Acridine orange (AO), agarose, bafilomycin, bufalin, dimethyl sulfoxide (DMSO), 3-methyladenine (3-MA), propidium iodide (PI), RNase A, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), proteinase K, and Triton X-100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, LC3B antibody kit for autophagy, penicillin/streptomycin, RPMI-1640 medium, trypan blue solution and Trypsin-EDTA were purchased from Invitrogen/Life Technologies (Carlsbad, CA, USA). AKT kinase assay Kit was purchased from Cell Signaling Technology (Danvers, MA, USA). Caspase-3 activity assay kit was purchased from R&D Systems Inc. (Minneapolis, MN, USA). CDK1 kinase activity kit was purchased from Medical & Biological Laboratories International (Nagoya, Japan). Tdt-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay kit was purchased from Roche Diagnostics (GmBH, Mannheim, Germany). Primary antibodies (anti-cyclin A, anti-cyclin B, anti-CDK1, anti-Cdc25c, anti-Chk1, anti-Weel, anti-PARP, anti-mTOR and anti-GAPDH), and second antibodies for western blot analysis were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The primary antibodies [anti-phospho-CDK1 (Thr161), phospho-Cdc25c (Ser198), anti-phospho-AKT (Thr308), anti-phospho-AKT (Ser473), anti-phospho-mTOR (Ser2481) and anti-caspase-3, anti-LC3-II, anti-Beclin-1, anti-Atg 5, anti-Atg 7 and anti-Atg 12] were obtained from Cell Signaling Technology. Antibody against β-actin was purchased from Sigma Chemical Co. All peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc. The enhanced chemiluminescence (ECL) detection kit was purchased from Pierce Chemical (Rockford, IL, USA).

The human hepatocellular carcinoma cell line (SK-HEP-1) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in 75-cm² tissue culture flasks at 37°C under a humidified 5% CO₂ atmosphere in RPMI-1640 medium containing 10% FBS, 2 mM of L-glutamine, 100 U/ml of penicillin and 100 μg/ml of streptomycin.

Cell viability was evaluated using the MTT assay (35). SK-HEP-1 cells (1×10⁴ cells) in 96-well plates were incubated with 0, 25, 50, 100 and 200 nM of bufalin or 0.1% DMSO as a vehicle control for 24 h. For incubation with the inhibitors, cells were pretreated with 3-MA (10 mM) or bafilomycin (10 nM) for 1 h, followed by treatment with or without bufalin (100 nM). After washing the cells, RPMI-1640 medium containing MTT (0.5 mg/ml) of was added to each well. The cells were incubated for 4 h at 37°C the supernatant was removed. The formed formazan crystals in viable cells were dissolved with isopropanol in 0.04 N HCl. The absorbance of each well was measured at 570 nm with ELISA reader with a reference wavelength of 620 nm. All experiments were performed in triplicate and the cell viability was expressed as percentage of the control as previously described. Cell morphological examination of autophagic vacuoles was determined utilizing a phase-contrast microscope.

Cell death was evaluated using a trypan blue assay (36,37). SK-HEP-1 cells (2.5×10⁵ cells) in 24-well plates were incubated with 0, 25, 50, 100 and 200 nM of bufalin for 24 h. At the end of incubation, cells were stained in the 0.25% trypan blue solution and then determined cell number by Countess Automated Cell Counter (Invitrogen/Life Technologies) as previously described.

The 2.5×10⁵ cells of SK-HEP-1 cells in 24-well plate were exposed to bufalin (50 and 100 nM) for 24 h. At the end of incubation, cells were then collected, fixed in 70% ethanol overnight, then washed in PBS once, and re-suspended in 500 μl of Na₂HPO₄ (192 mM), citric acid (4 mM) pH 7.8 at 25°C for 30 min. The cells were stained with 0.5 ml of PBS containing RNase (1 mg/ml), PI (10 μg/ml) for 30 min in the dark, and then analyzed by flow cytometry as previously described (38,39).

CDK 1 kinase activity was performed according to the manufacturer’s protocols (CycLex^® Cdc2-Cyclin B Kinase Assay Kit; MBL International Corporation, Japan) (40). SK-HEP-1 cells were seeded into 75-T flasks (1×10⁷/each). Bufalin (100 nM) were added and incubated for 0, 6, 12 and 24 h. At the end of incubation, cells were harvested, washed twice with ice-cold PBS, and then re-suspended the cell pellet with 500 μl of extraction buffer (20 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0.2% NP-40, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 1 μg/ml pepstatin, 0.5 μg/ml leupeptin, 5 mM β-glycerophosphate, 5 mM NaF, 1 mM Na₃VO₄, 5 mM β-mercaptoethanol). Cell extracts were diluted in a 1:5 ratio with Q-buffer (20 mM Tris-HCl, pH 8.5, 0.2 mM EDTA, 1 mM EGTA, 1 μg/ml pepstatin, 0.5 μg/ml leupeptin, 0.2 mM Na₃VO₄, 5 mM β-mercaptoethanol). The CDK1 activity was measured absorbance using ELISA reader at OD450. All results were performed in three independent experiments.

SK-HEP-1 cells were seeded into 75-T flasks (1×10⁷/each). Bufalin (0, 50 and 100 nM) were added and incubated for 24 h. At the end of incubation, cells were harvested and washed twice with ice-cold PBS. The cell pellets were re-suspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.2% Triton X-100). Cell lysates were treated with 0.1 mg/ml proteinase K at 50°C for 12 h, followed by 50 μg/ml RNase A at 37°C for 30 min. After precipitation, the DNA was subjected to electrophoresis in a 1.5% agarose gel (Sigma-Aldrich Corp.). DNA fragmentation on agarose gel was stained with 1 μg/ml ethidium bromide (EtBr, Invitrogen/Life Technologies) in 0.5X TBE buffer and examined in photographs taken under UV light as previously described (13,41).

TUNEL staining was performed according to the manufacturer’s protocol (in situ cell death detection kit; Roche Diagnostics) (13,41). The 2.5×10⁵ cells of SK-HEP-1 cells seeded in each well of 24-well plates were exposed to bufalin (25, 50 and 100 nM) or paclitaxel (100 nM) for 24 h. At the end of incubation, cells were collected then fixed in 70% ethanol then washed twice with ice-cold PBS, and incubated in the dark for 30 min at 37°C in 100 μl of TdT-containing solution. Following TUNEL staining, all samples were washed once. TUNEL positive cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). The median fluorescence intensity was quantities by CellQuest software. TUNEL assays were performed in triplicate on three independent experiments.

SK-HEP-1 cells (1×10⁷ cells/75-T flask) were treated with bufalin (25, 50 and 100 nM) or paclitaxel (100 nM) for 24 h. At the end of incubation, cells were harvested and re-suspended in a lysis buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT]. Cell lysates (50 μg protein) were incubated with caspase-3 specific substrates (Ac-DEVD-pNA) (R&D Systems Inc.) for 1 h at 37°C. The caspase-3 activity was determined by measuring OD₄₀₅ of the released pNA (42,43).

SK-HEP-1 cells (1×10⁶ cells/6-well) were treated with bufalin (100 nM) for 24 h. At the end of incubation, cells washing three times with PBS, cells were fixed for 30 min at room temperature in 2% paraformaldehyde and 2.5% glutaraldehyde in PBS buffer. The cells were rinsed twice in the same buffer and subsequently post fixed in 1% osmium tetraoxide. After rinsing followed by dehydration in graded alcohol series, the cells were embedded in LR white resin and polymerized at 70°C overnight. Ultrathin sections were then cut with a diamond knife and loaded onto TEM grids. The sections were examined by a Philips CM10 electron microscope at accelerating voltage of 120 kV and micrographs were taken (44).

SK-HEP-1 cells (1×10⁶ cells/6-well) were treated with bufalin (100 nM) for 24 h. At the end of incubation, cells were harvested. To detect and quantify acidic vesicular organelles (AVO), cells were stained with acridine orange (AO). The number of acridine orange positive cells was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). Cell morphology was examined using phase-contrast and fluorescence microscopy (Nikon, Melville, NY, USA). All results were performed in three independent experiments (45).

LC-3 staining was performed according to the manufacturer’s protocol (LC3 antibody kit for autophagy; Invitrogen/Life Technologies) (46,47). SK-HEP-1 cells (1×10⁶ cells/6-well) in 6-well flask were treated with bufalin (100 nM) for 24 h. At the end of incubation, cells were harvested then fixed with 3.7% formaldehyde in PBS for 15 min. Cells were washed three times with PBS, added 0.2% Triton X-100 in PBS to the cells, and incubated for 15 min at room temperature. Permeabilized cells were incubated with an anti-LC3 antibody (1:150) at 4°C overnight, and followed by incubation with an Alexa Fluor 488-conjugated secondary antibody (1:200; Invitrogen) for 1 h. The images were taken under a fluorescence microscopy (Nikon). The number of LC-3 positive cells was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). All results were performed in three independent experiments (48).

SK-HEP-1 cells (1×10⁷ cells/75-T flask) were treated with bufalin (100 nM) for 0, 2, 4, 6, 8, 12, 18 and 24 h. At the end of incubation, cytosolic or total proteins were prepared and determined as previously described. Protein lysates were sonicated and the supernatants were boiled in SDS sample buffer for 5 min. The protein concentration was measured by using a BCA assay kit (Pierce Chemical). Equal amounts of cell lysates were run on 10 to 12% SDS-polyacrylamide gel electrophoresis and electro-transferred to a nitrocellulose membrane by using the iBot Dry Blotting System (Invitrogen/Life Technologies). The transferred membranes were blocked for 1 h in 5% nonfat dry milk in Tris buffered saline/Tween-20 and incubated with primary antibodies at 4°C overnight. Membranes were washed three times with Tris-buffered saline/Tween-20 for 10 min and incubated with secondary HRP-conjugated antibody. The blots were developed by using an ECL kit and Kodak Bio-MAX MR film (Eastman Kodak, Rochester, NY, USA) (35).

In vitro AKT kinase assay was performed following the protocol of the manufacturer’s (Cell Signaling Technology) (46,49). In brief, SK-HEP-1 cells (1×10⁷ cells/75-T flask) were treated with bufalin (100 nM) for 0, 2, 4, 6 and 8 h. At the end of incubation, cells were lysed in ice-cold lysis buffer provided by the kit. The 200 μg of protein from each time point treatment was immuno-precipitated with 2 μg of anti-AKT antibody overnight. Immuno-precipitates were extensively washed, and then incubated with 1 μg of glycogen synthase kinase-3 α/β (GSK-3 α/β) fusion protein substrate in 50 μl of kinase buffer for 30 min at 30°C. Reactions were stopped by SDS loading buffer and samples were separated on 12% SDS-PAGE. The phospho-GSK-3 α/β (Ser219) was detected by immunoblotting.

All the statistical results were expressed as the mean ± SEM of triplicate samples. Statistical analyses of data were done using one-way ANOVA followed by Student’s t-test and ^*P<0.05 were considered significant.

SK-HEP-1 cells were treated with bufalin (0, 25, 50, 100 and 200 nM) for 24 h. Bufalin reduced cell viability of SK-HEP-1 cells in a concentration-dependent manner as measured by the MTT assay (Fig. 1A). The half maximal (50%) inhibitory concentration (IC₅₀) for a 24-h treatment of bufalin in SK-HEP-1 cell was 110.33±5.32 nM. Fig. 1B shows that bufalin increased cell death in a concentration-dependent manner by the trypan blue exclusion assay. We then examined cell cycle distribution in SK-HEP-1 cells exposed to 100 nM of bufalin for 24 h. These results showed G₂/M accumulation after SK-HEP-1 cells were treated with 100 nM of bufalin (Fig. 1C). Interestingly, SK-HEP-1 cells exposed to 50 and 100 nM of bufalin have undergone cell death, but did not detect significant sub-G₁ DNA content, a typical apoptosis. Our results suggested that bufalin effectively caused G₂/M arrest and then induced cell death.

We investigated the G₂/M phase specific protein expression levels by western blot analysis. As shown in Fig. 2A, bufalin (100 nM) caused an increase in the protein level of Chk1 and Wee1, and a decrease in the protein levels of Cyclin A, Cyclin B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198) for 6, 12 and 24 h treatment in SK-HEP-1 cells. We examined the CDK1 activity in bufalin-treated SK-HEP-1 cells. Fig. 2B depicted that bufalin (100 nM) caused a significant decrease in CDK1 activity for 0, 6, 12 and 24-h treatment in SK-HEP-1 cells. Our data indicated that bufalin-treated SK-HEP-1 cells downregulated CDK1 activity and caused G₂/M phase arrest.

To examine whether bufalin induced apoptosis in SK-HEP-1 cells, cells were treated with bufalin (0, 50 and 100 nM) for 24 h, and analyzed DNA fragmentation by DNA gel electrophoresis. Fig. 3A showed that no DNA fragmentation was observed in SK-HEP-1 cells treated with bufalin for 24 h. This result suggested that bufalin did not induce apoptosis in SK-HEP-1 cells. Similar result was obtained from TUNEL staining (Fig. 3B) when SK-HEP-1 cells were treated with bufalin (0, 50 and 100 nM) or paclitaxel (100 nM; an apoptotic agent) for 24 h. Fig. 3B showed that the percentage of TUNEL positive cells in bufalin-treated SK-HEP-1 cells was less than 5% compared with the paclitaxel-treated cells (37.26±4.34%). To investigate whether bufalin-induced cell death is mediated through caspase-3 activation, SK-HEP-1 cells were treated with bufalin (0, 50 and 100 nM) or paclitaxel (100 nM; an apoptotic reagent) for 24 h and then analyzed the protein levels of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), downstream target of caspase-3. As shown in Fig. 3C, the protein levels of cleaved caspase-3 or cleaved PARP were not increased in bufalin-treated SK-HEP-1 cells. In addition, the caspase-3 activity showed no change in bufalin-treated SK-HEP-1 cells. Our results indicated that bufalin-induced cell death did not proceed through apoptosis in SK-HEP-1 cells.

No apoptotic feature was detected in SK-HEP-1 cells. Next, we examine whether autophagic cell death is involved in bufalin-induced cell death. SK-HEP-1 cells were treated with bufalin (0, 25, 50 and 100 nM) for 24 h and the formation of autophagic vacuoles was examined by the phase microscopy. As shown in Fig. 4A, bufalin induced the formation of autophagic vacuoles in SK-HEP-1 cells in concentration-dependent manner. We further confirmed the formation of autophagosome vesicles in bufalin-treated SK-HEP-1 cells using transmission electron microscopic (TEM) analysis (Fig. 4B). Double- or multi-membrane structure characteristics of autophagosomes and autolysosomes were present in bufalin-treated SK-HEP-1 cells. The cytosolic acidic vesicular organelles (AVOs) are one of the hallmarks of autophagic cell death. In Fig. 5A, acridine orange (AO) staining of bufalin-treated SK-HEP-1 cells clearly showed AVOs within the cytoplasm compared to control by fluorescence microscopy. This observation was confirmed by FACS analysis, which showed a clear increase in red fluorescence (FL-3 positive) in concentration-dependent manner on bufalin-treated SK-HEP-1 cells (Fig. 5B). Furthermore, it has been shown that microtubule-associated protein 1 light-chain 3 (LC3) is an autophagic membrane marker for the detection of early autophagosome formation (50). We examined the LC3 distribution in bufalin-treated SK-HEP-1 cells by fluorescence microscopy and FACS analysis. As shown in Fig. 5C, bufalin treatment enhanced the punctate pattern of LC3-GFP in autophagic SK-HEP-1 cells. In addition, an increase in the green fluorescence (FL-1 positive) cells by FACS analysis showed that bufalin-treated SK-HEP-1 cells underwent autophagic cell death in a concentration-dependent manner (Fig. 5C, right). Our results suggested that bufalin-induced cell death in SK-HEP-1 cells is dependent on the induction of autophagy.

Induced autophagic cell death is associated with the elevated protein levels of autophagic proteins LC-3, Atg complex (Atg 5, Atg 7 and Atg 12) and Beclin-1. We investigated the autophagy-associated protein levels in bufalin-treated SK-HEP-1 cells by western blot analysis. As shown in Fig. 6A, bufalin (100 nM) increased the protein levels of LC-3 II, Atg 5, Atg 7 and Atg 12 and Beclin-1 in SK-HEP-1 cells. It is also reported that the AKT activity contributed to autophagic cell death. To examine whether bufalin-induced autophagic cell death is through the inhibition of AKT in SK-HEP-1 cells, cells were harvested after treatment with 100 nM of bufalin for 2, 4, 6 and 8 h, and then determined the protein levels by western blot analysis. Fig. 6B showed that bufalin (100 nM) significantly decreased the phospho-AKT (Thr308), phospho-AKT (Ser473), phospho-mTOR (Ser2481) protein levels in SK-HEP-1 cells. Bufalin decreased the AKT activity and this effect is time-dependent (Fig. 6C). Our results implied that bufalin induced autophagic cell death in SK-HEP-1 cells through interfering with the AKT/mTOR signaling pathway.

3-Methyladenine (3-MA), an inhibitor of class III phosphatidylinositol-3 kinase, is a reagent potently inhibiting autophagy-dependent protein degradation and suppressing the formation of autophagosomes (51), whereas bafilomycin A1, an inhibitor of the vacuolar proton pump of lysosomes and endosomes, appears to block the fusion of autophagosomes (52). In the present study, SK-HEP-1cells were pretreated with 3-MA (10 mM) or bafilomycin A1 (10 nM), and then exposed to 100 nM of bufalin before harvested for measuring the levels of autophagic vacuoles and cell viability by MTT assay. Both 3-MA (10 mM) and bafilomycin A1 (10 nM) inhibit autophagic vacuoles (Fig. 7A) and reduce cell viability (Fig. 7B) in bufalin-treated SK-HEP-1 cells. Furthermore, we examined effects of 3-MA (10 mM) and bafilomycin A1 on apoptosis by TUNEL assay (Fig. 7C). The results showed both 3-MA (10 mM) and bafilomycin A1 (10 nM) increased TUNEL positive apoptotic cells in bufalin-treated SK-HEP-1 cells. Taken together, inhibiting autophagic cell death by 3-MA or bafilomycin A1 facilitates bufalin-induced apoptosis in SK-HEP-1 cells.