RPMI-1640 medium, fetal bovine serum (FBS), and antibiotics were purchased from Welgene (Daegu, Korea). Baicalein (purity 98%, Fig. 1A), 4, 6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium (MTT), and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) were purchased from Calbiochem (San Diego, CA, USA). 2′,7′-dichlorofluorescein diacetate (DCF-DA) and fluorescein-conjugated Annexin V (Annexin V-FITC) were obtained from Molecular Probes (Eugene, OR, USA) and BD Biosciences Pharmingen (San Jose, CA, USA), respectively. An enhanced chemiluminescence (ECL) detection system and in vitro caspase colorimetric assay kits were purchased from Amersham Corp. (Arlington Heights, IL, USA) and R&D Systems (Minneapolis, MN, USA), respectively. The primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) used in this study were as follows: β-actin (1:1,000, sc-7120; rabbit polyclonal), DR4 (1:1,000, sc-7863; rabbit polyclonal), DR5 (1:1,000, sc-65314; mouse monoclonal), Fas (1:1,000, sc-715; rabbit polyclonal), FasL (1:1,000, sc-957; rabbit polyclonal), TRAIL (1:500, sc-7877; rabbit polyclonal), cIAP-1 (1:1,000, sc-7943; rabbit polyclonal), cIAP-2 (1:1,000, sc-7944; rabbit polyclonal), XIAP (1:1,000, sc-11426; rabbit polyclonal), caspase-3 (1:1,000, sc-7272; mouse monoclonal), caspase-8 (1:1,000, sc-7890; rabbit polyclonal), caspase-9 (1:1,000, sc-7885; rabbit polyclonal), PARP (1:1,000, sc-7150; rabbit polyclonal), Bcl-2 (1:1,000, sc-509; mouse monoclonal), Bax (1:1,000, sc-493; rabbit polyclonal), Bid (1:500, sc-11423; rabbit polyclonal). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin were purchased from Amersham Corp. All other chemicals were purchased from Sigma-Aldrich Chemical Co.

The 5637 human bladder cancer, Chang liver (an immortalized non-tumor cell line derived from normal liver tissue), and murine Raw 364.7 macrophage cell lines were obtained from American Type Culture Collection (Manassas, MD, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin) at 37°C in a humidified incubator under an atmosphere of 5% CO₂ in air. Baicalein was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 100 mM, which was then diluted with RPMI-1640 medium to the desired concentration prior to use.

The 5637 cells were seeded in 6-well plates at a density of 4.0×10⁵ cells per well. After a 24-h incubation, the cells were treated with various concentrations of baicalein for 24 h. Cell viability was determined using the MTT assay. In brief, an MTT working solution (0.5 mg/ml) was added to the culture plates and incubated for 3 h at 37°C. The culture supernatant was removed from the wells, and DMSO was added to dissolve the formazan crystals completely. The absorbance of each well was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA). Cell growth was assessed using the trypan blue dye exclusion assay. After treatment with the indicated concentrations of the baicalein for 24 h, the cells were trypsinized and viable cells were counted by trypan blue dye exclusion using a hemocytometer under an inverted microscope (Carl Zeiss, Jena, Germany). The effect of baicalein on cell viability and growth was assessed as the percentage of cell viability, in which the vehicle-treated cells were considered 100% viable.

For the assessment of apoptosis, the cells were washed with ice-cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The fixed cells were washed with PBS and stained with 2.5 μg/ml DAPI solution for 10 min at room temperature. The cells were then washed twice with PBS and analyzed with a fluorescence microscope (Carl Zeiss).

For the quantitative assessment of the induced cell apoptosis rate, an Annexin V-FITC staining assay was performed according to the manufacturer’s protocol. Briefly, the cells in each sample were stained with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI). After incubation for 15 min at room temperature in the dark, the degree of apoptosis was quantified by a flow cytometer (FACSCalibur, Becton-Dickinson, San Jose, CA, USA) as a percentage of the Annexin V-positive and PI-negative cells (25).

After removing the media, the cells were washed with ice-cold PBS and harvested and lysed with a lysis buffer [20 mM sucrose, 1 mM ethylendiaminetetraacetic acid, 20 μM Tris-Cl, pH 7.2, 1 mM dithiothreitol (DTT), 10 mM KCl, 1.5 mM MgCl₂, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, and 2 μg/ml aprotinin] for 30 min at 4°C. The supernatants were collected and protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. For western blotting, equal amounts of protein extracts were denatured by boiling at 95°C for 5 min in a sample buffer [0.5 M Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.1% bromophenol blue, and 10% β-mercaptoethanol] at a ratio of 1:1. Samples were stored at −80°C or immediately used for immunoblotting. The equal cellular proteins were separated via denaturing SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membranes (Amersham Corp.). The membranes were then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies, probed with enzyme-linked secondary antibodies for 1 h at room temperature, and detected using an ECL detection system.

The activities of the caspases were determined using colorimetric assay kits, which utilize synthetic tetrapeptides [Asp-Glu-Val-Asp (DEAD) for caspase-3; Ile-Glu-Thr-Asp (IETD) for caspase-8; and Leu-Glu-His-Asp (LEHD) for caspase-9] labeled with p-nitroaniline (pNA), which is linked to the end of the caspase-specific substrate. Briefly, the baicalein-treated cells and the untreated cells were lysed in the supplied lysis buffer. The supernatants were collected and incubated with the supplied reaction buffer containing DTT and DEAD-pNA, IETD-pNA, or LEHD-pNA as substrates at 37°C for 2 h in the dark. The reactions were measured by changes in absorbance at 405 nm using an ELISA reader (26).

The MMP values were determined using the dual-emission potential-sensitive probe, JC-1. Briefly, the cells were collected and incubated with 10 μM of JC-1 for 20 min at 37°C in the dark. After the JC-1 was removed, the cells were washed with PBS to remove unbound dye, and the amount of JC-1 retained by 10,000 cells per sample was measured at 488 and 575 nm using a flow cytometer (27).

To assess the generated ROS, the cells were treated with or without 10 mM NAC for 1 h before challenge with 250 μM baicalein for 30 min, and then stained with 10 μM DCF-DA and incubated at 37°C for 30 min in the dark to monitor ROS production. Later, the ROS production in cells was monitored by a flow cytometer (28).

All data are presented as mean ± standard deviation (SD). Significant differences among the groups were determined using the unpaired Student’s t-test. A value of p<0.05 was accepted as an indication of statistical significance. All of the data shown herein were obtained from at least three independent experiments.

To assess the effect of baicalein on 5637 cell viability and growth, equal numbers of cells were treated with various concentrations of baicalein (50–250 μM) for 24 h and the cell viability and growth were detected by the MTT and trypan blue dye exclusion assays. As shown in Fig. 1B and C, baicalein caused dose-dependent inhibition of cell viability and growth, with a significant reduction at 150 μM and an almost 60% reduction at 250 μM. The results of an additional experiment using Chang liver cells and Raw 264.7 macrophages, conducted to examine the effect of baicalein on the proliferation of normal cells, are shown in Fig. 1D and E. The MTT assay results indicated that baicalein concentrations ≤250 μM/ml did not induce cytotoxicity.

To determine whether baicalein-mediated inhibition of cell viability in 5637 cells is associated with induction of apoptosis, we examined apoptotic features by measuring the chromatin condensation of the nuclei and the amount of Annexin V-positive cells. As shown in Fig. 2A, the results of the DAPI reagent indicate that the nuclear fragmentation and chromatin condensation located in apoptotic cells were observed in baicalein-treated cells, with bright blue fluorescence. In addition, treatment with baicalein resulted in the increased accumulation of cells in the number of Annexin V-positive cells in a concentration-dependent manner (Fig. 2B and C). These findings suggest that baicalein suppresses 5637 cell viability by inducing cellular apoptosis.

In order to determine which apoptosis pathway contributes to baicalein-induced apoptosis, the levels of death receptors (DRs) and corresponding proapoptotic ligands were first examined by western blot analysis. After baicalein treatment, the protein levels of Fas were not altered; however, the expression of DR4, DR5, Fas ligand (FasL), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were increased (Fig. 3). Next, we examined the effects of baicalein on the levels of the inhibitor of apoptosis protein (IAP) family of proteins. The results of western blotting showed that baicalein treatment resulted in a concentration-dependent decrease in the expression levels of cIAP-1 and cIAP-2, but not XIAP (Fig. 3).

We then examined the expression levels and activities of caspases during baicalein-induced 5637 cell apoptosis. As shown in Fig. 4A, western blot analyses showed that the expression levels of pro-caspase-3 in cells treated with baicalein were concentration-dependently downregulated and the expression levels of active-caspase-3 were upregulated. In addition, the active forms of caspase-8 and -9 were increased, and the levels of the pro-forms of caspase-8 and -9, initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, were downregulated. Under the same conditions, the in vitro activities of these caspases were measured using substrates specific to each caspase, and we found that baicalein stimulates caspase-3, -8 and -9 activities in a concentration-dependent manner (Fig. 4B). Moreover, baicalein treatment leads to progressive proteolytic cleavage of poly(ADP-ribose)-polymerase (PARP), a substrate protein of active caspase-3 (Fig. 4A).

To further confirm the baicalein-induced apoptotic pathway, this study examined the effects of baicalein on the levels of Bcl-2 family expression and the MMP values. As shown in Fig. 5A, in response to baicalein treatment, the levels of proapoptotic Bax were upregulated, but those of antiapoptotic Bcl-2 were downregulated. Subsequent western blot analyses revealed progressive downregulation of total Bid protein and accumulation of truncated Bid (tBid). Moreover, baicalein treatment caused a concentration-dependent loss of MMP in comparison to the untreated control (Fig. 5B and C).

To confirm the involvement of baicalein-induced activation of caspases, the cells were pretreated with or without z-VAD-fmk, a pan-caspase inhibitor, for 1 h, followed by treatment with baicalein. Pretreatment with z-VAD-fmk resulted in significant prevention of the loss of MMP, expression of the active form of caspase-3, and cleavage of PARP (Fig. 6A and B). Furthermore, z-VAD-fmk also decreased the accumulation of Annexin V-FITC stained cells and increased cell viability in the presence of baicalein (Figs. 6C and D). These results provide evidence of baicalein-induced apoptotic cell death in association with the activation of caspases in 5637 cells.

Because the generation of intracellular ROS may be related to mitochondrial dysfunction and the induction of apoptosis in various cell types, we further investigated whether baicalein could stimulate ROS generation in 5637 cells. As shown in Fig. 7, the maximal generation of ROS was observed at 30-min treatment with baicalein; however, baicalein-induced ROS generation was effectively blocked by the antioxidant NAC. In addition, the loss of MMP, activation of caspase-3 and cleavage of PARP induced by baicalein were significantly attenuated by pretreatment with NAC (Fig. 8A and B). We also observed that baicalein-induced apoptosis and reduction of cell viability were markedly reduced by pretreatment with NAC (Fig. 8C and D), suggesting that baicalein induces apoptosis via ROS-dependent caspase activation mechanisms in 5637 cells.