Cephaeline attenuates malignant biological behaviors of colorectal cancer by regulating the Ras signaling pathway

doi:10.12122/j.issn.1673-4254.2026.05.16

Abstract

Abstract:

Objective To investigate the mechanism of cephaeline for suppressing malignant biological behaviors of colorectal cancer (CRC). Methods Network pharmacology was employed to identify the core targets and signaling pathways of cephaeline in CRC treatment. The binding between the key targets of CRC and cephaeline was simulated using molecular docking. CCK-8 assay was used to assess the viability of HCT116 and DLD-1 cells treated with 2-32 nmol/L cephaeline for 24, 48, and 72 h to determine the optimal treatment condition. The effects of cephaeline treatments (at 2 and 4 nmol/L in HCT116 cells and at 4 and 8 nmol/L in DLD-1 cells) for 48 h on cell proliferation, migration, invasion, epithelial‑mesenchymal transition (EMT), apoptosis, and expressions of RAS signaling pathway proteins were evaluated using colony-forming assay, wound healing assay, Transwell assays, and Western blotting. Results Network pharmacology analysis suggested that cephaeline inhibited CRC mainly by regulating the targets such as MAPK, VEGFA, and PDGFRB and modulating the Rap1, Ras, cAMP, and PI3K-Akt signaling pathways. Molecular docking results showed that the binding energies of cephaeline with PDGFRB and MAPK1 were less than -5 kcal/mol, indicating spontaneous and stable binding between them. In HCT116 and DLD-1 cells, treatment with cephaeline concentration-dependently suppressed the cell viability, proliferation, migration, and invasion, downregulated the expressions of PCNA, MMP9, VEGF-C, BCL-2, N-cadherin, vimentin, VEGF-A, PDGFRB, MYC, and MAPK1, and upregulated the expression levels of E-cadherin and BAX. Conclusion Cephaeline suppresses malignant biological behaviors of CRC possibly by regulating the RAS signaling pathway.

Key words: cephaeline, network pharmacology, colorectal cancer, Ras signaling pathway, molecular docking

Liang HAN, Baichao LI, Bingzhe WANG, Zhiwei ZHANG, Jiuna ZHANG. Cephaeline attenuates malignant biological behaviors of colorectal cancer by regulating the Ras signaling pathway[J]. Journal of Southern Medical University, 2026, 46(5): 1121-1130.

Figures/Tables 8

Fig.1 Results of network pharmacology. A: Venn diagram of the intersection between cephaeline and CRC Targets (Blue represents the drug, yellow represents the disease, and the numbers indicate the number of targets). B: Protein-protein interaction diagram (the darker the color, the stronger the correlation). C: PPI network diagram (the nodes represent the target proteins, and the thickness and density of the connections indicate the strength of interactions between proteins). D: GO enrichment analysis diagram. E: KEGG enrichment analysis diagram. F: The compound-disease-target-pathway regulatory network (Green represents compounds, red represents diseases, orange represents targets, and purple represents pathways).

Tab.1 Molecular binding energies

Target	PDB ID	Dock score(Kcal/mol)	Center (x, y, z)
KIT	1t46	-5.79	(31.925, 25.893, 35.097)
MAPK1	2y9q	-6.97	(59.373, 26.110, 6.762)
VEGFA	1vpp	-4	(3.908, 12.934, 12.916)
PDGFRB	1sha	-7.46	(47.460, 30.508, 3.349)

Fig.2 Visual analysis of molecular docking results.

Fig.3 Effects of cephaeline on survival rate of colorectal cancer cells. A: CCK-8 assay results of HCT116 cells. B: CCK-8 assay results in DLD-1 cells. *P<0.05, **P<0.01, ***P<0.001 vs control (0 nmol/L) (n=3).

Fig.4 Effect of cephaeline on the proliferative capacity of CRC cells. A: Colony formation of HCT116 cells. B: Colony formation of DLD-1 cells. C, D: Number of colonies formed by HCT116 and DLD-1 cells, respectively. *P<0.05, ***P<0.001 vs control (0 nmol/L) (n=3).

Fig.5 Effect of cephaeline on migration of CRC cells (Original magnification: ×4). A, C: Migration assay results of HCT116 cells. B, D: Migration assay results of DLD-1 cells. **P<0.01, ***P<0.001 vs control (0 nmol/L) (n=3).

Fig.6 Effect of cephaeline on invasion of CRC cells (Original magnification: ×10). A, C: Invasion assay results in HCT116 cells. B, D: Invasion assay results in DLD‑1 cells. **P<0.01 vs control (0 nmol/L) (n=3).

Fig.8 Effects of different concentrations of cephaeline on cell proliferation, apoptosis, EMT, RAS signaling pathway, and related protein expression in HCT116 and DLD‑1 cells. A: Expression levels of proliferation‑ and apoptosis‑related proteins in HCT116 and DLD‑1 cells. B: Expression levels of EMT‑related proteins. C: Expression levels of RAS signaling pathway proteins. D: Expression levels of MYC proto‑oncogene‑related proteins in both cell lines. *P<0.05,**P<0.01,***P<0.001 vs Control (0 nmol/L) (n=3).

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