Network pharmacology- and cell-based assessments identify the FAK/Src pathway as a molecular target for the antimetastatic effect of momordin Ic against cholangiocarcinoma

Abstract

Previous studies have indicated the efficacy of momordin Ic (MIc), a plant-derived triterpenoid, against several types of cancers, implying its potential for further development. However, comprehensive insights into the molecular mechanisms and targets of MIc in cholangiocarcinoma (CCA) are lacking. This study aimed to investigate the actions of MIc against CCA at the molecular level. Network pharmacology analysis was first employed to predict the mechanisms and targets of MIc. The results unveiled the potential involvement of MIc in apoptosis and cell migration, pinpointing Src and FAK as key targets. Subsequently, cell-based assays, in accordance with FAK/Src-associated metastasis, were conducted, demonstrating the ability of MIc to attenuate the metastatic behaviours of KKU-452 cells. The in vitro results further indicated the capability of MIc to suppress the epithelial-mesenchymal transition (EMT) process, notably by downregulating EMT regulators, including N-cadherin, vimentin, ZEB2 and FOXC1/2 expression. Furthermore, MIc suppressed the activation of the FAK/Src signalling pathway, influencing critical downstream factors such as MMP-9, VEGF, ICAM-1, and c-Myc. Molecular docking simulations also suggested that MIc could interact with FAK and Src domains and restrain kinases from being activated by hindering ATP binding. In conclusion, this study employs a comprehensive approach encompassing network pharmacology analysis, in vitro assays, and molecular docking to unveil the mechanisms and targets of MIc in CCA. MIc mitigates metastatic behaviours and suppresses key pathways, offering a promising avenue for future therapeutic strategies against this aggressive cancer.

Keywords: Antimetastasis; Cholangiocarcinoma; FAK; Momordin Ic; Network pharmacology; Src.

Fig. 1

Venn diagram displaying the intersection of MIc targets and CCA targets.

Fig. 2

GO and KEGG enrichment analysis of the potential targets of MIc in CCA. (A) KEGG enrichment analysis demonstrates the pathways involved. The y- and x-axes represent significant KEGG pathways and the percentage of fold enrichment, respectively. The gradient of colour represents target counts, and the size of nodes represents the false discovery rate. (B) Overview chart of GO enrichment analysis demonstrates the functions or signalling involved as the percentage of terms (function/signalling) per group.

Fig. 3

Protein-protein interaction (PPI) analysis. (A) PPI network of 37 potential MIc targets against CCA obtained from the STRING database. (B) The top 5 targets of potential MIc targets and (C) the top 5 targets of Src with only “physical interactions” were ranked using Cytoscape with cytoHubba plug-in. The higher degree value is represented by colours ranging from red to yellow.

Fig. 4

Effects of MIc on the metastatic behaviours of CCA cells. KKU-452 cells were exposed to different MIc concentrations. The initial evaluation included (A) cell viability and (B) colony forming ability. Subsequently, assessments consisting of (C) migration, (D) invasion, and (E) adhesion capabilities of MIc were performed. Representative figures and quantitative graphs are provided. Data are expressed as the mean ± SD from three separate experiments. *, p < 0.05 compared to the respective control.

Fig. 5

Effects of MIc on EMT in CCA cells. KKU-452 cells were chemically induced to undergo EMT (iEMT). (A) The effect of MIc on cell morphology at concentrations of 1 and 2.5 μM was determined. EMT marker expression, including N-cadherin, vimentin, ZEB2, and FOXC1/2, was further evaluated by (B) Western blot (Supplementary Fig. S1 Images of original blot 5B) and (C) immunocytofluorescence analyses. The data shown are representative of two reproducible experiments.

Fig. 6

Effects of MIc on p-FAK, p-Src, and their downstream effector molecules. KKU-452 cells were treated with MIc (0.5–2.5 μM) for 24 and 48 h. (A) The expression of p-FAK and p-Src, as well as their downstream effector molecules, including VEGF, ICAM-1, MMP-9, and c-Myc, was determined by Western blot analysis (Supplementary Fig. S2 Images of original blot 6A). (B) Immunocytofluorescence analysis was also performed in cells treated with MIc for 48 h. The data shown are representative of two reproducible experiments.

Fig. 7

Molecular interactions between MIc and FAK/Src. MIc binds to the kinase domain of (A) FAK (PDB ID: 3BZ3) and (B) Src (PDB ID: 4MOX) in a 3D representation. The amino acid residues occupied by MIc are highlighted in red on the surface. The 2D interactions of MIc with (C) FAK and (D) Src show hydrophobic interactions, including van der Waals (green) and alkyl to alkyl/pi (magenta), as well as a hydrophilic interaction involving hydrogen bonding (blue). The yellow box indicates residues shared by both MIc and FAK inhibitor (PF-562,271) or Src inhibitor (bosutinib).