Construction of an Ec‐LDP‐D5 fusion protein that targets human epidermal growth factor receptor and its anti‐pancreatic cancer activity

Abstract

AbstractObjectiveIt is well known that α‐defensins are mainly packaged in neutrophil granules (HNP1, HNP2, and HNP3) or secreted by intestinal Paneth cells (human defensin [HD]5 and HD6). HNP1–3 peptides are also secreted, and their accumulation in biological fluids was proposed as a tumor biomarker. It has been reported that α‐defensins promote tumor cell growth, but also provoke cell death at higher concentrations. The aim of the present study was to construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth factor receptor (EGFR), lidamycin apoprotein (LDP), and HD5, and investigate its anti‐pancreatic cancer activities.MethodsHD5 was fused to the Ec‐LDP protein to obtain the Ec‐LDP‐D5 fusion protein by DNA recombination. Then, immunofluorescence and enzyme‐linked immunosorbent assays were used to investigate the binding activities of Ec‐LDP‐D5 to EGFR‐overexpressed cancer cells. CCK‐8 assay was used to measure Ec‐LDP‐D5 in vitro cytotoxicity, and AnnexinV‐FITC/PI staining assay was used to analyze its apoptosis‐inducing efficacy.ResultsThe Ec‐LDP‐D5 fusion protein was constructed correctly and expressed in Escherichia coli in insoluble inclusion bodies. The production of Ec‐LDP‐D5 was 1 mg/L fermentation broth, and the purity of the fusion protein was 85% as analyzed by high‐performance liquid chromatography. Ec‐LDP‐D5 showed strong binding activities for pancreatic cancer cells that highly express EGFR, including PNAC‐1 and Aspc‐1 cells. The Ec‐LDP‐D5 fusion protein showed more potent cytotoxicity to cancer cells compared with Ec‐LDP. The results from the AnnexinV‐FITC/PI staining assay also revealed that Ec‐LDP‐D5 significantly induced cell apoptosis.ConclusionThe novel Ec‐LDP‐D5 fusion protein binds to EGFR specifically, and shows potent cytotoxicity and apoptosis‐inducing activities towards pancreatic cancer cells, which suggests that it would be a promising cancer therapeutics candidate.