The Mechanism Study On Schizandrin A Postponing The Resistance Of Gefitinib By Suppressing IKKβ/NF-κB Signaling In Non-small Cell Lung Cancer

Background:The emergence of resistance to epidermal growth factor receptor(EGFR)inhibitor therapy is a significant challenge for patients with non-small cell lung cancer(NSCLC).During the past few years,a correlation between EGFR TKIs resistance and dysregulation of IKKβ/NF-κB signaling has been increasingly suggested.However,few studies have focused on the effects of combining IKK/NF-κB and EGFR inhibitors to overcome EGFR TKIs resistance.Accumulating evidence demonstrated that Sch A effectively reverses chemotherapeutic drug resistance through various molecular mechanisms,but its impact on resistance to targeted therapies and the underlying mechanisms are still unknown.Therefore,the major purpose of this study was to explore the postponsal effect of Sch A on gefitinib resistance in NSCLC cells and the potential molecular mechanism,which may provide a candidate combination drug regimen to overcome the difficult problem of EGFR-TKIs resistance in NSCLC.Methods and results:1.In this study,we discovered that Schizandrin A(Sch A),a lignin compound isolated from Schisandra chinesnesis,showedfavourable proliferation inhibitory activity against Gefitinib-resistant HCC827 cells(HCC827GR).Through MTS assay,colony formation,and flow cytometry,the results demonstrated that Sch A could synergize with the EGFR inhibitor Gefitinib to inhibit cell growth(eg.Sch A could increased the cytotoxicity of gefitinib more than five times,inducingcell cycle arrest and apoptosis of HCC827/GR cells.2.By Western blot analysis,we found that Sch A effectively suppressed the phosphorylation of IKKβ and IκBα,as well as the nuclear translocation of NF-κB p65,and showed high and selective affinity for IKKβ in surface plasmon resonance(SPR)experiments,indicating that Sch A was a selective IKKβ inhibitor.3.To insight into the binding mode between Sch A and IKKβ,various simulation techniques were carried out,such as molecular docking,conventional molecular dynamics simulation and Gaussian accelerated molecular dynamics simulation.Molecular modeling between IKKβ and Sch A suggested that Sch A formed key hydrophobic interactions with IKKβ,which may contribute to its potent IKKβinhibitory effect.In addition,combined with structuralanalysis and binding free energy calculations,the relationship between structure and energy were analyzed to explore the dynamic behavir between Sch A and IKKβ.The initial conformation between Sch A and IKKβ were predicted by molecular docking and optimizd by conventional molecular dynamics simulation,our simulation results showed that Sch A can be stabilized in the IKKβ binding site and decrease the fluctuation of amino acid residues in the binding site,while enhancing fluctuation of the non-binding site.Conventional molecular dynamics simulation combined with free energy calculation and each energy term decomposition showed that the binding of Sch A and IKKβ were mainly contributed by van der Waals interaction,indicating complementary manner of structural shapes is the mainly binding meachism between them.Binding free energy decomposition to each amino acid residue revealed that most of key amino acid residues are hydrophobic amino acid residues,which weredistributed inside the IKKβ binding site.Gaussian accelerated molecular dynamics simulations further supported these observaions that the conformations of Sch A and IKKβ wererelatively stable and consistent with the structural conclusions from conventional molecular dynamics simulations.Sch A can decreasethe fluctuation of amino acid residues in the IKKβ binding site and enhance fluctuations in non-binding sites.4.To further validate our predictions from molecular modelings,we mutated,expressed and purified the IKKβ protein with the key amino acid residue Ile-165 mutated to alanine.SPR technique indicated thatSch A and IKKβ I165A did not bind,suggesting a direct binding between IKKβ and Sch A.5.Finally,we validated the inhibitory activity of Sch A on IKKβ/NF-κB signaling pathway at the animal level by the mouse model of drug-resistant nude mice xenografts.Indeed,Sch A play an essential role in postponingthe Gefitinib-resistance in nude mice xenografts.Our results confirmed that 100mg/kg of Sch A can reverse the Gefitinib-resistance in vivo without exacerbating its toxicityConclusions:Taken together,our study suggested that Sch A effectively suppressed the phosphorylation of IKKβ and IκBα,as well as the nuclear translocation of NF-κB p65,then dysregulatedthe IKKβ/NF-κB signaling and postponed the resistance of EGFR-TKIs.