The MKN28 human gastric cancer cell line and the GES-1 human gastric normal epithelial mucosa cell line were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/ml) and streptomycin (0.1 mg/ml; both Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. When the cells entered the logarithmic growth phase, they were washed with PBS three times, and were then digested by trypsin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Once the cells became rounded, the digestion was terminated and the cells were placed in culture medium, after which they were pipetted into 6-well plates for subsequent experiments. When cell confluence in the wells reached ~80%, the cells were treated with ISL (MedChemExpress, Monmouth Junction, NJ, USA) or 0.1% dimethyl sulfoxide (DMSO; Amresco, LLC, Solon, OH, USA) as a negative control (NC).

CCK-8 (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China) was used to assess the effects of ISL on MKN28 and GES-1 cell proliferation. MKN28 or GES-1 cells (100 µl) were seeded into 96-well plates at a cellular density of 1,000 cells/well. To the NC group, 0.1% DMSO was added, whereas 20 µM ISL was added to the experimental group. Cells were cultured for 0, 24, 48 and 72 h time intervals at 37°C, and the cell viabilities at each time interval were subsequently detected. Prior to detection, 10 µl CCK8 solution was added to each well for 1.5 h at 37°C. The optical density (OD) value was measured at 450 nm using a microplate reader to obtain the proliferation curve.

For the invasion assay, the inner layer of a 24-well Transwell system (EMD Millipore, Billerica, MA, USA) was pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at a 1:6 dilution. Cells treated with 20 µM ISL or 0.1% DMSO for 24 h were suspended in serum-free medium and 100-µl cell suspension (~1×10⁴ cells) was added to the upper chambers. The lower chambers were filled with culture medium. Following overnight incubation, cells on the surface of the upper chamber were removed with a cotton swab. Cells on the bottom were fixed in 4% paraformaldehyde for 0.5 h at room temperature, stained with 0.1% crystal violet dye for 20 min at room temperature and washed with PBS at room temperature. Subsequently, images were captured and the cells in five random fields were counted manually using light microscopy.

The migration assay was similar to the invasion assay, with the exception that the Transwell system was not coated with Matrigel, and the number of cells tested was 5,000.

Following treatment with 20 µM ISL or 0.1% DMSO (control group) for 24 h, cells were collected and digested with trypsin (Beijing Solarbio Science & Technology Co., Ltd.). Cells were then washed using precooled PBS (4°C). The cell density was adjusted to 1–5×10⁶/ml by adding 1X binding buffer. Subsequently, 100-µl cell suspension was placed in a 5 ml flow tube, and 5 µl Annexin V/fluorescein isothiocyanate (FITC; BD Biosciences, Franklin Lakes, NJ, USA) was added and mixed by flicking the tube for 5 min in the dark at room temperature, followed by incubation with 10 µl propidium iodide for 10–15 min in the dark room at room temperature. Following this, 400 µl PBS was added and mixed, and the tubes were subsequently placed on ice. The results were detected using a flow cytometer (BD Biosciences) in 1 h and analyzed using FlowJo v10.0 software (FlowJo LLC, Ashland, OR, USA).

To extract proteins, cells in the experimental and NC groups were added to 6-well plates and treated with radioimmunoprecipitation assay lysis buffer (CW Biotech, Beijing, China) plus protease inhibitor. Protein concentration was measured using a Bicinchoninic Acid Protein Assay kit (CW Biotech), after which the proteins were heated for 5 min at 95°C. Vertical electrophoresis was performed with ~20 µg protein in each lane. The proteins were isolated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1 h and were incubated with primary antibodies overnight at 4°C. The following rabbit anti-human primary antibodies were used: AKT (1:1,000; cat. no. 9272; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p-)AKT (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), mTOR (1:1,000; cat. no. 2972; Cell Signaling Technology, Inc.), p-mTOR (1:1,000; cat. no. 2971; Cell Signaling Technology, Inc.), B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. 4223; Cell Signaling Technology, Inc.), caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.), Bcl-2-associated X protein (Bax; 1:1,000; cat. no. 5023; Cell Signaling Technology, Inc.), microtubule-associated proteins 1A/1B light chain 3B (LC3; 1:1,000; cat. no. 14600-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), Beclin 1 (1:1,000; cat. no. 11306-1-AP; ProteinTech Group, Inc.), p62 (1:1,000; cat. no. 5114; Cell Signaling Technology, Inc.) and GAPDH (1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). The membranes were washed three times (5 min/wash) with Tris-buffered saline containing 0.05% Tween-20. The membranes were then incubated with goat anti-rabbit immunoglobulin G (H+L), horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) at 37°C for 1 h. After washing of the membranes, enhanced chemiluminescence (ProteinTech Group, Inc.) was used to detect the signals. Quantity One v4.6.9 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to scan the gray value of the blots, and GAPDH was used as an internal control. The relative expression levels were calculated by normalizing the target protein expression to the expression of the internal control.

Statistical analyses were performed using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). Data are expressed as the means ± standard deviation. Statistical comparison between two groups was carried out with Student's t-test and one-way analysis of variance followed by a Dunnett's post hoc test was used to compare >2 groups. P<0.05 was considered to indicate a statistically significant difference.

A CCK8 assay was performed to detect the potential effects of ISL on the proliferation of gastric cancer cells. ISL treatment decreased the proliferation of MKN28 cells compared with in the NC group in a concentration-dependent manner (Fig. 1A). In addition, MKN28 cells were treated with ISL (20 µM) for 0, 24, 48 and 72 h. The OD values at 48 and 72 h were significantly different between the ISL and NC groups (P<0.05; Fig. 1B), thus suggesting that ISL could effectively reduce MKN28 cell proliferation at 48 h. In addition, the effects of ISL on GES-1 cell proliferation were investigated. The results indicated that ISL exerted little inhibitory effect on GES-1 cell proliferation (Fig. 1C). Therefore, subsequent experiments were performed only using MKN28 cells. In summary, ISL clearly inhibited MKN28 cell proliferation but exhibited little effect on GES-1 cell proliferation.

A Transwell assay was used to determine the effects of ISL on the migration and invasion of MKN28 cells (Fig. 2A). The Transwell migration assay revealed that the number of migrated cells in the ISL group was significantly decreased compared with in the NC group (P<0.05; Fig. 2B). In addition, the Transwell invasion assay demonstrated that the number of invaded cells was reduced in the ISL group compared with in the NC group (Fig. 2C; P<0.05). These results indicated that ISL markedly reduced the invasion and migration of MKN28 cells.

The apoptosis of MKN28 cells was evaluated using the FITC Annexin V Apoptosis assay and flow cytometry, and apoptosis-associated proteins were detected by western blotting. The results demonstrated that treatment with ISL significantly increased cell apoptosis compared with in the NC group (Fig. 3A and B; P<0.05). Western blotting (Fig. 3C) indicated that the protein expression levels of Bcl-2 were reduced (P<0.05; Fig. 3D), whereas those of Bax were increased compared with in the NC group (P<0.05; Fig. 3E). Furthermore, the ratio of Bax/Bcl-2 was significantly increased following ISL treatment compared with in the NC group (Fig. 3F; P<0.05), and active caspase-3 expression was increased following ISL administration (Fig. 3C and G; P<0.05). These results suggested that ISL may promote the apoptosis of MKN28 cells.

Autophagy in MKN28 cells was detected by western blot analysis, which measured alterations in the protein expression levels of LC3I, LC3II, Beclin 1 and p62 (Fig. 4A). Compared with in the NC group, the ratio of LC3II/LC3I and the expression levels of Beclin 1 were clearly upregulated in response to ISL treatment, whereas p62 was downregulated (P<0.05; Fig. 4B-D). These results indicated that ISL may promote autophagy in MKN28 cells.

The effects of ISL on the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins in MKN28 cells were investigated by western blot analysis, which was performed to analyze AKT, p-AKT, mTOR and p-mTOR protein expression (Fig. 5A). The protein expression levels of p-AKT were significantly decreased in ISL-treated MKN28 cells, with no significant differences in the total expression levels of AKT (P<0.05; Fig. 5B and C). Furthermore, p-mTOR was significantly decreased following ISL treatment, with no significant differences in the total mTOR levels (P<0.05; Fig. 5D and E). These results suggested that the effects of ISL on MKN28 may be associated with downregulation of the PI3K/AKT/mTOR signaling pathway.