Pizotifen was purchased from MedChemExpress and its molecular structure is shown in Fig. 1A. The primary antibodies against Wnt3a (cat. no. ab28472; dilution, 1:1,000), active-caspase-3 (cat. no. ab2302; dilution, 1:1,000) were purchased from Abcam, and the primary antibodies against Cyclin D-1 (cat. no. 60186-1-lg; dilution, 1:1,000), Bax (cat. no. 50599-2-lg; dilution, 1:1,000), Bcl-2 (cat. no. 12789-1-AP; dilution, 1:1,000) and β-tubulin (cat. no. 10094-1-AP; dilution, 1:1,000) were purchased from ProteinTech Group, Inc. Primary antibodies against β-catenin (cat. no. 9562; dilution, 1:1,000), E-cadherin (cat. no. 14472; dilution, 1:1,000) and N-cadherin (cat. no. 4061; dilution, 1:1,000) were purchased from Cell Signaling Technology, Inc. Both secondary antibodies (horseradish peroxidase conjugated anti-rabbit, cat. no. SA00001-15; anti-mouse, cat. no. SA00001-1; both dilutions, 1:5,000) were purchased from ProteinTech Group, Inc. BML-284, a selective Wnt activator, was purchased from MedChemExpress. Unless specifically indicated, all other reagents were obtained from Sigma-Aldrich (Merck KGaA).

Human gastric cancer cell lines MNK45 and AGS were obtained from the Type Culture Collection of the Chinese Academy of Sciences. The cell lines were cultured in DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml), and streptomycin (100 mg/ml), and were maintained in a humidified atmosphere (95%) at 37°C under 5% CO₂. Cells in the logarithmic growth phase were treated with different concentrations of pizotifen (10, 20 and 40 µM) or BML-284 (10 µM) alone or co-treated with pizotifen (20 µM) and BML-284 (10 µM). The cells were treated for varying time periods, including 0, 24, 48 and 72 h, 0.1% DMSO was used as negative control.

The Cell Counting Kit-8 (CCK-8) assay was performed according to the manufacturer's protocol (Beijing Solarbio Science & Technology Co., Ltd.). Cells were seeded into 96-well plates at a density of 1×10³ cells/well and cultured overnight to allow for adherence. Cultures were subsequently treated with different concentrations of pizotifen (10, 20 and 40 µM). DMSO (0.1%) was used as the negative control. The cells were then cultured for 3 days and measurements were obtained every 24 h. When the cells were measured, CCK-8 solution (10 µl/well) was added to each well and incubated for an additional 1.5 h at 37°C. The final optical density was measured at a wavelength of 450 nm to estimate cell viability in the different treatment groups. Each experiment was performed in triplicate.

Cells were first seeded into six-well plates at a density of 5×10⁵ cells/well and incubated overnight at 37°C. The following day, when cells had reached ~95–100%confluency, wounds were generated using pipette tips. After being scratched with the pipettes, the cells were cultured in serum-free medium supplemented with 10 or 20 µM pizotifen or DMSO for 24 h. The wounds were photographed using a light microscope (magnification, ×40) and analyzed using ImageJ software v1.8.0 (National Institutes of Health). The percentage wound closure was calculated using the mathematical equation: Percentage wound closure (%) = (A₀-A_t)/A₀ × 100%, where A₀ is the initial wound area, and A_t is the wound area at 0, 24 h post-treatment.

Total protein was extracted from the cell cultures according to instructions from radioimmunoprecipitation assay Lysis Buffer (CWBIO Tech). Protein concentration was quantified using an Enhanced Bicinchoninic Acid Protein Assay kit. A total of 20 µg protein from each sample were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were incubated in blocking buffer [5% non-fat milk dissolved in TBS with Tween-20 (TBST)] at room temperature for 1-h and then probed at 4°C overnight with primary antibodies (dilution, 1:1,000). The membranes were subsequently rinsed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1-h at room temperature. The blots were visualized using the enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) and analyzed using the Quantity One 1-D software v4.6.7 (Bio-Rad Laboratories, Inc.). Each experiment was repeated at least three times.

Cells (5×10⁵) pretreated with 20 µM pizotifen or 0.1% DMSO were double-stained with fluorescein isothiocyanate-conjugated Annexin V (25 mg/ml) and propidium iodide (PI; 50 mg/ml) at 4°C for 15 min according to the manufacturer's protocol (4A Biotech Co., Ltd.), and then washed and analyzed using a BD FACSCalibur flow cytometry system (BD Biosciences). The obtained data was analyzed using FlowJo software v7.6.1 (FlowJo LLC).

For the Transwell migration assay, 5×10³ cells pretreated with pizotifen or BML-284 were seeded into the upper sections of the non-coated 96-well inserts (pore size, 8 µm; Corning, Inc.). For the invasion assay, Matrigel (BD Biosciences) was thawed at 4°C and diluted in serum-free medium at a ratio of 1:6, then added to the upper chamber of the Transwell chamber and maintained at 37°C for 4–6 h. A total of 1×10⁵ cells were seeded into the top chamber of 24-well inserts with 8 µm pore size and Matrigel-coated membranes. In both assays, cells were diluted in medium without FBS and seeded into the top chamber, whereas medium containing 10% FBS were introduced into the lower chamber in order to establish a chemoattractant gradient. Cells were incubated for 24 h, fixed with 4% paraformaldehyde at room temperature for 30 min, and stained with 0.1% crystal violet solution (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min. Then, 5 microscopic fields were used to count and photograph the stained cells under a light microscope (magnification, ×100).

Experimental data are presented as the mean ± SD. All statistical analysis was performed using SPSS 13.5 (SPSS, Inc.). Student's t-test and one-way ANOVA followed by Tukey's posthoc analysis were performed. P<0.05 was considered to indicate a statistically significant difference.

A CCK8 assay was carried out to determine the effect of pizotifen on the viability of gastric cancer cell lines MNK45 and AGS. As shown in Fig. 1B, pizotifen treatment of 48h or 72 h significantly inhibited the viability of MNK45 cells in a dose-dependent manner (P<0.05). Similarly, pizotifen also inhibited the viability of AGS cells (Fig. 1C).

Wound-healing and Transwell assays were performed to investigate whether pizotifen affects the migratory and invasive properties of gastric cancer cells. Since 10 and 20 µM pizotifen both caused appropriate inhibitory effect on MNK45 and AGS cells, whilst 40 µM pizotifen caused increased cell death, 10 and 20 µM of pizotifen were used in the rest experiments. As shown in Fig. 2A, 10 and 20 µM pizotifen both led to a significant decrease in the migratory abilities of MNK45 cells in a dose-dependent manner (P<0.05). A similar inhibitory effect of pizotifen was observed in AGS cells (P<0.05; Fig. 2B). Moreover, the results of the Transwell assay further suggested that the migratory abilities of MNK45 and AGS cells were significantly inhibited following treatment with pizotifen (P<0.05; Fig. 3A-C). In addition, the invasive abilities of MNK45 and AGS cells treated with pizotifen were significantly reduced (P<0.05; Fig. 3D-F). Furthermore, the data showed that BML-284, a selective Wnt signaling activator that has been revealed to induce β-catenin in vitro (24), significantly increased the migration and invasion of both MNK45 and AGS cells (both P<0.05; Fig. 3), and partially restored the migratory and invasive abilities of MNK45 and AGS cells inhibited by pizotifen (P<0.05; Fig. 3).

Annexin V-FITC/PI assay was performed to investigate whether pizotifen inhibited the growth of gastric cancer cells by triggering apoptosis. As shown in Fig. 4A, pizotifen (20 µM) significantly enhanced cell apoptosis in both MNK45 and AGS cells (both P<0.01) compared with the NC group (Fig. 4A). To further determine the mechanism of pizotifen-induced apoptosis, western blot analysis was performed to detect the expression of proteins associated with the mitochondrial apoptotic pathway in MNK45 and AGS cells treated with pizotifen for 48 h (Fig. 4B). The data demonstrated that the expression of the anti-apoptotic protein Bcl-2 was significantly reduced in pizotifen-treated cells, whereas the expression of pro-apoptotic proteins Bax and active caspase-3 were significantly increased (P<0.05; Fig. 4B). These data indicated that pizotifen may induce the apoptosis of gastric cancer cells via the intrinsic apoptotic pathway, specifically the Bax/Bcl-2 and caspase cascade.

The ability of pizotifen to inhibit the Wnt/β-catenin signaling pathway in MNK and AGS cells was investigated using western blot analysis. Pizotifen treatment significantly reduced the expression of Wnt3a and β-catenin in a dose-dependent manner in MNK45 and AGS cells (P<0.05; Fig. 5A). In addition, the expression of the epithelial marker E-cadherin was significantly upregulated by pizotifen treatment in MNK45 and AGS cells, while the expression of the mesenchymal marker N-cadherin was significantly downregulated (P<0.05; Fig 5A), suggesting that pizotifen regulated the expression of EMT markers proteins in a dose-dependent manner. This suggested that pizotifen can suppress the Wnt/β-catenin-EMT signaling pathway in MNK45 and AGS cells.

To further investigate the mechanism of action of pizotifen in the inhibition of gastric cancer cell invasion, BML-284 was used. It was found that the expression of β-catenin was significantly upregulated in MNK45 and AGS cells following BML-284 treatment compared with the NC group (Fig. 5B). In the presence of pizotifen, BML-284 treatment also partially restored β-catenin expression compared with the pizotifen-only group (Fig. 5B). In addition, BML-284 treatment partially reversed the effects induced by pizotifen on E-cadherin and N-cadherin expression in MNK45 and AGS cells compared with the pizotifen-treated group (Fig. 5B). Taken together, the results of the present study indicated that the pizotifen-induced inhibitory effects on gastric cancer cell migration and invasion may be due to the inhibition of the Wnt/β-catenin-EMT signaling pathway.