GC cell lines, NCI-N87 and HGC-27, and Jurkat T cells were purchased from the American Type Culture Collection. Cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 µg/ml penicillin, in an incubator with 5% CO₂ at 37°C. To evaluate the effect of solamargine (MedChemExpress) on GC cells, GC cells were induced with 20 ng/ml IL-6 (MilliporeSigma) for 24 h at 37°C, and then treated with 10 µM solamargine for 48 h at 37°C.

For CCK-8 assays, a total of 5×10³ HGC-27 or NCI-N87 cells were seeded in culture plates and incubated at 37°C overnight. Following treatment with 5, 10 or 20 µM solamargine at 37°C for 48 h, cells were then incubated with 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) at room-temperature for 4 h. Subsequently, the absorbance of each well was measured at OD450 using a plate microreader (Thermo Fisher Scientific, Inc.).

A total of 5×10⁴ HGC-27 or NCI-N87 cells were seeded into the upper chamber (serum-free RPMI 1640 medium) of the Transwell insert (3 µm, cat. no. 3414; Corning, Inc.). For the invasion assay, the upper chamber was precoated with 50 µl Matrigel for 3 h at 37°C. As for the migration assay, the upper chamber was not precoated with Matrigel. Following incubation for 12 h at 37°C, cells in the upper chamber were removed and those in the lower chamber (10% FBS RPMI 1640 medium) were fixed with 100% anhydrous ethanol for 30 min at room temperature, followed by staining with 1% crystal violet for 1 h at room temperature. The cells were then counted under an inverted light microscope (Leica Microsystems GmbH).

Following incubation overnight at 37°C, HGC-27 or NCI-N87 cells at a density of 4×10⁴ were treated with 50 µM EdU solution (cat. no. C10338; Thermo Fisher Scientific, Inc.) for 4 h at 37°C. Subsequently, cells were fixed with 4% formaldehyde for 24 h at room temperature, followed by permeabilization for 10 min in 0.5% Triton X-100. Cells were then incubated with 100 µl Apollo reaction cocktail (Guangzhou RiboBio Co., Ltd) for 30 min at room temperature and DNA was stained with 100 µl/well DAPI (Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature. The stained cells were observed using a fluorescence microscope (magnification, ×100). A total of three random fields were selected and the Edu-positive cells were counted.

GC cells were seeded in 24-well plates. After treatments with 5 or 10 µM solamargine for 48 h at 37°C, cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.5% Triton X-100 for 2 min at room temperature. After blocking with 5% BSA (MilliporeSigma) for 2 h at room temperature, the cells were then incubated with antibodies against phosphorylated (p)-STAT3 (1:200; cat. no. ab267373; Abcam), STAT3 (1:200; cat. no. ab68153; Abcam), cleaved caspase 3 (1:200; cat. no. ab32042; Abcam) or caspase 3 (1:200; cat. no. ab32351; Abcam) at 4°C overnight. Subsequently, cells were incubated with the goat anti-rabbit IgG (conjugated to Alexa Fluor^® 594) secondary antibody (1:500; cat. no. ab150080; Abcam) for 1 h at room temperature. The GC cells were stained with 500 µl DAPI for 10 min at room temperature Finally, images of the stained cells were captured using a confocal microscope (Carl Zeiss AG).

Total protein was extracted from HGC-27 and NCI-N87 cells using RIPA buffer (Beyotime Institute of Biotechnology) and protein concentration was quantified using a BCA kit (Beyotime Institute of Biotechnology). Subsequently, total proteins (20 µg/lane) were separated using SDS-PAGE on a 10% gel and transferred onto PVDF membranes. Following blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against PD-L1 (1:1,000; Abcam; cat. no. ab228415), c-Myc (1:1,000; Abcam; cat. no. ab32072) and β-actin (1:1,000; Abcam; cat. no. ab8227) overnight at 4°C. Subsequently, the membranes were incubated with the corresponding secondary goat anti-rabbit antibody (1:5,000; cat. no. ab288151; Abcam) at room temperature for 1 h. After which, the targeted proteins were visualized using with ECL kit (Beyotime Institute of Biotechnology). The Odyssey Imaging System (LI-COR Bio) was used to scan membranes and the data were analyzed using Odyssey version 2.0 software (LI-COR Bio).

HGC-27 or NCI-N87 cells were seeded into 24-well plates. After treatments with 5 or 10 µM solamargine for 48 h at 37°C, cells were fixed with 4% paraformaldehyde at room temperature for 15 min, before being permeabilized with 0.5% Triton X-100 for 2 min at room temperature. Subsequently, apoptotic cells were stained for 1 h at 37°C using the One Step TUNEL Apoptosis Assay kit (Beyotime Institute of Biotechnology). Cell nuclei were stained with 100 µl/well DAPI (10 µg/ml; Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature Finally, images of the positive apoptotic cells in three random fields were captured using a confocal microscope (Carl Zeiss AG).

Jurkat cells were stimulated with anti-CD3/CD28 antibodies (1:100; Thermo Fisher Scientific, Inc.; cat. no. 11161D) for 24 h at 37°C and then co-cultured with NCI-N87 or HGC-27 cells pretreated with 10 µM solamargine for 48 h at 37°C. To isolate the Jurkat cells, 5×10⁵ cells were centrifuged for 2 min at 500 × g at 4°C and incubated with APC anti-CD69 (dilution 1:100; Biolegend; cat. no. 985206) for 30 min at room temperature in the dark, followed washing with PBS for three times and centrifugation at 500 × g for 2 min at 4°C. The stained cells (HGC-27 or NCI-N87 cells) were analyzed using flow cytometry (BD Biosciences). Flow.JoX (version 10.0.7; FlowJo LLC) software was used for data analysis.

All data are expressed as the mean ± standard deviation. The results were analyzed using GraphPad Prism (version 7.0; Dotmatics). The differences between two groups were assessed using an unpaired Student's t-test, whilst those among multiple groups were compared using one-way ANOVA, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of solamargine on GC, GC cells were treated with 5, 10 or 20 µM solamargine for 48 h. It was demonstrated that solamargine decreased the viability of NCI-N87 (Fig. 1A) and HGC-27 cells (Fig. 1B), with all concentrations of solamargine significantly reducing the cell viability compared with that in the control group. In addition, the percentage of EdU-positive HGC-27 (Fig. 1C) and NCI-N87 cells (Fig. 1D) was found to be significantly decreased after treatment with 5 and 10 µM solamargine compared with that in the control group. These results suggest that solamargine can significantly attenuate the viability and proliferation of GC cells.

Subsequently, to evaluate the effect of solamargine on the migration and invasion of GC cells, Transwell assays were performed. The results indicated that the migration of NCI-N87 and HGC-27 cells were significantly decreased by 5 and 10 µM solamargine compared with that in the control group (Fig. 2A). Furthermore, the invasion of NCI-N87 and HGC-27 cells were also significantly reduced by 5 and 10 µM solamargine compared with that in the control group (Fig. 2B). Taken together, the aforementioned findings suggest that solamargine can reduce the migration and invasion of GC cells.

To assess the effect of solamargine on GC cell apoptosis, a TUNEL staining assay was next performed. Subsequently, 5 and 10 µM solamargine was found to significantly increase the percentage of TUNEL-positive NCI-N87 and HGC-27 cells compared with that in the control group (Fig. 3A). Furthermore, the relative fluorescence intensity of cleaved caspase 3 was significantly increased in 5 and 10 µM solamargine-treated NCI-N87 and HGC-27 cells (Fig. 3B). This suggests that solamargine notably induced GC cell apoptosis through the caspase-3 signaling pathway, which was demonstrated by the relative increase in fluorescence intensity of cleaved caspase 3.

To assess the effect of solamargine on STAT3/PD-L1 signaling, GC cells were treated with IL-6 before the expression levels of PD-L1 and c-Myc were investigated using western blotting. It was demonstrated that IL-6 significantly increased the protein levels of PD-L1 and c-Myc in HGC-27 (Fig. 4A) and NCI-N87 cells (Fig. 4B) compared with that in the control group. However, treatment with IL-6 and 5 or 10 µM solamargine significantly reversed this increased protein expression levels of PD-L1 and c-Myc in HGC-27 (Fig. 4A) and NCI-N87 cells (Fig. 4B) compared with those in the IL-6 treatment alone. Additionally, the IL-6-induced upregulation of STAT3 phosphorylation in HGC-27 (Fig. 4C) and NCI-N87 cells (Fig. 4D) was reversed by 5 and 10 µM solamargine treatment compared with that in the IL-6 treatment alone group. This data suggest that solamargine can reverse the IL-6-induced activation of STAT3/PD-L1 signaling.

A previous study reported that PD-L1 upregulation can promote the escape of tumor cells from the attack of T cells (20). Furthermore, the aforementioned results of the present study demonstrated that solamargine can reduce the protein expression levels of PD-L1. Therefore, to investigate the effect of solamargine on T cells, Jurkat T cells were stimulated with anti-CD3/CD28 antibodies. Subsequently, Jurkat T cells were co-cultured with non-treated or solamargine-treated NCI-N87 or HGC-27 cells. The results demonstrated that solamargine could increase the expression of CD69 (T cell activation marker) in Jurkat T cells (Fig. 5A and B). In summary, solamargine may activate T cells.