Human CFPAC-1 pancreatic ductal adenocarcinoma and PANC-1 pancreatic cancer cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The 2 cell lines were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin in a 37°C humidified atmosphere containing 5% CO₂/95% air.

CFPAC-1 or PANC-1 cells were cultured in 96-well plates at 1×10⁵ cells per well. After 24 h, the cells were treated with multiple concentration of BAI alone (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50 and 100 µM), GEM alone (0.03, 0.06, 0.125, 0.25, 0.5 and 1 µg/ml) or combination treatment (0.03, 0.06, 0.125, 0.25, 0.5 and 1 µg/ml GEM with either 1.6 or 6.25 µg BAI) for 48 h, the control group was treated with PBS (pH 7.4). Subsequently, 50 µl MTT (1 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to the cell media and incubated for 4 h at 37°C. MTT was discarded and 150 µl DMSO was loaded in each well. The spectrophotometric absorbance of the samples was measured using a microplate reader (Bio-Rad Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 570 nm with a reference wavelength of 655 nm. The percentage of cell survival was calculated using the following formula: Cell survival=absorbance value of infected cells/absorbance value of uninfected control cells. Six reduplicate wells were measured at each concentration and every experiment was performed at ≥3 times.

CI was used to evaluate the pharmacological interactions of different combinations of BAI and GEM (15). Briefly, synergism, additivity or antagonism in the BAI and GEM combinations was calculated on the basis of the multiple drug effect equation and quantitated by CI, where CI=1 indicates that the two drugs have additive effects, CI<1 indicates synergism effects and CI>1 indicates antagonism effects. The CI was calculated based on: CI=(D)1/(Dx)1+(D)2/(Dx)2+(D)1(D)2/=(Dx)1(Dx)2, where (Dx) is the dose of the drug, inhibiting ‘x%’.

Following GEM (0.125 µg/ml), BAI (1.60 or 6.25 µM) or combination treatment, the control group was treated with PBS, the cells were fixed and stained using Hoechst 33258 for 10 min at room temperature and observed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) at ×20 magnification. A total of 6 fields per treatment were assessed. The stained cells were identified as apoptotic if they were highly condensed with brightly stained nuclei, or non-apoptotic if they were stained pale blue.

CFPAC-1 or PANC-1 cells treated with 0.125 µg/ml GEM or 1.6/6.25 µM BAI alone or a combination treatment were incubated for 48 h at 37°C, the control group was treated with vehicle solution, and then cells were collected and washed twice with cold PBS and subsequently incubated with Annexin V-FITC/7-ADD double staining solution at room temperature for 15 min. Then, the stained cells were analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA). The cell apoptotic rates were analyzed using ModFIT software (version 3.2; Verity Software House, Inc., Topsham, ME, USA). Apoptotic events were recorded as a combination of Annexin V⁺/7-AAD⁻ (early apoptotic) and Annexin V⁺/7-AAD⁺ (late apoptotic/dead) events. This experiment was conducted 3 times.

Whole cell lysates from CFPAC-1 cells treated with vehicle control or drugs for 48 h, then were washed using PBS once and lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche Diagnostics), and sonicated for 15 min on ice. Protein concentration was measured using a BCA protein assay (Bio-Rad Laboratories, Inc.). A total of 30 µg samples were separated by SDS-PAGE on a 10% gel and transferred to polyvinylidene difluoride membranes using the Bio-Rad electrotransfer system (Bio-Rad Laboratories, Inc.). The membrane was blocked using 5% fat-free milk for 1 h at room temperature and then incubated with appropriate primary antibody diluted in 3% BSA solution at 4°C overnight. For immunodetection, the following antibodies were used for our analysis: anti-Bax (cat no. 5023), -Bcl-2 (cat no. 15071), -Caspase-3 (cat no. 9662), -PARP (cat no. 9532) and -Survivin (cat no. 2808) antibodies (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), followed by incubation with DyLight dye-conjugated secondary antibodies (cat nos. 35502 and 35568; 1:10,000; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Blots were scanned using the Odyssey Western Detection system (LI-COR Biosciences, Lincoln, NE, USA), followed by quantification using ImageStudio software (version 5.2.5; LI-COR Biosciences). Anti-β-actin (cat no. sc-47778; Santa Cruz Biotechnology, Inc.; 1:1,000 dilution) was used as a loading control.

A total of 24 female BALB/c nude mice (4–5 week-old, 15–16 g) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). Mice were allowed free access to sterilized water and food and were housed in individual ventilated cages at 23±5°C under a 12-h light/dark cycle. CFPAC-1 cells were resuspended in serum-free DMEM at a concentration of 1×10⁷ cells/ml. A total volume of 0.2 ml cell suspension (total 2×10⁶ cells) was then injected subcutaneously into the right anterior armpit of nude mice to establish a xenograft model. The 24 injected mice were randomly divided into 4 groups: control, BAI, GEM and combination treated groups (n=6 per group). Control group was treated with vehicle solution. The mice were sacrificed at day 28 or when the tumor grew to a maximum of 1,500 µM³. The Animal Care and Use Committee of Nanjing University of Chinese Medicine ethically approved the experimental protocol.

Tumor xenografts were fixed in formalin solution overnight at room temperature and embedded in paraffin blocks, which were sliced into 4-µm thick sections for immunohistochemical staining. Following deparaffinization, the sections were incubated at 4°C overnight with antibodies of Bcl-2 (cat no. 15071), Bax (cat no. 5023), caspase-3 (cat no. 9662) and survivin (cat no. 2808) (1:500; Cell Signaling Technology, Inc.). The sections were washed 3 times with PBS with 0.1% Tween-20 (PBST), and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody (1:200; cat no. sc-2031; Santa Cruz Biotechnology, Inc.). The slices were washed with PBST 3 times, and incubated in diaminobenzidine (Sangon Biotech Co., Ltd., Shanghai, China) for 5 min at room temperature and counterstained with hematoxylin (Sangon Biotech Co., Ltd.) for 1 min at room temperature. Images were captured using a light microscope (Olympus Corporation) at a magnification of ×20. Five sections per tumor tissue were examined.

All experiments were repeated at ≥3 times and all data are presented as the mean ± the standard error of the mean. Graph Pad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA) was use to perform statistical analysis. Comparisons among multiple groups were determined using a one-way or two-way analysis of variance test (two-way analysis of variance was used for tumor volume analysis) followed by Dunnett's or Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To test the potential of the cytotoxicity of BAI, the human pancreatic cancer cell line CFPAC-1 was incubated with a range concentrations (0.1–100 µM) of BAI for 48 h and then the number of cells in each group was determined. Treatment with higher concentrations of BAI (3.2–100.0 µM) significantly inhibited the viability of CFPAC-1 cells compared with the control group (P<0.001; Fig. 1A). For the cells treated with 0.125 µg/ml GEM, the number of viable cells decreased by 32% compared with the control group (Fig. 1B). Treatment with BAI in addition to GEM or GEM alone revealed that the viability of CFPAC-1 was inhibited in a concentration-dependent manner. Subsequently, to assess the synergistic effect of GEM in combination with BAI on cell proliferation, CFPAC-1 cells were incubated with a range of concentrations of GEM, in combination with 1.6 or 6.25 µg BAI for 48 h. MTT assays revealed that combined treatment of either concentration of BAI with 0.125 µg/ml GEM was significantly more cytotoxic in CFPAC-1 cells compared with the same concentration of GEM alone (CI (0.125 ug/ml GEM and 1.6 µM BAI)=0.181; CI (0.125 ug/ml GEM and 6.25 µM BAI)=0.465; Fig. 1B). A similar effect was identified in the PANC-1 cell line, where 0.125 µg/ml GEM in combination with 6.25 µM BAI significantly decreased the cell viability compared with 0.125 µg/ml GEM treatment alone (Fig. 1C).

To determine if GEM and BAI treatment induces apoptosis, Hoechst 33258 staining and flow cytometric analysis were used. As indicated in Fig. 2, apoptosis analysis revealed that treatment with GEM or BAI alone induced minimal apoptosis; however, the combined treatment of GEM with 1.6 or 6.25 µM BAI resulted in a significant increase of the number of apoptotic cells was observed in CFPAC-1 and PANC-1 cell lines compared with the control (P<0.0001; Fig. 2). In addition, there was a significant increase in apoptosis in cells treated with GEM in combination with 6.25 µM BAI compared with GEM alone in CFPAC-1 and PANC-1 cell lines (P<0.001; Fig. 2). Furthermore, western blot analysis demonstrated that the expression levels of Bax, caspase-3 and PARP were significantly increased, and Bcl-2 and survivin were significantly decreased, in the combination BAI (1.6 or 6.25 µM) and GEM (0.125 ug/ml) treated group compared with the control group (P<0.001; Fig. 3), and compared with the BAI-alone treated group (P<0.01; Fig. 3). These results provide evidence that the synergistic cytotoxic effects of GEM and BAI against pancreatic cancer cells may be attributed to the induction of apoptosis.

In the CFPAC-1 xenograft nude mouse model, all mice survived the duration of the experiment with no observable toxic effect. Mice were sacrificed after 28 days and the weights and volume of the xenografts were measured. Compared with the control group, xenografts from the mice treated with BAI or GEM therapy were decreased in volume and weight (Fig. 4A and B). Furthermore, the combination therapy consisting of 6.25 µM BAI and 0.125 ug/ml GEM induced a significant decrease in tumor volume and weight compared with the control and with BAI treatment alone (P<0.0001; Fig. 4C and D). Immunohistochemical assays additionally revealed that the protein levels of Bcl-2, survivin, Bax and caspase-3 followed a similar pattern to that of the in vitro experiment, since Bcl-2 and survivin were significantly decreased and Bax and caspase-3 were significantly increased in the combined treatment group compared with the control group (P<0.0001; Fig. 4E-H). Furthermore, survivin, Bax and caspase-3 levels were significantly altered in the combination therapy group compared with the GEM-alone treated group (P<0.01; Fig. 4E-H).