HepG2 and QGY7701 cells were provided by The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum plus 100 U/ml penicillin and 100 µg/ml streptomycin (all Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C, in a humidified atmosphere of 5% CO₂. Chrysin was diluted to various concentrations with cell culture media. After the cells had reached 60% confluence, various concentrations of chrysin were added (0, 10, 15, 20, 25, 30, 40 and 50 µg/ml).

Cells were seeded in 96-well plates at a density of 5,000–10,000 cells/well. Different concentrations of chrysin (0, 10, 15, 20, 25, 30, 40 and 50 µg/ml; 5 replicates per concentration group) were added to the cell cultures for 24 h, followed by 20 µl MTT solution (5 mg/ml; Beyotime Institute of Biotechnology, Shanghai, China). After incubation for 4 h at 37°C in an atmosphere of 5% CO₂, supernatants were removed and 150 µl dimethyl sulfoxide (Beyotime Institute of Biotechnology) was added. The plates were then placed on an orbital shaker for 10 min. Subsequently, the absorbance at 490 nm was measured using a spectrophotometer (EnSpire 2300 Multilabel reader; PerkinElmer Inc., Waltham, MA, USA). The inhibitory rate was calculated according to the following formula: (A490_control-A490_treated)/(A490_control-A490_blank) ×100%.

Apoptotic cells were detected using an Annexin V-FITC/PI kit (BioVision Inc., Milpitas, CA, USA). HepG2 and QGY7701 cells were seeded in 6-well plates at a density of 1×10⁵ cells/well. According to the results of the MTT assay, the half maximal inhibitory concentration (IC₅₀) concentration of chrysin was acquired for each cell line using GraFit-Erithacus IC₅₀ software (Erithacus Software Ltd., Horley, UK). Lower (20 µg/ml) and higher dosages (40 µg/ml) were selected for the cell apoptosis assay. Cells were incubated in different concentrations of chrysin (0, 20 and 40 µg/ml) for 24 h. The samples were then washed twice with cold D-Hank's buffer solution and resuspended in binding buffer (1×10⁶ cells/ml). Subsequently, 100 µl cell supernatants were transferred to a tube with 5 µl Annexin V-FITC (BD Biosciences, Franklin Lakes, NJ, USA) and 5 µl PI (Beyotime Institute of Biotechnology). Following incubation for 15 min at room temperature in the dark, the apoptotic cells were detected using flow cytometry (FACS Canto II; BD Biosciences), and analyzed using Modfit and CellQuest 5.1 software (BD Biosciences). Cells located in the Q4 zone were deemed to be in the early apoptosis stage, whereas cells in the Q2 zone were in the late apoptosis stage.

Drug-induced cell apoptosis is due to mitochondrial dysfunction and apoptotic signaling pathway activation (13). p53, B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter (Bad), Bcl-2-associated X (Bax), Bcl-2 homologous antagonist/killer (Bak), caspase-9 and caspase-3 are associated with apoptosis-induced mitochondrial damage (14); therefore the expression levels of these proteins were investigated. Cultured cells were collected and total protein was extracted using radioimmunoprecipitation assay lysis buffer [50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM EDTA and 40 µg.ml leupeptin], containing 1 mM phenylmethylsulfonyl fluoride (both Beyotime Institute of Biotechnology). After laying the extraction on ice for 30 min, cell lysate was centrifuged for 12,000 × g for 10 min at 4°C and the supernatants were collected. The total protein concentration in each supernatant sample was measured using a BCA protein assay kit (P0012; Beyotime Institute of Biotechnology). Subsequently, proteins (20 µg/lane) were separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Following blocking with 5% non-fat milk, the membrane was incubated with specific primary antibodies at a dilution of 1:1,000 for 2 h at room temperature. The following primary antibodies were used: p53 (2527), Bcl-2 (2870), Bax (5023), Bak (6947), Bad (9239), caspase-3 (9665), caspase-9 (9505) and GAPDH (2118; all Cell Signaling Technology, Beverly, CA, USA). The membrane was then washed three times in Tris-buffered saline-Tween 20 (TBST) for 5 min, and incubated with a goat anti-rabbit horseradish peroxidase-labeled secondary antibody (E030120-01; EarthOx Life Sciences, Millbrae, CA, USA) at a dilution of 1:5,000 for 1 h at room temperature. Following three washes in TBST (10 min each), the membranes were exposed to an enhanced chemiluminescence buffer (EMD Millipore, Billerica, MA, USA) and measured on an Sally Sue western blot imaging system (ProteinSimple, San Jose, CA, USA). Gel images were then analyzed using ImageJ (National Institutes of Health, Bethesda, MA, USA) to calculate the gray value of each band.

Experiments were repeated ≥3 times and all data were analyzed using Graphpad Prism 5.0 software. Between-group differences were analyzed using Student's t-test. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference (*P<0.05; **P<0.01; ***P<0.001).

Following treatment of the QGY7701 and HepG2 cells with chrysin, the cells were found to slow growing, distorted, round in shape and detached from the bottom of the plate. Furthermore, the numbers of detached cells increased with increasing drug concentration (Figs. 2A and B). MTT assay was used to evaluate the viability of QGY7701 and HepG2 cells treated with chrysin. The present results revealed that, after 24 h of treatment, chrysin significantly inhibited cell viability in the HCC cell lines in a dose-dependent manner (P<0.001; Figs. 2C and D). IC₅₀ is an index for the evaluation of drug sensitivity. The IC₅₀ values of chrysin in QGY7701 and HepG2 cells were measured using GraFit-Erithacus IC₅₀ software and were found to be 18 and 25 µg/ml, respectively.

The apoptosis of the QGY7701 and HepG2 cells was detected using Annexin V/PI double staining and flow cytometric analysis. The results indicated that chrysin induced HCC cell apoptosis. Chrysin induced-apoptosis in HepG2 and QGY7701 cells was found to significantly increase in a concentration-dependent manner (P<0.001; Fig. 3). QGY7701 cells were found to be more sensitive to chrysin treatment, with higher levels of apoptosis observed compared with the HepG2 cells.

HCC cells were treated with various concentrations of chrysin for 24 h. Apoptosis protein expression levels of cleaved caspase-3 and caspase-9 were found to significantly increase in a concentration-dependent manner following chrysin treatment in the QGY7701 and HepG2 cell lines (P<0.001; Fig. 4). These results indicated that the caspase-9/caspase-3-associated apoptosis pathway was activated. In addition, the possible activation of the p53/Bcl-2 signaling pathway was also investigated based on the protein expression levels of p53, Bcl-2, Bax, Bad and Bak. The results demonstrated that this intrinsic apoptosis pathway was also activated following chrysin treatment in the two HCC cell lines (P<0.001; Fig. 4).