Exploration of the multi-component, multi-target, and multi-pathway mechanism of Rubia yunnanensis in inhibiting breast cancer based on network pharmacology, molecular docking, and experimental validation

Background: Breast Cancer (BC) is a malignancy associated with high morbidity and mortality. Rubia yunnanensis (RY), a traditional Chinese medicinal herb, has demonstrated inhibitory effects against various tumor types, yet its role in BC remains undefined.

Objective: This study investigated the effect of RY on BC and elucidated its underlying molecular regulatory mechanisms.

Methods: The role of RY in BC was assessed through CCK-8, Transwell, flow cytometry, Western blot, xenograft modeling, and immunohistochemistry assays. We employed ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS), the PubChem and TCMSP database, and literature mining to identify the active ingredients of RY. Potential targets for these active ingredients were predicted using TargetPrediction database, while BC-regulated targets were collected from the GeneCards, DrugBank, and TTD databases. A protein-protein interaction network analysis identified core targets, followed by GO and KEGG enrichment analyses using the DAVID database. Molecular docking was performed to validate the binding affinity of the active ingredients for these core targets. Finally, Western blot analysis was used to detect the relative protein expression levels of the core targets.

Results: RY significantly reduced the viability of MDA-MB-231 and BT-549 cells, inhibited their migratory capacity, promoted Apoptosis, and upregulated the expression of the E-cadherin, while downregulating the expression of N-Cadherin and vimentin. These effects were concentration-dependent. In vivo studies demonstrated that RY treatment inhibited BC growth, reduced the Ki67-positive rate and the expression of N-Cadherin and vimentin in mouse BC tissues, and promoted the expression of E-cadherin. Furthermore, UPLC-Q-TOF/MS analysis and database mining identified 26 active components in RY and 312 potential targets related to its action against BC. The core targets for RY in BC treatment include TP53, Akt1, IL-6, TNF, STAT3, CTNNB1, EGFR, MYC, ESR1, and CASP3. Enrichment analysis indicated significant involvement of the PI3K-AKT and MAPK signaling pathways. The key active components of RY demonstrated strong binding potential with these core target genes, namely TNF, ESR1, CASP3, EGFR, STAT3, MYC, CTNNB1, Akt1, TP53, and IL-6.

Conclusion: This study reveals that RY exhibits therapeutic potential on BC through multi-ingredient, multi-target, and multi-pathway mode of action.