Construction of an enediyne-energized fusion protein IGF-LDP-AE and its antitumor activity on non-small cell lung cancer
- Author:
Xiao-Fang GUO
1
Author Information
- Publication Type:Journal Article
- Keywords: Fusion protein; Insulin-like growth factor 1 (IGF-1); Lidamycin; Non-small cell lung cancer
- From: Chinese Pharmaceutical Journal 2016;51(12):985-993
- CountryChina
- Language:Chinese
- Abstract: OBJECTIVE: To construct a novel fusion protein contained insulin-like growth factor 1 (IGF-1) and lidamycin (LDM) and evaluate its antitumor activity on non-small cell lung cancer (NSCLC). METHODS: DNA fragment coding for fusion protein (ldp-igf) was synthesized by linking apoprotein of lidamycin (ldp) with igf-1, and then was cloned into the plasmid pET30a. Fusion protein LDP-IGF was expressed in E.coli as inclusion bodies and was purified by Ni2+ affinity chromatography. Binding affinity of LDP-IGF to NSCLC cells was evaluated by immunofluorescence assay and flow cytometry-based binding assay. MTT assay was used to measure the in vitrocytotoxicity of LDP-IGF and its 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. RESULTS: Active 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 flow cytometry-based binding assay showed that LDP-IGF has strong binding activity 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. Furthermore, fusion protein LDP-IGF without active enediyne was also cytotoxic to A549 cells at high concentrations (50 and 100 μg·mL-1). 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. CONCLUSION: Fusion protein LDP-IGF-AE shows potent antitumor efficacy in vitro on NSCLC, suggesting it could be a promising candidate for targeted therapy.
