The Study Of Computer-aided Screening The PI3K Inhibitor In The Treatmment Of Lung Cancer

Lung cancer is the most common malignancy worldwide and the leading cause of cancer-related death,and one of the most dangerous cancers of human health and life.The traditional treatments for non-small cell lung cancer(NSCLC)including surgery,chemotherapy and radiotherapy have reached the bottlenneck stage.Genome wide map reveals that NSCLC is no longer regarded as a disease,but a heterogeneous disease composed of different molecular subtypes.Targeted drugs for different molecular subtypes significantly improved the survival and prognosis of NSCLC patients.The phosphatidylinositol-3-kinase(PI3K)/AKT signaling pathway plays a pivotal role in many cellular processes,including the proliferation,survival and differentiation of lung cancer cells.Thus,PI3K is a promising therapeutic target for lung cancer treatment.The computer-aided screening techniques research the drugs approved by FDA,which enrich active compounds,reduce costs and time,and find these new indications.In this study,idock software simulated the molecular docking between the PI3K target protein and ligand,then screened the PI3K inhibitor from the drug database approved by FDA.The inhibition of the new PI3K inhibitor,the anti-cancer mechanism and the combination of PI3K inhibitor and chemotherapy have been studied by vitro and vivo experiments.The new PI3K inhibitors can effectively inhibit PI3K/AKT signaling pathway,inhibit cell growth and promote apoptosis,exhibite additive and synergistic effects with chemotherapy.A new PI3K inhibitor may be served for treatment of lung cancer and reevaluated the new anti-cancer value.objective:To identify the new PI3K inhibitor can inhibit the growth and proliferation of lung cancer cells,inhibit the PI3K/AKT signaling pathway,and exhibite additive and synergistic effects with chemotherapy,and explore the underlying mechanisms.Methods:(1)The idock softwrare simulated the molecular docking between the PI3K target protein and ligand,then screened the PI3K inhibitors.(2)MTT assay was used to identify the PI3K inhibitors that inhibits the lung cancer cell proliferation.(3)Westem blotting was performed to examine the expression of the critical PI3K/AKT signaling pathway proteins and other pathway protein of ERK after the treatment of PI3K inhibitor.(4)Flow cytometry was used to measure apoposis of lung cancer cells induced by PI3K inhibitor.Western blotting was performed to examine the apoptosis related proteins expression of PARP and Caspase-3.(5)Lung cancer xenografts models in nude mice were used to evaluate the effects of PI3K inhibitor in vivo.(6)The combination of PI3K inhibitor and chemotherapeutic drugs have been studied by MTT,western blotting and flow cytometry.Results:(1)The database including 3167 FDA-approved drugs were docked to the ATP binding pocket of PI3Ka and rarked according to their average predicted binding affinity across structures of PI3Ka.The visual doeking results are available at http://istar.cse.cuhk.edu.hk/idock/iview/?4JPS-dbap+fda+npc.Eight compounds were selected for subsequent investigation,including Econazole,Fupentixol dihydrochloride,Ezetimibe,Talniflumate,Alvimopan,Mizolastine,Doxazosin mesylate and Apixaban.Their idock scores were-7.3?-8.97 kcal/mol,and RF-scores were 7.07?7.67 pKd.(2)Eight compounds were tested the anticancer effects by MTT assay,and five compounds decreased lung cancer cell viability.Among them,econazole exhibited the strongest anticancer effect in a dose-dependent manner(IC50=5.98 ?M),and the inhibitory effects in multiple NSCLC cell lines in a dose-dependent manner(IC50=13.5 ?M in A549,22.2 ?M in SK-SEM-1,and 22.3 ?M in NCI-H520)but had little cytotoxicity in BEAS-2B cells(a normal lung bronchial epithelial cell line).Moreover,econazole(CC50=5.98 ?M)was more potent than two known PI3K inhibitors BLY719(IC50=45.9 ?M)and BKM120(IC50=63.9 ?M)in terms of cytotoxicity for 24 hours.(3)The predicted conformation of econazole in complex with PI3Ka protein was shown that econazole binds to PI3Ka by forming a hydrogen bond with Ser854 and a halogen bond with Asp810 and hydrophobic contacts with Ile848 and Ile932.(4)Econazole decreased the expression of PI3K p110a ana AKT phosphorylation(Thr308 and Ser473)in a dose-dependent manner by western blotting.Econazole did not decrease the phosphorylation levels of ERK,indicating the specificity of econazole to the PI3K pathway.Econazole also specifically decreased the expression levels of Bcl-2 but not MMP-9 or VEGF.(5)Econazole significantly and dose-dependently increased the cell apoptosis percentage of lung cancer cells by Annexin V staining.Econazole induced PARP and caspase-3 cleavage in a dose-dependent manner by western blotting.(6)In vivo,A549 lung cancer cells were subcutaneously injected in BALB/C nude mice.When tumors grow to about 70-80 mm3(day 7),econazole(50 mg/kg daily)was administrated I.P.for 21 days.Econazole significantly suppressed tumor growth compared to the control group.The tumor weight was no difference and no nude mice death in the two groups.(7)The combination treatment exhibited synergistic and additive killing effects in H661 and A549 cells,respectively by MTT assay.In addition,the combination of econazole with cisplatin significantly increased the percentage of apoptotic cells compared to either econazole or cisplatin alone by flow cytometry analysis.Consistently,the combination treatment also induced more PARP and caspase-3 cleavage than single drug treatments.Conclusion:Econazole nitrate,an antifungal agent,was identified as a promising PI3K inhibitor.Econazole exhibited the highest activity in decreasing cell viability in pathological types of NSCLC cell lines.Furthermore,econazole significantly inhibited downstream target of the PI3K/AKT signaling pathway and promoted apoptosis.The combination of econazole and cisplatin exhibited a stronger effect than either econazole or cisplatin did alone.More importantly,econazole(50 mg/kg)significantly suppresses A549 tumor growth in nude mice.Econazole may serve as a new PI3K inhibitor for treatment of lung cancer and reevaluate the new anti-cancer value of an old medicine.