Exploration of key pathogenic mechanisms and potential intervention targets of the traditional Chinese medicine Coptis chinensis in the treatment of cervical cancer based on network pharmacology and molecular docking techniques

Introduction

According to the GLOBOCAN 2020 statistics, cervical cancer is now the fourth leading cause of cancer-related deaths among women (1). In 2020, there were 604,127 new cases of cervical cancer and 341,831 cervical cancer-related deaths worldwide (2). Persistent infection with high-risk human papillomavirus (HPV) is the primary risk factor for cervical cancer (3). In recent years, early screening for cervical cancer precursors, improvements in clinical diagnosis and treatment methods, increased HPV vaccination, and early screening have effectively reduced the incidence and mortality of cervical cancer (4). However, low- and middle-income countries still have low HPV vaccination coverage and inadequate cervical cancer screening, resulting in higher incidence and mortality rates compared to developed countries (5).

For non-metastatic cervical cancer patients, the main treatments remain surgery and radiation therapy, while for metastatic patients, the treatment is typically chemoradiotherapy. New, effective, low-cost, and low-toxicity therapeutic agents urgently need to be identified (6). Traditional Chinese medicine (TCM) has been widely used in the treatment of cancer, and has been shown to improve drug sensitivity, regulate immune function, enhance chemotherapy effects, and improve patient prognosis (7). The advantages of TCM lie in its multi-targeted and multi-pathway effects, which result in broad biological activity and lower toxicity, making it a viable alternative therapy for various cancers, including cervical cancer.

From the perspective of TCM theory, cervical cancer falls under categories like “abnormal bleeding” and “leukorrhea”, and its etiological factors are often related to kidney deficiency, dampness-heat accumulation, and the disruption of the Chong and Ren meridians. Thus, TCM treatment for cervical cancer focuses on syndrome differentiation to restore the internal environment of the body, alleviate symptoms, control the disease, and prolong survival (8). Coptis chinensis, a TCM herb, is bitter and cold, affecting the heart, spleen, stomach, liver, gallbladder, and large intestine channels, and possesses heat-clearing, dampness-drying, and detoxifying properties. Based on its pharmacological properties, Coptis chinensis exerts multiple pharmacological actions, including antibacterial, antiviral, anti-inflammatory, anti-arrhythmic, antihypertensive, and anti-tumor effects (9). The choice to study Coptis chinensis for cervical cancer treatment is grounded in TCM theory and the pharmacological characteristics of the herb. Its heat-clearing and detoxifying effects align with the TCM causation of cervical cancer, while its anti-tumor properties offer significant advantages. Therefore, this study aimed to systematically explore the potential efficacy of Coptis chinensis on cervical cancer by using network pharmacology methods and preliminarily reveal its mechanism of action through bioinformatics means. Our research results not only provide a scientific basis for clarifying the role of Coptis chinensis in cervical cancer but also lay a theoretical foundation for the research and development and clinical application of new anti-cervical cancer drugs based on Coptis chinensis. We present this article in accordance with the REMARK reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2608/rc).

Methods

Screening of active components and target genes

The active components of Coptis chinensis were screened using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform (http://tcmspw.com/) based on the following criteria: drug-like properties (DLP) ≥0.18 and oral bioavailability (OB) ≥30%. Predicted target genes for effective compounds were selected from the DrugBank (https://www.drugbank.ca/) database. The target information was also compared using the UniProt (https://www.uniprot.org/) database.

Screening of differentially expressed genes (DEGs)

DEGs from cervical cancer tumor and adjacent tissue samples were obtained from the Gene Expression Omnibus (GEO) dataset GSE7803 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7803), and the Coptis chinensis target genes and DEGs in the GSE7803 dataset were intersected to identify the final candidate target genes.

Data analysis using The Cancer Genome Atlas (TCGA) database

The expression of the key genes in the cervical cancer tumor tissues and their correlation with patient prognosis were explored using TCGA (https://portal.gdc.cancer.gov/) database.

Molecular docking

The three-dimensional (3D) structures of the effective compounds were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The receptor proteins for the key targets genes were obtained from PDB (http://www.rcsb.org/pdb/home/home.do). The 3D structures of the active components and key target proteins were uploaded to the online analysis platform DockThor (https://dockthor.lncc.br/v2/) for molecular docking. Based on the DockThor calculations, a lower binding energy during ligand-receptor binding indicated a more stable and effective interaction. Finally, visualization was performed using the CB-DOCK2 (https://cadd.labshare.cn/cb-dock2/) database.

Gene set enrichment analysis (GSEA)

A functional analysis of the DEGs was conducted using Metascape (https://metascape.org/) online. A GSEA was conducted to determine whether the entire genomic set was significantly enriched or depleted in terms of the biological processes, which in turn helped to identify the common signaling pathways and regulatory network modules, and to understand the biological differences between the samples (10).

Study subjects and grouping

Cervical cancer patients admitted to Nanjing Lishui People’s Hospital from June 2021 to June 2022 were selected as the study subjects. To be eligible for inclusion in this study, the patients had to meet the following inclusion criteria: (I) have been pathologically diagnosed with primary cervical cancer; (II) have not undergone any prior chemotherapy or radiotherapy before study enrollment; (III) provide informed consent or have a family member provide informed consent; (IV) have a Karnofsky Performance Status score >70; and (V) have complete medical records. Patients were excluded from the study if they met any of the following exclusion criteria: (I) had other malignant tumors; (II) had an estimated survival time of less than 3 months; (III) had severe heart, liver, or kidney dysfunction; (IV) had abnormalities in their complete blood count or electrocardiogram results; and/or (V) had an inability to comply with treatment and follow-up assessments.

A total of 60 patients were included in the study and randomly divided into the control and study groups. The control group received a chemotherapy regimen of docetaxel combined with carboplatin, while the study group was given berberine tablets on the basis of the control group (specification: 0.1 g, Hangzhou Sanofi Minsheng Health Pharmaceutical Co., Ltd., Hangzhou, China). Patients had to take berberine tablets twice a day, 5 tablets each time (11). Each treatment cycle lasted 21 days, and both groups underwent continuous treatment for three cycles. The general clinical data of the patients were collected, including age, body mass index (BMI), menstrual status, histological classification, clinical stage, lymph node metastasis, and maximum tumor diameter. The patients were followed up via telephone and outpatient interviews, with the follow-up period ending on patient death, resulting in a follow-up rate of 100%. The final cut-off date for follow-up was June 2024.

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of the Nanjing Lishui People’s Hospital (No. 2024KY1105-02), and informed consent was taken from all the patients or their family.

Detection of MMP1

Blood samples (3 mL) were collected from the elbow vein of patients in a fasting state both before and after treatment. After the addition of a coagulant, the samples were allowed to stand at room temperature for 2 hours, and then centrifuged at 2,500 rpm for 10 minutes to separate the serum. The serum levels of matrix metalloproteinase 1 (MMP1) were measured by enzyme-linked immunosorbent assay (ELISA) in strict accordance with the instructions provided in the reagent kit (CSB-E04672h, Wuhan CUSABIO Biotech Co., Ltd., Wuhan, China). The patients were classified into high- and low-expression groups based on the median value of the MMP1 expression levels among all patients.

Statistical analysis

The statistical analysis and visualization were performed using R software (version 3.6.3). A GSEA was conducted to explore the potential cellular mechanisms of CCNA2. The Kaplan-Meier method was used to assess patient survival, and the log-rank test was employed for significance testing. Graphs were created using GraphPrism (version 8.0, GraphPad Software, La Jolla, CA, USA). For the continuous data that conform to a normal distribution, comparisons between two groups were performed using the independent samples t-test, and the results are expressed as the mean ± standard deviation (x¯±s). For the categorical data, comparisons between groups were conducted using the χ² test, and the results are expressed as the frequency and rate [n (%)]. The factors potentially influencing the prognosis of cervical cancer patients were analyzed by univariate and multivariate Cox analyses. A P value <0.05 or a log-rank P value <0.05 was considered statistically significant.

Results

Screening the active components and target genes of Coptis chinensis

Using the TCMSP database, and a DLP ≥0.18 and OB ≥30% as the screening criteria, the study identified ten effective compounds in Coptis chinensis (see Table 1), and a total of 181 target genes were selected from the DrugBank database.

Screening of DEGs

The DEGs from the GSE7803 dataset in the GEO database were selected using the following criteria: |log fold change| >2 and P<0.05. This stringent threshold was chosen to ensure that the selected genes have robust and statistically significant differential expression, minimizing potential false positives. Volcano plots, principal component analysis (PCA) plots, heatmaps, and normalized box plots were generated (Figure 1A-1D). The effective target genes of Coptis chinensis were intersected with the DEGs from this dataset, resulting in the identification of the following seven candidate target genes: AR, MAOB, CDKN2A, TOP2A, CXCL8, MMP1, and SPP1 (Figure 1E).

Figure 1 Screening of DEGs. (A-D) Volcano plot, PCA plot, heatmap, and normalized box plot of the GSE7803 dataset. (E) Venn diagram of target genes of effective compounds from Coptis chinensis and DEGs in the GSE7803 dataset. DEGs, differentially expressed genes; PCA, principal component analysis.

Expression of DEGs in cervical cancer tissues from TCGA database and their predictive value

In TCGA database, the expression levels of the aforementioned seven genes were examined. Compared to normal tissues, in the non-paired cervical cancer tumor tissues, the expression of AR and MAOB was downregulated, while the expression of CDKN2A, TOP2A, MMP1, and SPP1 was upregulated, and the differences were statistically significant (P<0.05). Only CXCL8 showed no statistically significant difference in terms of expression between the cancerous and adjacent tissues (Figure 2A). The receiver operating characteristic (ROC) curve analysis in TCGA database indicated that except for CXCL8, the area under the curve (AUC) for the remaining six key genes was greater than 0.8, suggesting that these genes had good predictive discriminative value (Figure 2B).

Figure 2 Expression of DEGs in cervical cancer tissues from TCGA database and their predictive value. (A) Expression of DEGs in non-paired cervical cancer tissues (306 cases of cancer tissue, 3 cases of adjacent tissue); (B) ROC curves of DEGs in TCGA database. *, P<0.05; **, P<0.01. TPM, transcripts per million; TPR, true positive rate; FPR, false positive rate; AUC, area under the curve; CI, confidence interval; DEGs, differentially expressed genes; TCGA, The Cancer Genome Atlas; ROC, receiver operating characteristic.

Correlation between the expression of the key DEGs and the prognosis of cervical cancer patients

Kaplan-Meier survival curves were constructed in the TCGA database, and it was found that cervical cancer patients with low expression of MMP1 or SPP1 had better prognosis [overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI)] compared to those with high expression, and all the differences were statistically significant (P<0.05) (Figure 3A-3C).

Figure 3 Correlation between the expression of the key DEGs and the prognosis of cervical cancer patients in TCGA database. (A-C) OS, DSS, and PFI curves, respectively. P<0.05, differences are statistically significant. HR, hazard ratio; DEGs, differentially expressed genes; TCGA, The Cancer Genome Atlas; OS, overall survival; DSS, disease-specific survival; PFI, progression-free interval.

Molecular docking

During the DEG screening process, the expression of the candidate genes in the cervical cancer tumor tissues and their correlation with patient prognosis were examined. MMP1 and SPP1 were identified as candidate genes, and were both associated with quercetin, a compound found in Coptis chinensis. The 3D structure of quercetin was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/), while the receptor protein structures of the two key targets were obtained from PDB (http://www.rcsb.org/pdb/home/home.do). The 3D structures of the active ingredient and the receptor proteins were uploaded to the online analysis platform DockThor (https://dockthor.lncc.br/v2/) for molecular docking. The results showed that MMP1 had an affinity of –9.2 for quercetin, while SPP1 had an affinity of –6.3. Further visualization was conducted using the CB-DOCK2 database (https://cadd.labshare.cn/cb-dock2/) (Figure 4A,4B). Based on the affinity strength, MMP1 may be the key gene through which Coptis chinensis exerts its effects.

Figure 4 Molecular docking diagrams of the effective compound quercetin from TCM Coptis chinensis with the core targets MMP1 (A) and (B) SPP1. TCM, traditional Chinese medicine.

Prediction of signaling pathways based on GSEA

A functional GSEA was conducted using the Metascape online platform. The results showed that MMP1 appears to influence various biological processes in cervical cancer by participating in the regulation of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-β (TGF-β), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor 1 (HIF1) pathways, and apoptosis (Figure 5).

Figure 5 Statistically significant potential pathways in the GSEA. The gene set was sourced from Molecular Signatures Database (MSigDB, https://www.gsea-msigdb.org/gsea/msigdb). A total of 1,600 random-sample permutations were performed. NES, normalized enrichment score; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis.

Analysis of the clinical pathological characteristics of the control and study groups

There were no statistically significant differences in the clinical baseline characteristics between the two groups of patients (P>0.05, Table 2).

Expression of MMP1

There was no significant difference in MMP1 expression between the study group and the control group before treatment (9.12±5.00 vs. 9.38±4.51 ng/mL, P=0.83). However, post-treatment, MMP1 expression in the study group was significantly lower than that in the control group (5.05±2.74 vs. 9.00±4.70 ng/mL, P<0.001) (Figure 6).

Figure 6 Comparison of MMP1 expression levels between study and control groups. ***, P<0.001; ns, no significance.

Univariate and multivariate Cox regression analyses of prognosis in cervical cancer

Patients’ age, BMI, menstrual status, histological classification, clinical stage, lymph node metastasis, maximum tumor diameter, and MMP1 expression before and after treatment were used as independent variables, with follow-up time as the time variable and survival status as the dependent variable. The multivariate Cox regression analysis revealed that clinical stage III or IV, lymph node metastasis, high MMP1 expression before treatment, high MMP1 expression after treatment, and the absence of combined treatment with TCM Coptis chinensis were independent risk factors affecting the prognosis of cervical cancer patients, and the differences were statistically significant (P<0.05) (Table 3).

Discussion

TCM is a valuable treasure of medicine in China, with unique advantages in disease prevention and treatment. TCM aligns with the new generation of medical research paradigms that foster the development of network pharmacology (12,13). Network pharmacology is a multidisciplinary research method that integrates network science, systems biology, and pharmacology to reveal interactions between drugs and diseases (14-16). It can aid in understanding the multi-target and multi-component synergistic effects of drugs, providing new ideas and methods for the development and clinical application of drugs (17). Network pharmacology and TCM promote and complement each other, driving the development of drug research and clinical practice.

This study, which was based on network pharmacology and bioinformatics, identified core targets and mechanisms of Coptis chinensis in the treatment of cervical cancer. We verified the differential expression of the core target genes and their effects on the survival of cervical cancer patients from TCGA database, and further confirmed the direct interaction of the key gene MMP1 with the effective compound through molecular docking.

MMP1 is a zinc-dependent protease belonging to the MMP family. Its main substrate is fibrillar collagen, which can degrade collagen fibers and gelatin in the extracellular matrix, altering the cellular microenvironment, and facilitating tumor invasion and metastasis. MMP1 plays a critical role in the initial stages of tumorigenesis, promoting tumor formation (18,19). Zhao et al. (20) revealed that MMP1 could be a novel immunotherapy biomarker for prognosis in cervical cancer patients. Li et al. (21) found that the protein-ligand complex between MMP1 and a specific compound was the most stable target in exploring the pharmacological effects of a traditional Chinese herb on cervical cancer, and observed a potential link between increased MMP1 expression and interlukin-17 pathway activation. These studies support the research value of MMP1 in cervical cancer.

The present study identified quercetin (a flavonoid compound found in many plants) as a compound binding to MMP1, and quercetin is known for its antioxidant, anti-inflammatory, anti-tumor, and antibacterial activities. Its molecular structure features a phenolic and a pyran ring that grant it unique chemical and biological properties. Previous studies have also highlighted the significance of quercetin in cervical cancer research (20-22); for example, quercetin can induce cell death in cervical cancer by reducing the O-GlcNAcylation of AMP-activated protein kinase, and exerts a synergistic inhibitory effect with cisplatin on cervical cancer cells (22). Quercetin may inhibit cancer cell proliferation, migration, and invasion, and promote apoptosis by downregulating the expression of MMP2, ezrin, METTL3, and P-Gp, thereby enhancing the anti-tumor effect of cisplatin (23). Additionally, quercetin can modify 5’CpG promoter methylation in human cervical cancer cells, and reactivate various tumor suppressor genes through epigenetic regulation (24).

The GSEA suggested that MMP1 may influence different biological processes in cervical cancer through pathways, including the PI3K/AKT, JAK/STAT, TGF-β, MAPK, and HIF1 pathways, and apoptosis. The PI3K/AKT pathway plays a crucial role in cell survival, proliferation, migration, and invasion. Quercetin may inhibit cervical cancer cell proliferation and metastasis by suppressing the activation of the PI3K/AKT pathway, and reducing the expression of downstream growth-promoting and invasive factors (25). The JAK/STAT pathway participates in the signaling transduction of various cytokines, and regulates cell growth, differentiation, apoptosis, and the immune response, which are often abnormally activated in tumors, thus promoting tumor development and metastasis (26). The TGF-β pathway is significant for cell growth, differentiation, apoptosis, and immune regulation. In tumors, this pathway may have dual roles, inhibiting early tumor formation and promoting invasion and metastasis in late-stage tumors (27). The MAPK pathway is involved in transducing various extracellular signals, regulating cell growth, differentiation, apoptosis, and the stress response, and is frequently abnormally activated in tumors (28). The apoptosis pathway is the main route for programmed cell death, and it is crucial for maintaining homeostasis and inhibiting tumor development (29). The HIF1 pathway is one of the primary mechanisms by which cells adapt to hypoxic environments, and this pathway plays an essential role in tumor occurrence, development, and metastasis. HIF1 can regulate the expression of various genes associated with tumor invasion and metastasis (30). These studies provide insights into the potential mechanisms by which Coptis chinensis works in cervical cancer treatment. These pathways may interconnect and work together to form a complex regulatory network. For example, PI3K/AKT and the HIF1 pathway may jointly regulate the adaptation of tumor cells to the hypoxic microenvironment, enhancing tumor cell survival and invasiveness by promoting angiogenesis and metabolic reprogramming. In addition, the cross-action of TGF-β with the MAPK pathway may further promote tumor progression by enhancing inflammation and cell migration. This multi-channel synergy effect may be one of the key mechanisms of MMP1 regulation. Quercetin, the main active component of Coptis chinensis, may achieve its significant anti-tumor effect through multi-target and multi-pathway.

To further validate MMP1 as a potential target for Coptis chinensis treatment in cervical cancer, this study conducted a retrospective analysis of MMP1 expression in the peripheral blood of cervical cancer patients. The results indicated that the MMP1 expression levels were significantly lower in patients receiving conventional chemotherapy combined with Coptis chinensis than those receiving conventional chemotherapy alone. Multivariate Cox regression analysis revealed that high MMP1 expression and the absence of combined treatment with Coptis chinensis were independent risk factors affecting the prognosis of cervical cancer patients. This further suggests that MMP1 may serve as an effective evaluation indicator for predicting the prognosis of cervical cancer patients. Additionally, the combination of Coptis chinensis with conventional chemotherapy could improve the prognosis of cervical cancer patients, possibly through mechanisms related to targeting MMP1.

Although this study confirms the critical role of MMP1 in cervical cancer and explores its potential as a therapeutic target for Coptis chinensis, this study still has limitations. First, the retrospective analysis time of this study is short, which may not fully reflect the long-term effect of Coptis chinensis in cervical cancer treatment, and the peripheral blood samples are limited in representation, which may not fully reflect the molecular changes in the tumor microenvironment. Second, the specific molecular mechanism by which Coptis chinensis downregulates MMP1 needs to be further investigated. Furthermore, our study is based on retrospective analysis and molecular docking techniques, which can only reveal correlations and cannot directly prove causation. Retrospective data can provide valuable observational evidence, but its results are susceptible to patient selection bias, incomplete data, and other potential confounding factors. Finally, although molecular docking experiments revealed that MMP1 has a strong binding force to quercetin in Coptis chinensis, the molecular docking results only provide theoretical binding checkpoints and affinity, and there is a lack of sufficient in vivo and clinical experimental evidence to verify the actual biological role between these compounds and MMP1. Future studies will extend the study time, combine the analysis of peripheral blood and tissue samples, and systematically verify the specific molecular mechanism and clinical efficacy of Coptis chinensis down-regulating MMP1 through in vitro cell experiments, in vivo animal models, and prospective multicenter clinical trials. At the same time, the molecular docking results will be further verified to provide a more solid scientific basis.

Conclusions

Using network pharmacology and bioinformatics analysis, this study explored the potential mechanisms through which Coptis chinensis inhibits cervical cancer via multiple targets and pathways. The preliminary validation of the interaction between Coptis chinensis and cervical cancer targets via molecular docking provides a theoretical basis for further in vitro experiments. In addition, the study also found that Coptis chinensis combined with conventional chemotherapy significantly reduced the expression of MMP1 in the peripheral blood of patients with cervical cancer. High expression of MMP1 is significantly associated with a poor prognosis in patients. These findings provide important support for the clinical application of Coptis chinensis as an adjuvant treatment for cervical cancer and offer new ideas for further research on its anti-tumor mechanism in the future. In the future, more extensive experimental validation and clinical trials are still needed to confirm its therapeutic effect and a more comprehensive mechanism.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2608/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2608/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2608/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2608/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of the Nanjing Lishui People’s Hospital (No. 2024KY1105-02), and informed consent was taken from all the patients or their family.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)