Chrysosplenetin B induces apoptosis and inhibits metastasis of gastric cancer AGS cell by regulating reactive oxygen species-mediated signaling pathways

Chrysosplenetin B induces apoptosis and inhibits metastasis of gastric cancer AGS cell by regulating reactive oxygen species-mediated signaling pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chrysosplenetin B induces apoptosis and inhibits metastasis of gastric cancer AGS cell by regulating reactive oxygen species-mediated signaling pathways Hui Xue, Shu-Mei Li, Yan-Jun Tang, Jing-Long Cao, Wen-Shuang Hou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3796020/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chrysosplenetin B (CHR) is a flavonoid compound with various pharmacological activities. This study aimed to investigate the effect and mechanism of CHR on gastric cancer (GC). A cell counting Kit 8 assay results showed that CHR had a good cytotoxic effect in twelve types of GC cell lines. Annexin-V/PI staining, flow cytometry, and western blot analysis results showed that CHR induced mitochondrial-dependent apoptosis of AGS cells by decreasing mitochondrial membrane potential and increasing the expression levels of Bad/Bcl-2 homologous dimer proteins. Network pharmacological analysis results showed that there were twenty high-value signaling pathways correlated with CHR and GC, among which AKT, MAPK, and STAT3 signaling pathways were closely related to the CHR induced apoptosis signaling pathways on AGS cells. Further through western blot analysis results showed that the protein expression levels of p-AKT, p-ERK, and p-STAT3 were significantly decreased, while the protein expression levels of p-JNK and p-p38 were significantly increased. Moreover, reactive oxygen species (ROS) analysis results showed that CHR induced ROS accumulation on AGS cells as an initial signal to regulate downstream signaling pathways. Cell cycle results showed that CHR arrested the AGS cell cycle in the G2/M phase by regulating the ROS/AKT signaling pathway. Transwell and wound healing assay results showed that CHR inhibited the invasion and migration of AGS cells by regulating ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway. In conclusion, CHR inhibited cell proliferation, induced cell apoptosis, arrested the cell cycle in the G2/M phase, and inhibited invasion and migration on AGS cells. Chrysosplenetin B Gastric cancer Cell apoptosis Cell cycle Cell metastasis Reactive oxygen species Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Gastric cancer (GC) is a malignancy of the gastrointestinal tract and ranks among the top five new cancer incidence types worldwide [ 1 ]. In recent years, the prevalence and mortality of GC have increased significantly, and the number of new cases of GC in the world ranks top five [ 2 ]. Due to the lack of specific symptoms, signs, and low detection rate of early GC, patients have delayed treatment and their symptoms have worsened [ 3 ]. According to the onset period of GC, there are various treatments to choose. At present, in addition to surgical treatment for GC, chemotherapy and radiotherapy can maximize tumor control and prolong the survival of patients [ 4 – 7 ]. Nonetheless, these treatments are poorly targeted and have multiple side effects [ 8 , 9 ]. Therefore, it is urgent to find a kind of anticancer drug with good anticancer effects, few side effects, and cheapness. Reactive oxygen species (ROS) originates from the bottom of the respiratory chain of the inner mitochondrial membrane and it is related to biological activities such as mitochondria-dependent apoptosis [ 10 ]. As a second messenger, ROS controls a variety of signaling cascades that induce and maintain tumorigenic phenotypes in cancer cells [ 11 , 12 ]. The ROS-mediated AKT signaling pathway is a key medium for growth factory-induced cell survival and can be activated by various growth signals to regulate downstream protein functions [ 13 ]. Mitogen-activated protein kinase (MAPK) signaling pathway is mainly composed of JNK, ERK, and p38. The ROS-mediated MAPK signaling pathway regulates many key functions such as cell proliferation, cell apoptosis, and cell senescence [ 14 – 16 ]. ROS-mediated the transcription-3 signal transduction and activator (STAT3) signaling pathway to regulate the basic functions of cells and the expression levels of related genes, and is one of the central communication nodes for many cellular functions, including apoptosis [ 17 , 18 ]. Crosstalk between AKT, MAPK, and STAT3 signaling pathways has been described in multiple cancer types [ 19 – 21 ]. The abundant resources of natural traditional Chinese medicine (TCM) with minimal side effects offer a good source of candidate substances for the development of anticancer drugs [ 22 , 23 ]. CHR is mainly extracted from Composite plants and has a variety of pharmacological activities, including antiviral and antibacterial [ 24 , 25 ]. Previous research has shown that CHR be verified to have an inhibitory effect on neuraminidase and its antiviral effect was comparable to that of the traditional inhibitor oseltamivir phosphate [ 24 ]. In vitro antibacterial assay has confirmed that CHR has a strong inhibitory effect on staphylococcus aureus [ 25 ]. However, the mechanism of action of CHR on GC cells remains unclear. Network pharmacology is an analysis that directly demonstrates the interaction between drugs and biomolecules using network database. It is based on a large amount of bioinformatics, systems biology data, and network technologies to explore the mechanism of drug action, predict targets, and related signaling pathways [ 26 ]. Network pharmacology can link the biomolecular networks of cancer and anticancer drugs, and then systematically explore the overall regulatory mechanism of anticancer drugs on cancer [ 27 ]. This study evaluated the pharmacological effects of CHR on GC and the associated molecular mechanisms. The effects of CHR on cell apoptosis, cell cycle, and cell migration of GC cells were also verified. Materials and methods Cell culture The twelve types of human GC cells (AGS, SNU-484, MKN-45, MKN-28, KATO-3, SNU-5, NCI-N87, YCC-1, SNU-216, SNU-668, YCC-6, and YCC-16) and human normal lung IMR-90 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), the human gastric epithelial GES-1 cells and human renal epithelial 293T cells were purchased from Saga Biotechnology Co. (Shanghai, China). The human liver THLE-2 cells were purchased from Otwo Biotech (Shenzhen, China). All types of cells were grown into DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin and streptomycin (P/S, Gibco). All types of cells were placed in an incubator at 5% CO 2 and 37°C (Sanyo, Osaka, Japan). Cell viability assay The survival rates of twelve types of human GC cell lines and four types of human normal cell lines were determined by Cell Counting Kit-8 (CCK-8) (Solarbio, Beijing, China). All cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well and cultured for 24 h. Cells were treated with CHR (Herbpurify, Chengdu, China) and 5-fluorouracil (5-FU) (Med Chem Express, Princeton, NJ, USA) at different concentrations (20, 40, 60, 80, and 100 µM) and at different time gradients (6, 12, 18, 24, and 30 h). Solutions of 10 µL of CCK-8 were added to each well and incubated away from the light for 3 h. Cell viability after CHR treatment were measured by a multifunctional microplate reader at 495 nm wavelength (Tecan, Mannedorf, Switzerland). Concentrations that caused 50% cell growth inhibition (IC 50 ) values of cells treated with CHR were calculated using Sigma Plot 15.0 software. Cell apoptosis assay The induction of apoptosis effect of CHR on AGS cell was determined by Annexin V-FITC/PI Apoptosis Kit (4A Biotech, Beijing, China). AGS cells were seeded in 6-well plates at a density of 1 × 10 6 cells/well. AGS cells were treated with CHR (the IC 50 values of 40.03 µM) and 5-FU (40.03 µM) at different times (3, 6, 12, and 24 h). Operate according to the instructions, the collected AGS cells were mixed with 200 µL 1× binding buffer, 5 µL Annexin V-FITC, and 3 µL Propidium Iodide (PI) successively. AGS cells were incubated in a refrigerator at 4°C for 20 min. The morphological changes of AGS cells after CHR treatment were determined by fluorescence microscopy (MSHOT, Guangzhou, China). The proportion of apoptosis was observed by flow cytometry (Sysmes Co., Kobe, Japan). Associated targets of CHR and GC analysis The 2D structure of CHR was determined by the PubChem database ( https://pubchem.ncbi.nlm.nih.gov ) and submitted to the Swiss ADME platform ( http://www.swisssimilarity.ch/ ) for drug property prediction of CHR, and the prediction target gene of CHR was obtained using Target Prediction database ( http://www.swisstargetprediction.ch/ ). The genetic target of GC was obtained using GeneCards database ( https://www.genecards.org/ ). Overlapping target protein-protein interaction networks analysis The overlapping targets of CHR and GC were predicted on VENNY 2.1 website ( http://www.liuxiaoyuyuan.cn/ ). The target of the intersection number obtained was the common potential target of CHR and GC. Submit the resulting common targets to the STRING database ( https://string-db.org/ ) for the protein-protein interaction (PPI) network. Visual analysis of PPI networks using Cytoscape 3.9.1 software. GO and KEGG enrichment analysis The PPI overlapping targets predicted import DAVID Bioinformatics Resources 6.8 ( https://david.ncifcrf.gov/ ) in the database for enrichment analysis. Gene Ontology (GO) enrichment was used to analyze the biological process (BP), molecular function (MF), and cellular component (CC) of the cross-linked targets, while Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was used to analyze the involved signaling pathways. Finally, the functions were sorted according to p -value, and it is considered that the GO and KEGG channels with p -value < 0.05 is significant. Molecular docking analysis The PDB format of core targets was obtained through RSCB-PDB database ( https://www.rcsb.org/ ). Water removal molecules and ligands were performed at the core target in PyMOL software. The 3D structure of CHR was obtained through PubChem database and processed in Open Babel software. The core target was hydrogenated in the AutoDocks software, the charge number was calculated, and the rigid structure of the atom was determined. The docking tool processed in the AutoDockTools-1.5.6 software perform molecular docking and selects the site with the lowest energy for subsequent operations. Next, the docking site visualization of CHR and core target was created in PyMOL software and corresponding protein residues, binding bonds, and distances were displayed. Mitochondrial membrane potential analysis The mitochondrial membrane potential (MMP) of AGS cell after CHR treatment was determined by JC-1 Detection Kit (Solarbio, Beijing, China). AGS cells were seeded in 3.5 cm cell culture dish at a density of 1 × 10 5 cells/well. AGS cells were treated with CHR (40.03 µM) at different times (3, 6, 12, and 24 h). 1 mL JC-1 staining solution was added to the collected AGS cells and heated in a 37°C water bath for 30 min. Then, 1 mL 1× JC-1 binding buffer and 500 µL of phosphate-buffered saline (PBS) were added to AGS cells treated with CHR. The MMP of AGS cell was measured by flow cytometry. Western blot analysis The expression levels of AGS cells related proteins after CHR treatment were determined by Western Blot Analysis. AGS cells were seeded in a 6 cm cell culture dish at a density of 1 × 10 6 cells/well. AGS cells treated with CHR (40.03 µM) at different times (3, 6, 12, and 24 h), and protein extracts were obtained with 100 µL lysis buffer. The same amount of total protein was isolated on 12% SDS-PAGE, then the gel is electrically transferred to the nitrocellulose membrane. 5% skimmed milk was added and closed at 25°C for 2 h to avoid non-specific binding. The primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated with the film in a refrigerator at 4°C away from the light. Next, the membrane was incubated with the secondary antibody (ZSBG-BIO, Beijing, China) at room temperature for 2 h. The enhanced chemiluminescence (Tanon, Shanghai, China) solution-binding proteins. Images were exposed using multifunction imager (Analytik Jena AG, Jena, Germany), and the bands were analyzed with the ImageJ version 1.42. ROS levels analysis The ROS levels in AGS cells treated with CHR were determined by ROS Detection Kit (Beyotime Institute Biotechnology, Shanghai, China). AGS cells were seeded in a 3.5 cm cell culture dishes at a cell density of 1 × 10 5 cells/well and treated with CHR (40.03 µM) at different times (3, 6, 12, and 24 h). AGS cells was combined with 10 µL 2'7'-dichlorofluorescein diacetate (DCFH-DA) and incubated at 37°C for 30 min away from the light. ROS levels were determined by flow cytometry. Cell cycle analysis The cell cycle arrest of AGS after CHR treatment was determined by DNA Content Detection Kit (Solarbio, Beijing, China). AGS cells were seeded in a 3.5 cm cell culture dishes at a cell density of 1 × 10 5 cells/well and treated with CHR (40.03 µM) at different times (3, 6, 12, and 24 h). AGS cells were collected, fixed in pre-cooled 70% ethanol and stored overnight at 4°C. 100 µL RNase was added to the cells and incubated at 37°C away from the light for 30 min. Then 400 µL PI was added to the AGS cells and incubated at 4°C for 30 min. The changes in the number of CHR treated AGS cells in G0/G1 and G2/M phases were determined by flow cytometry. Cell invasion and migration analysis The inhibitory effect of CHR on AGS cells invasion was determined by Transwell Assay. AGS cells suspension containing serum-free DMEM medium was added to the upper chamber, and DMEM medium containing 10% FBS and 1% P/S was added to the lower chamber. The AGS cells treated with CHR (40.03 µM) at different times (3, 6, 12, and 24 h) were stained with 1% crystal violet (Solarbio, Beijing, China). The petri dish was placed under a fluorescence microscopy to observe the number of invading cells. In addition, the inhibitory effect of CHR on the migration of AGS cells was determined by Wound-Healing Assay. AGS cells were seeded in 6-well plates at a cell density of 1 × 10 6 cells/well and treated with CHR (40.03 µM) at different times (3, 6, 12, and 24 h). After the treatment time was reached, vertical labeling lines were taken. After 2 h, the culture medium was aspirated and the healing of cells was observed under fluorescence microscopy. Statistical analysis The data were all repeated three times and assessed by mean ± standard deviation. Sigma Plot 15.0 software was used to calculate IC 50 . SPSS 29.0 software was used for Tukey’s postmortem test, and p < 0.05 was statistically significant. Results Killing effect of CHR on GC cell As shown in Fig. 1 a and b, CHR had a good killing effect on all twelve types of GC cells at a dose and time-dependent manner. The survival rate of twelve types of GC cells after CHR treatment were significantly lower than that of 5-FU treatment group. The effects of CHR and 5-FU on survival rate of four types of normal human cells (GES-1, 293T, THLE-2, and IMR-90) were measured by a CCK-8 assay at dose gradient and time gradient, respectively. As shown in Fig. 1 c and d, the survival rate of the four types of normal cells after CHR treatment were higher than that in the 5-FU treatment group. It was proved that CHR had no significant toxic and side effects on normal human gastric, liver, lung, and renal cells. As shown in Table 1 , IC 50 value of twelve types of GC cells by CHR and 5-FU were summarized, and the sensitivity of AGS cells to CHR was higher than that of other eleven types of GC cells. AGS can be used as a target for subsequent detection of the anti-GC efficacy of CHR. Table 1 IC50 values of CHR and 5-FU in gastric cancer cells. Number Cell Name 5-FU (µM) CHR (µM) 1 AGS 80.21 ± 1.33 40.03 ± 1.23 2 MKN-45 77.48 ± 0.93 23.78 ± 1.13 3 MKN-28 79.32 ± 1.22 56.55 ± 1.12 4 KATO-3 76.88 ± 0.93 73.46 ± 1.01 5 NCI-N87 80.33 ± 1.03 71.02 ± 0.98 6 SNU-5 90.22 ± 1.1 76.54 ± 0.68 7 SNU-216 89.67 ± 0.88 79.65 ± 0.84 8 SNU-484 83.47 ± 0.99 77.56 ± 1.34 9 SNU-668 83.54 ± 0.87 65.22 ± 1.21 10 YCC-1 76.88 ± 0.93 73.46 ± 1.01 11 YCC-6 79.2 ± 1.33 64.85 ± 1.22 12 YCC-16 81.13 ± 0.83 65.76 ± 1.41 CHR induces apoptosis of AGS cell in mitochondria dependent manner As shown in Fig. 2 a, with the extension of CHR treatment time, the fluorescence intensity of Annexin-FITC/PI double staining was observed under the fluorescence microscopy to increased continuously, and morphological changes such as AGS cells becoming round and shrinking could be observed under the light field. As shown in Fig. 2 b, flow cytometry results showed that with the extension of CHR treatment time, the apoptosis rate of early and late AGS cells increased from 0.56 to 27.65%. As shown in Fig. 2 c, with the extension of CHR treatment time, the intracellular MMP levels decreased from 99.10% at the beginning to 43.97%. The expression levels of apoptotic proteins in mitochondrial pathway were analyzed by western blot. As shown in Fig. 2 d, the protein expression levels of Bad, cytochrome c (cyto-c), cleaved-caspase-3 (cle-caspase-3), and cleaved-PARP (cle-PARP) increased, while the protein expression levels of Bcl-2 decreased. It was further confirmed that CHR could induced apoptosis of AGS cells through endogenous mitochondrial pathway. Construction of protein interaction networks As shown in Fig. 3 a, Venny 2.1 obtained a total of fifty-four predicted targets for GC processing by CHR. As shown in Fig. 3 b, the interaction diagram of protein targets predicted by the common target of CHR and GC through the String website. As shown in Fig. 3 c, the interaction diagram of the PPI network was further screened by Cytoscape software. As shown in Fig. 3 d, sixteen core protein targets with high degree value were further screened in Cytoscape software. The results showed that CHR interacted with different targets of GC, among which AKT 1 and EGFR were the first two degrees. Target biological function analysis As shown in Fig. 3 e, the BP-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig. 3 f, the CC-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig. 3 g, the MF-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig. 3 h, it was a collection of BP, CC, and MF enrichment analysis of fifty-four cross targets by using DAVID Bioinformatics Resources 6.8. As shown in Fig. 3 i and j, KEGG enrichment analysis showed that twenty signal pathways that were closely related to CHR and GC crosslinking. The results showed that the protein phosphorylation degree value was higher, the activity of protein kinase B was stronger. Moreover, the degree values of AKT signaling pathway and cancer signaling pathway were in the top two places in KEGG analysis. CHR may play an anti-GC role by regulating protein kinase B phosphorylation. Molecular docking analysis As shown in Fig. 3 k, the grid tool in AutoDockTool-1.5.6 software was used to build a complete CHR binding site network map, and the molecular docking between CHR and EGFR was completed by docking tools. The corresponding protein residues were visualized in rod shape by PyMOL software, and the residues were distinguished by different colors. CHR binds to the GLN-791 residue on EGFR by hydrogen bonding. As shown in Fig. 3 l the results of the docking site of CHR and AKT. CHR forms three hydrogen bonds with GLU-9, ARG-41 and PRO-42 on AKT. Molecular docking results showed that CHR and AKT had good binding ability. CHR induces apoptosis of AGS cell by regulate AKT, MAPK, and STAT3 signaling pathways As shown in Fig. 4 a, the expression levels of p-AKT, p-ERK, and p-STAT3 protein were decreased, while the expression levels of p-JNK and p-p38 protein were increased. In addition, to verify the upstream and downstream relationship between MAPK and STAT3 signaling pathways, pretreated AGS cells with a 10 µM FR180204 (ERK inhibitor), a 10 µM SP600125 (JNK inhibitor), and a 10 µM SB203580 (p38 inhibitor). As shown in Fig. 4 b-d, the MAPK inhibitor + CHR co-treatment group significantly reversed the expression levels of corresponding proteins in the CHR alone treatment group. Among them, the CHR + ERK inhibitor co-treatment group promoted the expression levels of p-ERK and p-STAT3 protein compared with the CHR alone treatment group, CHR + JNK inhibitor and CHR + p38 inhibitor co-treatment groups inhibited p-JNK and p-p38 proteins expression levels, and promoted p-STAT3 proteins expression levels compared with CHR alone treatment group. The results confirmed that CHR could induces apoptosis of AGS cell by regulating AKT, MAPK, and STAT3 signaling pathways, with MAPK located upstream of STAT3 signaling pathway. CHR induces apoptosis of AGS cell by up-regulating ROS levels As shown in Fig. 5 a, with the extension of CHR treatment time, ROS levels on AGS cells continuously accumulated. To further verify the relationship between apoptosis of AGS cells induced by CHR and ROS accumulation, NAC was added to AGS cells for pre-treatment before CHR treatment. As shown in Fig. 5 b, after NAC treatment, the apoptosis rate of AGS cells treated with CHR decreased from 42.24 to 3.4%. As shown in Fig. 5 c, the expression levels of apoptosis-related signaling pathway proteins were reversed in CHR + NAC co-treatment group compared with CHR alone treatment group. The results of ROS analysis showed that CHR played an anticancer role by up-regulating the ROS levels on AGS cells. CHR arrests AGS cell cycle in G2/M phase As shown in Fig. 6 a, with the extension of CHR treatment time, the number of AGS cells in G0/G1 phase decreased from 71.47 to 44.94%, while the number of AGS cells in G2/M phase increased from 2.15 to 16.23%. In addition, to investigate the relationship between cell cycle arrest and ROS accumulation, the AGS cells were pretreated with NAC before CHR treatment. As shown in Fig. 6 b, NAC significantly inhibited the number of G2/M phase cells after CHR treatment. As shown in Fig. 6 c, with the increased of CHR treatment time, the expression levels of p-AKT, CDK 1/2, and cyclin B1 proteins decreased, while the expression levels of p27 and p21 proteins increased. In addition, as shown in Fig. 6 d, the expression levels of cycle-related proteins were reversed in CHR + NAC co-treatment group compared with CHR alone treatment group. The results suggest that CHR inhibited AGS cell cycle in G2/M phase by regulating ROS/AKT signaling pathway. CHR inhibits the invasion and migration of AGS cell As shown in Fig. 7 a, with the extension of CHR treatment time, the number of AGS cell invasion from the upper cavity to the lower cavity significantly decreased compared with the control group. As shown in Fig. 7 b, with the increased of CHR treatment time, compared with the control group, the migration of AGS cell was significantly inhibited. As shown in Fig. 7 c, with the increased of CHR treatment time, the expression levels of E-cadherin protein increased, while the expression levels of N-cadherin, Wnt-3a, β-catenin, and p-GSK-3β protein decreased. As shown in Fig. 7 d, the expression levels of migration-related signaling pathway proteins were reversed in CHR + NAC co-treatment group compared with CHR alone treatment group. The results suggest that CHR inhibits the invasion and migration of AGS cell by regulating the ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway. Discussion CHR is an active isoflavone extracted from Chamomile plant [ 28 ]. Previous studies have shown that isoflavones such as soy isoflavones and barbigerone induces apoptosis of cancer cells through MMP pathway [ 29 , 30 ]. As a polymethoxy-flavonoid, CHR has been verified to have dose-dependent inhibitory effects on lung cancer A549 and cervical cancer Hela cells, but has low toxicity to normal Vero and EVC304 cells [ 31 ]. The effects of CHR on the anticancer activity of GC were verified by CCK-8 assay in this study. The experimental data of this study showed that CHR had good toxic effects on twelve types of GC cells, and the survival rate of GC cells in the CHR treatment group was lower than that in the traditional chemotherapy drug 5-FU treatment group in both the concentration gradient and the time gradient. However, the toxic effect of CHR on gastric, liver, renal, and lung cells in normal human is lower than that of 5-FU. The above results have shown that CHR has anticancer activity on GC cells, and then this study explored its specific anti-GC mechanism through a series of experiments. There are many ways of cell death, among which apoptosis is the most typical programmed cell death. Apoptosis controls the spontaneous and orderly death of cells by regulating the expression levels of signaling pathways and plays a crucial role in the normal growth and development of cells [ 32 ]. The main source of intracellular ROS is the substrate end of the respiratory chain in the mitochondrial inner membrane [ 33 ]. When cancer cells receive death signals such as oxidative stress, mitochondria-dependent apoptosis dependent on caspases will be triggered [ 34 ]. The Bcl-2 family proteins located upstream of caspases regulate the mitochondrial apoptosis pathway, and the pro-apoptotic proteins will homodimerization or heterodimerize with the anti-apoptotic proteins to regulate the downstream apoptotic signal cascade [ 35 , 36 ]. The experimental results of this study showed that when CHR induced AGS cells apoptosis, the MMP decreased, the proportion of Bad/Bcl-2 protein expression levels increased, and cyto-c was released from mitochondria into the cytoplasm to activate the downstream apoptosis effecting factor caspase-3, and further shear the downstream substrate PARP to complete the mitochondria-dependent apoptosis pathway. Network pharmacology can screen and visually analyze the network relationships among TCM molecules, targets, and diseases through a high-throughput database [ 37 ]. TCM therapy has the characteristics of multi-target and multi-signal pathway, which is consistent with the comprehensiveness and systematicness of network pharmacology [ 38 , 39 ]. Therefore, network pharmacology is suitable for predicting the molecular mechanisms and signaling pathways of TCM compounds [ 40 ]. In this study, through network pharmacological predictive analysis and molecular docking, we explored the relevant targets (AKT and EGFR) and signaling pathways (AKT/MAPK/STAT3) of CHR action on GC. Further western blot analysis confirmed that CHR induced apoptosis of AGS cells in human GC by regulating ROS/AKT/MAPK/STAT3 signaling pathway. ROS is a natural by-product of normal oxygen metabolism, which mainly includes highly active heterogeneous molecules such as peroxides, superoxides, and hydroxyl radicals [ 41 , 42 ]. The generation and accumulation of ROS plays a key role in tumor transformation, cell proliferation, and apoptosis [ 43 ]. When ROS levels in the body fall below the ROS threshold, ROS activates oncogenes, inducing DNA repair and cancer cell survival [ 44 ]. When the ROS accumulation levels is higher than the threshold, ROS will trigger apoptosis signals and regulate downstream signaling pathways to induce cancer cell death [ 45 ]. In this study, flow cytometry analysis results showed that the ROS accumulation levels in AGS cells after CHR treatment were increased. Further, NAC pretreatment of AGS cells reversed verified that CHR mediated ROS accumulation regulated AKT, MAPK, and STAT3 signaling pathways induced apoptosis of AGS cell. Cell cycle progression is mediated by cyclin-dependent kinases (CDKs) and their cyclins, which strictly regulate the processes of cell expansion, genetic material replication, and cell division [ 46 ]. There are G1/S phase checkpoints and G2/M phase DNA damage checkpoints in the cell cycle, and the smooth progress of the cell cycle can be judged by the binding activation of CDKs and cyclin chaperone [ 47 ]. The network pharmacology analysis results of this study showed that AKT is the core target of GC cell cycle regulation by CHR, the p21/p27 protein is an inhibitor of the cyclin-CDK complex and can arrest the cell cycle by inhibiting the activity of the cyclin-CDK complex [ 48 – 51 ]. The results of this study showed that CHR induced an increase in the expression levels of p21 and p27 proteins by regulating of the ROS/AKT signaling pathway, which inhibited the expression levels of CDK 1/2 and cyclin B1 complex proteins, and finally CHR arrested the AGS cycle in G2/M phase. Cancer cell metastasis refers to when the primary tumor cells are shed and free in the body, they will engulf healthy cells in the body organs, resulting in the collapse of the immune system defense line, and the growth of malignant tumors of the same nature everywhere in the body [ 52 ]. Inhibiting cancer cell migration lessen the emergence of highly malignant cancer cells, delay the invasion of white blood cells during inflammation, or promote wound healing [ 53 ]. In the process of malignant tumor development, epithelial-mesenchymal transformation (EMT) usually reduces the adhesion between cells and enhances the invasion and migration of cancer cells [ 54 ]. Wnt-3a/β-catenin signaling pathway is a crucial pathway to regulate EMT [ 55 ]. Among them, β-catenin is an important biological indicator to detect whether the Wnt-3a signaling pathway is activate and stabilize intercellular adhesion by intracellular binding to cadherin [ 56 , 57 ]. GSK-3β, located upstream of β-catenin, acts as the main regulatory enzyme of Wnt-3a/β-catenin signaling pathway and negatively regulates wnt-3a signaling pathway by promoting β-catenin degradation [ 58 , 59 ]. In this study, Transwell and wound healing assays confirmed at the cellular levels that CHR inhibits the invasion and migration of AGS cells. Further western blot analysis showed that CHR could regulate the expression levels of ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway proteins, thereby inhibiting the metastasis of AGS cells. Conclusion In summary, this study showed that CHR regulates AKT/MAPK/STAT3 signaling pathway by up-regulating ROS levels on AGS human GC cells, leading to G2/M cell cycle arrest and inducing cell apoptosis. Moreover, CHR inhibited the invasion and migration of AGS cells on GC by regulating ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway (Fig. 8). Declarations Conflict of interest: The authors declare no competing interests. Funding: This research was funded by Heilongjiang Province Key Research and Development Plan Guidance Project (No. GZ20220039), Central Government Supports Local College Reform and Development Fund Talent Training Project (No. 2020GSP16), and Heilongjiang Touyan Innovation Team Program (No. 2019HTY078). Author Contribution H. Xue. Conceptualization and writing-original draft preparation; S.-M. Li and Y.-J. Tang. writing-review and editing; J.-L. Cao and W.-S. Hou. methodology; A.-Q. Wang. software; W.-X. Ren. data analysis; C.-H. Jin. supervision. All authors have read and agreed to the published version of the manuscript. References Correa P. Gastric cancer: overview. Gastroenterol Clin North Am. 2013;42(2):211–7. https://doi.org/10.1016/j.gtc.2013.01.002 . Morgan E, Arnold M, Camargo MC, et al. The current and future incidence and mortality of gastric cancer in 185 countries, 2020-40: A population-based modelling study. EClinicalMedicine. 2022;47:101404. https://doi.org/10.1016/j.eclinm.2022.101404 . Machlowska J, Baj J, Sitarz M, et al. Gastric Cancer: Epidemiology, Risk Factors, Classification, Genomic Characteristics and Treatment Strategies. Int J Mol Sci. 2020;21(11):4012. https://doi.org/10.3390/ijms21114012 . Zeng Y, Jin RU. Molecular pathogenesis, targeted therapies, and future perspectives for gastric cancer. Semin Cancer Biol. 2022;86(Pt3):566–82. https://doi.org/10.1016/j.semcancer.2021.12.004 . Tan Z. Recent Advances in the Surgical Treatment of Advanced Gastric Cancer: A Review. Med Sci Monit. 2019;25:3537–41. https://doi.org/10.12659/MSM.916475 . Wang Y, Zhang L, Yang Y, et al. Progress of Gastric Cancer Surgery in the era of Precision Medicine. Int J Biol Sci. 2021;17(4):1041–9. https://doi.org/10.7150/ijbs.56735 . Patel TH, Cecchini M. Targeted Therapies in Advanced Gastric Cancer. Curr Treat Options Oncol. 2020;21(9):70. https://doi.org/10.1007/s11864-020-00774-4 . Schwarz RE. Current status of management of malignant disease: current management of gastric cancer. J Gastrointest Surg. 2015;19(4):782–8. https://doi.org/10.1007/s11605-014-2707-x . Hoya Y, Mitsumori N, Yanaga K. The advantages and disadvantages of a Roux-en-Y reconstruction after a distal gastrectomy for gastric cancer. Surg Today. 2009;39(8):647–51. https://doi.org/10.1007/s00595-009-3964-2 . Srinivas US, Tan BWQ, Vellayappan BA, et al. ROS and the DNA damage response in cancer. Redox Biol. 2019;25:101084. https://doi.org/10.1016/j.redox.2018.101084 . Tuli HS, Kaur J, Vashishth K, et al. Molecular mechanisms behind ROS regulation in cancer: A balancing act between augmented tumorigenesis and cell apoptosis. Arch Toxicol. 2023;97(1):103–20. https://doi.org/10.1007/s00204-022-03421-z . Li Y, Xia J, Jiang N, et al. Corin protects H 2 O 2 -induced apoptosis through PI3K/AKT and NF-κB pathway in cardiomyocytes. Biomed Pharmacother. 2018;97:594–9. https://doi.org/10.1016/j.biopha.2017.10.090 . Fattahi S, Amjadi-Moheb F, Tabaripour R, et al. PI3K/AKT/mTOR signaling in gastric cancer: Epigenetics and beyond. Life Sci. 2020;262:118513. https://doi.org/10.1016/j.lfs.2020.118513 . Yong HY, Koh MS, Moon A. The p38 MAPK inhibitors for the treatment of inflammatory diseases and cancer. Expert Opin Investig Drugs. 2009;18(12):1893–905. https://doi.org/10.1517/13543780903321490 . Hendrikse CSE, Theelen PMM, van der Ploeg P, et al. The potential of RAS/RAF/MEK/ERK (MAPK) signaling pathway inhibitors in ovarian cancer: A systematic review and meta-analysis. Gynecol Oncol. 2023;171:83–94. https://doi.org/10.1016/j.ygyno.2023.01.038 . Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Cell Biol. 2007;19(2):142–9. https://doi.org/10.1016/j.ceb.2007.02.001 . Ma JH, Qin L, Li X. Role of STAT3 signaling pathway in breast cancer. Cell Commun Signal. 2020;18(1):33. https://doi.org/10.1186/s12964-020-0527-z . Yu H, Lee H, Herrmann A, et al. Revisiting STAT3 signaling in cancer: new and unexpected biological functions. Nat Rev Cancer. 2014;14(11):736–46. https://doi.org/10.1038/nrc3818 . Zhang X, Zeng Y, Qu Q, et al. PD-L1 induced by IFN-γ from tumor-associated macrophages via the JAK/STAT3 and PI3K/AKT signaling pathways promoted progression of lung cancer. Int J Clin Oncol. 2017;22(6):1026–33. https://doi.org/10.1007/s10147-017-1161-7 . Liu Y, Lv H, Li X, et al. Cyclovirobuxine inhibits the progression of clear cell renal cell carcinoma by suppressing the IGFBP3-AKT/STAT3/MAPK-Snail signaling pathway. Int J Biol Sci. 2021;17(13):3522–37. https://doi.org/10.7150/ijbs.62114 . Yao Y, Yang G, Lu G, et al. Th22 Cells/IL-22 Serves as a Protumor Regulator to Drive Poor Prognosis through the JAK-STAT3/MAPK/AKT Signaling Pathway in Non-Small-Cell Lung Cancer. J Immunol Res. 2022;2022:8071234. https://doi.org/10.1155/2022/8071234 . Thomas E, Stewart LE, Darley BA, et al. Plant-Based Natural Products and Extracts: Potential Source to Develop New Antiviral Drug Candidates. Molecules. 2021;26(20):6197. https://doi.org/10.3390/molecules26206197 . Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, et al. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv. 2015;33(8):1582–614. https://doi.org/10.1016/j.biotechadv.2015.08.001 . Mbarga PE, Fouotsa H, Ndemangou B, et al. Two new secondary metabolites with antibacterial activities from Conyza aegyptiaca (Asteraceae). Nat Prod Res. 2023;37(11):1806–15. https://doi.org/10.1080/14786419.2022.2122965 . Duan X, Li J, Cui J, et al. Chemical component and in vitro protective effects of Matricaria chamomilla (L.) against lipopolysaccharide insult. J Ethnopharmacol. 2022;296:115471. https://doi.org/10.1016/j.jep.2022.115471 . Nogales C, Mamdouh ZM, List M, et al. Network pharmacology: curing causal mechanisms instead of treating symptoms. Trends Pharmacol Sci. 2022;43(2):136–50. https://doi.org/10.1016/j.tips.2021.11.004 . Wang W, Yao Q, Teng F, et al. TCM network pharmacology: A new trend towards combining computational, experimental and clinical approaches. Chin J Nat Med. 2021;19(1):1–11. https://doi.org/10.1016/S1875-5364(21)60001-8 . Kim C, Kim B. Anti-Cancer Natural Products and Their Bioactive Compounds Inducing ER Stress-Mediated Apoptosis: A Review. Nutrients. 2018;10(8):1021. https://doi.org/10.3390/nu10081021 . Li G, Ding K, Qiao Y, et al. Flavonoids Regulate Inflammation and Oxidative Stress in Cancer. Molecules. 2020;25(23):5628. https://doi.org/10.3390/molecules25235628 . Alharbi Y, Kapur A, Felder M, et al. Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase. Sci Rep. 2020;10(1):19585. https://doi.org/10.1038/s41598-020-76342-5 . Cao C, Liu B, Shen W, et al. Research on antiproliferative effect of flavones isolated from Laggera pterodonta. Zhongguo Zhong Yao Za Zhi. 2010;35(16):2171–4. Chinese. Yang Y, Karakhanova S, Hartwig W, et al. Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy. J Cell Physiol. 2016;231(12):2570–81. https://doi.org/10.1002/jcp.25349 . Jeong SY, Seol DW. The role of mitochondria in apoptosis. BMB Rep. 2008;41(1):11–22. https://doi.org/10.5483/bmbrep.2008.41.1.011 . Ashkenazi A, Fairbrother WJ, Leverson JD, et al. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat Rev Drug Discov. 2017;16(4):273–84. https://doi.org/10.1038/nrd.2016.253 . Nemec KN, Khaled AR. Therapeutic modulation of apoptosis: targeting the BCL-2 family at the interface of the mitochondrial membrane. Yonsei Med J. 2008;49(5):689–97. https://doi.org/10.3349/ymj.2008.49.5.689 . Lopez A, Reyna DE, Gitego N, et al. Co-targeting of BAX and BCL-XL proteins broadly overcomes resistance to apoptosis in cancer. Nat Commun. 2022;13(1):1199. https://doi.org/10.1038/s41467-022-28741-7 . Zhang S, Lu Y, Chen W, et al. Network Pharmacology and Experimental Evidence: PI3K/AKT Signaling Pathway is Involved in the Antidepressive Roles of Chaihu Shugan San. Drug Des Devel Ther. 2021;15:3425–41. https://doi.org/10.2147/DDDT.S315060 . Wu N, Yuan T, Yin Z, et al. Network Pharmacology and Molecular Docking Study of the Chinese Miao Medicine Sidaxue in the Treatment of Rheumatoid Arthritis. Drug Des Devel Ther. 2022;16:435–66. https://doi.org/10.2147/DDDT.S330947 . Li S, Zhang B. Traditional Chinese medicine network pharmacology: theory, methodology and application. Chin J Nat Med. 2013;11(2):110–20. https://doi.org/10.1016/S1875-5364(13)60037-0 . Guo W, Huang J, Wang N, et al. Integrating Network Pharmacology and Pharmacological Evaluation for Deciphering the Action Mechanism of Herbal Formula Zuojin Pill in Suppressing Hepatocellular Carcinoma. Front Pharmacol. 2019;10:1185. https://doi.org/10.3389/fphar.2019.01185 . Sarniak A, Lipińska J, Tytman K, et al. Endogenous mechanisms of reactive oxygen species (ROS) generation. Postepy Hig Med Dosw (Online). 2016;70(0):1150–65. https://doi.org/10.5604/17322693.1224259 . Moloney JN, Cotter TG. ROS signaling in the biology of cancer. Semin Cell Dev Biol. 2018;80:50–64. https://doi.org/10.1016/j.semcdb.2017.05.023 . Yang S, Lian G. ROS and diseases: role in metabolism and energy supply. Mol Cell Biochem. 2020;467(1–2):1–12. https://doi.org/10.1007/s11010-019-03667-9 . Slika H, Mansour H, Wehbe N, et al. Therapeutic potential of flavonoids in cancer: ROS-mediated mechanisms. Biomed Pharmacother. 2022;146:112442. https://doi.org/10.1016/j.biopha.2021.112442 . Jelic MD, Mandic AD, Maricic SM, et al. Oxidative stress and its role in cancer. J Cancer Res Ther. 2021;17(1):22–8. https://doi.org/10.4103/jcrt.JCRT_862_16 . Wang Z. Cell Cycle Progression and Synchronization: An Overview. Methods Mol Biol. 2022;2579:3–23. https://doi.org/10.1007/978-1-0716-2736-5_1 . Wood DJ, Endicott JA. Structural insights into the functional diversity of the CDK-cyclin family. Open Biol. 2018;8(9):180112. https://doi.org/10.1098/rsob.180112 . Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 2013;140(15):3079–93. https://doi.org/10.1242/dev.091744 . Lee D, Hokinson D, Park S, et al. ER Stress Induces Cell Cycle Arrest at the G2/M Phase Through eIF2α Phosphorylation and GADD45α. Int J Mol Sci. 2019;20(24):6309. https://doi.org/10.3390/ijms20246309 . Vassilev LT. Cell cycle synchronization at the G2/M phase border by reversible inhibition of CDK1. Cell Cycle. 2006;5(22):2555–6. https://doi.org/10.4161/cc.5.22.3463 . Suski JM, Braun M, Strmiska V, et al. Targeting cell-cycle machinery in cancer. Cancer Cell. 2021;39(6):759–78. https://doi.org/10.1016/j.ccell.2021.03.010 . Mestre-Farrera A, Bruch-Oms M, Peña R, et al. Glutamine-Directed Migration of Cancer-Activated Fibroblasts Facilitates Epithelial Tumor Invasion. Cancer Res. 2021;81(2):438–51. https://doi.org/10.1158/0008-5472.CAN-20-0622 . Um E, Oh JM, Granick S, et al. Cell migration in microengineered tumor environments. Lab Chip. 2017;17(24):4171–85. https://doi.org/10.1039/c7lc00555e . Pastushenko I, Blanpain C. EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol. 2019;29(3):212–26. https://doi.org/10.1016/j.tcb.2018.12.001 . Tian S, Peng P, Li J, et al. SERPINH1 regulates EMT and gastric cancer metastasis via the Wnt/β-catenin signaling pathway. Aging. 2020;12(4):3574–93. https://doi.org/10.18632/aging.102831 . Li Y, Bavarva JH, Wang Z, et al. HEF1, a novel target of Wnt signaling, promotes colonic cell migration and cancer progression. Oncogene. 2011;30(23):2633–43. https://doi.org/10.1038/onc.2010.632 . Wang J, Cai H, Liu Q, et al. Cinobufacini Inhibits Colon Cancer Invasion and Metastasis via Suppressing Wnt/β-Catenin Signaling Pathway and EMT. Am J Chin Med. 2020;48(3):703–18. https://doi.org/10.1142/S0192415X20500354 . Yan Y, Zhao P, Wang Z, et al. PRMT5 regulates colorectal cancer cell growth and EMT via EGFR/Akt/GSK3β signaling cascades. Aging. 2021;13(3):4468–81. https://doi.org/10.18632/aging.202407 . Wu H, Lu XX, Wang JR, et al. TRAF6 inhibits colorectal cancer metastasis through regulating selective autophagic CTNNB1/β-catenin degradation and is targeted for GSK3B/GSK3β-mediated phosphorylation and degradation. Autophagy. 2019;15(9):1506–22. https://doi.org/10.1080/15548627.2019.1586250 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3796020","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":262675867,"identity":"93012601-719c-482d-acee-24c9b10a4af4","order_by":0,"name":"Hui Xue","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Xue","suffix":""},{"id":262675868,"identity":"b9c3bd12-0f75-4f77-9c4c-98fceacf6b23","order_by":1,"name":"Shu-Mei Li","email":"","orcid":"","institution":"Hemodialysis Center, Daqing Oilfield General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shu-Mei","middleName":"","lastName":"Li","suffix":""},{"id":262675869,"identity":"955c130e-42e8-45ae-884f-30146e774b25","order_by":2,"name":"Yan-Jun Tang","email":"","orcid":"","institution":"Department of Food Science and Engineering, College of Food Science, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan-Jun","middleName":"","lastName":"Tang","suffix":""},{"id":262675870,"identity":"90a906d8-970d-4f86-b093-326c5790a0eb","order_by":3,"name":"Jing-Long Cao","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing-Long","middleName":"","lastName":"Cao","suffix":""},{"id":262675871,"identity":"5b0d11a8-6428-46f4-be42-d2ed6a325753","order_by":4,"name":"Wen-Shuang Hou","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen-Shuang","middleName":"","lastName":"Hou","suffix":""},{"id":262675872,"identity":"cd55a23b-0109-4c7f-aba7-4466db49d0d7","order_by":5,"name":"An-Qi Wang","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"An-Qi","middleName":"","lastName":"Wang","suffix":""},{"id":262675873,"identity":"3027be16-405f-40de-b3c1-7062d45b040b","order_by":6,"name":"Wan-Xia Ren","email":"","orcid":"","institution":"Hospital Clinical Laboratory, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wan-Xia","middleName":"","lastName":"Ren","suffix":""},{"id":262675874,"identity":"d89d85cb-6846-41cc-8e06-070afa7eef7e","order_by":7,"name":"Cheng-Hao Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACxmYGBmYQwwBIP/5T8V+OJC1sBjxnmI2JsgmmhUGCt405sYGg8nbeg58LKu7YbWc/fMBAgo0tve94AuOHjzn4HMaXLD3jzLPknT1pCQ8MeHhyZ555wCw5cxs+LTxmzLxth5MNDuQYGCRISORuuJHAxsxLUMs/oJbzbwwkDhgYpBsQp6XhsJ3BjRwDyYaEhARitBhL8xw7DFT5LM2Y4cABw5lnHjbj9Yth/xnDzzw1h+0Nzicffsz474A83/Hkgx8+4tPSAKGRouMAgaiRh9L2CKEDCXh1jIJRMApGwcgDALLuVKH7blvIAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Heilongjiang Bayi Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Cheng-Hao","middleName":"","lastName":"Jin","suffix":""}],"badges":[],"createdAt":"2023-12-23 10:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3796020/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3796020/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49099449,"identity":"3457e151-563b-4b77-a561-bd02253df49f","added_by":"auto","created_at":"2024-01-03 05:12:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":373093,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effects of CHR and 5-FU were determined using a CCK-8 assay. \u003cstrong\u003ea.\u003c/strong\u003e Gastric cancer cell lines (AGS, SNU-484, MKN-45, MKN-28, KATO-3, SNU-5, NCI-N87, YCC-1, SNU-216, SNU-668, YCC-6, and YCC-16) were treated with various concentrations (20, 40, 60, 80, and 100 µM) of CHR or 5-FU for 24 h, and then their cell viabilities were determined by a CCK-8 assay. \u003cstrong\u003eb.\u003c/strong\u003e Gastric cancer cell lines (AGS, SNU-484, MKN-45, MKN-28, KATO-3, SNU-5, NCI-N87, YCC-1, SNU-216, SNU-668, YCC-6, and YCC-16) were treated with 40.03 μM (IC\u003csub\u003e50 \u003c/sub\u003evalue) of CHR or 5-FU at time gradients (6, 12, 18, 24, and 30 h), and then their cell viabilities were determined by a CCK-8 assay. \u003cstrong\u003ec.\u003c/strong\u003e Human normal cell lines (GES-1, 293T, THLE-2, and IMR-90) were treated with different concentration gradients (20, 40, 60, 80, and 100 µM) of CHR or 5-FU for 24 h, and then their cell viabilities were determined by a CCK-8 assay. \u003cstrong\u003ed.\u003c/strong\u003e Human normal cell lines (GES-1, 293T, THLE-2, and IMR-90) were treated with 40.03 μM (IC\u003csub\u003e50 \u003c/sub\u003evalue) of CHR or 5-FU at time gradients (6, 12, 18, 24, and 30 h), and then their cell viabilities were determined by a CCK-8 assay. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 5-FU group.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/d78d6e8b4c51f3c0b4d7a054.png"},{"id":49099185,"identity":"dc6c230a-ee8b-42ea-9c30-957d095dad64","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2396728,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic effects of CHR on AGS cells. AGS cells treated with 40.03 μM CHR for 3, 6, 12, and 24 h. \u003cstrong\u003ea.\u003c/strong\u003e AGS cells were stained with annexin V-FITC/PI, and the fluorescence intensity and morphology were observed by using a fluorescence microscope (original magnification 400×). \u003cstrong\u003eb. \u003c/strong\u003eThe percentage of apoptotic cells were determined by flow cytometry. \u003cstrong\u003ec. \u003c/strong\u003eMitochondrial membrane potentials were determined by flow cytometry. \u003cstrong\u003ed.\u003c/strong\u003e Expression levels of apoptosis-related proteins detected by Western blotting analysis. α-Tubulin used as internal reference. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0 h.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/f9bc1de1c84813505c28e6c4.jpg"},{"id":49099181,"identity":"f403f667-f37c-472e-9d46-06914439aaa3","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1746628,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork pharmacological prediction analysis of cross-linking targets and signaling pathways between CHR and gastric cancer. \u003cstrong\u003ea.\u003c/strong\u003e Venny\u003csup\u003e2.1 \u003c/sup\u003emap of a total of fifty-four cross-linked targets in CHR and gastric cancer cells. \u003cstrong\u003eb.\u003c/strong\u003e Cross-linked target mapping obtained from STRING database analysis. \u003cstrong\u003ec.\u003c/strong\u003e Cross-mapping of core targets obtained by Cytoscape software according to significance analysis. \u003cstrong\u003ed.\u003c/strong\u003e PPI interaction maps further selected according to significance via Cytoscape software. \u003cstrong\u003ee.\u003c/strong\u003e BP of the top twenty overlapping targets were obtained by GO enrichment analysis. \u003cstrong\u003ef.\u003c/strong\u003e The CC of the top twenty overlapping targets were obtained by GO enrichment analysis. \u003cstrong\u003eg.\u003c/strong\u003e MF of the top twenty overlapping targets were obtained by GO enrichment analysis. \u003cstrong\u003eh.\u003c/strong\u003e GO enrichment analysis obtained a triad map of the top twenty overlapping targets. \u003cstrong\u003ei.\u003c/strong\u003e The top twenty overlapping target signal pathways were obtained by KEGG enrichment analysis. \u003cstrong\u003ej. \u003c/strong\u003eKEGG enrichment analysis obtained all overlapping target signal pathways. \u003cstrong\u003ek. \u003c/strong\u003eMolecular docking diagram of CHR and EGFR. \u003cstrong\u003el. \u003c/strong\u003eMolecular docking diagram of CHR and AKT. Among them, CHR is molecularly docked with the molecules of EGFR. CHR binds to a protein residue in EGFR (GLN-791) by hydrogen bonding. CHR is molecularly docked with AKT. CHR binds to three protein residues in AKT (GLU-9), (ARG-41), and (PRO-42) by hydrogen bonding.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/7a9d8c4fee9ab983c4c43fca.jpg"},{"id":49099182,"identity":"4f7b393c-b56b-4d0d-9244-75a7c8783c04","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1920498,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CHR on AKT/MAPK/STAT3 signaling pathway. AGS cells treated with 40.03 µM CHR for 3, 6, 12, and 24 h. \u003cstrong\u003ea.\u003c/strong\u003e Expression levels of AKT, MAPK, and STAT3 signaling pathway-related proteins determined by Western blotting analysis. α-Tubulin used as internal reference. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0 h. \u003cstrong\u003eb.\u003c/strong\u003e Expression levels of proteins treated with 40.03 μM CHR and 10 μM FR180204 (ERK inhibitors). \u003cstrong\u003ec.\u003c/strong\u003e Expression levels of proteins treated with 40.03 μM CHR and 10 μM SP600125 (JNK inhibitors). \u003cstrong\u003ed. \u003c/strong\u003eExpression levels of proteins treated with 40.03 μM CHR and 10 μM SB203580 (p38 inhibitors). α-Tubulin used as internal references. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control or CHR + MAPK inhibitor groups.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/6543cbd3c1417b26019f3036.jpg"},{"id":49099184,"identity":"599026a4-95a5-4977-9acb-9f84387d4e96","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2471080,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CHR on ROS accumulation in AGS cells. AGS cells treated with 40.03 µM CHR for 3, 6, 12, and 24 h. \u003cstrong\u003ea.\u003c/strong\u003e DCFH-DA staining and flow cytometry analysis of ROS levels. AGS cells treated with 40.03 µM CHR and/or 10 mM NAC for 24 h. \u003cstrong\u003eb.\u003c/strong\u003e Annexin V-FITC/PI staining and flow cytometry analysis of apoptosis. \u003cstrong\u003ec.\u003c/strong\u003e Expression levels of AKT, MAPK, and STAT3 signaling pathway-related proteins, cle-caspase-3, and cle-PARP proteins detected by Western blot analysis. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control or CHR + NAC co-treatment groups.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/51f13601d136b7141e2f37e3.jpg"},{"id":49099186,"identity":"afde327f-e2ae-4909-8fb0-a1f777b1c572","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1959305,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CHR on cell cycle arrest of AGS cells. AGS cells treated with 40.03 µM CHR for 3, 6, 12, and 24 h. \u003cstrong\u003ea.\u003c/strong\u003e The percentages of cell cycle treated with CHR were detected using flow cytometry. \u003cstrong\u003eb.\u003c/strong\u003e The percentage of cell cycle treated with 10 mM NAC and 40.03 µM CHR were determined using flow cytometry. \u003cstrong\u003ec.\u003c/strong\u003e Expression levels of G2/M cell cycle-related proteins after 40.03 µM CHR treatment were detected using western blotting. α-Tubulin used as internal reference. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0 h. \u003cstrong\u003ed.\u003c/strong\u003e Expression levels of G2/M cell cycle-related proteins after 10 mM NAC and 40.03 µM CHR treatment were detected using western blotting. α-Tubulin served as an internal reference. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control or CHR + NAC co-treatment groups\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/f27ad5ba112fa4299d9f0bc2.jpg"},{"id":49099187,"identity":"aa920ec4-adf0-4747-93ef-cb87a60e8784","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2447192,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CHR on invasion and migration of AGS cells. AGS cells treated with 40.03 µM CHR for 3, 6, 12, and 24 h, followed by fluorescence microscopy.\u003cstrong\u003e a.\u003c/strong\u003e Transwell assay analysis for cell invasion (original magnification, 100×). \u003cstrong\u003eb.\u003c/strong\u003e Wound-healing assay analysis of cell migration rate (original magnification, 200×). \u003cstrong\u003ec. \u003c/strong\u003eExpression levels of migration related proteins after CHR treatment were detected by Western blotting analysis. α-Tubulin used as internal reference. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e\u003cstrong\u003e***\u003c/strong\u003e\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0 h. (d) Expression levels of migration related protein after 10 mM NAC and 40.03 µM CHR treatment were detected by Western blotting analysis. α-Tubulin served as an internal reference.\u003cstrong\u003e *\u003c/strong\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cstrong\u003e**\u003c/strong\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003cstrong\u003e***\u003c/strong\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control or CHR + NAC co-treatment groups.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/dab87e6df2044af589b91db2.jpg"},{"id":49099188,"identity":"fd47dd00-56f6-4dde-b283-b62b53626db7","added_by":"auto","created_at":"2024-01-03 05:04:26","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2515950,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-cancer mechanism of CHR on human gastric cancer AGS cells. CHR induces apoptosis through ROS mediated AKT, MAPK, and STAT3 signaling pathways. CHR arrests the cell cycle in the G2/M phase through ROS mediated AKT, CDK 1/2, and cyclin B1 signaling pathways. CHR inhibits the invasion and migration of AGS cells through ROS mediated Wnt-3a, GSK-3β, and β-catenin signaling pathways.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/e059e8fefecbaa583e903f33.jpg"},{"id":59254010,"identity":"0f76d788-de8f-469f-bfee-38b73d0eb129","added_by":"auto","created_at":"2024-06-28 08:20:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16635498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3796020/v1/fe8306e6-7a0a-468a-848a-0877cd5a8252.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chrysosplenetin B induces apoptosis and inhibits metastasis of gastric cancer AGS cell by regulating reactive oxygen species-mediated signaling pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is a malignancy of the gastrointestinal tract and ranks among the top five new cancer incidence types worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In recent years, the prevalence and mortality of GC have increased significantly, and the number of new cases of GC in the world ranks top five [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to the lack of specific symptoms, signs, and low detection rate of early GC, patients have delayed treatment and their symptoms have worsened [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to the onset period of GC, there are various treatments to choose. At present, in addition to surgical treatment for GC, chemotherapy and radiotherapy can maximize tumor control and prolong the survival of patients [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nonetheless, these treatments are poorly targeted and have multiple side effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it is urgent to find a kind of anticancer drug with good anticancer effects, few side effects, and cheapness.\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS) originates from the bottom of the respiratory chain of the inner mitochondrial membrane and it is related to biological activities such as mitochondria-dependent apoptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As a second messenger, ROS controls a variety of signaling cascades that induce and maintain tumorigenic phenotypes in cancer cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The ROS-mediated AKT signaling pathway is a key medium for growth factory-induced cell survival and can be activated by various growth signals to regulate downstream protein functions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Mitogen-activated protein kinase (MAPK) signaling pathway is mainly composed of JNK, ERK, and p38. The ROS-mediated MAPK signaling pathway regulates many key functions such as cell proliferation, cell apoptosis, and cell senescence [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. ROS-mediated the transcription-3 signal transduction and activator (STAT3) signaling pathway to regulate the basic functions of cells and the expression levels of related genes, and is one of the central communication nodes for many cellular functions, including apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Crosstalk between AKT, MAPK, and STAT3 signaling pathways has been described in multiple cancer types [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe abundant resources of natural traditional Chinese medicine (TCM) with minimal side effects offer a good source of candidate substances for the development of anticancer drugs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. CHR is mainly extracted from \u003cem\u003eComposite\u003c/em\u003e plants and has a variety of pharmacological activities, including antiviral and antibacterial [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous research has shown that CHR be verified to have an inhibitory effect on neuraminidase and its antiviral effect was comparable to that of the traditional inhibitor oseltamivir phosphate [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In vitro antibacterial assay has confirmed that CHR has a strong inhibitory effect on staphylococcus aureus [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the mechanism of action of CHR on GC cells remains unclear.\u003c/p\u003e \u003cp\u003eNetwork pharmacology is an analysis that directly demonstrates the interaction between drugs and biomolecules using network database. It is based on a large amount of bioinformatics, systems biology data, and network technologies to explore the mechanism of drug action, predict targets, and related signaling pathways [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Network pharmacology can link the biomolecular networks of cancer and anticancer drugs, and then systematically explore the overall regulatory mechanism of anticancer drugs on cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study evaluated the pharmacological effects of CHR on GC and the associated molecular mechanisms. The effects of CHR on cell apoptosis, cell cycle, and cell migration of GC cells were also verified.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe twelve types of human GC cells (AGS, SNU-484, MKN-45, MKN-28, KATO-3, SNU-5, NCI-N87, YCC-1, SNU-216, SNU-668, YCC-6, and YCC-16) and human normal lung IMR-90 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), the human gastric epithelial GES-1 cells and human renal epithelial 293T cells were purchased from Saga Biotechnology Co. (Shanghai, China). The human liver THLE-2 cells were purchased from Otwo Biotech (Shenzhen, China). All types of cells were grown into DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin and streptomycin (P/S, Gibco). All types of cells were placed in an incubator at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C (Sanyo, Osaka, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eThe survival rates of twelve types of human GC cell lines and four types of human normal cell lines were determined by Cell Counting Kit-8 (CCK-8) (Solarbio, Beijing, China). All cells were seeded in 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well and cultured for 24 h. Cells were treated with CHR (Herbpurify, Chengdu, China) and 5-fluorouracil (5-FU) (Med Chem Express, Princeton, NJ, USA) at different concentrations (20, 40, 60, 80, and 100 \u0026micro;M) and at different time gradients (6, 12, 18, 24, and 30 h). Solutions of 10 \u0026micro;L of CCK-8 were added to each well and incubated away from the light for 3 h. Cell viability after CHR treatment were measured by a multifunctional microplate reader at 495 nm wavelength (Tecan, Mannedorf, Switzerland). Concentrations that caused 50% cell growth inhibition (IC\u003csub\u003e50\u003c/sub\u003e) values of cells treated with CHR were calculated using Sigma Plot 15.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis assay\u003c/h2\u003e \u003cp\u003eThe induction of apoptosis effect of CHR on AGS cell was determined by Annexin V-FITC/PI Apoptosis Kit (4A Biotech, Beijing, China). AGS cells were seeded in 6-well plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well. AGS cells were treated with CHR (the IC\u003csub\u003e50\u003c/sub\u003e values of 40.03 \u0026micro;M) and 5-FU (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h). Operate according to the instructions, the collected AGS cells were mixed with 200 \u0026micro;L 1\u0026times; binding buffer, 5 \u0026micro;L Annexin V-FITC, and 3 \u0026micro;L Propidium Iodide (PI) successively. AGS cells were incubated in a refrigerator at 4\u0026deg;C for 20 min. The morphological changes of AGS cells after CHR treatment were determined by fluorescence microscopy (MSHOT, Guangzhou, China). The proportion of apoptosis was observed by flow cytometry (Sysmes Co., Kobe, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAssociated targets of CHR and GC analysis\u003c/h2\u003e \u003cp\u003eThe 2D structure of CHR was determined by the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and submitted to the Swiss ADME platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisssimilarity.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisssimilarity.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for drug property prediction of CHR, and the prediction target gene of CHR was obtained using Target Prediction database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genetic target of GC was obtained using GeneCards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOverlapping target protein-protein interaction networks analysis\u003c/h2\u003e \u003cp\u003eThe overlapping targets of CHR and GC were predicted on VENNY\u003csup\u003e2.1\u003c/sup\u003e website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.liuxiaoyuyuan.cn/\u003c/span\u003e\u003cspan address=\"http://www.liuxiaoyuyuan.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The target of the intersection number obtained was the common potential target of CHR and GC. Submit the resulting common targets to the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the protein-protein interaction (PPI) network. Visual analysis of PPI networks using Cytoscape 3.9.1 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG enrichment analysis\u003c/h2\u003e \u003cp\u003eThe PPI overlapping targets predicted import DAVID Bioinformatics Resources 6.8 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in the database for enrichment analysis. Gene Ontology (GO) enrichment was used to analyze the biological process (BP), molecular function (MF), and cellular component (CC) of the cross-linked targets, while Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was used to analyze the involved signaling pathways. Finally, the functions were sorted according to \u003cem\u003ep\u003c/em\u003e-value, and it is considered that the GO and KEGG channels with \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking analysis\u003c/h2\u003e \u003cp\u003eThe PDB format of core targets was obtained through RSCB-PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Water removal molecules and ligands were performed at the core target in PyMOL software. The 3D structure of CHR was obtained through PubChem database and processed in Open Babel software. The core target was hydrogenated in the AutoDocks software, the charge number was calculated, and the rigid structure of the atom was determined. The docking tool processed in the AutoDockTools-1.5.6 software perform molecular docking and selects the site with the lowest energy for subsequent operations. Next, the docking site visualization of CHR and core target was created in PyMOL software and corresponding protein residues, binding bonds, and distances were displayed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential analysis\u003c/h2\u003e \u003cp\u003eThe mitochondrial membrane potential (MMP) of AGS cell after CHR treatment was determined by JC-1 Detection Kit (Solarbio, Beijing, China). AGS cells were seeded in 3.5 cm cell culture dish at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well. AGS cells were treated with CHR (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h). 1 mL JC-1 staining solution was added to the collected AGS cells and heated in a 37\u0026deg;C water bath for 30 min. Then, 1 mL 1\u0026times; JC-1 binding buffer and 500 \u0026micro;L of phosphate-buffered saline (PBS) were added to AGS cells treated with CHR. The MMP of AGS cell was measured by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe expression levels of AGS cells related proteins after CHR treatment were determined by Western Blot Analysis. AGS cells were seeded in a 6 cm cell culture dish at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well. AGS cells treated with CHR (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h), and protein extracts were obtained with 100 \u0026micro;L lysis buffer. The same amount of total protein was isolated on 12% SDS-PAGE, then the gel is electrically transferred to the nitrocellulose membrane. 5% skimmed milk was added and closed at 25\u0026deg;C for 2 h to avoid non-specific binding. The primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated with the film in a refrigerator at 4\u0026deg;C away from the light. Next, the membrane was incubated with the secondary antibody (ZSBG-BIO, Beijing, China) at room temperature for 2 h. The enhanced chemiluminescence (Tanon, Shanghai, China) solution-binding proteins. Images were exposed using multifunction imager (Analytik Jena AG, Jena, Germany), and the bands were analyzed with the ImageJ version 1.42.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eROS levels analysis\u003c/h2\u003e \u003cp\u003eThe ROS levels in AGS cells treated with CHR were determined by ROS Detection Kit (Beyotime Institute Biotechnology, Shanghai, China). AGS cells were seeded in a 3.5 cm cell culture dishes at a cell density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and treated with CHR (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h). AGS cells was combined with 10 \u0026micro;L 2'7'-dichlorofluorescein diacetate (DCFH-DA) and incubated at 37\u0026deg;C for 30 min away from the light. ROS levels were determined by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eThe cell cycle arrest of AGS after CHR treatment was determined by DNA Content Detection Kit (Solarbio, Beijing, China). AGS cells were seeded in a 3.5 cm cell culture dishes at a cell density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and treated with CHR (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h). AGS cells were collected, fixed in pre-cooled 70% ethanol and stored overnight at 4\u0026deg;C. 100 \u0026micro;L RNase was added to the cells and incubated at 37\u0026deg;C away from the light for 30 min. Then 400 \u0026micro;L PI was added to the AGS cells and incubated at 4\u0026deg;C for 30 min. The changes in the number of CHR treated AGS cells in G0/G1 and G2/M phases were determined by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion and migration analysis\u003c/h2\u003e \u003cp\u003eThe inhibitory effect of CHR on AGS cells invasion was determined by Transwell Assay. AGS cells suspension containing serum-free DMEM medium was added to the upper chamber, and DMEM medium containing 10% FBS and 1% P/S was added to the lower chamber. The AGS cells treated with CHR (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h) were stained with 1% crystal violet (Solarbio, Beijing, China). The petri dish was placed under a fluorescence microscopy to observe the number of invading cells.\u003c/p\u003e \u003cp\u003eIn addition, the inhibitory effect of CHR on the migration of AGS cells was determined by Wound-Healing Assay. AGS cells were seeded in 6-well plates at a cell density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well and treated with CHR (40.03 \u0026micro;M) at different times (3, 6, 12, and 24 h). After the treatment time was reached, vertical labeling lines were taken. After 2 h, the culture medium was aspirated and the healing of cells was observed under fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were all repeated three times and assessed by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Sigma Plot 15.0 software was used to calculate IC\u003csub\u003e50\u003c/sub\u003e. SPSS 29.0 software was used for Tukey\u0026rsquo;s postmortem test, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eKilling effect of CHR on GC cell\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b, CHR had a good killing effect on all twelve types of GC cells at a dose and time-dependent manner. The survival rate of twelve types of GC cells after CHR treatment were significantly lower than that of 5-FU treatment group. The effects of CHR and 5-FU on survival rate of four types of normal human cells (GES-1, 293T, THLE-2, and IMR-90) were measured by a CCK-8 assay at dose gradient and time gradient, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d, the survival rate of the four types of normal cells after CHR treatment were higher than that in the 5-FU treatment group. It was proved that CHR had no significant toxic and side effects on normal human gastric, liver, lung, and renal cells. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, IC\u003csub\u003e50\u003c/sub\u003e value of twelve types of GC cells by CHR and 5-FU were summarized, and the sensitivity of AGS cells to CHR was higher than that of other eleven types of GC cells. AGS can be used as a target for subsequent detection of the anti-GC efficacy of CHR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC50 values of CHR and 5-FU in gastric cancer cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5-FU (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHR (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e80.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e40.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMKN-45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e77.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMKN-28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e79.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e56.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKATO-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e76.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e73.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCI-N87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e80.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e71.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNU-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e90.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e76.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNU-216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e89.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e79.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNU-484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e83.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNU-668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e83.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e65.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYCC-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e76.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e73.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYCC-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e79.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e64.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYCC-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e81.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e65.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCHR induces apoptosis of AGS cell in mitochondria dependent manner\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, with the extension of CHR treatment time, the fluorescence intensity of Annexin-FITC/PI double staining was observed under the fluorescence microscopy to increased continuously, and morphological changes such as AGS cells becoming round and shrinking could be observed under the light field. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, flow cytometry results showed that with the extension of CHR treatment time, the apoptosis rate of early and late AGS cells increased from 0.56 to 27.65%. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, with the extension of CHR treatment time, the intracellular MMP levels decreased from 99.10% at the beginning to 43.97%. The expression levels of apoptotic proteins in mitochondrial pathway were analyzed by western blot. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, the protein expression levels of Bad, cytochrome c (cyto-c), cleaved-caspase-3 (cle-caspase-3), and cleaved-PARP (cle-PARP) increased, while the protein expression levels of Bcl-2 decreased. It was further confirmed that CHR could induced apoptosis of AGS cells through endogenous mitochondrial pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of protein interaction networks\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Venny\u003csup\u003e2.1\u003c/sup\u003e obtained a total of fifty-four predicted targets for GC processing by CHR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the interaction diagram of protein targets predicted by the common target of CHR and GC through the String website. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the interaction diagram of the PPI network was further screened by Cytoscape software. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, sixteen core protein targets with high degree value were further screened in Cytoscape software. The results showed that CHR interacted with different targets of GC, among which AKT 1 and EGFR were the first two degrees.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTarget biological function analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, the BP-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, the CC-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, the MF-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, it was a collection of BP, CC, and MF enrichment analysis of fifty-four cross targets by using DAVID Bioinformatics Resources 6.8. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei and j, KEGG enrichment analysis showed that twenty signal pathways that were closely related to CHR and GC crosslinking. The results showed that the protein phosphorylation degree value was higher, the activity of protein kinase B was stronger. Moreover, the degree values of AKT signaling pathway and cancer signaling pathway were in the top two places in KEGG analysis. CHR may play an anti-GC role by regulating protein kinase B phosphorylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek, the grid tool in AutoDockTool-1.5.6 software was used to build a complete CHR binding site network map, and the molecular docking between CHR and EGFR was completed by docking tools. The corresponding protein residues were visualized in rod shape by PyMOL software, and the residues were distinguished by different colors. CHR binds to the GLN-791 residue on EGFR by hydrogen bonding. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el the results of the docking site of CHR and AKT. CHR forms three hydrogen bonds with GLU-9, ARG-41 and PRO-42 on AKT. Molecular docking results showed that CHR and AKT had good binding ability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCHR induces apoptosis of AGS cell by regulate AKT, MAPK, and STAT3 signaling pathways\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the expression levels of p-AKT, p-ERK, and p-STAT3 protein were decreased, while the expression levels of p-JNK and p-p38 protein were increased. In addition, to verify the upstream and downstream relationship between MAPK and STAT3 signaling pathways, pretreated AGS cells with a 10 \u0026micro;M FR180204 (ERK inhibitor), a 10 \u0026micro;M SP600125 (JNK inhibitor), and a 10 \u0026micro;M SB203580 (p38 inhibitor). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d, the MAPK inhibitor\u0026thinsp;+\u0026thinsp;CHR co-treatment group significantly reversed the expression levels of corresponding proteins in the CHR alone treatment group. Among them, the CHR\u0026thinsp;+\u0026thinsp;ERK inhibitor co-treatment group promoted the expression levels of p-ERK and p-STAT3 protein compared with the CHR alone treatment group, CHR\u0026thinsp;+\u0026thinsp;JNK inhibitor and CHR\u0026thinsp;+\u0026thinsp;p38 inhibitor co-treatment groups inhibited p-JNK and p-p38 proteins expression levels, and promoted p-STAT3 proteins expression levels compared with CHR alone treatment group. The results confirmed that CHR could induces apoptosis of AGS cell by regulating AKT, MAPK, and STAT3 signaling pathways, with MAPK located upstream of STAT3 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCHR induces apoptosis of AGS cell by up-regulating ROS levels\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, with the extension of CHR treatment time, ROS levels on AGS cells continuously accumulated. To further verify the relationship between apoptosis of AGS cells induced by CHR and ROS accumulation, NAC was added to AGS cells for pre-treatment before CHR treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, after NAC treatment, the apoptosis rate of AGS cells treated with CHR decreased from 42.24 to 3.4%. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the expression levels of apoptosis-related signaling pathway proteins were reversed in CHR\u0026thinsp;+\u0026thinsp;NAC co-treatment group compared with CHR alone treatment group. The results of ROS analysis showed that CHR played an anticancer role by up-regulating the ROS levels on AGS cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eCHR arrests AGS cell cycle in G2/M phase\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, with the extension of CHR treatment time, the number of AGS cells in G0/G1 phase decreased from 71.47 to 44.94%, while the number of AGS cells in G2/M phase increased from 2.15 to 16.23%. In addition, to investigate the relationship between cell cycle arrest and ROS accumulation, the AGS cells were pretreated with NAC before CHR treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, NAC significantly inhibited the number of G2/M phase cells after CHR treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, with the increased of CHR treatment time, the expression levels of p-AKT, CDK 1/2, and cyclin B1 proteins decreased, while the expression levels of p27 and p21 proteins increased. In addition, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, the expression levels of cycle-related proteins were reversed in CHR\u0026thinsp;+\u0026thinsp;NAC co-treatment group compared with CHR alone treatment group. The results suggest that CHR inhibited AGS cell cycle in G2/M phase by regulating ROS/AKT signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eCHR inhibits the invasion and migration of AGS cell\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, with the extension of CHR treatment time, the number of AGS cell invasion from the upper cavity to the lower cavity significantly decreased compared with the control group. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, with the increased of CHR treatment time, compared with the control group, the migration of AGS cell was significantly inhibited. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, with the increased of CHR treatment time, the expression levels of E-cadherin protein increased, while the expression levels of N-cadherin, Wnt-3a, β-catenin, and p-GSK-3β protein decreased. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, the expression levels of migration-related signaling pathway proteins were reversed in CHR\u0026thinsp;+\u0026thinsp;NAC co-treatment group compared with CHR alone treatment group. The results suggest that CHR inhibits the invasion and migration of AGS cell by regulating the ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCHR is an active isoflavone extracted from \u003cem\u003eChamomile\u003c/em\u003e plant [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previous studies have shown that isoflavones such as soy isoflavones and barbigerone induces apoptosis of cancer cells through MMP pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As a polymethoxy-flavonoid, CHR has been verified to have dose-dependent inhibitory effects on lung cancer A549 and cervical cancer Hela cells, but has low toxicity to normal Vero and EVC304 cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The effects of CHR on the anticancer activity of GC were verified by CCK-8 assay in this study. The experimental data of this study showed that CHR had good toxic effects on twelve types of GC cells, and the survival rate of GC cells in the CHR treatment group was lower than that in the traditional chemotherapy drug 5-FU treatment group in both the concentration gradient and the time gradient. However, the toxic effect of CHR on gastric, liver, renal, and lung cells in normal human is lower than that of 5-FU. The above results have shown that CHR has anticancer activity on GC cells, and then this study explored its specific anti-GC mechanism through a series of experiments.\u003c/p\u003e\u003cp\u003eThere are many ways of cell death, among which apoptosis is the most typical programmed cell death. Apoptosis controls the spontaneous and orderly death of cells by regulating the expression levels of signaling pathways and plays a crucial role in the normal growth and development of cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The main source of intracellular ROS is the substrate end of the respiratory chain in the mitochondrial inner membrane [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. When cancer cells receive death signals such as oxidative stress, mitochondria-dependent apoptosis dependent on caspases will be triggered [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The Bcl-2 family proteins located upstream of caspases regulate the mitochondrial apoptosis pathway, and the pro-apoptotic proteins will homodimerization or heterodimerize with the anti-apoptotic proteins to regulate the downstream apoptotic signal cascade [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The experimental results of this study showed that when CHR induced AGS cells apoptosis, the MMP decreased, the proportion of Bad/Bcl-2 protein expression levels increased, and cyto-c was released from mitochondria into the cytoplasm to activate the downstream apoptosis effecting factor caspase-3, and further shear the downstream substrate PARP to complete the mitochondria-dependent apoptosis pathway.\u003c/p\u003e\u003cp\u003eNetwork pharmacology can screen and visually analyze the network relationships among TCM molecules, targets, and diseases through a high-throughput database [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. TCM therapy has the characteristics of multi-target and multi-signal pathway, which is consistent with the comprehensiveness and systematicness of network pharmacology [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, network pharmacology is suitable for predicting the molecular mechanisms and signaling pathways of TCM compounds [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, through network pharmacological predictive analysis and molecular docking, we explored the relevant targets (AKT and EGFR) and signaling pathways (AKT/MAPK/STAT3) of CHR action on GC. Further western blot analysis confirmed that CHR induced apoptosis of AGS cells in human GC by regulating ROS/AKT/MAPK/STAT3 signaling pathway.\u003c/p\u003e\u003cp\u003eROS is a natural by-product of normal oxygen metabolism, which mainly includes highly active heterogeneous molecules such as peroxides, superoxides, and hydroxyl radicals [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The generation and accumulation of ROS plays a key role in tumor transformation, cell proliferation, and apoptosis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. When ROS levels in the body fall below the ROS threshold, ROS activates oncogenes, inducing DNA repair and cancer cell survival [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. When the ROS accumulation levels is higher than the threshold, ROS will trigger apoptosis signals and regulate downstream signaling pathways to induce cancer cell death [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this study, flow cytometry analysis results showed that the ROS accumulation levels in AGS cells after CHR treatment were increased. Further, NAC pretreatment of AGS cells reversed verified that CHR mediated ROS accumulation regulated AKT, MAPK, and STAT3 signaling pathways induced apoptosis of AGS cell.\u003c/p\u003e\u003cp\u003eCell cycle progression is mediated by cyclin-dependent kinases (CDKs) and their cyclins, which strictly regulate the processes of cell expansion, genetic material replication, and cell division [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. There are G1/S phase checkpoints and G2/M phase DNA damage checkpoints in the cell cycle, and the smooth progress of the cell cycle can be judged by the binding activation of CDKs and cyclin chaperone [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The network pharmacology analysis results of this study showed that AKT is the core target of GC cell cycle regulation by CHR, the p21/p27 protein is an inhibitor of the cyclin-CDK complex and can arrest the cell cycle by inhibiting the activity of the cyclin-CDK complex [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The results of this study showed that CHR induced an increase in the expression levels of p21 and p27 proteins by regulating of the ROS/AKT signaling pathway, which inhibited the expression levels of CDK 1/2 and cyclin B1 complex proteins, and finally CHR arrested the AGS cycle in G2/M phase.\u003c/p\u003e\u003cp\u003eCancer cell metastasis refers to when the primary tumor cells are shed and free in the body, they will engulf healthy cells in the body organs, resulting in the collapse of the immune system defense line, and the growth of malignant tumors of the same nature everywhere in the body [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Inhibiting cancer cell migration lessen the emergence of highly malignant cancer cells, delay the invasion of white blood cells during inflammation, or promote wound healing [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In the process of malignant tumor development, epithelial-mesenchymal transformation (EMT) usually reduces the adhesion between cells and enhances the invasion and migration of cancer cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Wnt-3a/β-catenin signaling pathway is a crucial pathway to regulate EMT [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Among them, β-catenin is an important biological indicator to detect whether the Wnt-3a signaling pathway is activate and stabilize intercellular adhesion by intracellular binding to cadherin [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. GSK-3β, located upstream of β-catenin, acts as the main regulatory enzyme of Wnt-3a/β-catenin signaling pathway and negatively regulates wnt-3a signaling pathway by promoting β-catenin degradation [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In this study, Transwell and wound healing assays confirmed at the cellular levels that CHR inhibits the invasion and migration of AGS cells. Further western blot analysis showed that CHR could regulate the expression levels of ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway proteins, thereby inhibiting the metastasis of AGS cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study showed that CHR regulates AKT/MAPK/STAT3 signaling pathway by up-regulating ROS levels on AGS human GC cells, leading to G2/M cell cycle arrest and inducing cell apoptosis. Moreover, CHR inhibited the invasion and migration of AGS cells on GC by regulating ROS/Wnt-3a/GSK-3\u0026beta;/\u0026beta;-catenin signaling pathway (Fig. 8).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by Heilongjiang Province Key Research and Development Plan Guidance Project (No. GZ20220039), Central Government Supports Local College Reform and Development Fund Talent Training Project (No. 2020GSP16), and Heilongjiang Touyan Innovation Team Program (No. 2019HTY078).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH. Xue. Conceptualization and writing-original draft preparation; S.-M. Li and Y.-J. Tang. writing-review and editing; J.-L. Cao and W.-S. Hou. methodology; A.-Q. Wang. software; W.-X. Ren. data analysis; C.-H. Jin. supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCorrea P. Gastric cancer: overview. Gastroenterol Clin North Am. 2013;42(2):211\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gtc.2013.01.002\u003c/span\u003e\u003cspan address=\"10.1016/j.gtc.2013.01.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgan E, Arnold M, Camargo MC, et al. The current and future incidence and mortality of gastric cancer in 185 countries, 2020-40: A population-based modelling study. EClinicalMedicine. 2022;47:101404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.eclinm.2022.101404\u003c/span\u003e\u003cspan address=\"10.1016/j.eclinm.2022.101404\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachlowska J, Baj J, Sitarz M, et al. Gastric Cancer: Epidemiology, Risk Factors, Classification, Genomic Characteristics and Treatment Strategies. Int J Mol Sci. 2020;21(11):4012. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms21114012\u003c/span\u003e\u003cspan address=\"10.3390/ijms21114012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng Y, Jin RU. Molecular pathogenesis, targeted therapies, and future perspectives for gastric cancer. Semin Cancer Biol. 2022;86(Pt3):566\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.semcancer.2021.12.004\u003c/span\u003e\u003cspan address=\"10.1016/j.semcancer.2021.12.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan Z. Recent Advances in the Surgical Treatment of Advanced Gastric Cancer: A Review. Med Sci Monit. 2019;25:3537\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12659/MSM.916475\u003c/span\u003e\u003cspan address=\"10.12659/MSM.916475\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zhang L, Yang Y, et al. Progress of Gastric Cancer Surgery in the era of Precision Medicine. Int J Biol Sci. 2021;17(4):1041\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/ijbs.56735\u003c/span\u003e\u003cspan address=\"10.7150/ijbs.56735\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel TH, Cecchini M. Targeted Therapies in Advanced Gastric Cancer. Curr Treat Options Oncol. 2020;21(9):70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11864-020-00774-4\u003c/span\u003e\u003cspan address=\"10.1007/s11864-020-00774-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwarz RE. Current status of management of malignant disease: current management of gastric cancer. J Gastrointest Surg. 2015;19(4):782\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11605-014-2707-x\u003c/span\u003e\u003cspan address=\"10.1007/s11605-014-2707-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoya Y, Mitsumori N, Yanaga K. The advantages and disadvantages of a Roux-en-Y reconstruction after a distal gastrectomy for gastric cancer. Surg Today. 2009;39(8):647\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00595-009-3964-2\u003c/span\u003e\u003cspan address=\"10.1007/s00595-009-3964-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrinivas US, Tan BWQ, Vellayappan BA, et al. ROS and the DNA damage response in cancer. Redox Biol. 2019;25:101084. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.redox.2018.101084\u003c/span\u003e\u003cspan address=\"10.1016/j.redox.2018.101084\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuli HS, Kaur J, Vashishth K, et al. Molecular mechanisms behind ROS regulation in cancer: A balancing act between augmented tumorigenesis and cell apoptosis. Arch Toxicol. 2023;97(1):103\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00204-022-03421-z\u003c/span\u003e\u003cspan address=\"10.1007/s00204-022-03421-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Xia J, Jiang N, et al. Corin protects H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis through PI3K/AKT and NF-κB pathway in cardiomyocytes. Biomed Pharmacother. 2018;97:594\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2017.10.090\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2017.10.090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFattahi S, Amjadi-Moheb F, Tabaripour R, et al. PI3K/AKT/mTOR signaling in gastric cancer: Epigenetics and beyond. Life Sci. 2020;262:118513. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lfs.2020.118513\u003c/span\u003e\u003cspan address=\"10.1016/j.lfs.2020.118513\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYong HY, Koh MS, Moon A. The p38 MAPK inhibitors for the treatment of inflammatory diseases and cancer. Expert Opin Investig Drugs. 2009;18(12):1893\u0026ndash;905. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1517/13543780903321490\u003c/span\u003e\u003cspan address=\"10.1517/13543780903321490\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHendrikse CSE, Theelen PMM, van der Ploeg P, et al. The potential of RAS/RAF/MEK/ERK (MAPK) signaling pathway inhibitors in ovarian cancer: A systematic review and meta-analysis. Gynecol Oncol. 2023;171:83\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ygyno.2023.01.038\u003c/span\u003e\u003cspan address=\"10.1016/j.ygyno.2023.01.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Cell Biol. 2007;19(2):142\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ceb.2007.02.001\u003c/span\u003e\u003cspan address=\"10.1016/j.ceb.2007.02.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa JH, Qin L, Li X. Role of STAT3 signaling pathway in breast cancer. Cell Commun Signal. 2020;18(1):33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12964-020-0527-z\u003c/span\u003e\u003cspan address=\"10.1186/s12964-020-0527-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu H, Lee H, Herrmann A, et al. Revisiting STAT3 signaling in cancer: new and unexpected biological functions. Nat Rev Cancer. 2014;14(11):736\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrc3818\u003c/span\u003e\u003cspan address=\"10.1038/nrc3818\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Zeng Y, Qu Q, et al. PD-L1 induced by IFN-γ from tumor-associated macrophages via the JAK/STAT3 and PI3K/AKT signaling pathways promoted progression of lung cancer. Int J Clin Oncol. 2017;22(6):1026\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10147-017-1161-7\u003c/span\u003e\u003cspan address=\"10.1007/s10147-017-1161-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Lv H, Li X, et al. Cyclovirobuxine inhibits the progression of clear cell renal cell carcinoma by suppressing the IGFBP3-AKT/STAT3/MAPK-Snail signaling pathway. Int J Biol Sci. 2021;17(13):3522\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/ijbs.62114\u003c/span\u003e\u003cspan address=\"10.7150/ijbs.62114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Y, Yang G, Lu G, et al. Th22 Cells/IL-22 Serves as a Protumor Regulator to Drive Poor Prognosis through the JAK-STAT3/MAPK/AKT Signaling Pathway in Non-Small-Cell Lung Cancer. J Immunol Res. 2022;2022:8071234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2022/8071234\u003c/span\u003e\u003cspan address=\"10.1155/2022/8071234\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas E, Stewart LE, Darley BA, et al. Plant-Based Natural Products and Extracts: Potential Source to Develop New Antiviral Drug Candidates. Molecules. 2021;26(20):6197. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26206197\u003c/span\u003e\u003cspan address=\"10.3390/molecules26206197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtanasov AG, Waltenberger B, Pferschy-Wenzig EM, et al. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv. 2015;33(8):1582\u0026ndash;614. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2015.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2015.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMbarga PE, Fouotsa H, Ndemangou B, et al. Two new secondary metabolites with antibacterial activities from Conyza aegyptiaca (Asteraceae). Nat Prod Res. 2023;37(11):1806\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/14786419.2022.2122965\u003c/span\u003e\u003cspan address=\"10.1080/14786419.2022.2122965\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan X, Li J, Cui J, et al. Chemical component and in vitro protective effects of Matricaria chamomilla (L.) against lipopolysaccharide insult. J Ethnopharmacol. 2022;296:115471. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jep.2022.115471\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2022.115471\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNogales C, Mamdouh ZM, List M, et al. Network pharmacology: curing causal mechanisms instead of treating symptoms. Trends Pharmacol Sci. 2022;43(2):136\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tips.2021.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.tips.2021.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Yao Q, Teng F, et al. TCM network pharmacology: A new trend towards combining computational, experimental and clinical approaches. Chin J Nat Med. 2021;19(1):1\u0026ndash;11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1875-5364(21)60001-8\u003c/span\u003e\u003cspan address=\"10.1016/S1875-5364(21)60001-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim C, Kim B. Anti-Cancer Natural Products and Their Bioactive Compounds Inducing ER Stress-Mediated Apoptosis: A Review. Nutrients. 2018;10(8):1021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu10081021\u003c/span\u003e\u003cspan address=\"10.3390/nu10081021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G, Ding K, Qiao Y, et al. Flavonoids Regulate Inflammation and Oxidative Stress in Cancer. Molecules. 2020;25(23):5628. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules25235628\u003c/span\u003e\u003cspan address=\"10.3390/molecules25235628\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlharbi Y, Kapur A, Felder M, et al. Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase. Sci Rep. 2020;10(1):19585. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-76342-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-76342-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao C, Liu B, Shen W, et al. Research on antiproliferative effect of flavones isolated from Laggera pterodonta. Zhongguo Zhong Yao Za Zhi. 2010;35(16):2171\u0026ndash;4. Chinese.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Karakhanova S, Hartwig W, et al. Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy. J Cell Physiol. 2016;231(12):2570\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcp.25349\u003c/span\u003e\u003cspan address=\"10.1002/jcp.25349\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeong SY, Seol DW. The role of mitochondria in apoptosis. BMB Rep. 2008;41(1):11\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5483/bmbrep.2008.41.1.011\u003c/span\u003e\u003cspan address=\"10.5483/bmbrep.2008.41.1.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshkenazi A, Fairbrother WJ, Leverson JD, et al. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat Rev Drug Discov. 2017;16(4):273\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrd.2016.253\u003c/span\u003e\u003cspan address=\"10.1038/nrd.2016.253\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNemec KN, Khaled AR. Therapeutic modulation of apoptosis: targeting the BCL-2 family at the interface of the mitochondrial membrane. Yonsei Med J. 2008;49(5):689\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3349/ymj.2008.49.5.689\u003c/span\u003e\u003cspan address=\"10.3349/ymj.2008.49.5.689\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez A, Reyna DE, Gitego N, et al. Co-targeting of BAX and BCL-XL proteins broadly overcomes resistance to apoptosis in cancer. Nat Commun. 2022;13(1):1199. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-022-28741-7\u003c/span\u003e\u003cspan address=\"10.1038/s41467-022-28741-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Lu Y, Chen W, et al. Network Pharmacology and Experimental Evidence: PI3K/AKT Signaling Pathway is Involved in the Antidepressive Roles of Chaihu Shugan San. Drug Des Devel Ther. 2021;15:3425\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/DDDT.S315060\u003c/span\u003e\u003cspan address=\"10.2147/DDDT.S315060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu N, Yuan T, Yin Z, et al. Network Pharmacology and Molecular Docking Study of the Chinese Miao Medicine Sidaxue in the Treatment of Rheumatoid Arthritis. Drug Des Devel Ther. 2022;16:435\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/DDDT.S330947\u003c/span\u003e\u003cspan address=\"10.2147/DDDT.S330947\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Zhang B. Traditional Chinese medicine network pharmacology: theory, methodology and application. Chin J Nat Med. 2013;11(2):110\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1875-5364(13)60037-0\u003c/span\u003e\u003cspan address=\"10.1016/S1875-5364(13)60037-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo W, Huang J, Wang N, et al. Integrating Network Pharmacology and Pharmacological Evaluation for Deciphering the Action Mechanism of Herbal Formula Zuojin Pill in Suppressing Hepatocellular Carcinoma. Front Pharmacol. 2019;10:1185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2019.01185\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2019.01185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarniak A, Lipińska J, Tytman K, et al. Endogenous mechanisms of reactive oxygen species (ROS) generation. Postepy Hig Med Dosw (Online). 2016;70(0):1150\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5604/17322693.1224259\u003c/span\u003e\u003cspan address=\"10.5604/17322693.1224259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoloney JN, Cotter TG. ROS signaling in the biology of cancer. Semin Cell Dev Biol. 2018;80:50\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.semcdb.2017.05.023\u003c/span\u003e\u003cspan address=\"10.1016/j.semcdb.2017.05.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang S, Lian G. ROS and diseases: role in metabolism and energy supply. Mol Cell Biochem. 2020;467(1\u0026ndash;2):1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11010-019-03667-9\u003c/span\u003e\u003cspan address=\"10.1007/s11010-019-03667-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlika H, Mansour H, Wehbe N, et al. Therapeutic potential of flavonoids in cancer: ROS-mediated mechanisms. Biomed Pharmacother. 2022;146:112442. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2021.112442\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2021.112442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJelic MD, Mandic AD, Maricic SM, et al. Oxidative stress and its role in cancer. J Cancer Res Ther. 2021;17(1):22\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/jcrt.JCRT_862_16\u003c/span\u003e\u003cspan address=\"10.4103/jcrt.JCRT_862_16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z. Cell Cycle Progression and Synchronization: An Overview. Methods Mol Biol. 2022;2579:3\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-0716-2736-5_1\u003c/span\u003e\u003cspan address=\"10.1007/978-1-0716-2736-5_1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWood DJ, Endicott JA. Structural insights into the functional diversity of the CDK-cyclin family. Open Biol. 2018;8(9):180112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rsob.180112\u003c/span\u003e\u003cspan address=\"10.1098/rsob.180112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 2013;140(15):3079\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/dev.091744\u003c/span\u003e\u003cspan address=\"10.1242/dev.091744\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee D, Hokinson D, Park S, et al. ER Stress Induces Cell Cycle Arrest at the G2/M Phase Through eIF2α Phosphorylation and GADD45α. Int J Mol Sci. 2019;20(24):6309. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms20246309\u003c/span\u003e\u003cspan address=\"10.3390/ijms20246309\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVassilev LT. Cell cycle synchronization at the G2/M phase border by reversible inhibition of CDK1. Cell Cycle. 2006;5(22):2555\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4161/cc.5.22.3463\u003c/span\u003e\u003cspan address=\"10.4161/cc.5.22.3463\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuski JM, Braun M, Strmiska V, et al. Targeting cell-cycle machinery in cancer. Cancer Cell. 2021;39(6):759\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ccell.2021.03.010\u003c/span\u003e\u003cspan address=\"10.1016/j.ccell.2021.03.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMestre-Farrera A, Bruch-Oms M, Pe\u0026ntilde;a R, et al. Glutamine-Directed Migration of Cancer-Activated Fibroblasts Facilitates Epithelial Tumor Invasion. Cancer Res. 2021;81(2):438\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-20-0622\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-20-0622\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUm E, Oh JM, Granick S, et al. Cell migration in microengineered tumor environments. Lab Chip. 2017;17(24):4171\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c7lc00555e\u003c/span\u003e\u003cspan address=\"10.1039/c7lc00555e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePastushenko I, Blanpain C. EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol. 2019;29(3):212\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tcb.2018.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tcb.2018.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian S, Peng P, Li J, et al. SERPINH1 regulates EMT and gastric cancer metastasis via the Wnt/β-catenin signaling pathway. Aging. 2020;12(4):3574\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/aging.102831\u003c/span\u003e\u003cspan address=\"10.18632/aging.102831\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Bavarva JH, Wang Z, et al. HEF1, a novel target of Wnt signaling, promotes colonic cell migration and cancer progression. Oncogene. 2011;30(23):2633\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/onc.2010.632\u003c/span\u003e\u003cspan address=\"10.1038/onc.2010.632\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Cai H, Liu Q, et al. Cinobufacini Inhibits Colon Cancer Invasion and Metastasis via Suppressing Wnt/β-Catenin Signaling Pathway and EMT. Am J Chin Med. 2020;48(3):703\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1142/S0192415X20500354\u003c/span\u003e\u003cspan address=\"10.1142/S0192415X20500354\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan Y, Zhao P, Wang Z, et al. PRMT5 regulates colorectal cancer cell growth and EMT via EGFR/Akt/GSK3β signaling cascades. Aging. 2021;13(3):4468\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/aging.202407\u003c/span\u003e\u003cspan address=\"10.18632/aging.202407\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu H, Lu XX, Wang JR, et al. TRAF6 inhibits colorectal cancer metastasis through regulating selective autophagic CTNNB1/β-catenin degradation and is targeted for GSK3B/GSK3β-mediated phosphorylation and degradation. Autophagy. 2019;15(9):1506\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15548627.2019.1586250\u003c/span\u003e\u003cspan address=\"10.1080/15548627.2019.1586250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chrysosplenetin B, Gastric cancer, Cell apoptosis, Cell cycle, Cell metastasis, Reactive oxygen species","lastPublishedDoi":"10.21203/rs.3.rs-3796020/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3796020/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChrysosplenetin B (CHR) is a flavonoid compound with various pharmacological activities. This study aimed to investigate the effect and mechanism of CHR on gastric cancer (GC). A cell counting Kit 8 assay results showed that CHR had a good cytotoxic effect in twelve types of GC cell lines. Annexin-V/PI staining, flow cytometry, and western blot analysis results showed that CHR induced mitochondrial-dependent apoptosis of AGS cells by decreasing mitochondrial membrane potential and increasing the expression levels of Bad/Bcl-2 homologous dimer proteins. Network pharmacological analysis results showed that there were twenty high-value signaling pathways correlated with CHR and GC, among which AKT, MAPK, and STAT3 signaling pathways were closely related to the CHR induced apoptosis signaling pathways on AGS cells. Further through western blot analysis results showed that the protein expression levels of p-AKT, p-ERK, and p-STAT3 were significantly decreased, while the protein expression levels of p-JNK and p-p38 were significantly increased. Moreover, reactive oxygen species (ROS) analysis results showed that CHR induced ROS accumulation on AGS cells as an initial signal to regulate downstream signaling pathways. Cell cycle results showed that CHR arrested the AGS cell cycle in the G2/M phase by regulating the ROS/AKT signaling pathway. Transwell and wound healing assay results showed that CHR inhibited the invasion and migration of AGS cells by regulating ROS/Wnt-3a/GSK-3β/β-catenin signaling pathway. In conclusion, CHR inhibited cell proliferation, induced cell apoptosis, arrested the cell cycle in the G2/M phase, and inhibited invasion and migration on AGS cells.\u003c/p\u003e","manuscriptTitle":"Chrysosplenetin B induces apoptosis and inhibits metastasis of gastric cancer AGS cell by regulating reactive oxygen species-mediated signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 05:04:21","doi":"10.21203/rs.3.rs-3796020/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3908436-f104-41ef-aa7d-afbc22c127dc","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-28T08:12:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 05:04:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3796020","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3796020","identity":"rs-3796020","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}