Exploring the anti-cancer effect of taraxasterol using network pharmacology, molecular docking and in vitro experimental validation

Taraxasterol (TS) is a naturally occurring pentacyclic triterpenoid extracted from the traditional Chinese herb Taraxacum mongolicum . Previous studies have highlighted its significant roles in exhibiting anti-inflammatory, anti-oxidant, and liver protective effects. In the present study, the anti-cancer potential of TS against cervical cancer was investigated, employing network pharmacology techniques, molecular docking, and in vitro experimental validation. TS exhibits its anticancer properties by modulating multiple targets, pathways, and biological processes. In vitro experiments demonstrated the potent inhibitory effects of TS on cancer cell growth and migration, while no significant impact on apoptosis was observed. The primary objective was to elucidate the anti-cancer potential of TS, which is a crucial lead compound in the treatment of cervical cancer. The findings may serve as a basis for the development of novel anticancer therapeutics and medicine-based interventions for cervical cancer.


Introduction
As GLOBOCAN 2020 was released, about 19.3 million new cancer cases and 10 million cancer deaths were reported in 185 countries for the 36 documented cancers [1]. Based on the data provided by the World Health Organization, cancer is emerging as the primary cause of human fatalities worldwide, posing a significant threat to human physical well-being and escalating the financial strain of cancer management [1][2][3]. In addition to surgical resection, chemotherapy, radiotherapy, and immunotherapy are also gradually improving from lab research to clinical trials. The increased expenditure and economic load of cancer therapy collapsed most of the patient families in China [4,5]. For more than 3,000 years, traditional Chinese herbs and formulas have been extensively used in China for managing various ailments. Nowadays, they are garnering increasing attention for their promising potential in treating malignancies. With the expansion of basic research, numerous traditional Chinese herbs are being explored for the extraction of unidentified bioactive compounds with potential therapeutic applications [6].
In recent times, traditional Chinese medicine has drawn considerable attention for its potential in the management of cancer. Several hundreds of Chinese herbs have emerged as research hotspots for tackling the disease. Among them, Taraxacum mongolicum Hand Mazz, also commonly known as dandelion, has gained significant attention in the realm of cancer treatment [7][8][9]. Dandelion has featured in over 100 traditional Chinese prescriptions due to its inclusion in the 2015 edition of the Chinese Pharmacopeia [10]. Dandelion is well-recognized and has been long-term used as a dietary and medicinal plant in China, where it can treat almost 181 types of disease [9,10]. The huge medicinal functions of dandelion were based on its' constitution, including, for instance, flavonoids, sesquiterpene lactones, phenolic acids, vitamins, polysaccharides, amino acids, triterpenoids, pigments, coumarins, and sterols [11,12]. The active compounds present in dandelion authorize its beneficial effects in anti-cancer, anti-thrombosis, anti-inflammatory, hypoglycemia, anti-oxidation, and immune regulation [9,[13][14][15].
This study sought to elucidate the anti-cancer effects of TS in cervical cancer using network pharmacology, molecular docking, and in vitro experimental validation. The results highlight the significance of traditional Chinese herbs, precious active compounds, associated target genes, and novel ideas in cancer therapy.

Cancer-related targets identification
Cancer-related targets were obtained from the GeneCards database (https://www.genecards.org/), the Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/) and the Therapeutic Targets Database (TTD database, http://db.idrblab.net/ttd/) [28][29][30]. The organism was set to "Homo sapiens," and "cervical cancer" served as the keyword used to identify putative targets. Targets retrieved from GeneCards with a relevance score of 10 or greater were included in the analysis.

Protein-protein interaction (PPI) network construction
The STRING database (version 11.5) (https://cn.string-db.org/) was utilised to compute the PPI network for the shared targets of TS and cervical cancer [32]. The study organism was "Homo sapiens", and interactions with a minimum required score of ≥ 0.4 were deemed significant [33]. Cytoscape was employed to visualise and scrutinise the PPI network. Hub targets were selected based on previously reported criteria with node size corresponding to degree value, whereby larger nodes represent higher degree values [7,34,35].

Target clustering analysis
Target clustering was analyzed through the Cytoscape app plugin MCODE [36]. The criteria were referred to in our previous study [7].

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
Biological functions and pathways of targets were done through GO (http://geneontology.org/) and KEGG (https://www.genome.jp/kegg/) [37,38]. The visualization of terms was done through the imageGP online tools (http://www.ehbio.com/ImageGP/index.php/) [39]. The KEGG enriched pathways network was constructed through Cytoscape. The balls represented the targets, and the squares were enriched KEGG pathways.

Molecular docking
Molecular docking between TS and hub targets was performed to validate interaction through Autodock Vina (https://vina.scripps.edu/) software [40,41]. The TS-hub target association was visualized using PyMoL (version 2.2) (http://www.pymol.org/2/). All the performance details and parameters set were refered to in our previous report [7]. The 2D molecular docking interaction was conducted through the ProteinsPlus web service (https://proteins.plus) [42]. When the affinity score was ≤ − 5.0 kcal/mol, which represents strong interactions between the core targets and TS [43].

Expression pattern and overall survival analysis
Sanger-box 3.0 website tools (http://vip.sangerbox.com/home.html) collected a unified, standardized pan-cancer data set, TCGA TARGET GTEx from UCSC (https://xenabrowser.net/) database. Cancer species with fewer than 3 samples were excluded, and finally, we obtained the

Highlights
Taraxasterol is a naturally produced marker molecule of Taraxacum mongolicum, which contains nearly 100 ingredients and is used to treat 181 different kinds of diseases. Taraxasterol exerts an anti-cancer effect through multiple targets, pathways, and biological processes based on network pharmacology and molecular docking validation.
In vitro experiments confirmed that taraxasterol played significant roles in inhibiting cancer cell growth, and migration but not apoptosis. expression data in cervical squamous cell carcinoma and endocervical adenocarcinoma. Overall survival analysis was performed through GEPIA [44].

Cell culture
HeLa-S3 were purchased from ATCC and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were routinely maintained in 5% CO 2 at 37°C. The culture medium was refreshed every 48 hours. For cell passage, the cells were digested with 0.25% trypsin. When grown as an adherent monolayer with a logarithmic growth stage, the cells were used for experiments.

Cell viability assay
The MTT assay (Beyotime) was employed to analyze cell viability. HeLa-S3 cells were diluted to a density of (1 × 10 5 /mL), and 100 µL was seeded into a 96-well plate in quadruplicate. After 16 hours of incubation, TS was administered at final concentrations of 0, 0.5, 2.5, 5, 10, 15, and 20 µg/mL for 24, 48, and 72 hours, respectively. Following a 4-hour incubation with MTT reagent at 37°C, formazan solution was added into the wells. Once the formazan was completely dissolved, cell viability was measured at 570 nm using a microtiter plate reader (Molecular Devices).

Cell migration assay
To investigate the effect of TS on cell migration, a wound healing assay was conducted. HeLa-S3 cells were seeded into a 6-well plate at a density of 3 × 10 5 /mL. Once the cell confluence reached 90%, a straight scratch was made using tips. TS was administered at a final concentration of 15 µg/mL for 24, 48, and 72 hours, respectively. Subsequently, photos were captured and areas were calculated using Image J.

Colony-forming assay
A 6-well plate was utilized to seed cells at a density of 1 × 10 4 /mL. TS was administered at a final concentration of 15 µg/mL for 7 days, with the cell culture medium being refreshed every 2 days. Colonies were fixed with 4% (wt/vol) paraformaldehyde for 30 minutes and stained with 1% crystal violet for 15 minutes. Photos were taken and analyzed with Image J. Colonies with more than 50 cells but less than 1000 cells were counted.

Target genes screening
A total of 216 targets correlated with TS were achieved due to the intersection of Super-PRED, PharmMapper, and Swiss Target Prediction ( Figure 1). Then, we used "cervical cancer" as the keyword to search for associated genes in the GeneCards, OMIM, and TTD databases. GeneCards obtained 1303 targets with a relevance score ≥ 10,497 in OMIM, and 14 in TTD (up to November 1, 2022). Through Venny 2.1, 1694 intersected targets correlated with cervical cancer were achieved ( Figure 1). Moreover, 66 shared targets were obtained ( Figure 1), and the detailed information was listed in (Table 1). "TS-cervical cancer-targets" network construction and hub targets selection Using Cytoscape 3.8.0, the "TS-cervical cancer-targets" network was established and presented in Figure 2A. The STRING database (up to November 1, 2022) was employed to analyze PPI, and the PPI network was constructed and visualized using Cytoscape ( Figure 2B). The degree values were utilized to determine the significance of the targets, with the 65 nodes in the PPI network represented by colored solid balls. The network produced 477 edges with an average neighbor of 14.68 ( Figure 2B). The 12 hub targets were obtained through selection criteria, and PPI was calculated using STRING, while the PPI network was visualized using Cytoscape ( Figure 2C-D). The PPI network generated 12 nodes and 60 edges, with an average neighbor of 10 ( Figure 2D). Signal transducer and activator of transcription 3 (STAT3) gained the highest degree of the 66 targets (degree = 43), followed by estrogen receptor 1 (ESR1, degree = 40) and albumin (ALB, degree = 38).

Targets clustering
The nodes and scores of the assigned clusters were plotted in ( Figure  3A). Networks of clusters were generated using STRING predictions and visualized using Cytoscape ( Figure 3B-D).
In Figure 3B, Cluster 1 comprised of 19 nodes and 100 edges, with an average neighbor of 10.53 and a score of 11.111. The seed node of Cluster 1 was PGR, which plays a crucial role in regulating eukaryotic gene expression, cellular proliferation, and differentiation [45]. Cluster 2, on the other hand, consisted of 15 nodes and 35 edges, with an average neighbor of 4.66 and a score of 5, as shown in Figure 3C. The seed node of Cluster 2 was steryl-sulfatase, which is responsible for catalyzing the conversion of sulfated steroid precursors to the free steroid [46]. Lastly, Cluster 3 contained 9 nodes and 16 edges, with an average neighbor of 3.56 and a score of 4, as depicted in Figure 3D. The seed node of Cluster 3 was mitogen-activated protein kinase 8, which primarily phosphorylates a number of transcription factors, particularly components of AP-1 [47].

GO enrichment analysis
The top 20 enriched GO biological process terms with P-value < 0.001 were organized in ( Figure 4A). Of the 20 terms, 6 were corelated with gene expression regulation, while the rest were associated with cell proliferation, protein phosphorylation, apoptotic process, cell migration, inflammation and histone deacetylation.
The top 20 molecular function terms, with a P-value < 0.001, were primarily associated with protein/kinase binding, enzyme binding, and receptor binding. These functions were mainly linked to cellular signaling transduction and gene expression ( Figure 4B).
A "targets-pathways" network was constructed to better understand the relationships between the terms and target proteins ( Figure 5B). The network had 63 nodes and 217 edges, with an average degree (neighbor) of 6.89. Submit a manuscript: https://www.tmrjournals.com/pr

Molecular docking validation
Of the 12 hub targets, nine can interact with TS through molecular docking analysis, with affinity energy < − 5.0 kcal/mol, which indicated strong interaction between TS and hub targets. 2D and 3D binding structures of the top 4 were selected and shown in (Figure 6 A-D). More details of the nine binding complexes are listed in (Table  3).

Expression and overall survival analysis
Expression patterns were analyzed using Sanger-box 3.0 and shown in (Figure 7). We observed significant up-regulation of histone deacetylase 1 (HDAC1) and down-regulation of the estrogen receptor 1 (ESR1) and androgen receptor (AR) in cervical squamous cell carcinoma and endocervical adenocarcinoma ( Figure 7A, Figure 7E, Figure 7G). No significant difference was observed in mitogen-activated protein kinase 1 (MAPK1) ( Figure 7C). Overall survival analysis was performed through GEPIA (Figure 7, right panel). During the first 100 months, no differences were observed in ESR1 expression, but after that time, high ESR1 expression led to a lower survival percent ( Figure 7B). MAPK1 expression level did not influence the survival percent, which was parallel with the pan-cancer analysis result ( Figure 7B-C). High expression of HDAC1 and AR led to a lower survival percent, especially after 100 months ( Figure 7F-H).

Cell viability analysis
To further prove the significant roles of TS in cervical cancer cells, cell viability had been assessed through MTT analysis. Different concentrations and time courses were set up, and after being treated with 0.5 µg/mL TS for 24 hours, cell viability alleviated significantly ( Figure 8A-B). The TS treated concentration was proportionately increased, from 2.5 to 20 µg/mL, however, no visible difference was detected ( Figure 8C-E). The statistical analysis was done and shown in (Figure 8D-F).

Cell migration assay
To test the influence of TS on cell migration, a wound healing assay was done. After being treated with 15 µg/mL TS for 24, 48, and 72 hours, migration rates were calculated. In contrast to the control group, TS treated cells had significantly poorer wound healing ( Figure  9A-C).

Colony-forming assay
After being treated with 15 µg/mL TS for 7 days, cell colonies were fixed, stained, photographed, and calculated ( Figure 10A). To further check the impact of TS on colony-forming, pictures were magnified and shown in ( Figure 10B). Compared with the control group, cells treated with TS were harder to form colonies and more earsily to disperse ( Figure 10B). Significant differences were obtained through colony counting (Figure 10 C).

Discussion
According to GLOBOCAN 2020 statistical data, cervical cancer ranks fourth in both incidence and mortality among females. It poses a massive threat to women's health, and more effective therapies are urgently needed [1]. In this study, 66 shared targets of TS and cervical cancer were identified for further investigation. The "TS-targets-cervical cancer" network revealed well-established relationships between TS and cervical cancer. Based on clustering analysis, three distinct clusters with different functions were constructed. The clusters demonstrated distinct anti-cancer effects of TS. The biological process analysis indicated that the shared targets were primarily associated with gene expression, cell proliferation, signal transduction, protein phosphorylation, cell migration, and apoptosis. Molecular function analysis revealed that these targets were linked with ATP binding, enzyme binding, transcription factor binding, and kinase activity. Furthermore, 40% of the KEGG enriched pathways corresponded to cancer, underscoring the significant anti-cancer roles that TS can play.
Molecular docking was performed between TS and 12 hub targets. Out of the 12 targets, 9 exhibited strong interaction with TS, with affinity scores below − 5 kcal/mol, indicating a robust binding affinity between the targets and TS.  In order to achieve this objective, an analysis of pan-cancer and overall survival was conducted based on four selected targets: ESR1, MAPK1, HDAC1, and AR. ESR1 is an estrogen receptor that regulates the expression of eukaryotic genes and contributes to cellular proliferation and differentiation upon binding to steroid hormones [48]. MAPK1 plays a significant role in the MAPK/ERK cascade, which regulates various biological functions such as cell growth, adhesion, survival, and differentiation by controlling transcription, translation, and cytoskeletal rearrangements [49,50]. HDAC1 catalyzes the deacetylation of lysine residues on the N-terminal region of the core histones, which creates an epigenetic repression tag and is crucial in regulating transcription, cell cycle progression, and developmental events [51]. Similar to ESR1, AR also binds to steroid hormones and mediates eukaryotic gene expression, cell proliferation, and differentiation in target tissues [52]. Moreover, together with the illustrated core targets, these hub targets played significant roles in cell growth, proliferation, migration, invasion, adhesion, and apoptosis, transcriptional regulation, and cell cycle [53][54][55].
The pan-cancer and overall survival analyses of hub targets indicated the significant anti-cancer effects of TS, particularly in cervical cancer. To further substantiate this hypothesis, in vitro experiments were conducted. The MTT analysis showed that TS effectively inhibited cell growth. Moreover, through cell migration and colony-forming assays, it was observed that TS suppressed cell migration. Additionally, cell apoptosis was analyzed via flow cytometry in HeLa-S3 cells following TS treatment, but no significant difference was observed (data not shown).
TS has potent anti-inflammatory and anti-tumor activity, and it can strongly impede the proliferation, growth, and migration of cells. The anti-cancer effect of TS is mediated through various targets, pathways, and biological processes.

Conclusion
In our study, we employed a network-based pharmacology approach, molecular docking, and in vitro experimental validation to elucidate the anti-cancer effect of TS. GO enrichment and KEGG pathway analysis indicated that TS exerts an antagonistic effect on cervical cancer by regulating cellular processes such as cell proliferation, gene expression, protein kinase activity, and transcription. In vitro experiments provided confirmation of the inhibitory effects of TS on cell proliferation, growth, and migration. In summary, our report introduces a significant and valuable naturally occurring lead compound that demonstrates a potent anti-cancer effect in cervical cancer.