A MULTI-TARGET INVESTIGATION INTO THE THERAPEUTIC MECHANISMS OF ACTION OF LUTEOLIN IN COLORECTAL CANCER

Abstract

Colorectal cancer (CRC) is still one of the major causes of mortality, particularly in nations with low-to-middle income, due to late diagnosis and limited access to therapeutics that are effective, affordable and less toxic. Multi-target anticancer potential of luteolin has been reported, yet its mechanisms in CRC are not well understood because most of the studies rely on one-target or one-pathway analysis. Consequently, this study used a multipronged approach including network pharmacology (NP), molecular docking, and In vivo experimentation to investigate the therapeutic potential of luteolin against CRC. Targets of luteolin and CRC were retrieved from online databases with intention to identify shared interactome. Hub genes were identified from protein-protein interaction network using STRING and cytoscape. Stability of luteolin and its targets were validated by docking and subsequent 100ns simulation. In vivo efficacy was verified in an azoxymethane (AOM)-induced Drosophila model of CRC. NP identified 379 shared targets existed between luteolin and CRC, including hub genes like AKT1, mTOR, TP53 and BCL2. Following enrichment analysis, the main targets are those in PI3K/Akt, Wnt and MAPK signaling pathways. Molecular docking predicted high binding energies - caspase-3 (-9.761 kcal/mol), AKT1 (-8.613 kcal/mol) and mTOR (-7.92 kcal/mol). Complexes were predicted to be favourable using MMGBSA, the major complex being predicted for AKT1 (-54.22 kcal/mol). MD simulations (100ns) demonstrated high structural integrity as the mean RMSD of the caspase-3 complex with luteolin was 1.8 Å and the variation of residues was small. In vivo data revealed luteolin significantly reduced tumour burden in all the groups supplemented with the diet compared to the AOM-only groups, achieving an exceptional result. End-point PCR analysis verified the predicted mechanism, as luteolin significantly (p < 0.05) down regulated oncogenic markers including mTOR, AKT1, c-MYC, and the anti-apoptotic BCL2. Luteolin up regulated critical pro-apoptotic and regulatory genes, specifically TP53, CDKN1A (dap), CASP3 (drice), and CASP9 (dronc). Luteolin disrupts CRC hallmarks by simultaneously inhibiting survival signaling and triggering apoptosis through a stable, multi-target interactome. These data scientifically position luteolin as a potent, multi-target chemopreventive agent which should be further investigated in a more physiologically relevant mammalian model of CRC.

Key Words: Colorectal carcinogenesis, Luteolin, Network pharmacology, PI3K/Akt pathway, Drosophila.