Integrating network pharmacology and molecular modeling to decipher the anti-esophageal squamous cell carcinoma mechanisms of Bidens pilosa L

Abstract

Bidens pilosa L (BL), known for its anti-inflammatory, antioxidant, and anticancer properties across multiple malignancies, was investigated for its potential therapeutic effects against esophageal squamous cell carcinoma (ESCC). Through integrative network pharmacology and computational approaches, 10 bioactive compounds from BL were identified from HERB 2.0, symMap, BATMAN-TCM (target prediction score cutoff = 20, adjusted P-value = 0.05 for target analyses), TCMSP (oral bioavailability ≥ 30% and drug likeness ≥ 0.18), and ETCM 2.0 databases, while 3,993 ESCC-associated targets were retrieved from OMIM, GeneCards, and DisGeNET. Cross-analysis revealed 214 shared targets, with tyrosine-protein kinase, epidermal growth factor receptor, and alpha serine/threonine-protein kinase emerging as central hubs in protein-protein interaction networks. Functional enrichment analysis highlighted significant involvement in cellular responses to hormone stimuli, nitrogen compounds, and phosphorylation, with key pathways including cancer, PI3K-Akt, and Ras signaling. Molecular docking demonstrated high-affinity binding of (2E)-2-(3,4-Dihydroxybenzylidene)-6,7-Dihydroxy-Benzofuran-3-One (− 10.4 kcal/mol), Luteolin (− 10.1 kcal/mol), and Okanin (− 9.7 kcal/mol) to matrix metalloproteinase-9 (MMP9), specifically targeting catalytic residues GLU227 and TYR245. Quantum chemical calculations further revealed narrow HOMO-LUMO gaps for Luteolin (ΔE = 2.41 eV) and Okanin (ΔE = 2.93 eV), indicating high reactivity, while molecular dynamics simulations confirmed stable MMP9-ligand complexes (nearly − 200 kJ/mol), with Okanin exhibiting faster equilibration than Luteolin. The analysis of datasets from TCGA and GEO revealed that MMP9 is significantly upregulated in esophageal squamous cell carcinoma tissues compared to normal esophageal tissues. Meanwhile, Luteolin significantly inhibited the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) capabilities of ESCC in vitro experiments.These findings collectively suggest that BL exerts anti-ESCC effects in silico through multi-target synergy, with Luteolin and Okanin identified as promising MMP9 inhibitors, providing a mechanistic foundation for future drug development.

Supplementary Information: The online version contains supplementary material available at 10.1007/s12672-025-03441-y.

Keywords: Bidens pilosa L; Esophageal squamous cell carcinoma; Molecular docking; Molecular dynamics simulation; Molecular orbital calculations; Network pharmacology.

Fig. 1

Network pharmacology analysis of BL targets in ESCC pathogenesis. A Intersection analysis of BL-derived compounds and ESCC-associated targets. B Protein-protein interaction network of shared therapeutic targets. C Key hub targets identified through topological analysis (degree centrality ranking)

Fig. 2

Network pharmacology analysis of BL bioactive components in ESCC. The diagram illustrates the therapeutic relationships between active compounds (green hexagons), target proteins (light red circles), and relevant signaling pathways (pink quadrangles), with BL (dark blue V-shapes) and ESCC (yellow hexagons) as central elements

Fig. 3

Functional enrichment analysis of BL targets in ESCC. A Gene Ontology (GO) classification showing enriched terms in biological processes (BP), cellular components (CC), and molecular functions (MF). B Top 20 significantly enriched BP terms ranked by -log10(p-value). C String diagram displaying the top 40 enriched KEGG pathways. D Statistical significance (-log10(p-value)) of the enriched KEGG pathways

Fig. 4

Binding affinity profiles of bioactive compounds with hub therapeutic targets. The heatmap illustrates docking energy scores (kcal/mol), where the color spectrum denotes binding strength-progressing from high-affinity interactions (blue) to low-affinity bindings (red)

Fig. 5

Bioinformatics analysis of MMP9 mRNA expression in ESCC. A Heatmap of differentially expressed genes (DEGs) ranked by absolute log₂fold change (|logFC|). Blue and Red gradients denote low and high expression levels, respectively. B Volcano plot of DEGs between ESCC (n = 26) and normal tissues (n = 28). Pink dots represent upregulated genes (|logFC|≥2), green dots indicate downregulated genes (|logFC|≤2), and grey dots denote non-significant changes. C Differential MMP9 expression in ESCC versus normal tissues from the GEO dataset (GSE161533). D MMP9 mRNA levels in ESCC, esophageal adenocarcinoma (EAC), and normal tissues from the UALCAN database (TCGA-EC cohort, n = 195). Significance levels:*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Structural representations of the highest-affinity MMP9-ligand complexes identified by molecular docking analysis. A Three-dimensional binding pose of Luteolin (binding energy: − 10.1 kcal/mol) within the MMP9 active site. B Molecular interaction pattern between Okanin (binding energy: − 9.7 kcal/mol) and MMP9 catalytic domain. C Binding conformation of (2E)-2-(3,4-dihydroxybenzylidene)-6,7-dihydroxy-benzofuran-3-one (binding energy: − 10.4 kcal/mol) complexed with MMP9

Fig. 7

Molecular orbital analysis of three compounds. The figure displays the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distributions along with their corresponding energy gaps (ΔE) for Luteolin with ΔE = 2.41 eV (A), Okanin with ΔE = 2.89 eV (B), and (2E)-2-(3,4-Dihydroxybenzylidene)-6,7-Dihydroxy-Benzofuran-3-One with ΔE = 2.93 eV (C)

Fig. 8

Comparative molecular dynamics simulation profiles of MMP9 in complex with Luteolin or Okanin. (A, F) Time evolution of root-mean-square deviation (RMSD) for (A) MMP9-Luteolin and (F) MMP9-Okanin complexes. (B, G) Radius of gyration (Rg) profiles for (B) MMP9-Luteolin and (G) MMP9-Okanin complexes. (C, H) Residue-specific root-mean-square fluctuation (RMSF) of (C) MMP9-Luteolin and (H) MMP9-Okanin complexes. (D, I) Binding free energy trajectories for (D) MMP9-Luteolin and (I) MMP9-Okanin interactions. (E, J) Hydrogen bond formation dynamics between (E) MMP9-Luteolin and (J) MMP9-Okanin during 100 ns simulations

Fig. 9

Luteolin inhibits cell proliferation, migration, and invasion in ESCC cells. TE-13 and KYSE-510 cells were treated with different concentrations of Luteolin (10, 20, 40, 80, and 120 µM) for 24 h and 48 h, the cell proliferation was examined by CCK-8 assay (A). The migration rate and number of invading cells in TE-13 and KYSE-510 cells treated with 10 µM and 20 µM Luteolin were examined by Wound healing assay (B) and Transwell assay (C). (D) The protein levels of EMT-related proteins (Snail, MMP9, and N-cadherin) induced by 10 ng/mL TGF-β1 were assayed by Western Blot in TE-13 and KYSE-510 cells. **P < 0.0 L, ***P < 0.00 L