A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), penicillin and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-glucose Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS), SDS, dimethyl sulfoxide (DMSO), Tris and Tween-20 were purchased from Beijing Dingguo Changsheng Biological Technology Co., Ltd. (Beijing, China). The Bradford Protein Assay kit, radioimmunoprecipitation assay (RIPA) lysis buffer, Annexin V-fluorescein isothiocyanate (FITC) Apoptotic Detection kit and Caspase Activity Assay kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). The primary antibodies against apoptosis-inducing factor (AIF; mouse monoclonal; cat. no. sc-55519), COX-2 (rabbit polyclonal; cat. no. sc-7951), cPLA2 (rabbit polyclonal; cat. no. sc-438) and secondary antibodies (rabbit anti-mouse IgG-HRP, cat. no. sc-358914; goat anti-rabbit IgG-HRP, cat. no. sc-2004) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-caspase-3 (rabbit polyclonal; cat. no. 9662) and anti-caspase-9 (mouse monoclonal; cat. no. 9508) antibodies were from Cell Signaling Technology, Inc. (Shanghai, China). β-actin primary antibody (rabbit monoclonal, cat. no. 1854-1) and GAPDH primary antibody (rabbit monoclonal, cat. no. 2251-1) were obtained from Epitomics, Inc. (Burlingame, CA, USA). BBR (Purity >99%) was donated by the Northeast Pharmaceutical Group Co., Ltd. (Shenyang, China) and was dissolved with 0.2% DMSO culture medium to a final concentration of 200 µM as a stock solution. All other chemicals and reagents were of analytical grade.

H22, HepG2 and Bel-7404 hepatoma cell lines and normal hepatic embryo HL-7702 cells, were maintained in DMEM high glucose medium, supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, in an atmosphere of 95% air and 5% CO₂ at 37°C. An MTT assay was performed to assess cell viability, which was performed in 96-well plates in octuplicate. The cells were seeded at a density of 5×10³ cells/well overnight, and treated with BBR at final concentrations of 0, 12.5, 25, 50 and 100 µM for 24, 48 or 72 h. Subsequently 20 µl MTT (5 mg/ml) was added to each well for the final 3 h of the 24, 48 or 72 h BBR treatment periods. Following this, the cell supernatants were discarded, the MTT crystals were dissolved with DMSO and the optical density (OD) was measured at 490 nm wavelength (3360063; Tecan Austria GmbH, Grödig, Austria). The ratio of cell proliferation to control group was calculated from the MTT data. IC₅₀ values of BBR were calculated from the percentages of cell viability obtained from the MTT assay.

Cell death and apoptosis were detected using an Annexin V-FITC Apoptotic Detection kit by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). In brief, following treatment with 0, 50 and 100 µM BBR at 37°C for 24 h, the HepG2 cells plated in six-well plates at a density of 5×10⁶ cells/well were harvested and washed twice with cold phosphate-buffered saline. Following this, the cell pellets were suspended with binding buffer (Beyotime Institute of Biotechnology) at a density of 1×l0⁶ cells/ml. Following the addition of 5 µl FITC-conjugated annexin V to the suspension, the suspension was incubated for 15 min at 4°C in the dark, followed by the addition of 5 µl prop-idium iodide (PI; Beyotime Institute of Biotechnology) for 5 min. The samples were subsequently analyzed using flow cytometry (BD FACSCalibur; BD Biosciences).

The cells were collected by centrifugation at 12,000 × g for 15 min at 4°C and were then washed twice with cold PBS. The cell pellets were suspended in 200 µl RIPA lysis buffer for 30 min at 4°C, vortexed every 10 min, then centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was collected as total protein extract. In order to investigate AIF translocalization into the nucleus, the nuclear protein extract was collected using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The protein concentration was measured using a Bradford Protein Assay kit. The protein extract (50 µg) was aliquoted by 12% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The blots were blocked with Tris-buffered saline containing 0.05% Tween-20 and 5% non-fat dried milk for 1 h at room temperature, then they were incubated with rabbit anti-mouse primary anti-COX-2, anti-cPLA2, anti-caspase-3, anti-caspase-9 and anti-AIF antibodies (all 1:1,000). The reaction was incubated overnight at 4°C with gentle agitation, following which the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:2,000) for 2 h at room temperature with gentle agitation. The membranes were then washed and the bands were visualized using an Enhanced Chemiluminescence Western Blotting Detection system (GE Healthcare Life Sciences, Chalfont, UK).

The cells were plated in 96-well plates in RPMI-1640 medium containing 10% (v/v) FBS. When ~70% confluence was reached, the cells were treated with BBR at concentrations of 0, 12.5, 25, 50 or 100 µM for 24 h. At the end of treatment, the culture supernatant was collected and centrifuged (10,000 × g, 3 min at 25°C) to remove cells or cell debris. The concentrations of PGE2 and AA in the supernatant were measured using an enzyme immunoassay (PGE2 EIA and AA monoclonal EIA kits; Cayman Chemical Company, Ann Arbor, MI, USA), according to the manufacturer's instructions. The production of PGE2 (pg/ml) was normalized to the cell counts/well.

A total of 50 male BALB/c mice (6–8 weeks old, weighing 18–22 g) were purchased from the Experimental Animal Center of Norman Bethune College of Medicine, Jilin University (Changchun, China). The mice were housed 5/cage under a 12/12 h light/dark cycle with ad libitum access to food and water. The mouse production permit number was SCXK (JI) 2010-0001 and the usage license was SYXK (JI) 2010-0001. The animal experiments in the present study were performed in accordance with the Good Laboratory Practice Guidelines (18) and the experimental protocol was approved by the Ethics Committee of the Norman Bethune Health Science Center of Jilin University (Changchun, China). In brief, H22 cells (2×10⁶) suspended in 0.1 ml PBS were injected subcutaneously into the right side of the dorsal flank of each mouse. The mice were randomly divided into the following five groups, each containing 10 mice: Control group, positive control cyclophosphamide (CTX) group, 12.5 mg/kg BBR group, 25 mg/kg BBR group and 50 mg/kg BBR group. The mice in the control group were treated with vehicle (water; 2 ml/kg body weight). Mice in the BBR groups were administrated with a daily gavage of BBR (dissolved in water), beginning the day following transplantation of H22 mice hepatoma tumor cells. The tumor volumes were measured every day, calculated according to the following formula: Length × width² × 0.52. Body weights were also measured every day. The mice were sacrificed ~2 weeks subsequent to treatment.

All data are expressed as the mean ± standard error of the mean. The statistical significance of the data was compared using Student's t-test with SPSS software, version 14.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the anticancer characteristics of BBR, the viability of the cells was measured using an MTT assay following exposure to BBR for different time-periods (24, 48 or 72 h). It was found that BBR significantly reduced cell viability in mouse model and human hepatoma cell lines in a time- and dose-dependent manner. BBR also affected the viability of normal cells folloing longer durations of exposure (48 and 72 h), however, the IC₅₀ values in the HL-7702 hepatic embryonic cell line (122.4 µM) at 72 h were markedly higher than the Bel-7404, H22 and HepG2 HCC cell lines (9.21, 43.2 and 82.8 µM, respectively). The observed IC₅₀ values indicated that hepatoma cells are more susceptible to BBR, compared with normal cells (Fig. 1).

The HepG2 cells were treated with 50 and 100 µM of BBR for 24 h. Apoptosis was detected using flow cytometric analysis with annexin V-PI staining. As shown in Fig. 2, HepG2 cells without BBR treatment were observed in the quadrant (Q)3, however, BBR treatment significantly induced early apoptosis, indicated by the distribution of the cells in Q4, and late apoptotic cells, indicated by distribution of the cells in Q2, in a dose-dependent manner. The total apoptotic rates were 24.13±2.14 and 55.55±2.86% in the 50 and 100 µM BBR treatment groups, respectively. These results suggested that the BBR-induced apoptosis in the tumor cells contributed to its anti-proliferative and cytotoxic efficacies.

In order to elucidate how BBR exerts its effects on apoptosis in HepG2 cells, the effects of BBR on the intrinsic apoptotic pathways of caspase-3 and caspase-9 were investigated. The results demonstrated that the protein levels of total caspase-3 and total caspase-9 were not significantly altered following BBR treatment. In addition, the no protein expression of cleaved caspase-3 or cleaved caspase-9 proteins were detected using western blotting (Fig. 3). Similar results were observed in the caspase-3 activity assay, which demonstrated that BBR had no significant effect on the activity of caspase-3 (data not shown). To further examine the apoptotic mechanisms of BBR, total AIF and nucleic AIF were assessed using western blot analysis. The results demonstrated that a wide range of BBR concentrations between 12.5 and 100 µM, induced significant increases in the protein expression of nucleic AIF, however, the protein levels of total AIF were not affected. These results indicated that there is a potential translocation of AIF between the mitochondria and the nucleus, which is likely to be a pivotal factor of apoptosis in the BBR-treated HCCs.

To investigate how BBR affects the AA metabolic pathway, two key enzymes (cPLA2 and COX-2) in the pathway were assessed using western blot analysis. As shown in Fig. 4A, the protein expression levels of cPLA2 and COX-2 were suppressed significantly in a dose-dependent manner between concentrations of 12.5–100 µM BBR in the H22 and HepG2 cells. The contents of AA and PGE2 in the culture medium of the H22 and HepG2 cells treated with BBR for 24 h were then measured. As shown in Fig. 4B, the content of AA was significantly increased, and increased as the dose of BBR increased. The level of PGE2 was significantly reduced, even with treated with a low dose of BBR (12.5 µM). The concentration ratio of AA to PGE2 was increased following BBR treatment in the H22 and HepG2 cells, suggesting that the ratio may be important in BBR-induced apoptosis in the tumor cells.

To evaluate the efficacy of BBR on tumor growth in vivo, a H22 transplanted tumor model was established in BALB/c mice. As shown in Fig. 5A, the tumor volume in the control group reached the logarithmic growth phase 12 days following inoculation, and the same inhibitory effects were observed in the 50 mg/kg BBR and the CTX positive control group. Treatment with BBR reduced tumor volume in a dose-dependent matter (Fig. 5A), and this was confirmed by the ratios of tumor weight relative to mouse body weight (Fig. 5B).