Gastric carcinoma cell lines (HGC-27) (cat. No. CL-0107) and (SNU-1) (cat. No. CL-0474), normal human gastric epithelial cells (GES-1) (cat. No. CL-0563), and fetal bovine serum (FBS) were purchased from Wuhan Procell Life Technology, Wuhan, China. RPMI-1640 medium was purchased from Gibco (Thermo Fisher Scientific, Carlsbad, CA, United States). The Hoechst 33258 staining solution (C1017), TUNEL Apoptosis Assay Kit (C1088), and BCA Protein Assay Kit (P0012) were procured from Beyotime Institute of Biotechnology, Shanghai, China. Rabbit anti-human Cyclin B1 (1:1,000, cat. No. 12231S), Rabbit anti-human cell division cyclin 25 homolog C (Cdc25c) (1:1,000, cat. No. 4866S), Rabbit anti-human Cdk1(1: 1,000, cat. No. 77055S), Rabbit anti-human Caspase 3 (1: 1,000, cat. No. 9662S), Rabbit anti-human Bcl-2(1:1,000, cat. No. 4223S), Rabbit anti-human Bax (1: 1,000, cat. No. 2774S), Rabbit anti-human Bak (1:1,000, cat. No. 12105S), Rabbit anti-human PARP (1:1,000, cat. No. 9532S), Rabbit anti-human Caspase 8 (1:1,000, cat. No. 4790S), Rabbit anti-human death receptor-4 (DR4) (1:1,000, cat. No. 42533S), Rabbit anti-human death receptor-5 (DR5) (1:1,000, cat. No. 69400S), Rabbit anti-human Fas ligand (FasL) (1:1,000, cat. No. 68405S), Rabbit anti-human Fas (1: 1,000, cat. No. 4233S), Rabbit anti-human microtubule-associated protein1 light chain3B (LC3B) (1:1,000, cat. No. 3868S), Rabbit anti-human IRE1α (1:1000, cat. No. 3294S), Rabbit anti-human p62 (1:1,000, cat. No. 5114S), Rabbit anti-human Beclin (1:1,000, cat. No. 3495S), Rabbit anti-human PARP (1:1,000, cat. No. 9532S) antibody, rabbit anti-human AKT (1:1,000, cat. No. 9272S) antibody, rabbit anti-human phosphor-Akt (1:2,000, cat. No. 4060S) antibody, rabbit anti-human GSK3β (1:1,000, cat. No. 9315S) antibody, rabbit anti-human phosphor-GSK3β (Ser 9) (1:1,000, cat. No. 9322S) antibody, and rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, cat. No.5174S) antibody were obtained from Cell Signaling Technology, Cambridge, MA, United States. Rabbit anti-mouse Ki-67 (1:1,000, cat. No. ab16667) was obtained from Abcam, Cambridge, United Kingdom. A horseradish peroxidase (HRP)-labeled goat anti-rabbit Immunoglobulin G (IgG) antibody (1:2,000, cat. no. CW0103) was purchased from CWbio, Beijing, China. Cell Counting Kit-8 kits (CCK-8), ROS detection kits (S0033S), Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kits (C1062M), and cell cycle detection kits (C1052) were purchased from Beyotime. D-luciferin (122799) was from Perkin Elmer, Waltham, MA, United States. NAC (HY-B0215), SC79 (HY18749), 3-Methyladenine (3-MA) (HY-19312) were from MedChemExpress, Monmouth Junction, NJ, United States. 4% polyformaldehyde was from Solarbio, Beijing, China.

HGC-27 and SNU-1 were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) (containing 100 μg/ml streptomycin and 100 IU/ml penicillin) supplemented with 100 ml/L FBS and kept at 37°C with 5% CO₂ atmosphere.

HGC-27 and SNU-1 that are in the logarithmic growth phase were collected and inoculated into a 96-well plate at 5 × 10³ cells/well, cultured overnight at 37°C; then HGC-27 and SNU-1 were treated with Cos at different concentrations (0, 2.5, 5, 10, 20, 40, 80, and 160 μmol/L) in FBS-free RPMI 1640 for 24 and 48 h, and cell proliferation was detected by the Cell Counting Kit-8 assay. We added 10 μl of CCK-8 reagent to the cells in each well and incubated them at 37°C for 4 h; optical density values were measured with a microplate reader at 450 nm. After the half-maximum inhibitory concentration (IC50) was determined, the cells in four different Cos concentrations were selected according to the IC50, observed, and photographed under inverted light microscopy (Leica, DMIL, Germany × 200). The cells in five microscope fields of view were randomly selected for counting to evaluate the cell viability in each group.

HGC-27 and SNU-1 were seeded into the 60 mm dish at a density of 500 cells/well and cultured into RPMI 1640 containing 10% FBS for 24 h, then treated with various concentrations of Cos (0, 10, 20, and 40 μM). The treated cells were resuspended in RPMI 1640 containing 10% FBS and cultured in 5% CO₂ at 37°C for 15 days to form colonies. After the dish was washed with PBS, the colonies were fixed with 4% polyformaldehyde at room temperature then dyed with 1% crystal violet for 30 min at room temperature. Colonies comprising 50 cells or more were counted by microscope (Leica Microsystems, Wetzlar, Germany) as previously described (Chen et al., 2016). Each experiment was done thrice in this study. Colony formation rate = the number of each treatment/the number of control × 100%.

HGC-27 and SNU-1 were seeded into 12-well plates, cultured for 24 h, then treated with 0, 10, 20, and 40 μM Cos for 24 h. The adherent cells were washed twice with PBS, then stained with Hoechst 33258 (Beyotime) for 5 min at room temperature in the dark. After being washed twice, the blue-stained nucleus was observed under the BX41 fluorescence microscope (Olympus, Tokyo Japan; amplification: × 400). The nucleus of living cells presents diffuse and uniform fluorescence, and the characteristic of apoptotic cells was that the nucleus or cytoplasm presents dense granular and clumpy fluorescence. Images were captured to quantitatively analyze via Image Pro Plus analysis software 6.0 (Media Cybernetics Inc., Rockville, MD, United States).

The apoptosis of GC cells and animal tumors were evaluated via the Tunel Apoptosis Assay Kit (Beyotime). Firstly, the cell samples and paraffin-embedded tissue sections (4 μm thick) were treated by protein kinase K and 3% H₂O₂, respectively, and incubated with Tunel detection solution (the component of Tunel staining kit) for 1 h at 37°C, then incubated with Streptavidin-HRP working solution. At last, the DAB solution was added and the samples were observed and photographed under the BX41 fluorescence microscope (Olympus Corporation; amplification: × 400). Images were captured to quantitatively analyze the apoptosis of cells via Image-Pro Plus analysis software 6.0 (Media Cybernetics). The number of apoptotic cells and the total number of cells were counted, and the proportion of apoptosis was calculated. Apoptosis cell proportion = number of positive cells/total number of cells × 100%.

Cell cycle, apoptosis, and ROS level were measured by flow cytometry analysis. HGC-27 and SNU-1 (2.0 ml/well, 3 × 10⁵ cells/mL) were seeded and cultured into the six-well plate for 24 h. After aspiration, the cells were incubated with 2.0 ml of Cos at different concentrations (0, 10, 20, and 40 μmol/L) or treated with Cos before pretreating with NAC in FBS-free high-glucose DMEM for 24 h. The cell cycle detection kit, Annexin V-FITC apoptosis detection kit, and ROS detection kit were used for analysis according to the manufacturer’s instructions, respectively. Briefly, the collected cells were stained with 75% ethanol at 4°C overnight, propidium iodide (PI) for cell cycle analysis, and Annexin V-FITC and PI for 15 min at 37°C in a darkroom for apoptosis analysis, respectively. Then they were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 min at 37°C in a darkroom for ROS level analysis. The cells were analyzed via flow cytometry (BD FACSCalibur; Becton Dickinson, San Jose, CA, United States).

The levels of cell cycle-related protein (Cyclin B1, Cdc25c, Cdk1), intrinsic apoptosis-related proteins (Caspase 3, Bak, Bax, Bcl2, PARP), extrinsic apoptosis-related proteins (caspase 8, DR4, Fas, FasL), autophagy-related proteins (LC3B, beclin-1, p62), and signaling pathway-related proteins (AKT, P-AKT, GSK3β, and P-GSK3β) in HGC-27, SNU-1 were analyzed by Western blot analysis. Briefly, the protein of GC cell lines HGC-27 and SNU-1 was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors on ice, and quantified using the BCA Protein Assay Kit. The protein bands were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After being blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline with Tween (PBST) for 1 h, the membranes were incubated with primary antibodies at 4°C overnight, then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The SuperSignal ELISA Femto Substrate was added onto the membranes in a darkroom and was subsequently exposed to x-ray films. The intensity of the Western bands was determined by Image J software version 1.46 [National Institutes of Health (NIH), Bethesda, MD, United States].

The slides with the climbed cells in the culture plate were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 20 min. After being blocked with BSA, the cells were incubated with the LC3B primary antibody overnight at 4°C. At last, they were incubated with Alexa-Fluor 488-conjugated secondary antibodies in 1% bovine serum at 37°C for 1 h in the dark. Nuclei were counterstained with DAPI for 15 min in the dark. Images were photographed via a confocal laser scanning microscope (OLYMPUS FV3000; Olympus Corporation, Center Valley, PA, United States; amplification: × 1000), and endogenous LC3 puncta formation were analyzed using the FV10-ASW viewer software ver. 4.2b (Olympus).

We harvested the cells by centrifuging at 3000 r/min for 10 min, washing twice with cold PBS, aspirating the supernatant, and fixing with 2.5% glutaraldehyde along the tube wall. Then electron microscope slices were prepared according to conventional procedures. At last, cell ultrastructure in every group was observed under the electron microscope (HITACHI, HT7700-SS, Tokyo, Japan).

The procedures and ethics of animal use have been reviewed and approved by the Biomedical Ethics Committee of Shaanxi Provincial People’s Hospital (The Third Affiliated Hospital of Xi’an Jiaotong University) (approval no. 2021-155). The 16 female Balb/c nude mice (5–6 weeks old, 19.5 ± 2.6 g) were from the Animal Center of Shanghai Institute of Family Planning Science (Shanghai, China) [SCXK (Hu) 2018-0006].

Firstly, the HGC-27 cells with stable expression of luciferase were constructed by lentivirus. Secondly, luciferase-positive HGC-27 cells (5 × 10⁶ cells per mouse) were injected subcutaneously into the right flank of Balb/c nude mice, when the tumor volume reached 100 mm³ (Festing and Altman, 2002). The mice were randomly divided into four groups (n = 4/group), the negative control, the positive control, and the experimental group. In the experimental group, the mice were administered intraperitoneally with 30 mg/kg and 50 mg/kg Cos, respectively, and with the same volume of dimethyl sulfoxide (DMSO) in the negative control group, with cisplatin (2 mg/kg) in the positive control group. It was injected every 3 days. The weight of the animal was analyzed every 3 days, and the length (L) and width (W) of the tumor were measured with a caliper. The volume calculation formula is:

L × (W)²/2 (Du et al., 2012). Thirty days later, animals were sacrificed and the dissecting tumors, heart, liver, spleen, lungs, and kidneys were for corresponding analysis.

The tissues (containing tumors, hearts, livers, spleens, lungs, kidneys) were fixed in 4% paraformaldehyde for 24 h, dehydrated, and embedded in paraffin. Sections 4 μm thick were stained with hematoxylin and eosin (H&E) and Tunel for morphological observation, respectively. Images were observed and photographed under the BX41 fluorescence microscope (Olympus Corporation; amplification: ×200) and quantitatively analyzed via Image-Pro Plus analysis software 6.0 (Media Cybernetics).

The paraffin-embedded tissue sections (4 μm thick) were deparaffinized and rehydrated, incubated with rabbit polyclonal antibodies specific to Ki-67 and P-AKT at 4°C overnight, incubated with HRP-conjugated secondary antibody at room temperature for 2 h, and stained in hematoxylin for 3 min and observed under the BX41 fluorescence microscope (Olympus Corporation; amplification: ×200).

Bioluminescence imaging (BLI) was performed using an IVIS imaging system (Perkin Elmer, Waltham, MA, United States) after 15 and 24 days after drug intervention; 100 μl PBS containing 25 mM D-luciferin (Caliper Life Sciences, Hopkinton, MA, United States) was injected intraperitoneally 10 min before luciferase detection.

All data were represented as mean ± SEM. The biotechnology was repeated at least three times in vitro. The intergroup deviations were evaluated with a one-way analysis of variance (ANOVA) implemented in the GraphPad Prism 6.0 software, with P < 0.05 indicating a statistically significant difference.

CCK-8 and colony formation assay were used to analyze the effect of Cos (Figure 1A) on GC cell proliferation. As shown in Figure 1B, Cos could significantly inhibit the proliferation of HGC-27 and SNU-1 cells in a dose-dependent manner compared with that in the control group (p < 0.001), but the effect of Cos on normal gastric cells (GES-1) was not as sensitive as GC cells (p > 0.05) (Figure 1B). As shown in Figure 1B, the half-maximum inhibitory concentration (IC 50) for the two cells at 24 or 48 h is about 40 μM. Therefore, Cos concentrations of 0, 10, 20, and 40 μM were chosen in these assays. Phase-contrast microscope results showed that Cos induced shrinkage, deformation, and rupture and inhibited the proliferation in GC cell lines HGC-27 and SNU-1, but it had no effect on GES-1 (Figure 1C). In addition, colony formation assay further revealed that Cos obviously inhibited proliferation in a dose-dependent manner (p < 0.001) but, on GES-1, was not as sensitive as GC cells (Figure 1D).

To estimate the effect of Cos on the cell cycle, we performed flow cytometry and western blot analysis in HGC-27 and SNU-1 cells. The flow cytometry results suggested that Cos significantly induced cell cycle arrest in the G2/M phase in HGC-27, SNU-1 cells with obvious dose-dependency (p < 0.001), but the effect of Cos on GES-1 was not as sensitive as GC cells (p > 0.05) (Figure 2A), and Western blot showed that the expression levels of cell cycle-related proteins (Cdc25c, Cdk1, Cyclin B1) in GC cells were significantly downregulated by Cos, especially in the 40 μM Cos group (p < 0.001), but the effect of Cos on GES-1 cells was not significant (p > 0.05) (Figure 2B).

Hoechst 33258, Tunel staining, and flow cytometry were used to evaluate the effect of Cos on apoptosis in GC cells. Hoechst 33258 and Tunel staining showed that along with Cos concentration increase, the rate of apoptosis cell increased (p < 0.001) (Figures 3A,B). The flow cytometry results revealed that Cos could dose-dependently lead to the apoptosis of GC cell lines HGC-27 and SNU-1 in the Cos treatment compared with the control group (p < 0.001) (Figure 3C).

To further explore the mechanism of Cos-inducing apoptosis, we analyzed intrinsic and extrinsic apoptotic. Western blot revealed that the levels of intrinsic apoptotic proteins [Cleaved-Caspase 3 (Cle-Caspase 3), Bax, Bak, Cleaved-PARP (Cle-PARP)] were upregulated with dose-dependency, but Bcl-2 was downregulated in HGC-27 and SNU-1 cells in the Cos treatment compared with the control group (p < 0.001) (Figure 4A). However, the activities of extrinsic apoptosis proteins [Cleaved-Caspase 8 (Cle-Caspase 8), DR4, Fas, FasL] did not change significantly between the Cos treatment group and the control group (p > 0.05) (Figure 4B).

To demonstrate the effect of Cos on autophagy in GC cells, autophagic activity and autophagy-related proteins were analyzed in HGC-27 and SNU-1. Transmission electron microscopy results showed that the formation of autophagic vacuoles in HGC-27 and SNU-1 significantly increased after Cos treatment (Figure 5A). The confocal microscopy results showed that treatment with Cos could lead to the aggregation of autophagosomes both in HGC-27 and SNU-1 (p < 0.001) (Figure 5B). Autophagy markers (LC3B, beclin-1, IRE1α) were increased and p62 was decreased after Cos treatment with dose-dependent manner (p < 0.001) (Figure 5C).

To investigate the mechanism of Cos-induced cell cycle arrest, apoptosis, and autophagy of GC cell, the levels of ROS were detected. The flow cytometry results showed that Cos could boost ROS generation both in HGC-27 and SNU-1 cells in a dose-dependent manner compared with the control group (p < 0.001) (Figure 6A). HGC-27 and SNU-1 cells were first treated with 4 mmol/L NAC (an ROS scavenger) before the cells being incubated with 40 μM Cos. The flow cytometry results showed NAC could not reverse G2/M arrest (p > 0.05) (Figure 6B), and the results of cell cycle protein markers in HGC-27 and SNU-1 also showed the same trend (p > 0.05) (Figure 6C).

The flow cytometry results indicated NAC could significantly reduce Cos-induced apoptosis (p < 0.001) (Figure 7A). The Western blot results showed that the ratio of P-AKT/AKT and P-GSK3β/GSK3β markedly downregulated in the Cos treatment groups with dose-dependent manner compared with the control group (p < 0.001) (Figure 7B). GC cells were pretreated with 4 mmol/L NAC for 1 h before the cells were treated with 40 μmol/L Cos for 24 h. The ratio of P-AKT/AKT and P-GSK3β/GSK3β in the Cos and NAC co-treated group markedly upregulated higher than that of Cos alone (p < 0.05) but downregulated lower than that of NAC alone. In addition, apoptosis-associated protein PARP and autophagy-associated protein LC3BII in the Cos and NAC co-treated group downregulated higher than that of Cos alone (p < 0.05) (Figure 7C).

The flow cytometry results revealed SC79 (an AKT activator) could not reverse G2/M arrest in HGC-27 and SNU-1 cells treated with Cos (p > 0.05) (Figure 8A), and the results of cell cycle protein markers in HGC-27 and SNU-1 also showed the same trend (p > 0.05) (Figure 8B). However, SC79 could reverse apoptosis and autophagy in HGC-27 and SNU-1 cells (p < 0.05) (Figure 8C).

To study the relationships between autophagy and ROS-AKT/GSK3β pathway, and between Cos-induced apoptosis and autophagy, we pretreated HGC-27 and SNU-1 with 4 mmol/L 3-MA (an autophagy inhibitor) for 1 h before the cells were incubated with 40 μM Cos. The results revealed 3-MA could reverse the downregulation of cell viability after Cos treatment in HGC-27 and SNU-1 cells (Figure 9A). The flow cytometry results showed that 3-MA did not reverse the upregulation of ROS after Cos treatment in HGC-27 and SNU-1 cells (Figure 9B), and the Western blot results showed 3-MA also did not reverse the upregulation of P-AKT and P-GSK3β (Figure 9C), which meant autophagy was downstream to ROS-AKT/GSK3β pathway. Western blot results showed that 3-MA could reverse the upregulation of autophagy-related and intrinsic apoptosis-related proteins after Cos treatment in HGC-27 and SNU-1, while extrinsic apoptosis-related proteins were not significantly altered among these groups. This indicated that Cos induced intrinsic apoptosis via activating pro-death autophagy (p < 0.05, Figure 9D).

To estimate the anti-tumor growth effect of Cos in vivo, HGC-27 tumor-bearing xenograft nude mouse models were established and treated. The results showed that tumor volume and weight in 30 mg/kg and 50 mg/kg Cos were significantly reduced compared with the DMSO group, especially in the 50 mg/kg group (p < 0.01), but both of them increased compared to the Cisplatin group (Figures 10A–C). IVIS images showed the same change after Cos treatment for 15 and 24 days (p < 0.01) (Figure 10D). In addition, the HE staining results of tumor tissue revealed the number of tumor cells in tissue sections was decreased by Cos administration in mice and was even less in the 50 mg/kg Cos group. As shown in Ki-67 and P-AKT immunohistochemical staining results, Ki-67 and P-AKT positive ratios were obviously inhibited in the 30 mg/kg and 50 mg/kg Cos group, especially in the 50 mg/kg group, compared with the DMSO group (p < 0.01). In contrast, the Tunel staining was increased in Cos-treated mice, especially in the 50 mg/kg Cos group (p < 0.01) (Figure 10E).

Western blot results confirmed that intrinsic apoptotic associated proteins (Cle-Caspase 3, Bak, Bax, Cle-PARP) (Figure 11A) and autophagy-associated protein LC3BII (Figure 11B) were upregulated in Cos treatment groups, and was higher in the 50 mg/kg Cos-treated group compared with DMSO group, while apoptosis-related protein Bcl-2, autophagy-related protein p62, and the ratio of P-AKT/AKT and P-GSK3β/ GSK3β (Figure 11C) were significantly decreased in the Cos treatment group, especially in the 50 mg/kg treatment group, compared with DMSO group (p < 0.001).

The results showed no significant change in body and liver weight between the Cos treatment group and the DMSO group (p > 0.05) (Figures 12A,B), and HE staining of pathological sections elucidated that Cos treatment had no evident damage to the major organs (heart, liver, spleen, lung, and kidney) of mice (Figure 12C), which confirmed the safety of Cos in vivo.