The human gastric mucosal cell line GES-1 and human GC cells (AGS and HGC-27) were purchased from Huatuo Biotechnology Co., Ltd. (HTX1964, HTX1739, HTX1740, Shenzhen, China). After routine resuscitation from cryogenic storage, the cells were inoculated in Dulbecco’s Modified Eagle Medium (DMEM) (C2703, Beyotime, Shanghai, China) containing 10% fetal bovine serum (FBS) (C0226, Beyotime) and 1% penicillin/streptomycin (P1400, Solarbio, Beijing, China) and cultured in an incubator (MiDi-40, Thermo Scientific, Waltham, MA, USA) under standard conditions (37°C, 5% CO₂). When the cell confluence reached 70%–80%, the cells were passaged. All cells were tested for mycoplasma contamination by the mycoplasma kits (CA1080, Solarbio), and the cells without mycoplasma were used in subsequent experiments.

GES-1, AGS, and HGC-27 cells were seeded in 96-well plates at 1 × 10⁴ cells/well (100 μL per well) and treated with 0, 100, 200, 400, 800, or 1000 μg/mL DOM (B21803, Yuanye Biotechnology Co., Ltd., Shanghai, China), respectively. GES-1 cells were cultured for 24 or 48 h. AGS and HGC-27 cells were cultured for 24 h. Then, 10 μL of CCK-8 working solution (CA1210, Solarbio) was added to each well and gently mixed with the medium. The cells were returned to the cell incubator and incubated for 2 h. A microplate reader (Synergy H1, Agilent, Palo Alto, CA, USA) was used to detect the OD₄₅₀ value of each well, and the appropriate dosage and time of DOM were selected according to the OD value.

The growth of cells was evaluated with an EdU kit (C10310-1, RiboBio, Guangzhou, China). AGS and HGC-27 cells were divided into a control group and 200, 400, and 800 μg/mL DOM treatment groups. The GC cells in the DOM treatment groups were treated with the corresponding doses of DOM for 24 h. Then, the GC cells were incubated with 10 μM EdU staining mixture in the dark for 1 h, washed with phosphate buffer saline (PBS), fixed, and permeabilized with 4% paraformaldehyde (PFA) and 0.3% Triton X-100. After the cells were subjected to EdU and 4^′,6-diamidino-2-phenylindole (DAPI) staining, they were coverslipped with an antifluorescence quenching sealing agent. A fluorescence microscope (MF52-N, Guangzhou Ming-Mei Technology Co., Ltd., Guangzhou, China) was used to take photos. Three fields of vision were randomly selected to calculate the percentage of EdU-positive cells in each group.

After AGS and HGC-27 cells were cultured for 24 h, the cells were fixed with an appropriate amount of 4% PFA for 20 min and then stained with Hoechst 33258 staining solution (C0021, Solarbio) at room temperature for 20 min. In normal cells, the nucleus was light blue, and its shape was clearly visible; in apoptotic cells, uneven nuclear staining was bright blue with strong fluorescence. The apoptosis rate was calculated as the percentage of apoptotic cells divided by the total number of cells.

Ninety microliters of diluted Matrigel (DMEM:Matrigel = 8:1) was added to the top Transwell chamber and allowed to gel for 3 h in the incubator. GC cells were digested with trypsin and centrifuged. The cell density was adjusted to 1 × 10⁵ cells/mL. Two hundred microliters of cell suspension without FBS was added to the upper chamber of the Transwell, and 500 μL of complete medium (containing 10% FBS) was added to the lower chamber. After 24 h of culture, the cells on the surface of the lower chamber membrane were fixed with methanol at room temperature for 15 min and then incubated with 1% crystal violet (G1063, Solarbio) staining solution at room temperature for 20 min. Finally, the chamber was observed under an optical microscope (CX23, Olympus, Tokyo, Japan), and images of the cells were collected. ImageJ software (ImageJ 1.8.0, National Institute of Mental Health, Bethesda, MD, USA) was used to analyze the number of cells that invaded through the Matrigel.

GC cells were divided into the control group, RIPK2 overexpression (oe-RIPK2) group, and negative control (oe-NC) group. The oe-RIPK2 (Gene ID: NM_003821.6) and negative control oe-NC plasmids were purchased from Ruibo Biological Co., Ltd. (Guangzhou, China). The control group was transfected with liposomes without any treatment. The oe-NC group was transfected with oe-NC, and the oe-RIPK2 group was transfected with oe-RIPK2. The transfection procedure was performed with Lipofectamine^TM 3000 (L3000001, Invitrogen, Austin, TX, USA) according to the instructions of the manufacturer. The cells were seeded in 6-well plates at 1 × 10⁵ cells/well 24 h before transfection. During transfection, the corresponding reagents were added and mixed evenly. The cell culture medium was replaced with serum-free medium. After the cells were cultured in an incubator for 6 h, the medium was replaced with fresh medium, and then the subsequent experiment was carried out.

A total of 200 AGS or HGC-27 cells were inoculated into dishes containing complete medium. To evenly distribute the cells, the culture dish was slowly rotated for 1 min after the cells were inoculated. After the cells had adhered, they were treated with 200, 400, or 800 μg/mL DOM. The cell growth status and density were observed during the culture process. When the cells had formed colonies visible to the naked eye, the supernatant was discarded, and the cells were fixed with 4% PFA and stained with 0.1% crystal violet for 10 min (G1063, Solarbio). The cells were observed under a microscope, and colonies formed number was calculated using ImageJ software.

AGS and HGC-27 cells were seeded in 6-well plates at 5 × 10⁵ cells/well. When the cell density was close to 100%, the plate was scratched with a 20 μL pipette tip. The cells were incubated with DOM-containing medium for 24 h. The scratch area was photographed with a microscope at 0 and 24 h.

An Annexin V-FITC/PI apoptosis detection kit (CA1020, Solarbio) detected apoptosis. AGS and HGC-27 cells were digested with trypsin (T1350, Solarbio) without EDTA. The cells were seeded in 6-well plates at a density of 1 × 10⁵ cells/well. After 48 h of culture, the different levels of DOM were added, and the culture was continued for 24 h. After centrifugation, the cells were collected in a flow cytometry tube. Then, 100 μL of 1× binding buffer was added to resuspend the cells, and 2.5 μL of Annexin V and 5 μL of PI were added at the same time. After mixing, the reaction was carried out for 15 min, and the apoptosis rate was determined using FCM (BD FACSCalibur^TM, BD Biosciences, San Diego, CA, USA).

AGS and HGC-27 cells were treated with DOM for 24 h, washed with PBS, centrifuged, and collected in a flow cytometry tube. The cells were fixed with 70% ethanol and incubated at 4°C overnight. The ethanol was discarded, and PBS was added to resuspend the cells. Then, 100 μL of PI reagent was added to each tube, and the reaction was carried out for 30 min in the dark. The cell cycle distribution after each drug treatment was detected by FCM.

AGS and HGC-27 cells (3 × 10⁴ cells/well) were cultured in 12-well plates and transfected. After 6 h, the GC cells were treated with DOM for 24 h, fixed with 4% PFA for 10 min, and infiltrated with 0.5% Triton X-100 (IR9073, Solarbio) for 15 min. The cells were blocked with blocking solution (P0260, Beyotime) for 1 h and then incubated with primary anti-NF-κB p65 (ab32536, 1:100, Abcam, Cambridge, UK) and anti-RIPK2 (ab8428, 1:100, Abcam) antibodies overnight at 4°C. The secondary antibody, goat anti-rabbit IgG (GB22303, GB21303, Servicebio, Wuhan, China), was added and incubated for 1 h in the dark. The nuclei were stained with DAPI staining solution (C0065, Solarbio) for 10 min, and the sealing agent (S2110, Solarbio) was added. The samples were coverslipped and photographed using the fluorescence microscope.

The culture medium of GC cells was transferred to sterile centrifuge tubes, and the supernatants were collected after centrifugation. Interleukin (IL)-6 (SEKH-0013, Solarbio), IL-1β (SEKH-0002, Solarbio), and tumor necrosis factor-α (TNF-α, SEKH-0047, Solarbio) levels were determined by ELISA. The supernatant and antibody mixture were incubated in an ELISA plate for 1 h. One hundred microliters of substrate was included and incubated in the dark for 10 min, and then 100 μL of termination reaction mixture was added. The OD value was detected by a microplate reader (Synergy H1, Agilent) to evaluate the contents of inflammatory factors.

After intervention, the GC cells were collected, and the proteins were extracted using a protein extraction kit (R0010, Solarbio). After centrifugation, the protein concentration was detected with a BCA kit (PC0020, Solarbio). After separation by gel electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (G6047, Servicebio) and blocked with 5% skim milk at room temperature for 2 h. Primary antibodies against rabbit anti-Ki-67 (ab92742, 1:5000, Abcam), proliferating cell nuclear antigen (PCNA) (ab92552, 1:10000, Abcam), matrix metalloproteinase (MMP)-2 (ab181286, 1:1000, Abcam), IκBα (9242, 1:1000, Cell Signaling, Boston, MA, USA), MMP-9 (ab137867, 1:1000, Abcam), cysteinyl aspartate-specific proteinase (caspase) 3 (ab184787, 1:2000, Abcam), cleaved-caspase 3 (ab2302, 1:500, Abcam), cyclin D1 (ab134175, 1:10000, Abcam), cyclin E1 (ab33911, 1:1000, Abcam), Bax (ab32503, 1:10000, Abcam), Bcl2 (ab182858, 1:2000, Abcam), RIPK2 (ab8428, 1:1000, Abcam), p-IκBα (2859, 1:1000, Cell Signaling), NF-κB p65 (ab32536, 1:5000, Abcam), p-NF-κB p65 (ab239882, 1:1000, Abcam), and β-actin (ab8226, 1:1000, Abcam) were added and incubated overnight at 4°C. The samples were exposed to a diluted HRP-labeled secondary antibody (ab205718, 1:10000, Abcam) at room temperature for 2 h. The ECL chemiluminescence solution (PE0010, Solarbio) was added, and a gel imaging system (Tanon 5200 Multi, Tianneng Technology Co., Ltd., Shanghai, China) was used for imaging. The protein gray values were analyzed by ImageJ software.

Statistical analysis was performed using SPSS 26.0 software (SPSS Inc., Chicago, IL, USA), and the data were normally distributed according to the Kolmogorov–Smirnov test and are presented as the means ± standard deviations. One-way ANOVA was used for comparisons among multiple groups. p < 0.05 was considered statistically significant.

RIPK2 is relatively highly expressed in AGS and HGC-27 cells [30]; thus, these cells were selected for experiments, and GES-1 cells were used as controls. Different concentrations of DOM had no substantial effect on the cell viability of GES-1 cells at 24 or 48 h (Fig. 1A), indicating that DOM was not toxic to normal gastric cells. Moreover, after 24 h of DOM exposure, the cell viability of AGS and HGC-27 cells decreased with increasing dose, but when the DOM concentration was 1000 μg/mL, the viability was less than 50% (Fig. 1B). The IC₅₀ of AGS cells was 858 μg/mL, and that of HGC-27 cells was 835.6 μg/mL. Therefore, we selected 200, 400, and 800 μg/mL DOM for subsequent experiments.

On this basis, after treatment with DOM for 24 h, the percentage of EdU-positive cells and colony formation ability were significantly reduced (Fig. 1C–F), indicating that DOM could reduce the proliferation ability of GC cells. Since the cell cycle regulates cell fate and plays a key role in cancer progression [31], the FCM results revealed that after DOM treatment, the proportion of AGS and HGC-27 cells in the G0–G1 phase raised markedly, whereas that in the S phase lessened significantly (Fig. 1G–H). Thus, DOM could significantly increase the number of GC cells in the G0–G1 phase and reduce the number of S-and M-phase cells.

Cyclin D1 and cyclin E1 can regulate the cell cycle, and reducing their expression can hinder tumor growth [32,33]; Ki67 and PCNA can be used as markers of cell proliferation activity [34]. Ki67, PCNA, cyclin D1, and cyclin E1 expressions were markedly declined after DOM treatment (Fig. 1I–K), indicating that DOM inhibited GC cell proliferation and encouraged cell cycle arrest. Overall, the above experiments showed that DOM could restrain GC cell proliferation and promote G0/G1 cycle arrest, suggesting that DOM had an anti-GC effect.

We also found that untreated GC cells grew well and adhered firmly, and that the cells were closely connected. The nuclei were evenly stained blue with Hoechst 33258, resulting in uniform blue fluorescence. After DOM treatment, the GC cells became round and detached, and the cell spacing became significantly greater. The cells were stained bright blue with Hoechst 33258. With increasing drug concentration, the blue fluorescence became stronger; that is, the phenomenon of apoptosis became more obvious. After quantification, the apoptosis rates of the GC cells rose significantly in response to DOM treatment (Fig. 2A,B). FCM experiments also revealed the same results. The apoptosis rate increased significantly with increasing DOM concentration (Fig. 2C,D), indicating that DOM promoted GC apoptosis.

Subsequently, the migration rate and invasive cell number were significantly reduced after DOM treatment (Fig. 2E–H), indicating that DOM could reduce the migration and invasion of GC cells. MMP-2 and MMP-9 are associated with cell migration and invasion [35]; Bax, Bcl2, and caspase are apoptosis indicators [36]. After DOM treatment, MMP-2, MMP-9, and Bcl2 levels were significantly lessened, and Bax and cleaved-caspase 3 expressions were markedly raised (Fig. 2I–M). In conclusion, DOM promoted GC apoptosis and inhibited migration and invasion. These findings, combined with those of previous experiments, indicate that DOM inhibited the malignant progression of GC.

We also showed that RIPK2 was localized in the cytoplasm by immunofluorescence and WB. Compared to GES-1 cells, the fluorescence intensity and protein expression of RIPK2 in GC cells were substantially greater. The fluorescence intensity and protein expression of RIPK2 significantly decreased after DOM treatment (Fig. 3A–D), indicating that DOM significantly suppressed the expression of RIPK2 in GC cells.

The occurrence of gastric cancer is associated with the inflammatory response [37]. RIPK2 was overexpressed, and the transfection efficiencies were examined using immunofluorescence and WB. The fluorescence intensity and protein level of RIPK2 were markedly raised (Fig. 4A–D), indicating that RIPK2 was effectively highly expressed. The cells were then divided into the 800 μg/mL DOM treatment (DOM) group, the RIPK2 overexpression (DOM + oe-RIPK2) group, and the negative control (DOM + oe-NC) group. The contents of TNF-α, IL-1β, and IL-6 were notably elevated after RIPK2 overexpression (Fig. 4E–G), indicating that RIPK2 overexpression aggravated the inflammatory response of GC cells, whereas RIPK2 inhibition reduced the inflammatory response. These findings indicate that DOM might reduce the inflammation of GC cells by inhibiting RIPK2.

To further investigate the roles of RIPK2 in DOM’s effects on GC, after the overexpression of RIPK2, colony formation experiments revealed that colony formation ability was significantly increased (Fig. 5A,B), indicating that the proliferation of GC cells was increased; the migration rates of AGS and HGC-27 cells were also significantly increased (Fig. 5C,D). In addition, the proportion of cells in the G0–G1 phase was significantly decreased, the proportion of those in the S phase was markedly increased (Fig. 5E,F), and apoptotic cells were markedly decreased (Fig. 5G,H). These results indicated that overexpression of RIPK2 reduced the proportion of GC cells in the G0–G1 phase, increased the proportion in the S phase, promoted cellular growth, and inhibited apoptosis. In contrast, the inhibition of RIPK2 expression inhibited GC progression. In summary, DOM inhibited GC progression by inhibiting RIPK2 expression.

To explore the related pathways by which DOM affects GC cells, the cells were separated into a normal (Control) group, a DOM treatment (DOM) group, a RIPK2 overexpression (DOM + oe-RIPK2) group, and a negative control (DOM + oe-NC) group. The fluorescence intensity of NF-κB p65 located in the nucleus of GC cells decreased significantly after DOM treatment, and the fluorescence intensity increased significantly after RIPK2 overexpression (Fig. 6A,B), indicating that RIPK2 overexpression promoted the activation of the NF-κB signaling pathway; that is, the suppression of RIPK2 expression inhibited the activation of the NF-κB signaling pathway. Moreover, the WB experimental results confirmed this conclusion. After DOM treatment, RIPK2, p-IκBα, and p-NF-κB p65 expressions of AGS and HGC-27 cells were significantly decreased, and they were significantly increased after RIPK2 overexpression (Fig. 6C–E). In conclusion, DOM might inhibit RIPK2, which inhibits the activation of the NF-κB signaling pathway.