Oridonin suppresses gastric cancer SGC‐7901 cell proliferation by targeting the TNF‐alpha/androgen receptor/TGF‐beta signalling pathway axis

Abstract Statistics provided by GLOBOCAN list gastric cancer as the sixth most common, with a mortality ranking of third highest for the year 2020. In China, a herb called Rabdosia rubescens (Hemsl.) H.Hara, has been used by local residents for the treatment of digestive tract cancer for hundreds of years. Oridonin, the main ingredient of the herb, has a curative effect for gastric cancer, but the mechanism has not been previously clarified. This study mainly aimed to investigate the role of TNF‐alpha/Androgen receptor/TGF‐beta signalling pathway axis in mediating the proliferation inhibition of oridonin on gastric cancer SGC‐7901 cells. MTT assay, cell morphology observation assay and fluorescence assay were adopted to study the efficacy of oridonin on cell proliferation. The network pharmacology was used to predict the pathway axis regulated by oridonin. Western blot assay was adopted to verify the TNF‐α/Androgen receptor/TGF‐β signalling pathway axis regulation on gastric cancer by oridonin. The results showed Oridonin could inhibit the proliferation of gastric cancer cells, change cell morphology and cause cell nuclear fragmentation. A total of 11signaling pathways were annotated by the network pharmacology, among them, Tumour necrosis factor alpha (TNF‐α) signalling pathway, androgen receptor (AR) signalling pathway and transforming growth factor (TGF‐β) signalling pathway account for the largest proportion. Oridonin can regulate the protein expression of the three signalling pathways, which is consistent with the results predicted by network pharmacology. These findings indicated that oridonin can inhibit the proliferation of gastric cancer SGC‐7901 cells by regulating the TNF‐α /AR /TGF‐β signalling pathway axis.


| INTRODUC TI ON
Oridonin (Figure 2A), a 7,20-Epoxy-ent-Kauren Diterpenoid, is an effective ingredient with anti-cancer activity. 2,3 The chemical formula is C 20 H 28 O 6 , the molecular weight is 364. 44, the melting point is between 248 and 250°C, the compound is colourless or light yellow and displays a physical structure similar to needle-like crystals. The compound tastes bitter, is slightly soluble in water and completely soluble in methanol, ethanol, dimethyl sulfoxide and other organic solvents.
The enkeone system, the D-ring core pharmacophore, is closely related to the biological activity of ordonin and its anticancer activity may be lost if the enkeone system is removed. 4 In order to improve the bioavailability of oridonin, a variety of preparations such as liposomes 5,6 microspheres 7 and nanoparticles 8,9 have been developed.
Network pharmacology is a theory developed by Hopkins, 32,33 a British pharmacologist, in 2007 and is considered a new interdisciplinary subject. Based on systems biology and multidirectional pharmacology, network pharmacology gives a new idea for drug research from the perspective of a multi-target strategy. 11 Through the analysis and integration of big data, potential drug targets and signalling pathways can be predicted more comprehensively. In this study, we first confirmed that oridonin could inhibit the proliferation of gastric cancer cells. Then, the network pharmacology method was adopted to predict the signal pathway of oridonin for gastric cancer. Finally, experimental studies were carried out to study the predicted pathways and comprehensively reveal the mechanism of action of oridonin on gastric cancer.
The research flow chart of this paper is shown in Figure 1.

| Cell line
Human gastric cancer SGC-7901 cells were obtained from Heilongjiang Provincial Key Laboratory of Tumour Prevention and Antitumor Drug Research.

| In vitro cell viability assay
SGC-7901 cells at the logarithmic growth stage were added into a 96-well plate with 3.0 × 10 3 cells/well. After incubation in the carbon dioxide incubator with 5% CO 2 for 24 h, oridonin were added to cell suspensions and then incubated for another 72 h. This was followed by adding 0.5 mg/mL MTT solution to the suspensions, then incubated for 4 h in the carbon dioxide incubator. After this time, the liquid was poured out and 150 μL of DMSO was added to the suspensions, after which the optical density (OD) was detected by a microplate reader. The wavelength used for measurement was 570 nm, the IC 50 value and inhibition rate were calculated. 31,34

| Protein-protein interaction analysis(PPI) and hub gene screening
The PPI of targets was generated by Bisogenet, a plug-in of Cytoscape. Hub gene was screened using six parameters, including betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), local average connectivitybased method (LAC) and network centrality (NC).

| Pathway and biological functional enrichment analysis of Hub targets
Enrichment analysis of the Hub targets was performed by ClueGo, a plug-in of Cytoscape (https://cytos cape.org), pV <0.05, Pathway = wikipathway. Visualisation was organized and imported using Cytoscape. Biological processes (BP) and pathways were visualized to show the relations between oridonin and gastric cancer targets.
Protein concentration of the lysates were detected by Bradford assay. SDS-PAGE was performed until the protein bands were completely separated, and then the proteins were transferred onto NC membranes. After blocking, the corresponding primary and secondary antibodies were added. The bands were measured by Gel Imaging System (GE, ImageQuant LAS500), and all the bands were repeated for three times.

| Statistical analysis
Data are represented by mean ± standard deviation. The statistical analysis of data were calculated using Student's t-test. p < 0.05 is believed as statistically significant.

| Potential targets of oridonin
The Pharmapper Database was adopted to identify the potential targets of oridonin, and the top 300 targets were selected as relevant.
After processing, a total of 293 targets meeting the standards were obtained (Table S1).

| Gastric cancer targets
Through five different databases, 180 targets relating to gastric cancer was identified (Table S3). A total of 18 targets were found by Drugbank, 56 targets were sought out from GAD, 103 targets were got from KEGG, 12 targets were mined from OMIM and 7 targets were fished using TTD.

| Common targets fishing of oridonin in the treatment of gastric cancer and Hub target screening
By means of PPI, 7046 potential targets of oridonin (Figure3Aa, Table S2) and 5613 targets of gastric cancer related diseases (Figure 3Ab,   NC and other parameters were adopted to find 4342 common targets of oridonin for gastric cancer successively and finally 358 oridonin Hub targets for gastric cancer were obtained (Figure 3Ae, Table S6).

| Oridonin-Targets-Gastric Cancer network construction
A network depicting Oridonin-Targets-Gastric Cancer was constructed to observe the anti-gastric cancer mechanism of oridonin ( Figure 4). The network consists of 453 nodes (273 oridonin targets, 160 gastric cancer-related targets and 20 overlapping targets) and 473 edges. The 20 overlapped targets are the potential targets for the treatment of gastric cancer with oridonin, which suggested a possible mechanism for the treatment of on gastric cancer with oridonin.

| Pathway enrichment analysis of the Hub targets
The 358 Hub targets of oridonin on gastric cancer were adopted to fish related pathways by wikipathway. 11 related pathways were enriched, among which the three pathways with the largest weight were TNFα signalling pathway, androgen receptor signalling pathway and TGFβ signalling pathway (Figure 3B b). The results showed the anti-gastric cancer effect of oridonin is potentially associated with the three signalling pathways.  Figure 5E).

| Effects of oridonin on AR signalling pathway in SGC-7901 cells
The protein expression of AR in cytoplasm was increased, while it was decreased in the nucleus, and the protein expression of STEAP1 in cells was also decreased, suggesting that oridonin could promote the flow of AR protein in the nucleus to the cytoplasm. It demonstrated the translocation of AR was induced by oridonin ( Figure 6).

| Effects of oridonin on the TGFβ signalling pathway in SGC-7901 cells
The expression levels of TGFβ and p-Smad 2/3 protein were decreased and the Smad4 protein were increased, but the Smad2/3 protein were not changed by oridonin, The results showed that oridonin could regulate down the TGFβ expression, inhibiting the activation of Smad2/3 protein and activating Smad4 protein ( Figure 7). The observation of nuclear morphology proved that oridonin caused the typical nuclear morphology of cell death, such as nuclear fragmentation and dense staining.
In this study, through the study of network pharmacology, it was predicted that the proliferation inhibition effect of oridonin was related to the regulation of TNFα, AR and TGFβ signalling pathways.
Therefore, this finding was then experimentally verified in the following study.
TNFα is involved in many physiological and pathological pro- factor of NF-κB, so the activity of NF-κB was inhibited, and the activity of promoting proliferation and inhibiting apoptosis was decreased.
These findings showed that the inhibitory effect of oridonin on SGC-7901 cells is related to TNFα signalling pathway, which is achieved by regulating caspase family proteins, NF-κB signalling family proteins and promoting the protein expression of JNK signalling pathway.
AR is the androgen receptor, and AR signalling pathway is related to many hormone-dependent diseases such as prostate cancer, 49 breast cancer [50][51][52] and ovarian cancer, 53 78 Hence, SGC-7901 cell proliferation inhibited is related to its regulation of the TGFβ signalling pathway.
Tumour-related signalling pathways are not only independent but also indivisible. They interweave, interact and influence each other to jointly affect the tumours and participate in the regulation of tumour cell proliferation, differentiation, invasion, metastasis, angiogenesis, immune response as well as other mechanisms.
JNK is not only activated by TNFα, but also closely related to the caspase family, NF-κB pathway and TGFβ pathway. JNK mediates the exogenous death receptor pathway initiated by caspase-8, and its active substance promotes the release of mitochondrial proapoptotic protein jBID, which promotes the release of caspase-8 inhibitors on the TNFR1 complex. Thus, the inhibition of caspase-8 was decreased, and apoptosis was induced. 79 JNK is also activated by TGFβ in many cancer cell lines, and the TGFβ/Smad is upstream of JNK. MEKK1, a member of the MAPKKK family, directly binds to and activates the NF-κB inhibitory kinase signals IKKα and IKKβ, leading to phosphorylation. MEKK1 is also an upstream kinase of IκB, selectively phosphorylating the Ser32 and Ser36 sites of IκB, which together activate the downstream NF-κB to accelerate proliferation and reduce apoptosis. 80,81 That is, TNFα pathway, androgen receptor pathway and TGFβ pathway are independent but related to each other and the three pathways constitute a TNFα/AR/TGF-β signalling pathway axis.

| CON CLUS IONS
In conclusion, in this study, the signal pathway of oridonin inhibiting gastric cancer proliferation was predicted by network pharmacology. Experimental studies confirmed that the proliferation inhibition of oridonin on SGC-7901 cell was related to TNFα/AR/TGF-β signalling pathway axis, which confirmed the prediction of network pharmacology, and elucidated the mechanism of oridonin on gastric cancer ( Figure 8).

ACK N O WLE D G E M ENTS
No applicable.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article O RCI D Shiyong Gao https://orcid.org/0000-0002-8499-9485