SGC7901 cells were purchased from the Chinese Academy of Medical Sciences' tumor cell library (Beijing, China). The primary antibodies against p38MAPK (dilution, 1:200; catalog no. sc-7972), phosphorylated (p-)p38MAPK (dilution, 1:200; catalog no. sc-17852-R), MMP-2 (dilution, 1:200; catalog no. sc-13594), MMP-9 (dilution, 1:200; catalog no. sc-21733) and -actin (dilution, 1:3,000; catalog no. 130300) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G(IgG) (dilution, 1:2,000; catalog no. ZDR-5306) and the HRP-conjugated goat anti-mouse IgG (dilution, 1:2,000; catalog no. ZDR-5307) secondary antibodies were purchased from Beijing Golden Bridge Biotechnology Co., Ltd. (Beijing, China). The silibinin compound was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The RPMI-1640 culture medium and fetal calf serum were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The Transwell chamber and the TRIzol reagent were obtained from Corning Incorporated (Corning, NY, USA) and Invitrogen; Thermo Fisher Scientific, Inc., respectively.

The gastric cancer SGC7901 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

The cells were seeded on 6-well plates at a density of 2×10⁵ cells/ml per well. The complete culture medium was replaced by culture medium without FBS subsequent to cell seeding for 48 h to induce cell synchronization. A 200-µl pipette tip was used to draw a straight line in the longitudinal center of each well, and then each well was washed with PBS twice. The cells were cultured in RPMI-1640 medium containing 50, 100 or 200 µM silibinin for 24, 48 and 72 h. The image was captured by measuring the width of a scratch using the IPP 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The cell migration distance formula used was: Cell migration distance (µm)=(initial scratch width-drug treatment scratch width)/². In the control group, complete medium was added only and it considered 0 µM.

The Transwell assays were performed in Transwell chambers with 8-µm pore size. The SGC7901 cells were trypsinized (catalog no. 25200056; Gibco; Thermo Fisher Scientific, Inc.) and suspended with 1% FBS. Subsequent to counting cells at a density of 2×10⁶ cells/ml, a 100-µl cell suspension was plated into the upper chamber, and 600 µl medium supplemented with 20% FBS was placed into the lower chamber. Subsequent to incubation at 37°C for 48 h, the cells on the upper surface of the filters were removed and the cells adhering to the undersurface of the filter membrane were fixed with 4% paraformaldehyde for 20 min. Subsequently, Giemsa stain (2%) was added to stain the transferred cells for 30 min at room temperature, cells were then washed with PBS three times. The cells on the lower chamber were counted under an inverted microscope in five random fields. The mean cell numbers were recorded and analyzed. The experiment was repeated three times. For the control group, complete medium was added only.

The Matrigel assays were performed in Transwell chambers with 8-µm pore size coated with Matrigel at 1:9 dilution in RPMI-1640 medium for 4 h at 37°C (to allow the Matrigel to solidify prior to plating cells). The experimental processes were performed as described above for the Transwell assays.

Equal numbers of cells were lysed in lysis buffer composed of 0.6 M Tris-HCl (pH 6.8), 10% SDS and a protease inhibitor cocktail (catalog no. 78430; Thermo Fisher Scientific, Inc.). Samples were incubated at 4°C for 10 min and then centrifuged at 10,000 × g for 15 min at 4°C. The supernatants were transferred, mixed and boiled in sample buffer (10 ml glycerol, 15 ml 20% SDS, 12.5 ml 4X upper tris, 12.5 H₂O). The supernatants were then separated by SDS-PAGE (12% gel) and transferred to a polyvinylidene fluoride membrane. The membrane was then incubated at room temperature in a blocking buffer composed of 5% fat-free milk dissolved in 1X 10 mM Tris (pH 7.5), 100 mM NaCl and 1% Tween 20 (TBST) for 1 h, followed by incubation with the blocking buffer containing the primary antibodies against p38MAPK, p-p38MAPK, MMP-2, MMP-9 and β-actin at 4°C overnight. The membrane was next washed with TBST and incubated with the secondary antibodies for 1 h at room temperature. The blot was exposed to Pierce^™ ECL Plus Western Blotting Substrate (catalog no. 32132X3; Pierce; Thermo Fisher Scientific, Inc.) electrochemiluminescence subsequent to TBS washing. The blots were analyzed using ImageJ software 1.4 (National Institutes of Health, Bethesda, MD, USA).

All statistical analyses were carried out using the SPSS 13.0 statistical software package (SPPS, Inc., Chicago, IL, USA). Either a two-tailed Student's t-test or a one-way analysis of variance, followed by the Tukey's test, was performed. P<0.05 was considered to indicate a statistically significant difference.

The wound healing assay indicated that silibinin significantly reduced the migration distance of SGC7901 cells at 50, 100 and 200 µM silibinin, in a dose- and time-dependent manner (Fig. 1).

The result of the Transwell migration assay confirmed that silibinin significantly reduced the motility of SGC7901 cells, since the number of migrated cells decreased markedly in a dose-dependent manner (Fig. 2A and B). The Matrigel-based invasion assay also indicated that silibinin significantly decreased the invasive ability of SGC7901 cells in a dose-dependent manner (Fig. 2C and D). These data suggested that silibinin may inhibit cell motility and affect the activity of MMPs.

Subsequent to treatment with 50, 100 and 200 µM silibinin for 48 h, the total p38MAPK protein levels remained unchanged, but the expression of p-p38MAPK significantly decreased in a dose-dependent manner (Fig. 3A and B). Additionally, it was demonstrated that silibinin decreased MMP-2 and MMP-9 protein expression levels (Fig. 3C and D), in a dose-dependent manner.

The results of the Transwell migration and Matrigel-based invasion assays (Fig. 4) simultaneously indicated that 100 µM silibinin and 20 µM SB203580 (a MAPK inhibitor) (25), independently and in combination, significantly decreased the migration (Fig. 4A and D) and invasion (Fig. 4C and E) of SGC7901 cells, particularly when administered in combination. Additionally, it was observed that silibinin treatment combined with SB203580 decreased the protein expression levels of MMP-2 and MMP-9 concomitantly (Fig. 4B and F).