In a previous report, we have indicated that butin protected against H₂O₂-induced apoptosis [15]. Change in Δψ_m was examined to improve understanding of butin’s protection mechanism for H₂O₂-induced apoptotic process in terms of mitochondrial involvement. JC-1 is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green (~525 nm) to red (~590 nm). As shown in Figure 2A, control cells and butin-treated cells exhibited strong red fluorescence (JC-1 aggregated form, indicative of mitochondrial polarization) in the mitochondria. However, H₂O₂ resulted in reducing red fluorescence and increasing green fluorescence (JC-1 monomer form, indicative of mitochondrial depolarization) in the mitochondria. Butin treatment blocked reducing red fluorescence and increasing green fluorescence in H₂O₂-treated cells. Image analysis data was consistent with flow cytometric data; the level of Δψ_m loss was increased in H₂O₂-treated cells, as substantiated by an increase in fluorescence with JC-1 dye. However, butin recovered the level of Δψ_m loss (Figure 2B), suggesting that butin partially inhibited loss of Δψ_m in response to H₂O₂ treatment.

In order to confirm the cytoprotective impact of butin on H₂O₂-induced apoptosis, cell nuclei were stained with Hoechst 33342 for visualization by microscopy. The microscopic images in Figure 3A demonstrate that the control cells had intact nuclei, whereas H₂O₂-treated cells showed significant nuclear fragmentation, a characteristic of apoptosis. However, butin-pretreated cells exhibited a dramatic decrease in nuclear fragmentation induced by H₂O₂ treatment. In addition to morphological evaluation, the protective effect of butin against apoptosis was also confirmed by apoptotic sub-G₁ DNA analysis. As shown in Figure 3B, an analysis of DNA content in H₂O₂-treated cells revealed a 36% increase in the apoptotic sub-G₁ DNA content. However, butin decreased the apoptotic sub-G₁ DNA content to 16%. Furthermore, H₂O₂-treated cells increased the levels of cytoplasmic histone-associated DNA fragmentations as compared to control, and butin significantly decreased the level of DNA fragmentation (Figure 3C).

To further understand the protection mechanism of butin on H₂O₂-induced apoptotic process, we detected the protein expressions involved in mitochondria related apoptosis. Beforehand, changes in Bcl-2 expression, an anti-apoptotic protein, and Bax expression, a pro-apoptotic protein, were examined. As shown in Figure 4A, butin showed an increase in Bcl-2 expression and a decrease in Bax expression in H₂O₂-treated cells. It has been reported that Bcl-2 fails to inhibit cell apoptosis when inactivated via phosphorylation [9]. We noticed that butin also decreased phosphorylation of Bcl-2 (Ser 87) induced by H₂O₂ treatment. During the apoptotic process, Bcl-2 prevented the opening of the mitochondrial membrane pore, whereas Bax induced the opening of membrane pore [21]. Pore opening induces loss of Δψ_m, which in turn induces the release of cytochrome c from the mitochondria [22]. As shown in Figure 4B, butin inhibited the release of mitochondrial cytochrome c. Next, caspase 9 activity was examined by Western blot since it is known that this enzyme is activated due to mitochondrial membrane disruption [23]. As shown in Figure 3C, treatment of cells with butin inhibited H₂O₂-induced active form of caspase 9 (39 and 37 kDa) and caspase 3 (19 and 17 kDa), a target of caspase 9. These results suggest that butin protects cells from apoptosis by inhibiting the caspase dependent pathway via mitochondria.

The JNK signal pathway plays an important role in oxidative stress-induced apoptosis [24] and JNK translocates to the mitochondrial, then phosphorylates Bcl-2, and presumably inactivates them [25]. In addition, JNK induces the mitochondrial pathway of apoptosis by activating Bax [26], thus we tested whether butin regulates this signaling pathway. As shown in Figure 5A, butin inhibited JNK activation in H₂O₂-treated cells at 12 h. Moreover, SEK1 is known to be an upstream component in the JNK signaling pathway [27].

To investigate whether this upstream kinase plays a role in H₂O₂-induced JNK activation, SEK1 phosphorylation was determined by Western blot analysis. As shown in Figure 5B, SEK1 phosphorylation levels were increased in H₂O₂-treated cells at 6 h. However, treatment of cells with butin inhibited H₂O₂-induced SEK1 phosphorylation. AP-1 is a downstream target of the phospho JNK pathway, and activated AP-1 is involved in cell death including apoptosis [28]. Subsequently, we examined the effect of butin pretreatment on the DNA binding activity of AP-1 after H₂O₂ treatment at 24 h. As shown in Figure 5C, AP-1 DNA binding activity was increased in H₂O₂ treated cells, whereas treatment of cells with butin inhibited AP-1 activity.

The transcriptional activity of AP-1 was also assessed using a promoter construct containing AP-1 binding DNA consensus sequences, which were linked to a luciferase reporter gene. As shown in Figure 5D, butin inhibited the transcriptional activity of AP-1 induced by H₂O₂. These results suggest that butin inhibits H₂O₂-induced apoptosis via suppression of the SEK1-JNK-AP-1 pathway.

Butin was purchased from Wako Pure Chemical Ind., Ltd. (Tokyo, Japan). 5,5′,6,6′-Tetrachloro- 1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was purchased from Invitrogen (Carlsbad, CA, USA). The primary anti-B-cell lymphoma 2 (Bcl-2), -Bcl-2-associated x protein (Bax), -phospho Bcl-2, and -cytochrome c antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Primary anti-caspase 9, -caspase 3, -c-Jun N-terminal kinases (JNK), -phospho JNK, -mitogen-activated protein kinase kinase-4 (SEK1), and -phospho SEK1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). A plasmid containing activator protein-1 (AP-1) binding site-luciferase construct was a generous gift from Professor Young Joon Surh of Seoul National University (Seoul, Korea). Propidium iodide and Hoechst 33342 were purchased from the Sigma Chemical Company (St. Louis, MO, USA).

Chinese hamster lung fibroblasts (V79-4 cells) from the American type culture collection were maintained at 37 °C in an incubator, with a humidified atmosphere of 5% CO₂ and cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 μg/mL) and penicillin (100 units/mL).

Δψ_m analysis was determined by confocal image analysis and flow cytometer. The V79-4 cells were seeded at a concentration of 1 × 10⁵ cells/mL, and 16 h after plating, were treated with butin at 10 μg/mL, and after 1 h, 1 mM of H₂O₂ was added to the plate, and the mixture was incubated for 12 h. Cells were then harvested, and after changing the media, JC-1 was added to each well and was incubated for an additional 30 min at 37 °C. After washing with PBS, the stained cells were mounted onto microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on confocal microscope [29]. In addition, Δψ_m analysis was also determined by flow cytometer. The cells were harvested, washed and suspended in phosphate buffered saline (PBS) containing JC-1 (10 μg/mL). After incubation for 15 min at 37 °C, the cells were washed and were suspended in PBS and were analyzed by flow cytometer [30].

Cells were seeded at a concentration of 1.5 × 10⁵ cells/mL and 16 h after plating, cells were treated with butin at 10 μg/mL, and after 1 h, 1 mM of H₂O₂ was added. After 6, 12 or 24 h, cells were harvested, washed twice with PBS, lysed on ice for 30 min in 100 μL of a lysis buffer (120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40) and then centrifuged at 13,000 × g for 15 min. The supernatants were collected from the lysates and the protein concentrations determined. Aliquots of the lysates (40 μg of protein) were boiled for 5 min and electrophoresed in 10% sodium dodecysulfate-polyacrylamide gel. The blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then incubated with the primary antibodies. The membranes were further incubated with the secondary immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA). Protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK), and then exposed onto X-ray film.

Cells were seeded at a concentration of 1 × 10⁵ cells/mL, and 16 h after plating, were treated with butin at 10 μg/mL. After 1 h, 1 mM of H₂O₂ was added to the plate and the mixture was incubated for 24 h. 1.5 μL of Hoechst 33342 (stock 10 mg/mL), a DNA specific fluorescent dye, was added to each well and incubated for 10 min at 37 °C. The stained cells were then observed under a fluorescent microscope, which was equipped with a CoolSNAP-Pro color digital camera, in order to examine the degree of nuclear condensation. The percentage of apoptotic cells (apoptotic index) was assessed by counting 3 random fields in triplicate wells.

The amount of apoptotic sub-G₁ hypodiploid cells was determined using flow cytometer [31]. Cells were seeded at a six-well plate at a concentration of 1 × 10⁵ cells/mL, and 16 h after plating, were treated with butin at 10 μg/mL. After 1 h, 1 mM of H₂O₂ was added to the plate and the mixture was incubated for 24 h. Cells were harvested and fixed in 1 mL of 70% ethanol for 30 min at 4 °C. The cells were then washed twice with PBS, and incubated for 30 min in the dark at 37 °C in 1 mL of PBS containing 100 μg of propidium iodide and 100 μg of RNase A. A flow cytometric analysis was performed using a FACS Calibur flow cytometer. Sub-G₁ hypodiploid cells were assessed based on histograms generated by the Cell Quest and Mod-Fit computer programs.

Cells were seeded at a concentration of 5 × 10⁴ cells/mL, and 16 h after plating, cells were treated with butin at 10 μg/mL. After 1 h, 1 mM of H₂O₂ was added to the plate and the mixture was incubated for 24 h. Cellular DNA-fragmentation was assessed by analyzing cytoplasmic histone-associated DNA fragmentation, using a kit from Roche Diagnostics according to the manufacturer’s protocol.

Cells were seeded at a concentration of 1.5 × 10⁵ cells/mL, and 16 h after plating, cells were treated with butin at 10 μg/mL. After 1 h, 1 mM of H₂O₂ was added to the plate and the mixture was incubated for 24 h. After 24 h, cells were harvested, and subsequently lysed on ice with 1 mL of lysis buffer (10 mM Tris-HCl, pH 7.9, 10 mM NaCl, 3 mM MgCl_2, and 1% NP-40) for 4 min. After 10 min of centrifugation at 3000 × g, the pellets were re-suspended in 50 μL of extraction buffer (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, and 1 mM PMSF), incubated on ice for 30 min and centrifuged at 13,000 × g for 5 min. The supernatant (nuclear protein) was stored at −70 °C after determining the protein concentration. Oligonucleotides containing transcription factor AP-1 consensus sequence (5′-CGC TTG ATG ACT CAG CCG GAA-3′) were annealed, labeled with [γ-³²P] ATP using T4 polynucleotide kinase and used as probes. The probes (50,000 cpm) were incubated with 6 μg of the nuclear extracts at 4 °C for 30 min, to reach a final volume of 20 μL, containing 12.5% glycerol, 12.5 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, and 1 mM DTT with 1 μg of poly (dI-dC). The binding products were resolved on 5% polyacrylamide gel and the bands were visualized by autoradiography.

Cells were seeded at a concentration of 1.0 × 10⁵ cells/mL, and 16 h after plating, cells were transiently transfected with plasmid harboring the AP-1 promoter using DOTAP as the transfection reagent, according to the manufacturer’s protocol (Roche Diagnostics, Portland, OR, USA). Following overnight transfection, cells were treated with 10 μg/mL of butin, and after 1 h, 1 mM of H₂O₂ was added to the plate for 24 h. Cells were washed twice with PBS and lysed with reporter lysis buffer (Promega, Madison, WI, USA). Following vortex mixing and centrifugation at 12,000 × g for 1 min at 4 °C, the supernatant was stored at −70 °C for the luciferase assay. After mixing 20 μL of cell extract with 100 μL of luciferase assay reagent at room temperature, the mixture was placed in an illuminometer to measure the light produced.

All measurements were performed in triplicate and all values were represented as the mean ± standard error of the mean (SEM). The results were subjected to an analysis of variance (ANOVA) using the Tukey’s test to analyze difference. P < 0.05 were considered statistically significant.