PO-407 Exploring the inhibitory potential of napthoquinones against NF-κB pathway

Abstract

Introduction NF-κB is constitutively activated in various cancers and regulates cell proliferation, apoptosis, tumour angiogenesis, tumour metastasis and remodels tumour metabolism. Naphthoquinones are constituents of various natural and synthetic drugs widely used to treat many diseases. However, the molecular mechanisms by which they may exert anticancer effects remain to be elucidated. In this study, we have evaluated the inhibitory effects of naphthoquinones on NF-κB pathway using molecular docking and other experiments. Material and methods The crystal structure of NF-κB p50 (1SVC) was extracted from Protein Data Bank and ligands were retrieved from PubChem Compound Database. 3D structures of ligands were deduced using Molegro Molecular Viewer, energy minimization of the molecules was done by UCSF Chimaera 1.12, binding site of p50 protein was identified using RaptorX and Autodock 4.2 was used for molecular docking simulations. MTT assay,luciferase assay and JC1 assay was performed to check the IC50 values, NF-κB inhibiton and apoptosis, respectively. Reactive species generation was measured using Dichlorofluorescin diacetate (DCFDA) and Dihydroethidium (DHE) probes. Results and discussions Among all the 23 naphthoquinones docked with NF-κB p50 protein, Naftazone had the lowest binding energy of −8.60 Kcal and inhibition constant (Ki) of 495.63 nM followed by AC1O9F6K and Sudan I with a binding energy of −8.51 (Ki 574.77 nM) and −7.96 (Ki 1.46 uM), respectively.Since complete or highly potent inhibition of NF-κB is detrimental to normal cells, menadione with medium binding affinity was chosen for experimental work.IC50 of menadione (for 24 hour of treatment) was found to be 16 µM, 20 µM and 24 µM in SiHa, C-33A and HeLa cells, respectively. NF-κB luciferase activity and mitochondrial membrane potential were significantly decreased in the HeLa and SiHa cells treated with menadione as compared to untreated cells. Menadione increased the level of reactive species, but fold change was more in DCFDA than that in DHE when treated for different time intervals. Taken together, these data suggest that menadione predominantly works by non-superoxide anion radical generation in cancer cells. Conclusion Naftazone seems to be a potent NF-κB inhibitor having the highest binding affinity followed by AC1O9F6K and Sudan I. Menadione induces reactive species and death majorly by induction of non-superoxide anions and inhibition of NF-κB pathway in cervical cancer cells and thus qualifies to be a potential anticancer agent.