Z-guggulsterone (Fig.1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The stock solution was prepared in dimethyl sulfoxide (DMSO; Solar Biotechnology, Inc., Shanghai, China) and then diluted with serum-free medium. RPMI-1640 and heat-inactivated fetal calf serum were obtained from Gibco Industries (Tulsa, OK, USA). The XTT proliferation kit and Hoechst 33258 were purchased from Boehringer Ingelheim (Mannheim, Germany). The Caspase Activity Colorimetric Assay kit, pan-caspase inhibitor z-VAD-fmk, capase-8 inhibitor z-IETD-fmk and caspase-9 inhibitor z-LEHD-fmk were purchased from Assay Designs (Ann Arbor, MI, USA). The following primary antibodies were also used: Rabbit anti-cleaved poly (adenosine diphosphate-ribose) polymerase (PARP; cat no. G3411; Promega, Madison, WI, USA); mouse anti-human survivin (cat no. sc-65610; 1:300 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-human B-cell lymphoma 2 (Bcl-2; cat no. sc-377576; 1:500 dilution; Santa Cruz Biotechnology, Inc.); and goat anti-human Bcl-2-associated X protein (cat no. sc-20287; 1:1,100 dilution; Santa Cruz Biotechnology, Inc.). The rabbit anti-mouse (cat no. sc-358961; 1:1,000 dilution), bovine anti-rabbit (cat no. sc-362290; 1:1,000 dilution) and bovine anti-goat (cat no. sc-362284; 1:1,000 dilution) secondary antibodies were all obtained from Santa Cruz Biotechnology, Inc. The present study was approved by the ethics committee of Anhui Medical University (Hefei, China).

The human immortalized cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell lines were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/l L-glutamine and 5 U/ml penicillin (Solar Biotechnology, Inc.)in a humidified incubator with a 5% CO₂ atmosphere at 37°C.

The effects of guggulsterone on the viability of Sk-ChA-1 and Mz-ChA-1 cells were determined by the XTT assay. In brief, 1×10⁴ cells/well were seeded into a 96-well plate and incubated for 24 h, and these cells were then treated with 0, 20, 40 and 60 µmol/l Z-guggulsterone. Cells treated with only DMSO were used as the control. At the indicated time points, 50 µl of XTT/phenazine methosulfate (PMS) mixture (Solar Biotechnology, Inc.), consisting of 50 µmol/l PMS and 0.1% XTT in medium, was added to the cell culture media and incubated continuously for 4 hours. The absorbance at 450 nm was then measured using a Synergy™ HT multi-mode microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

The cholangiocarcinoma cells were seeded into a six-well plate and routinely cultured overnight in RPMI-1640 medium containing 10% fetal calf serum. These cells were then treated with 60 µmol/l Z-guggulsterone for 72 h. Subsequently, the cells were fixed with 4% formaldehyde for 15 min at room temperature. The cells were then washed twice with 1X phosphate-buffered saline (PBS) and stained with 10 mg/l Hoechst 33258 for 1 h at room temperature. Finally, the alterations in nuclear morphology were observed under fluorescence microscopy (Nikon TE 2000-U; Nikon, Tokyo, Japan).

In total, 1×10⁶ cells were harvested and washed once in 1X Ca₂⁺- and Mg₂⁺-free PBS. The cells were then suspended in 400 µl cell lysis buffer consisting of 10 mM Tris-HCl (pH 7.4; Solar Biotechnology, Inc.), 10 mM EDTA (pH 8.0) and 0.5% Triton X-100 (Solar Biotechnology, Inc.). The suspension was incubated at 4°C for 15 min. Following centrifugation of the cell lysates at 15,000 × g for 20 min, the supernatants were collected and incubated with 40 µg/ml proteinase K and 40 µg/ml RNase A (SBS Genetech Co., Beijing, China) at 37°C for 3 h. The lysate was isolated using 0.5 M NaCl and 50% 2-propanol and incubated at −20°C overnight. Subsequent to centrifugation at 15000 × g for 20 min, the pellets were suspended in Tris-EDTA buffer consisting of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA (pH 8.0). The genomic DNA was separated using a 2% agarose gel and stained with 0.1 µg/ml ethidium bromide (19).

Flow cytometry analysis was performed as previously described (18). In brief, the cells treated with Z-guggulsterone or DMSO were harvested and fixed with 75% ethanol at −20°C. Subsequent to washing twice with cold PBS, the fixed cells were then suspended in 100 µl RNase A solution (1 mg/ml) and incubated at 37°C for 1 h. Next, 400 µl propidium iodide solution (50 µg/ml) was added to the cells, and the cells were subsequently incubated for 30 min in the dark. Following incubation, the apoptotic status of these cells was measured using a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The data were further analyzed using CellQuest Software (Becton-Dickinson). In the caspase inhibitor assay, the cells were treated with 60 µmol/l Z-guggulsterone for 72 h, with or without 2-h pre-treatment in 100 µM of the specific caspase inhibitors. Cellular apoptosis was then measured as aforementioned.

The activity of caspases-3, -8 and -9 was determined using the colorimetric assay kit, according to the manufacturer's instructions. In brief, the cells were lysed in buffer containing 5 mM DTT (Solar Biotechnology, Inc.), 10 µg/ml aprotinin (Santa Cruz Biotechnology, Inc.), 20 mM EDTA (Solar Biotechnology, Inc.), 50 mM HEPES (SBS Genetech Co.), 0.2% Triton X-100 (Santa Cruz Biotechnology, Inc.), 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology, Inc.) at 4°C for 10 min. Following centrifugation at 10,000 × g for 10 min at 4°C, the supernatant was collected and the protein concentration was measured. Subsequently, 100 µg protein was incubated with 0.2 mM DEVD-pNA, IETD-pNAu or LEHD-pNA (Santa Cruz Biotechnology, Inc.), which are substrates of caspases-3, -8 and -9, respectively. The caspase activity was determined from the absorbance at 405 nm using the microplate reader.

Total protein was extracted using the RIPA lysis buffer containing protease inhibitors. Equal amounts of protein (20–50 µg) were then loaded and separated by 12% SDS-PAGE and transferred onto the nitrocellulose membranes. For the blocking procedure, the membranes were incubated in 5% non-fat milk for 1 h. Appropriate primary antibodies were added to the membrane and the membrane was then incubated at room temperature for 2 h. Next, the membranes were incubated with the appropriate secondary antibodies for 2 h at room temperature. The specific protein bands were then developed using a commercial enhanced P1701 chemiluminescence kit (Applygen Technologies Inc., Beijing, China). The staining density was measured using the GS-710 Imaging Densitometer, which was obtained from Bio-Rad Laboratories (Hercules, CA, USA).

All experiments were performed in triplicate and repeated at least three times. The data were expressed as the mean ± standard deviation. Student's t-test was used to determine the significance of the differences between groups. SPSS software (version 15.0; SPSS, Inc., Chicago, IL, USA) was used to perform all statistical analyses and P<0.01 was considered to indicate a statistically significant difference.

To investigate the cytotoxicity of guggulsterone on cholangiocarcinoma cells, the Sk-ChA-1 and Mz-ChA-1 cells were incubated with Z-guggulsterone for 24, 48 and 72 h. As demonstrated by the present data, treatment with Z-guggulsterone resulted in significant growth suppression of the two cell lines in a dose- and time-dependent manner (P<0.001 vs. control; Fig. 2). In addition, the Mz-ChA-1 and Sk-ChA-1 cells were sensitive to treatment with Z-guggulsterone. In the group of cells treated with 60 µmol/l Z-guggulsterone for 72 h, the cell viability of Sk-ChA-1 and Mz-ChA-1 cells was 18.78 and 26.98%, respectively.

Hoechst 33258 staining and DNA fragmentation analysis were then performed to investigate whether cytotoxicity induced by guggulsterone resulted from cellular apoptosis. According to the results of Hoechst staining, 72-h treatment with Z-guggulsterone led to notable morphological alterations, such as chromatin condensation and nuclear fragmentation, which were observed in each of the two cholangiocarcinoma cell lines (Fig. 3A). In the agarose gel electrophoresis, a DNA fragment ladder was detected subsequent to 48-h treatment with guggulsterone, which was more notable subsequent to 72-h treatment in each of the two cell lines (Fig. 3B). These findings indicated that that guggulsterone may lead to the death of cholangiocarcinoma cells by inducing cellular apoptosis. In addition, flow cytometry was performed to quantify the apoptosis induced by guggulsterone. As revealed in Fig. 3C, there was a significant dose- and time-dependent increase in the proportion of Sk-ChA-1 and Mz-ChA-1 cells in the sub-G1 phase subsequent to treatment with ≥20 µmol/l Z-guggulsterone (P<0.001 vs. control). In addition to the increase of cell growth suppression, cellular apoptosis was also upregulated gradually and the sub-G1 population of the two cells was >50% when the cells were treated with 60 µmol/l Z-guggulsterone for 72 h.

Considering that caspases play critical roles in cellular apoptosis, the effects of caspase inhibition on Z-guggulsterone-induced apoptosis were then explored. Three caspase inhibitors, consisting of pan-caspase, caspase-8 and caspase-9 inhibitors, were used in the present study. As shown in Fig. 4A, these inhibitors significantly impaired the apoptosis induced by Z-guggulsterone in Sk-ChA-1 (P<0.01) and Mz-ChA-1 (P<0.001) cells, compared with the cells treated with Z-guggulsterone.

The activity of caspases-3, -8 and -9 following treatment with 60 µmol/l Z-guggulsterone was then assessed using colorimetric assay kits. In the Sk-ChA-1 and Mz-ChA-1 cell lines, the highest activity of caspases-8 and -9 was detected at subsequent to treatment for 48 h, which was followed by a further enhancement of caspase-3 activity subsequent to 72 h of treatment (Fig. 4B). In addition, the level of cleaved PARP, a hallmark of apoptosis, significantly increased following treatment with 60 µmol/l Z-guggulsterone in a time-dependent manner (Fig. 4C). Overall, the present findings indicated that guggulsterone may induce cytotoxicity in cholangiocarcinoma cells through caspase-dependent apoptosis.

According to the results of western blot analysis, the protein levels of survivin and Bcl-2 were markedly downregulated in a time-dependent manner, while the expression of Bax remained constant (Fig. 5), indicating the important roles of survivin and Bcl-2 during the apoptosis induced by guggulsterone.