Construction Of An Enediyne-energized Fusion Protein IGF-LDP-AE And Its Antitumor Activity On Non-small Cell Lung Cancer

BACKGROUNDInsulin-like growth factor receptor 1 over-expressed on non-small cell lung cancer which making it a promising target for cancer therapy.Lidamycin(LDM)composed of apoprotein(LDP)and active enediyne chrompore(AE)is an antibiotic with highly potent antitumor activity.The conjugates or fusion proteins which composed of antibody/ligand and lidamycin displayed potent antitumor activity.OBJECTIVETo construct a novel fusion protein composed of insulin-like growth factor 1(IGF-1)and lidamycin(LDM)and measure its antitumor effects on non-small cell lung cancer(NSCLC).METHODSGene coding for fusion protein(ldp-igf)was synthesized by linking apoprotein of lidamycin(ldp)with igf-1,and then was cloned into the plasmid pET30 a.Fusion protein LDP-IGF was expressed in E.coli as inclusion bodies and was purified by Ni2+ affinity chromatography.Binding affinity of LDP-IGF with NSCLC cells was evaluated by immunofluorescence assay and fluorescence activated cell sorting(FACS)-based binding assay.MTT assay was used to measure the in vitro cytotoxicity of LDP-IGF enediyne-energized analogue LDP-IGF-AE.PI staining assay and Annexin V-FITC/PI staining assay were used to analyze the cell cycle arrest and cell apoptosis after treatment with LDP-IGF-AE,respectively.RESULTSActive soluble LDP-IGF protein was prepared by isolation,purification,denaturation and refolding,and the production of LDP-IGF was 12 mg per liter fermentation broth.Both of immunofluorescence assay and FACS-based binding assay showed that LDP-IGF has strong binding affinity to NSCLC cells.Enediyne-energized fusion protein LDP-IGF-AE exhibited potent cytotoxicity to NSCLC cells in vitro,and it is more potent than that of LDM.LDP-IGF-AE could cause significant G2-M arrest in A549 and H460 cells,and it also induced the apoptosis in NSCLC cells in a concentration-dependent manner.CONCLUSIONFusion protein LDP-IGF-AE showed potent antitumor efficacy in vitro on NSCLC,suggesting it could be a promising candidate for targeted therapy.