SFN (purity, 98.3%) was purchased from Alexis Biochemicals; Enzo Life Science (Farmingdale, NY, USA). The following additional materials were obtained: Adriamycin (ADR; 110826; Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou, China); RPMI-1640 culture medium and pancreatin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA); Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit, rabbit anti-human binding immunoglobulin protein (Bip)/glucose-regulated protein 78 (GRP78) antibody (cat. no. AF0171), prestained protein molecular weight marker (cat. no. P0062), cell lysis buffer, and alkaline phosphatase-labeled sheep anti-rabbit immunoglobulin (Ig) G (catalog no. A0239) (Beyotime Institute of Biotechnology, Shanghai, China); mouse anti-β-actin antibody (cat. no. TA-09), peroxidase-conjugated affinipure goat anti-mouse IgG (H+L; cat. no. ZB-2305; Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China); Tween 20 and bovine serum albumin (BSA; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany); Coomassie Brilliant Blue G250, Triton X-100 and paraformaldehyde (Beijing Chemical Reagents Company, Beijing, China); rabbit anti-human X-box binding protein-1 (XBP-1; cat. no. bs-1668R), C/EBP homologous protein (CHOP)/growth arrest- and DNA damage-inducible gene 153 (GADD153; cat. no. bs-8875R), BH3 interacting domain death agonist (Bid; cat. no. bs-1153R), caspase-12 (cat. no. bs-23014R), and FITC-conjugated goat anti-rabbit antibodies (cat. no. bs-0295G-FITC; BIOSS, Beijing, China).

The following laboratory equipment was used throughout the experiments of the present study. Super-clean bench (JJT-900/1300; Suzhou SuJie Purifying Equipment Co., Ltd., Suzhou, China); microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA); electrophoresis apparatus (DYY-24D, Beijing Liuyi Instrument Factory, Beijing, China); high-speed centrifuge at low temperature (Beckman Coulter, Inc., Brea, CA, USA); flow cytometer (COULTER^® EPICS^® XL™; Beckman Coulter, Inc.); CO₂ incubator (MC0175; SANYO Electric Co., Ltd., Moriguchi, Japan); and gel imaging system (GIS-2019, Tanon Science & Technology Co., Ltd., Shanghai, China).

The HepG2 human hepatocellular carcinoma cell line was obtained from the Research Center of Life Sciences and Environmental Sciences, Harbin University of Commerce (Harbin, China). HepG2 cells were maintained in culture flasks containing RPMI-1640 supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). The cultures were incubated at 37°C in an atmosphere containing 5% CO₂ with saturated humidity. Cells were transferred to fresh medium once every 2 to 3 days.

Logarithmic phase HepG2 cells were seeded in a 96-well plate at a density of 5×10⁴ cells/ml (100 µl/well). Following a 24 h incubation at 37°C, 100 µl of the drugs were added to each well. The final concentrations of SFN used were 2.5, 5, 10, 20, 40 and 80 µmol/l; the final concentrations of ADR were 0.01, 0.1, 1, 10 and 100 µmol/l. Following treatment with SFN or ADR for 24, 48 and 72 h, drugs were discarded and the cells were incubated with 100 µl MTT (0.5 mg/ml) for 4 h at 37°C. After 4 h, the supernatant was aspirated, and 200 µl dimethyl sulfoxide was added. Finally, the absorbance was measured at 570 nm using a microplate reader, which was used to calculate the rate of inhibition and the half maximal inhibitory concentration (IC₅₀).

After placing coverslips in a 6-well plate, 3×10⁵ HepG2 cells (1 ml) were seeded in each well and allowed to attach overnight. Cells were cultured with various concentrations of SFN (10, 20 and 40 µmol/l). ADR was added as a positive control at a final concentration of 0.5 µmol/l, and the control group was supplemented with equal volumes of RPMI-1640 culture medium. Following 48 h of treatment with SFN or ADR at 37°C, the cells were digested with pancreatin and washed once with phosphate-buffered saline (PBS). The cells were then fixed with a fixing solution composed of methyl alcohol and glacial acetic acid (3:1) for 10 min at 4°C. After further washing with PBS, 5 mg/l Hoechst 33258 fluorescent probe was added to the cells and incubated for 15 min. The cover slips were hand washed with PBS and placed on glass slides containing drops of glycerin. Finally, the cells were visualized and images were captured under an inverted fluorescence microscope.

Logarithmic phase HepG2 cells were seeded in 6-well plates at a density of 3×10⁵ cells/ml (1 ml/well) and were allowed to attach overnight. SFN was added to the wells (1 ml per well) at a final concentration of 10, 20 or 40 µmol/l. ADR was added as a positive control at a final concentration of 0.5 µmol/l. An equal volume of medium was added to the wells of the control group. The plates were incubated at 37°C in an atmosphere containing 5% CO₂ for 48 h. The cells were digested with pancreatin, collected and washed with PBS (4°C), and adjusted to 1×10⁵ cells/ml. The cells were resuspended in binding buffer and then stained with Annexin V-FITC and propidium iodide (PI) solution in an ice bath in the dark, according to the Annexin V-FITC Apoptosis Detection kit instructions. A nylon mesh filter (300 µm) was used to filter the cell samples to ensure cells were detected at single cell suspension. Following filtration, the cell samples of each group were analyzed by a flow cytometer at a wavelength of 488 nm and the results were analyzed by Expo 32 ADC Analysis software (Beckman Coulter, Inc.).

The HepG2 cells were plated in culture flasks and allowed to attach overnight. The cells were treated with various concentrations of SFN (10, 20 and 40 µmol/l) or ADR (0.5 µmol/l). The cells of the control group were treated with an equal volume of medium. After 48 h of treatment, cells were collected, lysed, and proteins were extracted. The Bradford method was used to quantify protein content. Equal amounts of protein (2 µg/ml; 20 µl loading volume) from the various groups were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto nitrocellulose membranes at 200 mA for 30 min. The membranes were then incubated in blocking buffer [5% nonfat dry milk in Tris-buffered saline containing Tween-20 (TBST)] for 2 h at room temperature, and the blots were incubated with rabbit anti-human Bip/GRP78, XBP-1 and Bid antibodies (1:200) and β-actin antibody (1:5,000) overnight at 4°C. The membranes were rinsed twice with TBST, undergoing 10 min of oscillation with each wash, and were rinsed once with TBS for 10 min. Subsequently, the membranes were incubated with alkaline phosphatase-labeled anti-mouse IgG antibody (1:2,000 dilution) for 2 h at room temperature. The membranes were rinsed three times as aforementioned and were stained with a diaminobenzidine chromogenic system. Finally, images were captured using the gel imaging system, and the protein content was quantified and analyzed using a GIS- 2019 gel imaging system software, version 3.14 (Tanon Science & Technology Co., Ltd.).

Logarithmic phase HepG2 cells were seeded in 6-well plates at a density of 3×10⁵ cells/ml (1 ml/well) and were allowed to attach overnight. Following treatment with various concentrations of SFN (10, 20 and 40 µmol/l) or ADR (0.5 µmol/l) for 48 h, the cells were collected by centrifugation for 10 min at 4°C at a speed of 558 × g and washed twice with PBS. The cells were then fixed with paraformaldehyde (500 µl) for 30 min at room temperature, collected by centrifugation for 5 min at room temperature, at a speed of 558 × g and then washed with PBS for 45 min. The cells were permeabilized with 200 µl PBS containing 0.1% Triton X-100 for 10 min and blocked with 200 µl blocking solution (PBS containing 5% BSA) for 60 min. After rinsing once with PBS, the cells were incubated with rabbit anti-human CHOP/GADD153 or rabbit anti-human caspase-12 antibodies (1:400) for 2 h at room temperature. Subsequently, the cells were washed once with PBS and incubated with FITC-labeled goat anti-rabbit secondary antibody for 60 min in the dark at room temperature. The samples were finally resuspended in PBS and filtered via a 300 µm nylon mesh filter. The protein content was analyzed using flow cytometry and Expo 32 ADC Analysis software (Beckman Coulter Inc.).

Data are presented as the mean ± standard deviation of three independent experiments. Data were analyzed using SPSS Software for Windows Version 11.5 (SPSS, Inc., Chicago, IL, USA). Data from the various groups were compared using one-way analysis of variance and Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

Following treatment with various concentrations of SFN for 24, 48 and 72 h, the proliferation of HepG2 cells was effectively inhibited in a dose- and time-dependent manner. The IC₅₀ values of SFN treatment were 32.03±0.96, 20.90±1.96 and 13.87±0.44 µmol/l following treatment for 24, 48 and 72 h, respectively (Fig. 1). The IC₅₀ values of ADR were 0.78±0.12, 0.43±0.09 and 0.32±0.40 µmol/l following treatment for 24, 48 and 72 h, respectively.

Following exposure to various concentrations of SFN or 0.5 µmol/l of ADR for 48 h, HepG2 cells displayed typical apoptotic morphology, including chromatin condensation and the formation of apoptotic bodies (Fig. 2). These results indicated that HepG2 cells were undergoing apoptosis.

FCM demonstrated that treatment with 10, 20, 40 µmol/l SFN or 0.5 µmol/l of ADR significantly increased the apoptotic rate of HepG2 cells (Annexin V⁺ PI⁻) compared with that of the control group (Table I and Fig. 3).

Following treatment with 20 or 40 µmol/l SFN or 0.5 µmol/l ADR for 48 h, the protein expression levels of Bip/GRP78, Bid and XBP-1 were significantly increased (P<0.01; Fig. 4).

The expression levels of CHOP/GADD153 and caspase-12 were significantly higher in HepG2 cells following exposure to increasing concentrations of SFN or 0.5 µmol/l of ADR for 48 h compared with control cells (P<0.01 or P<0.05). Protein quantities relative to the control were calculated and plotted as histograms. The results are presented in Figs. 5 and 6.