Esculetin (6,7-dihydroxycoumarin) was purchased from Sigma Chemical Co. (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO, vehicle), and adjusted to final concentrations using complete culture medium. The final DMSO concentration was <0.1% in all experiments. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were obtained from WelGENE Inc. (Daegu, Korea). H₂O₂, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC), 4′,6-diamidino-2- phenylindole (DAPI), propidium iodide (PI) and zinc protoporphyrin IX (ZnPP) were also purchased from Sigma Chemical Co. PD98059, an ERK inhibitor, was purchased from Calbiochem, Inc. (San Diego, CA, USA). 2′,7′-Dichlorofluorescein diacetate (DCFDA) and an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit were purchased from Molecular Probes, Inc. (Eugene, OR, USA) and R&D Systems Inc. (Minneapolis, MN, USA), respectively. Primary antibodies (Table I) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), Cell Signaling Technology, Inc. (Danvers, MA, USA) and Abcam, Inc. (Cambridge, MA, USA). An enhanced chemiluminescence (ECL) kit and horseradish (HRP)-conjugated secondary antibodies were obtained from Amersham Life Science (Arlington Heights, IL, USA). All other chemicals not specifically mentioned here were purchased from Sigma Chemical Co.

C2C12 myoblasts obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 100 µg/ml of penicillin/streptomycin antibiotics in a humidified atmosphere containing 5% CO₂ and 95% air at 37°C. The cells were pre-treated with various concentrations (0–5 µM) of esculetin for 1 h and then incubated with or without 1 mM H₂O₂ for 6 h in the absence or presence of 5 mM NAC or 50 µM PD98059. To measure cell viability, the cells were maintained with MTT at a final concentration of 0.5 mg/ml for 3 h, and the formazan that formed was dissolved in DMSO. Optical density was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA). The optical density of the formazan formed in the control (untreated) cells was used to represent 100% viability (36).

To measure ROS levels, the cells were washed twice with phosphate-buffered saline (PBS) and lysed with 1% Triton X-100 in PBS for 10 min at 37°C. The cells were pre-treated with 5 µM esculetin for 1 h and then incubated with or without 1 mM H₂O₂ for 6 h in the absence or presence of 5 mM NAC or 50 µM PD98059. The cells were then stained with 10 µM DCFDA for 20 min at room temperature in the dark. The green fluorescence of DCF was recorded at 515 nm using a flow cytometer, and 10,000 events were counted per sample. The results are expressed as the percentage of increase relative to the non-treated cells.

A comet assay was performed to detect DNA migrating from single cells in the gel, following a previously described method (37). Briefly, the cells were exposed to 1 mM H₂O₂ for 6 h in the presence and absence of 5 µM esculetin for 1 h. The cells were suspended in 1% low melting point agarose and aliquoted onto glass microscope slides. The slides were placed in single rows and electrophoresed at 30 V (1 V/cm) and 300 mA for 20 min to draw negatively charged DNA toward the anode. Finally, the slides were washed with 0.4 M Tris (pH 7.5) at 4°C and stained with 20 µg/ml PI. The slides were examined under a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany), and the resulting images were analyzed.

The cells were pre-treated with 5 µM esculetin for 1 h and then incubated with or without 1 mM H₂O₂ for 6 h, or pre-treated with or without 50 µM PD98059 for 1 h and then treated with 5 µM esculetin for 4 h. The cells were harvested, washed with PBS, and lysed on ice for 30 min in lysis buffer (20 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 20 µM Tris-HCl, pH 7.2, 1 mM dithiothreitol, 10 mM KCl, 1.5 mM MgCl₂, and 5 µg/ml aprotinin). Subsequently, an equal amount of protein for each sample was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Schleicher & Schuell, Inc., Keene, NH, USA). The membranes were blocked with 5% skim milk and then incubated overnight at 4°C with desired primary antibodies. The membranes were further incubated with corresponding HRP-conjugated secondary antibodies for 2 h at room temperature. The proteins of interest were visualized using an ECL detection system.

The cells were pre-treated with 5 µM esculetin for 1 h and then incubated with or without 1 mM H₂O₂ for 6 h. The detection of chromatin condensation and nuclear fragmentation in the nuclei of apoptotic cells was performed using DAPI staining. The cells were harvested, washed with PBS twice, and fixed with 3.7% paraformaldehyde in PBS for 10 min at 25°C. The fixed cells were washed with PBS and stained with 1 mg/ml DAPI solution for 10 min. The cells were then washed twice with PBS and observed under a fluorescence microscope (38).

The cells were pre-treated for 1 h with the indicated concentrations of esculetin and then incubated for 6 h with or without 1 mM H₂O₂ in the absence or presence of 50 µM PD98059. The rate of apoptosis was determined using an Annexin V-FITC apoptosis detection kit. After treatment with the agents, the cells in each sample were stained with Annexin V-FITC and PI in accordance with the manufacturer's instructions. After a 15-min incubation at room temperature in the dark, the degree of apoptosis was quantified as a percentage of the Annexin V-positive and PI-negative (Annexin V⁺/PI⁻ cells) cells using a flow cytometer (Becton Dickinson, San Jose, CA, USA) (39).

Unless specified otherwise, data are expressed as the means ± standard deviation (SD) of at least three independent experiments. A one-way analysis of variance (SPSS version 12.0 software) and a Scheffe's test were used to determine the significance of differences between groups. P<0.05 was considered to indicate a statistically significant difference.

To determine the protective effects of esculetin against H₂O₂-induced cytotoxicity, C2C12 cells were pre-treated with various concentrations of esculetin (1.25–5 µM) for 1 h and then exposed to H₂O₂ (1 mM) for a further 6 h. The concentrations of esculetin that did not have any measurable adverse effects on the cells were selected (data not shown). As shown in Fig. 1A, exposure to H₂O₂ alone significantly reduced cell viability (more than 60%) when measured using the MTT assay, whereas the H₂O₂-induced reduction in cell viability was prevented by pre-treatment with esculetin in a concentration-dependent manner. In addition, H₂O₂ stimulation significantly induced morphological changes, including extensive cytosolic vacuolization and the presence of irregular cell membrane buds, which were effectively attenuated by esculetin pre-treatment (Fig. 1B).

The intracellular ROS generation was monitored to investigate whether esculetin can prevent H₂O₂-induced ROS generation. The results of the flow cytometric analysis using DCFDA as a fluorescence probe demonstrated that the intensity of the DCF-liberated fluorescent signal from the H₂O₂-exposed cells was significantly increased; however, the signal was markedly reduced in the presence of 5 µM esculetin (Fig. 2). As a positive control, the ROS scavenger NAC at 5 mM also markedly attenuated H₂O₂-induced ROS generation, indicating that esculetin scavenged H₂O₂-induced ROS accumulation.

We then examined the effects of esculetin on H₂O₂-mediated DNA damage in C2C12 cells. Fig. 3A shows the results of the comet assay performed to evaluate the protective effect of esculetin against H₂O₂-induced DNA damage. Exposure to H₂O₂ leads to the loss of membrane integrity; therefore, the fragmented DNA appeared outside the cell as comet-like structures; however, this adverse effect was markedly inhibited by esculetin. In addition, treatment of the C2C12 cells with H₂O₂ upregulated the level of the phosphorylated histone variant H2AX at serine 139 (p-γH2AX), a sensitive marker of DNA double-strand breaks (40) (Fig. 3B). However, pre-treatment with esculetin significantly decreased H₂O₂-induced p-γH2AX expression.

To evaluate the potential effect of esculetin on H₂O₂-induced C2C12 cell apoptosis, we examined apoptotic features by measuring chromatin condensation in the nuclei, poly(ADP ribose) polymerase (PARP) cleavage, and Annexin V-positive cells. DAPI staining revealed increased nuclei with chromatin condensation and the formation of apoptotic bodies, characteristic morphological changes of apoptosis, in cells cultured with 1 mM H₂O₂. However, the control and esculetin (5 µM)-treated groups showed few apoptotic cells, and pre-treatment of the cells with esculetin significantly abrogated the H₂O₂-induced apoptotic characteristics (Fig. 4A). The results of western blot analysis also indicated a marked increase in the level of cleaved PARP, an apoptotic marker protein, in the H₂O₂-treated cells compared with the control group, and treatment with esculetin significantly decreased the levels of cleaved PARP (Fig. 4B). Furthermore, the results of flow cytometric analysis revealed an increase in the percentage of Annexin-positive C2C12 cells exposed to H₂O₂ compared with the cells in the control group. By contrast, treatment of the cells with esculetin prior to exposure to H₂O₂ strongly protected the C2C12 cells against apoptosis in a concentration-dependent manner (Fig. 4C). These results clearly indicated that esculetin inhibited H₂O₂-induced apoptotic signaling in the H₂O₂-exposed C2C12 cells.

To determine whether the protective effects of esculetin against H₂O₂-induced oxidative stress and apoptosis result from the induction of the expression of antioxidant genes, such as HO-1 and NQO1, and their transcription factor Nrf2, western blot analysis was performed. As shown in Fig. 5, the esculetin-treated cells exhibited a significant increase in the protein levels of NQO1 compared to these levels in the control group; however, no changes were observed in the levels of HO-1. Furthermore, esculetin enhanced the phosphorylated levels of endogenous Nrf2 in a time-dependent manner without affecting their total steady-state levels and obtained greatest induction at 5 µM after 4 h. By contrast, esculetin reduced the levels of Keap1 under the same conditions.

To investigate whether Nrf2 phosphorylation by esculetin in C2C12 cells is affected by the activation of MAPKs as upstream signaling mediators, we assessed the phosphorylated forms of ERK, JNK and p38 MAPK. As shown in Fig. 6A, although the total protein levels of ERK did not show notable changes, esculetin markedly increased the phosphorylation of ERK within 1 h of treatment, while the phosphorylation levels of JNK and p38 MAPK remained unaltered. The dependence of the phosphorylation of Nrf2 challenged with esculetin upon the activation of ERK was confirmed using a specific inhibitor of ERK, PD98059. For this experiment, the C2C12 cells were pre-treated with 50 µM PD98059 for 1 h and then treated with esculetin for 4 h. We found that treatment with PD98059 effectively reduced the esculetin-induced phosphorylation of Nrf2, with a resulting decrease in the expression of NQO1 (Fig. 6B). In addition, co-pre-treatment with PD98059 and esculetin prior to exposure to H₂O₂ markedly abrogated the protective effects of esculetin against H₂O₂-induced ROS generation and apoptosis as well as growth reduction (Fig. 7).