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

## TL;DR

This study shows that Rhoifolin, a natural compound, can fight lung cancer by targeting EPHB2, a protein linked to poor survival, and it works well in lab and animal tests.

## Contribution

The study identifies EPHB2 as a novel therapeutic target for Rhoifolin in lung cancer and validates its anti-tumor effects through multiple experimental and computational methods.

## Key findings

- Rhoifolin inhibits lung cancer cell growth, migration, and invasion in vitro.
- EPHB2 is a key target of Rhoifolin, with high diagnostic value and poor survival correlation in patients.
- Rhoifolin suppresses tumor growth in mice without toxicity, confirming its therapeutic potential.

## Abstract

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.

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 in vivo antitumor efficacy and the safety of ROF were assessed in an H358 xenograft mouse model.

ROF potently inhibited lung cancer cell proliferation (IC50: 15.35–33.84 µM), migration, and invasion, while inducing G2/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 in vitro. 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 in vivo without discernible toxicity.

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.

Diagram illustrating methods for efficacy verification, mechanisms exploration, and mechanism verification of rhoifolin on cancerous cells and animals. Includes platforms like PharmMapper, Super-PRED, TargetNet, and SwissTargetPrediction for exploration. Uses machine learning, bioinformatics, molecular docking, molecular dynamics, Western blot, qRT-PCR, and immunofluorescence for verification processes.