Integrated network pharmacology, bioinformatics, and experimental approach to explore the mechanism of honokiol liposomes against glioblastoma

Introduction: Honokiol (HK), a bioactive compound isolated from Magnolia officinalis Rehd. et Wils. (commonly called Houpu Magnolia or Magnolia-bark), has demonstrated significant anticancer potential across various malignancies. Despite its promising therapeutic effects, the clinical application of honokiol has been hampered by its low bioavailability and poor aqueous solubility. To address these limitations, we have investigated the use of liposomal drug delivery systems. Preliminary results from our phase I clinical trials have shown encouraging outcomes: patients with recurrent glioblastoma multiforme (GBM) treated with honokiol liposomes exhibited a longer median overall survival (15 months) compared to those receiving bevacizumab (9.3 months), a standard treatment option. While these initial findings are promising, the precise mechanisms underlying the enhanced efficacy of honokiol when delivered via liposomes remain to be fully elucidated. Methods: Various databases including TCMSP, CTD, BATMAN-TCM, PharmMapper, and SwissTargetPrediction were searched to identify honokiol targets and GBM targets were obtained from GeneCards, OMIM, and DisGeNET disease databases. The component-disease intersection target protein-protein interaction (PPI) network was constructed using String database and Cytoscape3.9.1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of major intersection targets was performed using DAVID. Differential expression targets among the major intersection targets were analyzed using GEPIA, HPA, ROC Plotter, and TIMER databases. Finally, molecular simulation docking verification was conducted using Auto Dock4.2. Finally, we conducted targeted experiments to validate and assess key biological processes, molecular functions, and signaling pathways identified through our comprehensive network pharmacology analysis. Our approach included in vitro studies focusing on proliferation, cell cycle progression, and apoptosis, as well as in vivo experiments examining inflammatory responses. Results: Network pharmacological screening identified 31 major intersecting targets of honokiol and GBM. GO analysis revealed significant enrichment (P < 0.05) in 316 biological processes, 38 cellular components, and 70 molecular functions. KEGG pathway analysis highlighted significant correlations with cancer pathways, metabolic disease pathways, and inflammatory signaling pathways. Further bioinformatic analysis pinpointed 16 core targets significantly affecting GBM, with molecular docking confirming honokiol's spontaneous binding to these targets. Both in vitro and in vivo experiments substantiated these findings, demonstrating that honokiol and its liposomal formulation effectively suppress GBM cell proliferation, induce cell cycle arrest and apoptosis, while significantly reducing inflammatory cytokine expression in GBM-bearing mice. Conclusion: This comprehensive investigation uncovers the sophisticated anti-GBM mechanisms of honokiol and its liposomal formulation. Our findings demonstrate that honokiol and honokiol liposomes effectively target multiple pathways and modulate diverse cellular components within the tumor microenvironment. The elucidated molecular mechanisms not only advance our understanding of honokiol's therapeutic potential but also establish a solid foundation for future clinical development of honokiol liposomes.