Eupatilin inhibits non-small cell lung cancer metastasis by suppressing Netrin-1-mediated epithelial-mesenchymal...

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality, primarily attributed to distant metastasis. The trimethoxyflavone eupatilin (Eup), a major flavonoid derived from the leaves of Artemisia argyi, exhibits potential antitumor properties. However, its effects and underlying mechanisms in NSCLC have been inadequately elucidated.

To investigate the efficacy of Eup against NSCLC and elucidate its molecular targets and mechanisms, particularly focusing on its effects on epithelial-mesenchymal transition (EMT).

A lung metastasis model was established via tail vein injection of NCI-H1299 cells. In vitro, Eup's effects on proliferation, migration, invasion, and cell cycle in NCI-H1299/H23 cells were assessed using CCK-8, colony formation, wound healing, Transwell, EdU, and flow cytometry assays. Western blotting was employed to analyze EMT markers (E-cadherin, N-cadherin, Vimentin, Snail), PCNA, and phosphorylation of PI3K/AKT pathway components (p85, Akt, GSK3β, S6). Target identification integrated molecular docking, RNA-seq, Cellular Thermal Shift Assay (CETSA), and Surface Plasmon Resonance (SPR). Co-immunoprecipitation validated Netrin-1-UNC5B and UNC5B-PI3K interactions, while Netrin-1 modulation and rescue experiments clarified its functional role. In vivo efficacy and safety were evaluated in NCI-H1299 xenograft models.

In the lung colonization and metastasis model, Eup significantly suppressed NCI-H1299 cell colonization and metastasis. In vitro assays demonstrated that Eup inhibited NSCLC cell proliferation and migration while inducing G0/G1 phase cell cycle arrest. Further analysis revealed that Eup reversed EMT by upregulating E-cadherin and downregulating N-cadherin, Vimentin, Snail, and PCNA. Mechanistically, recombinant Netrin-1 protein activated the PI3K/AKT pathway, an effect that was abrogated upon UNC5B knockdown. Eup binds to Netrin-1 and suppresses Netrin-1/UNC5B-mediated PI3K/Akt signaling and phosphorylation of downstream GSK3β (Ser9) and S6 (Ser235/236). Functional assays showed that Netrin-1 overexpression antagonized the antitumor effects of Eup. In the subcutaneous xenograft model, Eup significantly suppressed tumor growth and counteracted the pro-tumorigenic effect of Netrin-1 without inducing obvious systemic toxicity.

This study demonstrates that Eup binds to Netrin-1 to suppress Netrin-1/UNC5B-mediated PI3K/Akt signaling, thereby inhibiting EMT-driven metastasis in NSCLC, and provides preclinical evidence supporting its potential as a candidate therapeutic for metastatic NSCLC.