力达霉素-胰岛素样生长因子1融合蛋白的构建及其抗非小细胞肺癌活性研究被引量：1

Construction of an Enediyne-energized Fusion Protein IGF-LDP-AE and Its Antitumor Activity on Nonsmall Cell Lung Cancer

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摘要目的制备一种由胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)和力达霉素(lidamycin,LDM)构成的融合蛋白,并对其抗非小细胞肺癌(non-small cell lung cancer,NSCLC)活性进行检测。方法合成融合蛋白基因ldp-igf并将其插入到p ET30a载体中,再将获得的重组表达载体p ET30-ldp-igf转化至大肠杆菌中进行诱导表达,得到融合蛋白LDP-IGF。采用细胞免疫荧光实验和基于流式细胞术的亲和实验检测LDP-IGF蛋白与非小细胞肺癌细胞的结合能力;力达霉素的发色团(AE)与LDP-IGF蛋白在体外进行分子组装,获得强化融合蛋白LDP-IGF-AE。四甲基偶氮唑蓝法实验检测LDP-IGF-AE对不同非小细胞肺癌细胞的体外杀伤活性,PI染色结合流式细胞术检测LDP-IGF-AE对细胞周期的影响以及Annexin V-FITC/PI双染法检测LDP-IGF-AE对非小细胞肺癌细胞凋亡的诱导作用。结果经过诱导LDP-IGF蛋白在大肠杆菌中以包涵体形式表达。采用Ni^(2+)亲和层析技术对包涵体蛋白进行分离纯化,经过变性和分步透析复性后获得了高纯度、有活性的融合蛋白LDP-IGF。亲和实验结果显示,LDP-IGF蛋白与非小细胞肺癌细胞具有高度的亲和力。与AE分子进行组装后获得的强化融合蛋白LDPIGF-AE对不同的非小细胞肺癌细胞系均有非常强烈的杀伤活性,且其杀伤活性显著高于力达霉素。细胞周期阻滞和细胞凋亡实验结果显示,极低浓度的LDP-IGF-AE处理可使非小细胞肺癌细胞阻滞于G_2/M期,并可显著地诱导细胞发生凋亡。结论融合蛋白LDP-IGF-AE对非小细胞肺癌细胞有强大的体外抗肿瘤活性,具有发展成为非小细胞肺癌靶向治疗剂的潜能。 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 p ET30 a.Fusion protein LDP-IGF was expressed in E.coli as inclusion bodies and was purified by Ni^（2＋）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 vitro cytotoxicity of LDP-IGF and its enediyne-energized analogue LDP-IGF-AE.PI staining assay and Annexin VFITC/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·m L^（-1））.LDP-IGF-AE could cause significant G_2-M arrest in A549 and H460 cells,and it also induced the apoptosis in NSCLC cells in a concentration-dependent manner.CONCLUSION Fusion proteinLDP-IGF-AE shows potent antitumor efficacy in vitro on NSCLC,suggesting it could be a promising candidate for targeted therapy.

作者郭晓芳朱小飞曹海英钟根深甄永苏

机构地区新乡医学院河南省分子诊断与医学检验技术协同创新中心新乡医学院第一附属医院中国医学科学院北京协和医学院医药生物技术研究所

出处《中国药学杂志》 CAS CSCD 北大核心 2016年第12期985-993,共9页 Chinese Pharmaceutical Journal

基金国家自然科学基金资助项目(81202447)

关键词胰岛素样生长因子1 力达霉素融合蛋白非小细胞肺癌 insulin-like growth factor 1（IGF-1） lidamycin fusion protein non-small cell lung cancer

分类号 R965 [医药卫生—药理学]

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1BARESCHINO M A, SCHETTINO C, ROSSI A, et al. Treatment of advanced non small cell lung cancer [J]. J Thorac Dis, 2011, 3(2): 122-133.
2GOFFIN J, LACCHETTI C, ELLIS P M, et al. First-line systemic chemotherapy in the treatment of advanced non-small cell lung cancer: a systematic review [J]. J Thorac Oncol, 2010, 5(2): 260-274.
3GE L, SHI R. Progress of EGFR-TKI and ALK/ROS1 inhibitors in advanced non-small cell lung cancer [J]. Int J Clin Exp Med, 2015, 8(7):10330-10339.
4LARSEN J E, CASCONE T, GERBER D E, et al. Targeted therapies for lung cancer: clinical experience and novel agents [J]. Cancer J, 2011, 17(6): 512-527.
5RUSSO A, FRANCHINA T, RICCIARDI G R, et al. A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): old successes and future perspectives [J]. Oncotarget, 2015, 6(29):26814-26825.
6HAISA M. The type I insulin-like growth factor receptor signaling system and targeted tyrosine kinase inhibition in cancer [J]. J Int Med Res, 2013, 41(2): 253-264.
7YOU L, LIU C, TANG H, et al. Advances in targeting insulin-like growth factor signaling pathway in cancer treatment [J]. Curr Pharm Des, 2014, 20(17):2899-2911.
8KARAMOUZIS M V, PAPAVASSILIOU A G. Targeting insulin-like growth factor in breast cancer therapeutics [J]. Crit Rev Oncol Hematol, 2012, 84(1):8-17.
9CAPPUZZO F, TOSCHI L, TALLINI G, et al. Insulin-like growth factor receptor 1(IGFR-1) is significantly associated with longer survival in non-small-cell lung cancer patients treated with gefitinib [J]. Ann Oncol, 2006, 17(7): 1120-1127.
10LUDOVINI V, BELLEZZA G, PISTOLA L, et al. High coexpression of both insulin-like growth factor receptor-1 (IGFR-1) and epidermal growth factor receptor (EGFR) is associated with shorter disease-free survival in resected non-small-cell lung cancer patients [J]. Ann Oncol, 2009, 20(5):842-849.