phosphate buffer, pH 7.4 at 37°C for 1 h. The production of lipid peroxidation was assessed using thiobarbituric acid reactive substance (TBARS). TLCK and TPCK were added to reaction system at the beginning of incubation. The TBARS values represented in the data was corrected by subtracting the value obtained in the reaction system without ascorbic acid and FeCl₃ from total value.

The data are expressed as mean ± SD of at least three experiments. Student’s t-test was used to analyze statistical difference. P values less than 0.05 were considered significant.

We had previously reported that H₂O₂ induces hypertrophy in H9c2 cardiomyocytes and cell death in GSHdepleted H9c2 cells. An exposure to 100 µM H₂O₂ for more than 3 h induced an increase in the damages of the GSH-depleted H9c2 cells (Figure 1(a)), as assessed by the function of MTT reduction. The damages were inhibited by the addition of antioxidants such as Trolox and butylated hydroxyanisol (data not shown). H₂O₂ (100 µM) alone did not induce the damages in the cells pretreated without BSO. These results suggest that H₂O₂ induces the oxidative damages in the GSH-depleted H9c2 cells. Pretreatment with TLCK significantly inhibited the cell damages induced by H₂O₂ up to 6 h of test (Figure 1(a)). Although data not shown, the loss of MTT reductive activity was observed in the cells treated with H₂O₂ for 24 h in the presence of TLCK, suggesting that TLCK prevented oxidative damages 6 h but dit not the cell death 24 h after the addition of H₂O₂. Therefore, we selected 6 h as the duration of H₂O₂ treatment for the GSH-depleted cells in further experiments unless otherwise stated. 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; however, TLCK significantly prevented H₂O₂-induced cell damages caused by ~250 µM H₂O₂ (Figure 1(b)). TLCK at a concentration of more than 30 µM, particularly a concentration of 100 µM, significantly prevented the cell damages induced by H₂O₂ (Figure 1(c)). On the other hand, TLCK at a concentration of 300 µM exerted a deleterious effect in a preliminary experiment (data not shown). Therefore, we selected 100 µM TLCK as the dose in further experiments. An analysis using a phase contrast microscope was performed to observe the effect of TLCK on morphological changes in the H₂O₂-treated cells. Exposure to 100 µM H₂O₂ for 6 h resulted in a globular shape of the cells and detachment of the cells from the well in the GSH-depleted H9c2 cells (Figure

2(b)) in comparison with the H₂O₂-unexposed control cells (Figure 2(a)). Moreover, the cells remained attached had shrunken nuclei and cytoplasm (Figure 2(b)). When the cells were pretreated with TLCK, none of these morphological changes was observed in the cells even after incubation with H₂O₂ for 6 h (Figure 2(c)), however, marked decrease of cell numbers was observed for 24 h. These results show that TLCK protects the GSH-depleted H9c2 cells against the H₂O₂-induced cell oxidative damages and morphological changes, but not the cell death.

TLCK was added to the GSH-depleted H9c2 cells at different time intervals before or after the addition of H₂O₂. TLCK could prevent the H₂O₂-induced cell damages when it was added to the cells at the same time as H₂O₂, 15 h before the addition of H₂O₂, or 0.5 h after the addition of H₂O₂. However, it could not prevent the cell damages when added 1 h after the addition of H₂O₂ (Figure 3(a)). On the other hand, catalase completely prevented the cell damages when it was added at the same time as H₂O₂. However, it did not prevent cell damages when it was added 15 min after addition of H₂O₂ (Figure 3(b)). These results suggest that H₂O₂ induces the cell damages within 15 min after its addition and that TLCK may prevent the H₂O₂-induced cell damages by interfering with some steps in the signaling pathway that results in H₂O₂-induced cell damages, but not by quenching H₂O₂.

To clarify the mechanism through which TLCK protects the GSH-depleted H9c2 cells against the H₂O₂- induced cell damages, we first tested whether inhibitors of serine protease other than TLCK could prevent the cell damages as with TLCK. The inhibitors tested in the present study included TPCK, leupeptin, PMSF, and AEBSF; and their test concentrations are shown in the Figure 4(a). None of these four inhibitors could protect the cells against the H₂O₂-induced cell damages; in fact, higher concentrations of TPCK and AEBSF exerted by themselves a deleterious effect on the ability of the cells for MTT reduction (Figure 4(a)). These results suggest that the protection of cells by TLCK against the H₂O₂-induced cell damages could not be due to its inhibitory effect on serine protease.

TLCK blocks the activation of NF-kB by inhibiting

the proteolytic cleavage of IkB, a cytosolic inhibitor of NF-kB. Accordingly, we examined the effects of SN50, an inhibitor of NF-kB translocation into the nucleus, and MG-132, a proteasome inhibitor that inhibits NF-kB activation by preventing IkB degradation, on the H₂O₂- induced cell damages. Neither SN50 nor MG-132 could protect the cells against the H₂O₂-induced cell damages; however, at higher concentrations, both the inhibitors had a little tendency to increase the damages of the control cells (Figure 4(b)). These results indicate that the protection of cells by TLCK against the H₂O₂-induced cell damages may not be mediated by the inhibition of NF-kB activation.

TLCK prevents neuronal cell death, which is induced by UV irradiation or by treatment with camptothecin, through the inhibition of the induction and nuclear accumulation of p53 [14]. H₂O₂ induces phosphorylation of p53, leading to accumulation of p53 and transcription of its target gene p21^Cip1/WAF1 in endothelial cells [21]. If p53 participates in the H₂O₂-induced cell damages in our model, suppression of p53 should prevent the cell damages. Therefore, we examined the effect of pifithrin-a, an inhibitor of p53 [22], on the H₂O₂-induced cell damages. Figure 4(c) shows that pifithrin-a at a concentration of more than 5 µM protected the GSH-depleted H9c2 cells against H₂O₂-induced cell damages in a concentration-dependent manner, and 20 µM pifithrin-a almost completely blocked the cell damages. Pifithrin-a itself did not exert any deleterious effects on the ability of the cells for MTT reduction in the range of concentrations that were tested.

To evaluate the effect of TLCK on oxidative stress in the GSH-depleted H9c2 cells after the addition of H₂O₂, the generation of intracellular ROS was measured using a fluorescent probe DCFH₂-DA. DCFH₂ reacts with ROS to generate a highly fluorescent derivative DCF [23]. As shown in Figure 5(a), an increase in the intensity of DCF fluorescence was observed after the addition of H₂O₂ in the GSH-depleted H9c2 cells, while TLCK significantly inhibited it after addition of H₂O₂. In the cells preincubated with BSO, TLCK does not affect the intensity of DCF fluorescence. TLCK did not induce the increase of GSH levels in the cells pretreated with BSO, or rather TLCK slightly decreased the level (16 ± 8.0%, p > 0.05). To characterize the antioxidative action of TLCK, we evaluated the effect of TLCK on lipid peroxidation of rat liver microsomes in the reaction system of ascorbic acid and FeCl₃. As shown in Figure 5(b), TLCK significantly inhibited the production of TBARS in a dose-  dependent manner. However, TPCK did not inhibit the

increase in production of TBARS, suggesting that TLCK may have some antioxidant action in the cells.