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

doi:10.21203/rs.3.rs-3796020/v1

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

https://doi.org/10.21203/rs.3.rs-3796020/v1

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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

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.

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% CO₂ 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 × 10⁴ 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 (IC₅₀) 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 × 10⁶ cells/well. AGS cells were treated with CHR (the IC₅₀ 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 × 10⁵ 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 × 10⁶ 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 × 10⁵ 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 × 10⁵ 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 × 10⁶ 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 IC₅₀. SPSS 29.0 software was used for Tukey’s postmortem test, and p < 0.05 was statistically significant.

Killing effect of CHR on GC cell

As shown in Fig. 1a 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. 1c 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, IC₅₀ 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. 2a, 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. 2b, 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. 2c, 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. 2d, 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. 3a, Venny^2.1 obtained a total of fifty-four predicted targets for GC processing by CHR. As shown in Fig. 3b, the interaction diagram of protein targets predicted by the common target of CHR and GC through the String website. As shown in Fig. 3c, the interaction diagram of the PPI network was further screened by Cytoscape software. As shown in Fig. 3d, 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. 3e, the BP-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig. 3f, the CC-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig. 3g, the MF-related function clusters of the top twenty CHR processing GC were listed. As shown in Fig. 3h, 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. 3i 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. 3k, 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. 3l 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. 4a, 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. 4b-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. 5a, 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. 5b, after NAC treatment, the apoptosis rate of AGS cells treated with CHR decreased from 42.24 to 3.4%. As shown in Fig. 5c, 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. 6a, 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. 6b, NAC significantly inhibited the number of G2/M phase cells after CHR treatment. As shown in Fig. 6c, 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. 6d, 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. 7a, 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. 7b, with the increased of CHR treatment time, compared with the control group, the migration of AGS cell was significantly inhibited. As shown in Fig. 7c, 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. 7d, 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.

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.

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).

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.

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 H₂O₂-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.

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

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Abstract

Figures

Introduction

Materials and methods

Cell culture

Cell viability assay

Cell apoptosis assay

Associated targets of CHR and GC analysis

Overlapping target protein-protein interaction networks analysis

GO and KEGG enrichment analysis

Molecular docking analysis

Mitochondrial membrane potential analysis

Western blot analysis

ROS levels analysis

Cell cycle analysis

Cell invasion and migration analysis

Statistical analysis

Results

Killing effect of CHR on GC cell

CHR induces apoptosis of AGS cell in mitochondria dependent manner

Construction of protein interaction networks

Target biological function analysis

Molecular docking analysis

CHR induces apoptosis of AGS cell by regulate AKT, MAPK, and STAT3 signaling pathways

CHR induces apoptosis of AGS cell by up-regulating ROS levels

CHR arrests AGS cell cycle in G2/M phase

CHR inhibits the invasion and migration of AGS cell

Discussion

Conclusion

Declarations

Conflict of interest:

Funding:

Author Contribution

References

Additional Declarations

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