Human colon cancer cell line SW620 was purchased from American Type Culture Collection (Manassas, VA, USA). Fetal bovine serum (FBS), RPMI-1640 medium and pancreatin were purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). GA (purity, ≥95%) was purchased from Chengdu Pulis Biotech Co., Ltd. (Chengdu, China). MTT, dimethylsulfoxide (DMSO) and 5-Fu were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), while the Annexin V-fluorescein isothiocyanate (FITC)/propudium iodide (PI) apoptosis and cell cycle assay kits [Cell cycle and apoptosis (PI) kit] were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against PI3K (1:500; cat. no. ab32089), AKT (1:500; cat. no. ab8805), phosphorylated (p)-AKT (1:500; cat. no. ab8933), P21 (1:500; cat. no. ab109520), MMP-2 (1:500; cat. no. ab37150), MMP-9 (1:500; cat. no. ab73734) and GAPDH (1:1,000; cat. no. ab181602) were purchased from Abcam (Cambridge, MA, USA). Furthermore, a DM3000 microscope (Leica Microsystems GmbH, Wetzlar, Germany) and LightCycler^® 480 instrument (Roche Molecular Diagnostics, Pleasanton, CA, USA) were used in the present study.

SW620 cells were cultured in RPMI-1640 medium with 10% FBS in an incubator at 37°C and 5% CO₂ and passaged upon reaching 90% confluence. The culture was divided into five groups based on subculturing conditions: i) No-treatment control (NC) group cultured in RPMI-1640 medium with 10% FBS; ii) NC group medium + 10 µg/ml GA (low group); iii) NC group medium + 50 µg/ml GA (medium group); iv) NC group medium + 100 µg/ml GA (high group); and v) NC group medium + 10 µg/ml 5-Fu (5-Fu group). Cells were cultured in their respective experimental group media as described below for each specific assay. Cells were cultured until the logarithmic growth stage and digested with trypsin prior to seeding into culture plates for specific experiments.

A 200-µl cell suspension (4×10³ cells/ml) was cultured in 6-well-plates for 5 h to allow time for cell adherence, after which the medium was replaced with the group-specific experimental culture media, as described above. Each group was cultured in triplicate. After 48 h at room temperature, 20 µl MTT (5 g/ml) was added to each well and the plates were cultured for 30 h at room temperature. DMSO was added to each well to stop the reaction, and the OD value was measured at 490 nm on the plate reader to calculate cell proliferation.

SW620 cells (1×10⁶ cells/ml) were seeded in a 6-well plate (2 ml/well) and cultured for 24 h in group-specific media at room temperature. Media were removed and cells collected by resuspending in 300 µl binding buffer, to which 5 µl Annexin V-FITC was added according to the manufacturer's protocol. Suspensions were incubated at room temperature in the dark for 15 min, after which 5 µl PI was added. Apoptosis was detected by flow cytometry (FACSAria; BD Biosciences; wavelength, 488 nm) supplemented with 200 µl 1X binding buffer (diluted in double distilled water) at room temperature after 1 h. All tests were performed in triplicate. Data were analyzed using Diva software (version 8.0.1; FACSAria; BD Biosciences).

SW620 cells in the logarithmic growth phase were seeded in a 7.5-cm culture flask and cultured for 24 h, then digested by pancreatin and pelleted by centrifugation (1,000 × g at 4°C for 5 min). Cell cycle status was detected using the aforementioned cell cycle kit according to the manufacturer's protocol. Briefly, the cells of each group were treated with their respective aforementioned conditioned media for 24 h at room temperature. Afterwards, 5 µl RNase (10 mg/ml) was added to the PBS-resuspended cell pellet, and the cells were cultured at room temperature for 1 h. PI (100 µg/ml) was subsequently added and the cells incubated at room temperature for 30 min. The cell cycle status was measured in triplicate by flow cytometry (FACSAria; BD Biosciences) at a wavelength of 488 nm and data were analyzed using Diva software (version 8.0.1; FACSAria; BD Biosciences).

Matrigel (1:3) was diluted with serum-free medium (RPMI-1640) and placed in the upper transwell chamber (pore size, 8 µm) at 37°C for 4 h, after which the transwell chambers were placed in 24-well plates. SW620 cells (1×10⁴ cells/ml; 100 µl/well cell suspension) from each individual experimental group were added to the upper chamber. Complete culture medium (RPMI-1640) containing 10% FBS was added to the lower chamber, and the cells were cultured for 24 h. Subsequently, noninvasive cells were removed with cotton swabs. The transwell chambers were removed from the wells, washed with phosphate-buffered saline (PBS) three times and the remaining cells in the lower chamber were fixed with 4% paraformaldehyde-PBS solution for 10 min at room temperature. The chambers were subsequently washed with PBS as aforementioned, inverted and dried. Cells were stained with 0.1% cresyl violet solution for 20 min at room temperature and washed with PBS three times. Cells were subsequently counted under an inverted optical microscope.

SW620 cells (5×10⁴/well) from each experimental group were seeded into a six-well plate and cultured for 24 h at room temperature. When the cells formed a single, tightly adherent layer, a scraper with a 2-mm wide tip was used to scratch the confluent cells. Images of each scratch were captured under a light microscope at 0 h. Plates were cultured and images were captured again under the light microscope at 48 h. Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used to measure the scratch width in the 0- and 48-h images and to calculate the cell migration rate from the difference.

Cells were incubated for 24 h at room temperature in the aforementioned conditioned media and collected using lysis buffer (Beyotime Institute of Biotechnology) and the protein concentration was measured using a bicinchoninic acid Protein kit (Beyotime Institute of Biotechnology). Subsequently, cells of the different groups were washed three times with ice-cold PBS and lysed with buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 5 µg/ml aprotinin, 5 µg/ml leupeptin and 1 mM phenylmethylsulphonyl fluoride. Cell lysates were cleared via centrifugation at 12,000 × g at 4°C for 30 min and denatured by boiling in Laemmli buffer (Beyotime Institute of Biotechnology). Proteins were denatured by incubation in a boiling water bath for 5 min with 5X SDS gel buffer solution. A total of 15 µl/lane of each sample was electrophoresed on an 8% SDS-PAGE (110V; 4 h). The protein samples were transferred to polyvinylidene fluoride (PVDF) membranes using the semi-dry method. The PVDF membranes were blocked with 5% skim milk in PBS for 1.5 h at room temperature. The membranes were subsequently incubated with the aforementioned primary antibodies overnight at 4°C. Following this incubation, the membrane was washed three times in PBS and incubated in goat anti-rabbit second antibody (1:5,000; cat. no. ab205718; Abcam) solution at room temperature for 2 h. Anti-GAPDH antibody was used as an internal reference. Specific immune complexes were detected using the Western Blotting Plus Chemiluminescence Reagent (Thermo Fisher Scientific, Inc.). Band intensity was quantified via densitometry analysis using Image-Pro Plus version 4.5 software (Media Cybernetics, Inc.).

Data are presented as the mean ± standard deviation from three independent experiments. Quantitative results were analyzed via one-way ANOVA assay followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Cell proliferation rates of the GA-treated groups were significantly suppressed compared with the NC group (all P<0.05). The proliferation rate of 5-Fu treated cells was significantly suppressed when compared with the NC, low and medium groups (each, P<0.05). Furthermore, significant differences in proliferation rates were identified between the GA-treated groups (low, medium and high; all P<0.05; Fig. 1).

Compared with the NC group, apoptosis rates in all GA-treated groups were significantly upregulated (P<0.05; Fig. 2). The rates of apoptosis increased in a dose-dependent manner. The apoptosis rate of the 5-Fu group was significantly upregulated compared with the NC, low and medium groups (each, P<0.05; Fig. 2).

A significantly increased number of G1-phase cells was observed among GA treated groups compared with the NC group (all P<0.05; Fig. 3). The number of 5-Fu treated cells in the G1 phase were significantly increased compared with the NC, low and medium groups (each, P<0.05; Fig. 3). The increase in the number of G1-phase cells was dose dependent and the differences between GA-treated groups were statistically significant (P<0.05).

Counts of invasive cells were significantly lower in all GA groups compared with the NC group (all P<0.05; Fig. 4). The number of invasive cells in the 5-Fu group were significantly reduced when compared with the NC, low and medium groups (each, P<0.05). Significant differences were also observed between GA-treated groups (P<0.05).

The wound healing rates of GA-treated groups were significantly inhibited compared with the NC group (P<0.05; Fig. 5). The wound healing rate of the 5-Fu group was significantly reduced compared with the NC, low and medium groups. This effect was dose-dependent.

Compared with the NC group, protein expression levels of PI3K, AKT, p-AKT, MMP-2 and −9 significantly decreased, and the expression levels of P21 significantly increased following treatment with GA at low, medium and high doses (P<0.05; Fig. 6). Furthermore, the p-AKT/AKT ratio decreased in all GA-treated groups compared with the NC group and this effect was dose-dependent (P<0.05).