Network pharmacology, bioinformatics and in vitro/in vivo validation elucidate the anti-lung cancer activities and potential targets of Rhoifolin.

[BACKGROUND] Rhoifolin (ROF), a naturally occurring flavonoid, exhibits broad bioactivities, but its therapeutic potential and underlying mechanisms in lung cancer remain largely unknown. This study was designed to systematically investigate the anti-tumor effects of ROF and identify its key molecular targets.

[MATERIALS AND METHODS] Anti-tumor activities of ROF were assessed using CCK-8, colony formation, flow cytometry, wound healing, and Transwell assays, respectively. An integrated approach combining network pharmacology, transcriptomic analysis with machine learning was employed to identify primary targets. The Kaplan-Meier survival and ROC curve analyses also evaluated the targets' clinical outcomes and tumor microenvironment through the Cancer Genome Atlas (TCGA) data and single-cell RNA sequencing. The confirmed experimentally via RT-qPCR, Western blot, and immunofluorescence. The drug-target interaction was characterized by molecular docking and dynamics simulations. Finally, the antitumor efficacy and the safety of ROF were assessed in an H358 xenograft mouse model.

[RESULTS] ROF potently inhibited lung cancer cell proliferation (IC: 15.35-33.84 µM), migration, and invasion, while inducing G/M phase arrest and apoptosis (increased Bax/Bcl-2 ratio). ROF also impaired metastatic potential as evidenced by upregulated E-cadherin and downregulated N-cadherin . EPHB2 was identified as the most therapeutically relevant, showing high diagnostic value (AUC=0.856) and a significant correlation with poor patient survival. The experimental validation confirmed that ROF downregulates EPHB2 expression at both the mRNA and protein levels in a dose-dependent manner. Molecular docking and dynamics simulations predicted a stable, high-affinity interaction between ROF and the EPHB2 protein. Importantly, ROF treatment significantly suppressed tumor growth without discernible toxicity.

[CONCLUSION] Rhoifolin exerts potent and selective anti-lung cancer activity by directly targeting and downregulating EPHB2, providing a strong rationale for its further development as a novel therapeutic agent for lung cancer.