The MHCC-97H cell line was purchased from the American Type Culture Collection (Manassas, VA, USA), and was cultured in high-glucose Dulbeccos modified Eagles medium with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere with 5% CO₂ at 37°C. Emodin (1,3,8-trihydroxy-6-methyl-anthraquinine) was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Its molecular formula is C₁₅H₁₀O₅ and its molecular weight is 270.24 g/mol. It was dissolved at a concentration of 100 mmol/l in 100% dimethylsulfoxide (DMSO; Sigma-Aldrich; Merck Millipore) as a stock solution, stored at −20°C, and the following concentrations were prepared: 2.5, 5, 12.5, 25, 50 and 100 mmol/l. DMSO (0.2%) was used as vehicle control for all the experiments. MTT was purchased from Sigma-Aldrich (Merck Millipore). The Annexin V-FITC apoptosis detection kit (cat. no. KGA108) was purchased from KeyGen Bio-Tech Co., Ltd., Nanjing, China. Rabbit phosphorylated (p)-Akt (cat. no. 4060S) and total (t)-Akt (cat. no. 4691S) polyclonal antibody, rabbit p-extracellular-signal-regulated kinase (ERK)1/2 (cat. no. 4370S) and t-ERK1/2 (cat. no. 4695S) monoclonal antibody, rabbit p-p38 (cat. no. 4511S) and t-p38 (cat. no. 9212S) monoclonal antibody and mouse β-actin (cat. no. 3700) monoclonal antibody were all purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). Rabbit matrix metalloproteinase (MMP)-2 (cat. no. BS1236) and MMP-9 (cat. no. BS1241) polyclonal antibody were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA).

Cell viability was determined by MTT assay. Briefly, Cells were seeded into a 96-well plate at a density of 1×10⁴ cells/well and cultured for 24 h. Emodin was then added to the wells with final concentrations of 0, 5, 10, 25, 50, 100 and 200 µmol/l and incubated for 12, 24, 48 and 72 h at 37°C. DMSO (0.2%) was used as a negative control. Then, 20 µl MTT solution (5 mg/ml) was added to each well and incubated for an additional 4 h at 37°C. The medium was carefully removed and 150 µl DMSO was added to each well to dissolve the formazan crystals and the absorbance was detected at 570 nm using a multiskan spectrum microplate reader (Thermo Fisher Scientific, Inc.).

Cell apoptosis was detected using the Annexin V-FITC apoptosis detection kit according to the manufacturers instructions. In brief, a total of 3×10⁵ MHCC-97H cells were seeded into each well of the 6-well plates and various concentrations (10, 25, 50,100 µmol/l) of emodin were added to the wells after 24 h of incubation at 37°C. The control group was treated with the equivalent quantity of DMSO (0.2%). Cells of each sample were harvested after an additional 12 or 24 h and suspended in 500 µl of Annexin V binding buffer (1X). Annexin V-FiTC (5 µl) and 5 µl of propidium iodide (PI) were added and incubated for 15 min in dark. The stained cells were analyzed by flow cytometry using a FACS Calibur (BD Biosciences, San Jose, CA, USA).

Cell migration was analyzed with the aid of a Transwell chamber (Corning Incorporated, Corning, NY, USA) with 8-µm pores and a cell invasion assay was performed using a Corning^® Matrigel^® invasion chamber (Corning Incorporated). Cells suspended in 200 µl serum-free medium were seeded onto the upper chambers (1×10⁵ cells/chamber for migration and 2×10⁵ cells/chamber for invasion) and incubated with different concentrations of emodin (10, 25 and 50 µmol/l), and the chambers were placed into 24-well plates with medium containing 10% serum. DMSO (0.2%) was used as a negative control. After 24 h, the cells remaining in the upper chamber were removed and migrated or invaded cells on the lower membrane surface were fixed with 4% formaldehyde polymerisatum followed by staining with 0.1% crystal violet for 20 min. The migrated or invaded cells in six visual fields (magnification, ×200) selected randomly were counted in each Transwell chamber under a phase-contrast microscope.

After treatment with 50 µM emodin and DMSO (0.2%) as a negative control, the cells were washed twice using ice-cold phosphate-buffered saline (pH 7.4) and lysed in radioimmunoprecipitation assay protein lysis buffer containing 1 mM PMSF on ice. Total proteins were extracted by centrifuging the cell lysates at 12,000 × g for 15 min at 4°C and the protein concentration was determined using a BCA assay kit (cat. no. P0010) Beyotime Institute of Biotechnology, Shanghai, China). A total of 30 µg protein from every sample was separated using a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with Tris-buffered saline and Tween 20 (TBST) buffer containing 5% skimmed milk for 1 h at room temperature, the PVDF membrane was incubated with appropriate concentrations of primary antibodies (dilution, 1:1,000) at 4°C overnight. After washing the membrane with TBST three times for 15 min, the membrane was incubated with corresponding secondary antibody labeled with horseradish peroxidase-conjugated (goat anti-mouse, 1:5,000; goat anti-rabbit, 1:2,000) secondary antibody for 2 h at room temperature. Following three washes with TBST for 15 min, the immunoreactive bands were detected using an enhanced chemiluminescence detection kit (Sigma-Aldrich; Merck Millipore). β-actin was used as the internal control and the relative values of target protein were corrected in accordance with the absorbency of the internal control.

All results were expressed as the mean ± standard deviation of at least three independent experiments and were analyzed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). The statistical analysis was performed using a one-way analysis of variance and Dunnetts test. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the antiproliferative effect of emodin on MHCC-97H cells, MTT assay was used to quantify the effect following treatment with emodin at different concentrations (5, 10, 25, 50, 100 and 200 µmol/l) for 12, 24, 48 and 72 h. As shown in Fig. 1, when the treatment concentration was <100 µmol/l and the treatment time was <24 h, the viability of the cells changed very little. With increases in the emodin concentration and treatment time, cell viability decrease evidently in a concentration- and time-dependent manner.

To determine the effect of emodin on apoptosis induction in MHCC-97H cells, flow cytometry was used to assess the cell apoptosis rate. After treatment with various concentrations of emodin (0 µmol/l for the control, 10, 25, 50 and 100 µmol/l for the experimental groups) for 24 h, MHCC-97H cells were stained with FITC-Annexin V/PI. As shown in Fig. 2, the apoptosis rate of MHCC-97H cells was shown to gradually increase as the emodin concentration increased (1.73±1.53, 6.31±0.59, 10.16±2.21, 12.99±3.77 and 53.68±8.32%, respectively). These results suggest that emodin was able to induce apoptosis in MHCC-97H cells, but the change was mild when the emodin concentration was <50 µmol/l.

To determine the effect of emodin on cell migration and invasion, MHCC-97H cells treated with 0, 10, 25 and 50 µmol/l emodin were induced to migrate through the Transwell membranes and invade in Matrigel-coated Transwells for 24 h. The number of cells that migrated was reduced by emodin in a dose-dependent manner (Fig. 3). In the same manner, following treatment with emodin at concentration of 0, 10, 25 and 50 µmol/l, the numberof invasive cells was 26.67±3.51, 18.34±4.16, 13.33±1.53 and 7.33±1.53, respectively. These data clearly demonstrate that emodin treatment significantly inhibits the migration and invasion of MHCC-97H cells in a dose-dependent manner.

In order to investigate the probable mechanism of the inhibition of migration and invasion induced by emodin, the activation of metastasis-related signal pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways, were detected by western blotting. The results demonstrated that following treatment with 50 µM emodin, the phosphorylated (p)-extracellular signal regulated kinase (ERK1)/2 expression levels were significantly decreased in a time-dependent manner until 60 min, at which point the levels began to increase again. In addition, p-Akt expression levels were decreased in a time-dependent manner, whereas the tendency of p-p38 expression levels were inverse. However, the ERK1/2, p38 and Akt expression levels remained essentially unchanged, except for a mild decrease at 60 min in ERK1/2 expression. In addition, MHCC-97H cells were treated with 50 µmol/l emodin for 0, 3, 6, 9 and 12 h, and it was observed that the MMP-2 and MMP-9 expression levels decreased in a time-dependent manner (Fig. 4).