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

Background: 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.

Purpose: 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).

Methods: 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.

Results: 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.

Conclusion: 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.