Investigation on the mechanism of Ranunculus ternatus Thunb. against esophageal squamous cell carcinoma based on network pharmacology and experimental verification

doi:10.21203/rs.3.rs-4358038/v1

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Research Article

Investigation on the mechanism of Ranunculus ternatus Thunb. against esophageal squamous cell carcinoma based on network pharmacology and experimental verification

https://doi.org/10.21203/rs.3.rs-4358038/v1

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Purpose

This study was conducted to assess the pharmacological mechanisms of ethyl acetate extract of Ranunculus ternatus Thunb. (RTE) in combating esophageal squamous cell carcinoma (ESCC) through the integration of network pharmacology analysis and experimental validation.

Methods

Utilizing network pharmacology methodologies, potential targets of RTE and targets associated with ESCC were identified from public databases. Subsequently, protein-protein interaction (PPI) network analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted to ascertain the key targets and pathways through which RTE may exert its effects against ESCC. Finally, the putative mechanisms of action of RTE on ESCC, as predicted by network pharmacology analysis, were validated through in vitro experiments.

Results

A total of 274 potential targets were retrieved by searching the intersection of RTE and ESCC targets. 14 key genes of RTE acting on ESCC were obtained combined Component-Target-Disease Pathway and PPI network analysis, including AKT1, HSP90AA1, EGFR, MAPK1, and TNF.GO biological process analysis mainly involved in regulation of inflammatory response, response to lipopolysaccharide, and regulation of apoptotic signaling pathway, etc. KEGG signaling pathway analysis mainly related to MAPK signaling pathway, Relaxin signaling pathway, and PI3K/Akt pathway, etc. Then, the results of in vitro experiment indicated that RTE could inhibit proliferation of EC-109 and TE-13 cells. The in vitro experiments validated that RTE exhibited its therapeutic effects on ESCC mainly though the regulation of cell proliferation via MAPK/ERK and PI3K/Akt signaling pathways.

Conclusion

This study demonstrated that it may offer a useful tool to clear the molecular mechanism of RTE on ESCC by combination of network pharmacology prediction with experimental validation.

esophageal squamous cell carcinoma

Ranunculus ternatus Thunb.

network pharmacology

traditional Chinese medicine

PI3K/Akt signaling pathways

Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide[1]. Among these, esophageal squamous cell carcinoma (ESCC) accounts for ~ 90% of esophageal cancer worldwide[2]. Despite advancements in surgical interventions, chemotherapy, radiotherapy, and chemoradiotherapy, ESCC continues to exhibit a high fatality rate, with overall 5-year survival rates ranging from 15–25%. [3].

Traditional Chinese medicine (TCM) remedies, a form of complementary and alternative medicine, are frequently utilized in conjunction with antineoplastic drugs for the treatment of diverse cancer types. These remedies are increasingly acknowledged as potential therapeutic agents due to their favorable characteristics, including minimal side effects, low resistance development, and prolonged efficacy. Ranunculus ternatus Thunb. (Ranunculaceae) is a plant species belonging to the Ranunculus genus, which has been included in the Chinese pharmacopoeia since 1990. It is predominantly found in the Henan and Anhui regions of China and has been utilized in traditional Chinese medicine for the management of tuberculosis, faucitis, and neck scrofula[4, 5]. The ethyl acetate extract, saponins, and polysaccharides derived from Ranunculus ternatus Thunb. demonstrated anti-tumor effect both in vitro and in vivo in various cancer[6–14]. Ranunculus ternatus Thunb. is widely used clinically and has been approved for the treatment of cancer[15, 16]. Now, it has been further developed for medicinal use in Japan for the treatment of breast cancer, demonstrating significant efficacy [17]. Nevertheless, the specific active ingredients and molecular targets of this compound have yet to be fully elucidated.

Network pharmacology, which clarifies the synergistic effects and underlying mechanisms of multi-component and multi-target agents using the analysis of networks, is a suitable approach to reveal the functional mechanisms of multi-target drugs[18, 19]. In this study, we first screened the active ingredient in vitro, then through a network pharmacology to uncover the pharmacological mechanism of the compounds of Ranunculus ternatus Thunb. on ESCC and further verified through biological experiment.

Network Pharmacology-Based Analysis

Screening of bioactive components of RTE and collection targets

The active components of the RTE extract were identified through a review of the literature. The chemical composition information, such as molecular structures, canonical smiles, and 'SDF' files, was obtained from product databases including PubChem (https://pubchem.ncbi.nlm.nih.gov/) and ZINC (http://zinc15.docking.org). Subsequently, the targets of the bioactive components were predicted using public databases such as TCMSP (http://tcmspw.com), PubChem (https://pubchem.ncbi.nlm.nih.gov/), PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), TargetNet (http://targetnet.scbdd.com/) and Similarity Ensemble Approach database (SEA, http://sea.bkslab.org/). Additionally, the targets were standardized to unified gene names using the protein database UniProt (http://www.uniprot.org/uploadlists/). The target protein names were entered into UniProtKB, with the organism limited into “Homo sapiens,” prior to the retrieval of the official symbol.

Acquisition of intersection genes and network construction

The search for "esophageal squamous cell carcinoma" related genes was conducted using the GeneCard database (https://www.genecards.org/). The intersection of predicted RTE targets and ESCC-related genes was obtained through Venny 2.1.0. Subsequently, a network titled "Components-targets-ESCC" was constructed using Cytoscape software. Protein-protein interaction (PPI) analysis of the intersection genes was carried out using the String software, with a focus on data with a confidence level exceeding 0.9. The network was visualized and analyzed using Cytoscape software in this research study.

GO and KEGG Pathway Analysis

The key genes underwent GO and KEGG pathway enrichment analyses through the utilization of R software. Following the conversion of input molecular list IDs, the enrichment analysis was conducted using the clusterProfiler package (species: Homo sapiens, ID transformation library (package): org.Hs.eg.db), and the resulting enrichment analysis outcomes were visually represented using the ggplot2 package.

Preparation of R. ternatus Extracts

The dried root powder of R. ternatus (0.2 kg) was extracted 3 times with 95% ethanol at 78℃. Following filtration and evaporation, the residue was suspended in H₂O and subsequently fractionated with petroleum ether (60–90℃), ethyl acetate, and n-BuOH three times. This process yielded the R. ternatus petrloeum ether extract (RTP, 7.7 g), the R. ternatus ethyl acetate extract (RTE, 3.3 g), and the R. ternatus n-BuOH extract (RTB, 10.9 g). The three R. ternatus extracts were then dissolved in dimethyl sulfoxide at a concentration of 200 mg/mL.

Cell culture

The Human esophageal squamous cell carcinoma cell line EC-109, TE-1 and TE-13 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml of penicillin, and 100 µg/ml of streptomycin at 37°C in a humidified atmosphere containing 5% CO₂ .

Cell viability assay

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which relies on the metabolic activity of viable cells to convert MTT into a formazan product. Specifically, cells were plated at a density of 5000 cells per well in 96-well plates and allowed to adhere overnight before being treated with varying concentrations of three R. ternatus extracts for 48 hours. Subsequently, MTT was added to each well and the cells were incubated for an additional 4 hours. The colored formazan product was determined photometrically at 570 nm in a multi-well plate reader (Bio-Rad Laboratories, Hercules, CA).

Western blotting

EC-109 and TE-13 cells were seeded at a density of 5 × 10⁵ cells per dish in 60-mm culture dishes and incubated for 24 h. Following treatment with the IC₅₀ concentration of RTE for an additional 48 hours, the cells were lysed in RIPA lysis buffer supplemented with a protease-inhibitor cocktail on ice for 15 minutes. The lysed cell suspensions were then centrifuged at 12,000 ×g at 4 ℃ for 15 minutes, and the resulting supernatants containing soluble cellular proteins were collected and stored at -80 ℃ until further analysis. BCA protein assay kits were utilized for the quantification of protein concentration. Subsequently, 20 µg of protein was reconstituted in sample buffer and subjected to separation via 10% SDS-polyacrylamide gel electrophoresis. The resolved proteins were then transferred onto a to PVDF membrane, which was subsequently blocked using 5% milk in tris-buffered saline/0.1% Tween20. Following this, the membranes were probed overnight at 4°C and immunodetection was carried out using primary antibodies, including anti-rabbit Phospho-p44/42 MAPK antibody (1:1000), anti-rabbit Phospho-Akt antibody (1:1000), anti-rabbit c-Myc antibody (1:1000), anti-rabbit HSP90 antibody (1:1000) or anti-rabbit β-actin antibody (1:1000). The membranes were incubated a secondary antibody conjugated to peroxidase (1:2000, all from Cell Signaling Technology, Beverly, MA) and subsequently analyzed using the ECL Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) to visualize signals, which were captured on X-ray film (Fuji Photo Film, Tokyo, Japan).

Statistical Analysis

The experiments were conducted with a minimum of three repetitions each. Prism statistical software (Graph Pad Software, San Diego, CA) was utilized to calculate the half inhibitory concentration (IC₅₀) values of the drugs. The figures present data as mean ± S.E.M., while the tables display data as mean ± SD. Statistical analysis was performed using SPSS 25.0 software (SPSS Inc., Chicago, IL), with Student's two-tailed t test used for comparing data between two groups and Repeated measures ANOVA or One-way ANOVA for comparing data between three or more groups. P < 0.05 were considered statistically significant.

Identification of Potential Bioactive Compounds and Targets of RTE

29 active components from the RTE were searched in published literatures [15, 20–27] (Table S1). After eliminating the overlapping proteins, 461 associated proteins were obtained for subsequent analysis.

Construction of the Volatile Component-Target-Disease Pathway

A total of 4838 related targets were obtained from GeneCards (https://www.genecards.org/) and OMIM database using the search terms “esophageal squamous cell carcinoma”. Following the removal of duplicate proteins, a set of 274 shared targets between RTE and esophageal squamous cell carcinoma was identified. The regulatory network of RTE acting on ESCC was constructed using Cytoscape software version 3.7.2. The compound-compound target-disease network, illustrated in Fig. 1, consisted of 305 nodes (comprising 29 component nodes, 274 common targets, RTE, and ESCC) and 1640 edges. The network analysis indicates that a single component may interact with multiple targets, while multiple targets may be affected by a single component. Degree centrality is measure of how important a node is at linking poorly connected parts of the network [28]. Among the 84 nodes identified with an average degree value of ≥ 8.82, comprising 29 compounds and 55 genes (Table S2), hexadecanoic acid, amentoflavone, and phillygenin are likely key components of RTE responsible for its anti-tumor properties.

PPI Network Analysis

To elucidate the systemic and pharmacological mechanism of RTE in the treatment of ESCC, we initially constructed and visualized a protein-protein interaction (PPI) network comprising 274 putative targets. The network consist of 247 targets and 1279 edges (Fig. 2), and an average value of degree is 10.36. Finally, we identified 77 core targets in descending order (Table S3). These hub targets included AKT1、HSP90AA1、EGFR、MAPK1 and TNF, indicating that they serve as vital targets of RTE in treating ESCC.

Key Genes

Finally, combine above two network diagrams to obtain 14 key genes of RTE acting on ESCC (Fig. 3).

GO functional enrichment analysis and KEGG pathway enrichment analysis

To identify the biological characteristics of putative targets of RTE on ESCC in detail, the GO and pathway enrichment analyses of involved targets were conducted via R software (4.2.1 version). The results of GO enrichment analysis showed that the therapeutic effect of RTE on ESCC was mainly related to the biological processes such as regulation of inflammatory response, response to lipopolysaccharide, and regulation of apoptotic signaling pathway. The cellular components include vesicle lumen, secretory granule lumen, and cytoplasmic vesicle lumen. The molecular functions include RNA polymerase II-specific DNA-binding transcription factor binding, transcription coregulator binding, and ligand-activated transcription factor activity (Fig. 4). As shown in Fig. 5, the results of KEGG pathway analysis suggested that RTE may exert anti-tumor effects through MAPK signaling pathway, Relaxin signaling pathway, and PI3K/Akt pathway, etc.

The cytotoxicity of RTP and RTE on ESCC cells

In order to assess the cytotoxic potential of various extracts of R. ternatus, IC₅₀ values were determined. The inhibitory impact of RTB on the viability of three esophageal squamous cell carcinoma cell lines was found to be less than 50% at a concentration of 2000 µg/mL. Analysis using the MTT assay demonstrated that treatments with RTE and RTP resulted in suppression of ESCC cell viability, as indicated in Table 1. While there was no significant disparity between the effects of RTE and RTP treatments in each cell line, both treatments significantly impeded the proliferation of EC-109 cells (P < 0.05).

Table 1

RTE and RTP suppressed the viability of ESCC cells.
	IC₅₀(µg/mL)
	TE-1	EC-109
RTE	220.8 ± 61.9	90.2 ± 14.0^***
RTP	298.9 ± 32.2	100.2 ± 3.4^*
Each cell was treated for 48 h with three extracts of RT (0–2000 mg/mL). Cell viability was then determined by 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium assay. IC₅₀ values were calculated with Prism software. Data are presented as Mean ± SEM. (n = 3) of a representative experiment performed in triplicate. *** presented P value less than 0.001, * presented P value less than 0.05.

RTE suppresses MAPK/ERK pathway and PI3K/Akt pathway

Based on the results of network pharmacology, we believe that it is necessary to elucidate these three proteins related pathways. As the results showed, a significantly decreased expression of p-ERK1/2, p-Akt, HSP90 and c-Myc in both EC-109 and TE-13 were observed in RTE groups when compared with the control group. And no significant differences were observed in the expression of total ERK1/2 and Akt between groups (Fig. 6).

ESCC is a prevalent malignancy associated with various proteins and signaling pathways involved in its development and progression[29–32]. TCM, comprising a diverse array of compounds, demonstrates broad pharmacological effects targeting multiple pathways and molecular targets, potentially offering therapeutic benefits for ESCC treatment[33]. However, the complexity of TCM poses challenges for elucidating its underlying mechanisms. The application of network pharmacology, which combines systems biology and network analysis methodologies, may provide a promising approach for investigating the intricate mechanisms of action of TCM in treating ESCC. The extracts of Ranunculus ternatus Tunb. have shown their anti-tumor activity in many studies[7, 10, 11], but the anti-tumor mechanism of it has not yet been elaborated. The primary objective of this study was to investigate the impact of RTE on ESCC and validate the mechanisms proposed by network pharmacology through in vitro experimentation.

The network pharmacology results showed that hexadecanoic acid, amentoflavone, and phillygenin are key active ingredients of RTE in treating ESCC. Hexadecanoic acid, commonly known as palmitic acid, is a saturated long-chain fatty acid with a straight-chain structure consisting of sixteen carbon atoms. Research has shown that palmitic acid can induce cell cycle arrest and promote apoptosis in human neuroblastoma cells and breast cancer cells. Additionally, it has been found to inhibit the proliferation of hepatoma cells by altering membrane fluidity and interfering with glucose metabolism([34–36]. In prostate cancer cells, palmitic acid has been shown to suppress proliferation and metastasis by inhibiting the PI3K/Akt pathway[37]. Furthermore, palmitic acid has been demonstrated to inhibit the growth and metastasis of gastric cancer by blocking the STAT3 signaling pathway[38]. Amentoflavone, a biflavonoid compound, exhibits anti-tumor activity in various types of cancer. Studies have demonstrated its involvement in numerous biological processes, such as cell proliferation[39], apoptosis[40], cell cycle regulation[41, 42], migration, and invasion. Phillygenin has been reported to demonstrate anti-tumor activity in drug-resistant human esophageal cancer[43], pancreatic cancer[44], osteosarcoma[45], and non-small cell lung cancer[46].

The findings from the PPI network analysis revealed 77 core targets associated with RTE in the treatment of ESCC, such as AKT1, HSP90AA1, EGFR, MAPK1, and TNF. These targets are intricately linked to cellular processes including proliferation, cell cycle regulation, apoptosis, and are pivotal in the efficacy of RTE for ESCC treatment.According to the analysis of GO enrichment results in hub targets, RTE is mainly involved in biological processes such as regulation of inflammatory response, response to lipopolysaccharide, and regulation of apoptotic signaling pathway. Analysis of the KEGG pathway results showed that RTE is mainly involved in MAPK signaling pathway, Relaxin signaling pathway, and PI3K/Akt pathway, etc. These pathways are closely related to the growth and metabolism of tumor, indicating that RTE may regulate the above pathways through hub targets, particularly, MAPK1 and HSP90, thereby inhibiting tumor progression.

In order to elucidate the molecular mechanism of RTE in the treatment of ESCC, we conducted in vitro cell experiments to validate the predicted outcomes. MTT assays were performed on EC109 and TE-13 cells, revealing a dose-dependent anti-ESCC effect of both RTE and RTP. These findings suggest that RTE may hold promise as a potential therapeutic agent for ESCC. Previous research has indicated the anti-tumor properties of Ranunculus ternatus Thunb. extracts[7, 10, 11], prompting our selection of RTE for further investigation in this study.The network analysis revealed that the hub targets primarily participate in the MAPK signaling pathway. Considering the interconnectedness of signaling pathways and their downstream targets, as well as the relevance of closely associated targets identified in the literature on ESCC [47–53], ERK1/2, p-ERK1/2, AKT, p-AKT, HSP90 and c-Myc proteins were chosen for validation through Western blot experiments. In comparison to the control group and the group treated with RTE in Eca-109 and TE-13 cells, a significant decrease in the expression of p-ERK1/2 and p-Akt was observed. Additionally, a slight reduction in HSP90 and c-Myc expression was noted in the RTE-treated group. These findings suggest that RTE has the potential to inhibit the MAPK/ERK and PI3K/Akt pathways, as well as downregulate the expression of oncogenic factors HSP90 and c-Myc, ultimately leading to the inhibition of cell proliferation in ESCC..

MAPK1 is one of the hub targets in our analysis, which an important protein in the MAPK/ERK pathway. MAPK/ERK pathway is obviously the most well-studied signal transduction module, especially in the context of ESCC development[54]. Moreover, c-Myc is activated upon various mitogenic signals such as via the MAPK/ERK pathway [55]. By modifying the expression of its target genes, c-Myc activation results in numerous biological effects, including cell proliferation, apoptosis and differentiation. Under stimulation, ERK kinases migrate to the nucleus and phosphorylate their nuclear protein substrates. The anti-apoptosis response in many tumor cells is initiated through the ERK1/2-mediated pathway [56]. And the results showed that RTE significantly decreased the expression of p-ERK1/2. Therefore, RTE inhibited ESCC via MAPK/ERK pathway.

Besides, Hsp90 is another hub target, which stabilizes various growth factor receptors[57] and some signaling molecules including PI3K and Akt proteins. Thereby inhibition of Hsp90 downregulates the PI3K/Akt pathway resulting in apoptosis of cancerous and senescent cells[58]. PI3K/Akt pathway plays an important regulatory role in the process of tumor cell proliferation and anti-apoptosis[59]. Our results showed that RTE significantly down-regulated the expression of p-Akt and HSP90. So, RTE weaken the proliferation of ESCC cell by inhibiting PI3K/Akt pathway.

This study presents persuasive evidence of the anti-cancer properties of RTE on ESCC; however, it is important to recognize several limitations. Primarily, the reliance on in vitro assays, while informative, may not fully capture the intricacies of cancer within a living organism. Future investigations should incorporate in vivo studies to confirm the therapeutic efficacy of RTE in animal models of ESCC. Furthermore, a deeper understanding of the mechanisms through which RTE components interact with molecular targets in the context of ESCC is necessary.

In this study, the results of network pharmacology and PPI network showed that MAPK1 and HSP90 were the key targets of RTE against ESCC. Through network pharmacology and experiments in vitro, RTE was verified to against ESCC via MAPK/ERK pathway and PI3K/Akt pathway, which may offer a useful tool to clear the molecular mechanism of RTE on ESCC treatment.

Data availability statement

The original contributions presented in the study are included in the article/ supplementary material, further inquiries can be directed to the corresponding authors.

Author contributions

Michen Deng: Performing experiments, Data analysis and curation. Yanhong Deng: Data analysis and curation. Zu-gui Tang: Performing experiments. Wenqiang Nie: Data analysis. Yi Lu: Performing experiments. Zhe Wang: Writing-review and editing. Xicheng Wang: Writing-review and editing. Yi Kong: Writing original draft, Data analysis and curation.

Declaration of competing interest

We confirm that this manuscript has not been published by another journal. All authors have approved the manuscript and agree with its submission to your journal. The authors declare that there is no conflict of interests regarding the publication of this paper.

Funding

This work is supported by the Guangdong Provincial Natural Science Foundation (Grant No. 2019A1515011609), Shenzhen Science and Technology Program(NO.JCYJ20220530154215035), and the Department of Education of Guangdong province (2022KTSCX220) to Yi Kong.

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Investigation on the mechanism of Ranunculus ternatus Thunb. against esophageal squamous cell carcinoma based on network pharmacology and experimental verification

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Abstract

Purpose

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Results

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Introduction

Materials and Method

Network Pharmacology-Based Analysis

Screening of bioactive components of RTE and collection targets

Acquisition of intersection genes and network construction

GO and KEGG Pathway Analysis

Preparation of R. ternatus Extracts

Cell culture

Cell viability assay

Western blotting

Statistical Analysis

Results

Identification of Potential Bioactive Compounds and Targets of RTE

Construction of the Volatile Component-Target-Disease Pathway

PPI Network Analysis

Key Genes

GO functional enrichment analysis and KEGG pathway enrichment analysis

The cytotoxicity of RTP and RTE on ESCC cells

RTE suppresses MAPK/ERK pathway and PI3K/Akt pathway

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1