Human gallbladder cancer (GBC-SD) and human cholangiocarcinoma (RBE) cell lines were obtained from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). GBC-SD and RBE cells were maintained in RPMI-1640 (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C.

BSO was purchased from Sigma-Aldrich (St. Louis, MO, USA). Gemcitabine was purchased from Jiangsu Hansoh Pharmaceutical Co., Ltd. (Lianyungang, China), and cisplatin was obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China).

Human GBC-SD and RBE cells were pretreated with 50 µM BSO for 24 h before exposure to 4 or 8 µg/ml cisplatin or 0.5 mg/ml gemcitabine for 24 h. The cells were then collected and the cytotoxic effects examined.

Cell viability was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich), as previously described (26). Briefly, the cells were seeded in a 96-well plate at a density of 10,000 cells/well. Following overnight incubation in a humidified atmosphere of 5% CO₂ at 37°C, each well was refreshed with 0.2 ml serum-free medium (SFM) containing 50 µM BSO for a further day. The cells were then pretreated with 0.2 ml SFM containing 50 µM BSO for 24 h. Gemcitabine (500 µg/ml) or cisplatin (4 or 8 µg/ml) were subsequently added to the medium for an additional 24 h. Cells were not washed between treatments. Finally, cell viability was assessed with an MTT reagent and by measuring the absorbance at a wavelength of 570 nm using a VersaMax™ ELISA Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA). Relative viability was obtained from the absorbance of the drug-treated cells divided by that of the untreated cells. The same experiment was repeated three times.

Cell apoptosis was assessed using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Pharmingen, San Diego, CA, USA) and analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) (27). Briefly, the cells were seeded into 6-well plates and treated with BSO, gemcitabine, cisplatin, BSO/gemcitabine or BSO/cisplatin. The cells were collected 24 h later and washed twice using cold phosphate-buffered saline (Gibco; Thermo Fisher Scientific, Inc.). The cells were then stained using an Annexin V/PI double staining solution at room temperature. After 15 min, the Annexin V/PI-stained cells were analyzed by flow cytometry, and the percentage of apoptotic and necrotic cells was calculated. Cells that were positively stained by Annexin V-FITC only (early apoptosis), or positive for Annexin V-FITC and PI (late apoptosis/necrosis) were quantitated, and these two sub-populations were considered as the overall population of apoptotic cells.

GSH is a tripeptide with a free thiol group that functions as a major antioxidant in cells. Usually, cellular GSH exists predominantly in its reduced form, whereas GSSG is present in small amounts (28). The GSH/GSSG ratio is often used as an indicator of cellular redox status (28). Total GSH and GSSG levels were determined by colorimetric microplate assay kit (Beyotime Institute of Biotechnology, Haimen, China) as previously described (29,30). Following treatment with 50 µM BSO, cells were collected by centrifugation at 10,000 × g for 10 min at 4°C and re-suspended in 20 µl cell culture medium. Cells (10 µl) were mixed with 30 µl 5% metaphosphoric acid (Beyotime Institute of Biotechnology), then frozen and thawed twice in liquid nitrogen and 37°C water respectively. The samples were centrifuged again (using the same conditions) and the supernatant was used for GSH and GSSG assays. The total GSH level was measured by performing a DTNB-GSSG recycling assay (29). The GSSG level was quantified by the same method as for total GSH after the supernatant was treated with 1 mol/l 2-vinylpyridine solution to remove the reduced GSH. The quantity of reduced GSH was obtained by subtracting the quantity of GSSG from that of total GSH. The GSH/GSSG ratio was calculated using the following formula: Ratio = (total GSH − 2GSSG) / GSSG.

Myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma 2 (Bcl-2) protein expression was determined by western blot analysis as previously described (31). Cells were lysed in sample solution. Proteins were separated with 10% SDS-PAGE gels (Sangon Biotech, Shanghai, China) run at 80 V for 30 min, then 120 V for 60 min at room temperature, and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% milk in Tris-buffered saline in Tween 20 buffer (TBST; Gibco; Thermo Fisher Scientific, Inc.), the membrane was incubated overnight at 4°C with polyclonal rabbit anti-human Bcl-2 (1:200 dilution; cat no. sc-492) and Bcl-xl (1:200 dilution; cat no. sc-7195; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies, and monoclonal rabbit anti-human Mcl-1 antibody (1:800 dilution; cat no. ab32087; Abcam, Cambridge, UK). The membranes were also incubated with monoclonal mouse anti-human β-actin antibody (1:1,000 dilution; cat no. sc-47778; Santa Cruz Biotechnology, Inc.) as the loading control. Following incubation, the membranes were washed three times with TBST. The membranes were subsequently incubated with goat anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (cat nos. 111-035-003 and 115-035-003; Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) for 2 h at 37°C, prior to detection using ECL system (cat no. WBKLS0500; EMD Millipore).

Data are presented as the mean value ± standard deviation. SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Analysis of variance was applied for comparison of the means of two or multiple groups. A value of P<0.05 was considered to indicate statistically significant differences.

To examine the synergistic effect of BSO on the inhibition of cell viability by cisplatin, GBC-SD human gallbladder cancer cells and RBE cholangiocarcinoma cells were pretreated with 50 µM BSO for 24 h before exposure to cisplatin for 24 h. Cell metabolic activity was measured using an MTT assay. No significant difference in cell viability was detected between untreated cells and cells treated only with BSO (P>0.05). However, compared with the control, GBC-SD and RBE cells that were not pretreated with BSO experienced 20 and 12% decreases in viability, respectively, following 24-h exposure to cisplatin (P<0.0001 and P=0.0029, respectively). In addition, pretreatment of BTC cells with BSO followed by treatment with cisplatin resulted in reductions in cell viability of 44% and 43% in the GBC-SD and RBE cell lines, respectively (Fig. 1A and B).

To determine whether the reduction in cell viability could be attributed to an increase in apoptosis, Annexin V-FITC/PI double-labeling flow cytometry was conducted. Compared with cisplatin alone, pretreatment with BSO followed by cisplatin treatment resulted in an increase in apoptosis in GBC-SD cells (P=0.0013; Fig. 1C and D). Similarly, the BSO/cisplatin co-treatment also led to an increase in apoptosis in RBE cells (P=0.0374; Fig. 1E and F). Together, these data demonstrate that BSO is capable of enhancing cisplatin-induced apoptosis in BTC cells.

BSO has been demonstrated to deplete intracellular GSH and induce oxidative stress (19–27,29–32), and this may sensitize various types of cancer cells to certain drugs (33–36). To study the oxidative impact of BSO on the cellular redox state of BTC cells, intracellular GSH levels and the GSH/GSSG ratio were measured in GBC-SD cells treated with 50 µM BSO. Notably, BSO reduced the intracellular GSH levels in a time-dependent manner (P=0.0321; Fig. 2A). Furthermore, BSO decreased the GSH/GSSG ratio, a reflection of the cellular redox state (P=0.0016; Fig. 2B). These data provide evidence that the enhancement of BSO on cisplatin-induced apoptosis in BTC cells is associated with the depletion of GSH.

Antiapoptotic proteins, including Bcl-2, Bcl-xL and Mcl-1, are overexpressed in numerous types of cancer cells and contribute to tumor drug resistance (37). Therefore, inhibition of their expression may lead to an increased sensitivity to anticancer drugs. To better understand the mechanism responsible for the ability of BSO to overcome cisplatin resistance, the expression of these antiapoptotic proteins was analyzed in lysates from BTC cells treated with 50 µM BSO for 24 h. Results from western blot analyses revealed that BSO effectively downregulated Mcl-1, Bcl-2 and Bcl-xL expression (Fig. 3). The observation that BSO increases the cytotoxicity of cisplatin may be explained, at least partially, by downregulation of antiapoptotic proteins in BTC cells.

Patients with advanced BTC are currently being treated with a combination of cisplatin and gemcitabine (10). Therefore, the potential synergistic effect of BSO with gemcitabine was also examined. Cell viability was assessed following the treatment of BTC cells with BSO and/or gemcitabine. The results revealed that a dose of 500 µg/ml gemcitabine was significantly more effective at reducing cell viability when it was combined with 50 µM BSO in GBC-SD (P=0.0020) and RBE (P=0.0005) cells (Fig. 4A and B). These results suggest that the effects of BSO are not specific to cisplatin and that it may also enhance the antiproliferative effect of gemcitabine in BTC cells.

To determine whether BSO enhances the induction of apoptosis by gemcitabine, the induction of apoptosis in BTC cells treated with BSO and/or gemcitabine was investigated by Annexin V-FITC/PI flow cytometry. No increase in apoptosis was detected in the two cell lines following treatment with gemcitabine alone or treatment with BSO and gemcitabine (P>0.05; Fig. 4C and D).