Objective: Non-small cell lung cancer (NSCLC), a leading cause of cancer-related mortality, presents a poor prognosis due to its tendency to metastasize and its resistance to standard therapies. Montanine (MTN), a naturally occurring alkaloid, has recently shown potential as an anticancer agent. However, the specific mechanisms through which it exerts its anticancer effects are still largely unknown. This study investigates the anti-NSCLC potential of MTN by utilizing integrative network pharmacology alongside in vitro and in vivo experimental validations.

Methods: Genes targeted by MTN in NSCLC were identified and subjected to functional enrichment analysis to elucidate the most enriched signaling pathways. A protein-protein interaction network was constructed to identify the interactions of potential targets. Adrenoceptor β2 (ADRB2) expression in lung cancer was analyzed using data from The Cancer Genome Atlas and Gene Expression Omnibus datasets. The effects of MTN on NSCLC cell lines, including migration, proliferation, viability and apoptosis, were assessed in vitro. MTN-related mechanisms were investigated using quantitative reverse transcription-polymerase chain reaction and Western blotting. Furthermore, an in vivo lung cancer metastasis experiment was performed in rats using a tail vein assay.

Results: MTN at nontoxic doses significantly reduced cell migration and lung colonization in an in vivo metastasis study (P < 0.05). Network pharmacology revealed 29 targets of MTN in NSCLC, highlighting ADRB2 as a primary target linked to poor prognosis in lung cancer. In vitro studies confirmed that MTN notably upregulated ADRB2 expression and downregulated epithelial-mesenchymal transition (EMT) signaling pathways, which was further enhanced by terbutaline sulfate, an ADRB2 activator.

Conclusion: MTN is a promising therapeutic agent for NSCLC by suppressing the migration via ADRB2 and EMT mechanisms. Please cite this article as: Iksen I, Singharajkomron N, Wongtayan A, Wattanathamsan O, Choonhapan A, Wademonkolgorn S, Nguyen HM, Hoang THX, Nguyen HT, Pongrakhananon V. Unveiling the antimetastatic activity and molecular mechanism of montanine in lung cancer cells via the integration of network pharmacology approaches with in vitro and in vivo investigations. J Integr Med. 2026; 24(2):253-264.

Key words: Adrenoceptor β2, Epithelial-to-mesenchymal transition, Lung cancer, Metastasis, Network pharmacology