Human hepatocellular carcinoma cell line HepG2 was obtained from ATCC (Rockville, MD, USA). Cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO₂ and experiments were done using 70–80% confluent cultures.

HepG2 cell survival was evaluated using the MTT (3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl-tetrazolium bromide) (Sigma, USA) colorimetric assay. After different doses (vehicle, 10, 20, 40, 60 and 80 µM) of SFN treatment for 48 h, MTT assay was performed on hepatocellular carcinoma cell line HepG2 to evaluate cell growth ability and to calculate the 50% inhibitory concentration of SFN. Then, the cell proliferation in the presence of indicated SFN (IC₅₀) or vehicle control for indicated time-points. In brief, prior to the treatment, HepG2 cells were seeded in 96-well tissue culture plates at 2×10⁴ cells per well. After treatment, cells were washed with PBS and incubated in 100 µl of 5 mg/ml MTT solution (Invitrogen Inc., USA) for 3 h. MTT is converted into purple colored formazan in living cells which was then solubilized with dimethylsulfoxide (DMSO) (Invitrogen Inc.) and absorbance of solution was evaluated at 450 nm using the microplate reader Thermo Plate (Rayto Life and Analytical Science C. Ltd., Germany).

After treatment, cells were harvested, trypsinized and rinsed 3 times with buffer solution with adjusted concentration of 1×10⁶ cells/ml and prepared using Cycletest™ Plus DNA Reagent kit (Becton-Dickinson, USA) according to the manufacturer's instructions. Cell cycle status was analyzed by flow cytometer using propidium iodide (PI) as a specific fluorescent dye probe. The PI fluorescence intensity of 10,000 cells was measured for each sample using a Becton-Dickinson FACSCalibur flow cytometer.

Cell apoptosis was determined by Annexin V assay. After transfection for 48 h, cells were harvested by trypsinization and washed with PBS, and suspended in Annexin V binding buffer. FITC-conjugated Annexin V and propidium iodide (PI; Beyotime, China) were added to the cells successively. After incubation, Annexin V binding buffer was added, and cells were analyzed by a FACScan (Becton-Dickinson) flow cytometry. Annexin V(+)/P(−) and Annexin V(+)/P(+) represent the cells in early apoptosis and late apoptosis/necrosis, respectively.

Cell migration and invasion were assayed using a Transwell chamber (Millipore, USA). For the invasion assay, Transwell chamber was coated with 30 µl Matrigel and was placed into 24-well plate and incubated for 30 min at 37°C. After 48 h of transfection, HepG2 cells were trypsinized and seeded in chambers at the density of 8×10⁴ cells per well and cultured in medium with RPMI-1640 medium with 2% serum, while 600 µl of 10% FBS-RPMI-1640 with or without TGF-β (10 ng/ml) was added to the lower chamber. Twenty-four hours later, migrated cells were fixed with 100% methanol for 30 min and stained by crystal violet for 20 min. Non-migrated cells were removed by cotton swabs. Cell images were obtained under a phase-contrast microscope (Olympus, Tokyo, Japan).

Morphological changes in cells were emulated by scanning electron microscopy. Cells were grown on plastic cover slips. After indicated treatments, fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) for 2 h, rinsed with PBS, and dehydrated through graded ethanol (30, 50, 70, 80, 90 and 100% for 20 min each). Cells were transferred to amylacetate for 10 min, critical point dried, and then coated with gold. Cells were viewed in a scanning electron microscope (Olympus).

Western blotting was applied to detect markers of EMT at protein level. After indicated treatments, HepG2 cells cultured on the glass slide were washed twice in ice-cold PBS, and then lysed by RIPA buffer with 1% phenylmethanesulfonyl fluoride (PMSF) and complete™ protease inhibitor cocktail (Roche Molecular Biochemical, Indianapolis, IN, USA) for 30 min. After centrifugation at 12,000 rpm for 5 min, supernatant was collected and protein concentrations were determined using the Bio-Rad kit (Bio-Rad Laboratories, USA). Cell lysates were separated by SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF; Millipore). Membranes were blotted with 10% non-fat milk, washed in TBS-Tween and incubated with primary polyclonal antibodies anti-E-cadherin or anti-Vimentin (1:400; Santa Cruz, USA) overnight at 4°C. After washing with TBS-Tween, membranes were incubated with secondary antibody (horseradish peroxidase conjugated IgG) for 60 min at room temperature. Polyclonal anti-GAPDH (1:800; Bioss, China) was used as an internal control. Then, they were washed again with TBS-Tween before using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, USA). Blots were imaged by Molecular Image^® ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad Laboratories, Inc.).

Five-week-old female Balb/c athymic nude mice (Vitalriver Laboratory Animals, Beijing, China) were subcutaneously injected in the right flank with 3.0×10⁶ HepG2 cells in 0.1 ml PBS. Once tumors grew to~5 mm in diameter, tumor volume (V) was measured by caliper daily and calculated using the formula V=(L×W²)/2, where L was the length and W was the width of the tumor. The mice were randomly divided into two groups (n=6) for treatment with SFN (50 mg/kg) or vehicle control through i.p. injection every 2 days, respectively. Growth curves were plotted using average tumor volume within each experimental group every 2 days. Thirteen days later, the mice were euthanized, and the dissected tumors were collected and prepared for subsequent analyses. All animal experiments were approved by the animal center.

The data are presented as the mean ± SD from the three independent experiments. Immunoblot signals were quantitated using densitometry and ImageJ software version 1.4 (NIH, USA). Statistical analysis was performed by the SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) using analysis of variance (ANOVA) followed by the Tukey's t-test. P<0.05 was considered statistically significant.

The dose and time course of the effect of SFN on cell proliferation was examined by MTT assay (Fig. 1). We found that SFN significantly inhibited cell proliferation of HepG2 in a dose-dependent manner, with an IC₅₀ value of 40.05 µM. HepG2 also responded to SFN in a time-dependent manner. SFN significantly inhibited proliferation of HepG2 cells at 40.05 µM after 48 h. Thus, SFN inhibited the proliferation of hepatocellular carcinoma HepG2 cells both dose- and time-dependently.

As the growth inhibition of cancer cells is usually associated with cell cycle arrest, the effect of SFN on cell cycle distribution of HepG2 was examined (Fig. 2A and B). We found that cell cycle was arrested at the G0/G1 phase when HepG2 cells were treated with 40 µM SFN for 48 h.

Next, we tested the effect of SFN on HepG2 cell apoptosis (Fig. 2C and D). SFN markedly increased both the early and late apoptosis, compared with control.

Morphology of HepG2 cells was changed in the presence of SFN (Fig. 3A). SFN suppressed the typical morphology changes of EMT induced by TGF-β, and inhibited the formation of fibroblast-like mesenchymal cells in HepG2 cells.

Phenotypic transition of SFN-inhibited EMT was further evaluated by changes of the mesenchymal marker Vimentin, and the epithelial marker E-cadherin. As expected, SFN significantly suppressed the expression of Vimentin, and significantly elevated the expression of E-cadherin (Fig. 3B). The effect of SFN on the expression of these EMT markers was in a dose-dependent. Overall, SFN inhibited TGF-β-induced EMT of HepG2.

Using Transwell assay, the effects of SFN on cell migration and invasive ability was determined (Fig. 4A). We found a significantly decreased number of migrated HepG2 cells treated with 40 µM of SFN (Fig. 4B).

To evaluate whether ROS was involved in TGF-β-induced HepG2 cell migration and invasion, and EMT, ROS scavenger N-acetyl-L-cysteine (NAC, 20 mM) was added to treat the HepG2 cells for 24 h in the present of TGF-β (Fig. 4C–E). The inhibitory effect of SFN on migration and invasion were almost completely abolished by NAC in HepG2 cells (Fig. 4C and D). Also, NAC abolished the decrease of Vimentin, and the increase of E-cadherin (Fig. 4E). It was suggested that ROS-dependent mechanism mediated the effect of SFN on EMT of HepG2 cells.

We showed a significant inhibitory effect of SFN on cell growth of HepG2 cells. Here, we further determined the effect of SFN on the growth of HepG2 cell-derived xenograft tumors in nude mice (Fig. 5). HepG2 cell-derived xenograft tumor grew slowly in the mice treated with 50 mg/kg SFN, whereas grew progressively in the control mice with a larger tumor volume than the SFN group. Taken together, our results indicate that SFN is an effective agent for hepatocellular carcinoma treatment.