ANTP-SmacN7融合肽对H460细胞的辐射增敏作用

背景与目的肿瘤的辐射耐受制约了放疗疗效，第二个线粒体衍生的半胱氨酸蛋白酶激活剂（Second mitochondria-derived activator of caspase, Smac）蛋白类似物可明显提高辐射诱导的肿瘤细胞凋亡，有望成为新型肿瘤辐射增敏药物。本研究旨在探讨新型Smac蛋白类似物ANTP-SmacN7融合肽对肺癌细胞系H460的辐射增敏作用。方法合成ANTP-SmacN7融合肽，连接荧光素FITC以观察融合肽能否进入细胞。对数生长期H460细胞分为空白对照组、单纯照射组、ANTP-SmacN7组和照射联合ANTP-SmacN7组，单纯照射组给予0 Gy、2 Gy、4 Gy、6 Gy照射，照射联合ANTP-SmacN7组中ANTP-SmacN7的浓度为20μmol/L，WST-1测定H460细胞的增殖。流式细胞仪测定细胞处理后24 h和48 h的细胞凋亡率。Western blot实验检测caspase3和cleaved caspase3的表达水平。结果 ANTP-SmacN7融合能够顺利进入细胞，且能够增强H460细胞的辐射敏感性（F=25.1，P<0.01，增敏比为1.86），照射联合ANTP-SmacN7可明显降低H460细胞的克隆形成率（χ2=45.2,P<0.01；χ2=40.3,P<0.01），提高cleaved caspase3的表达量，促进caspase3的活化，增加辐射诱导的细胞凋亡率。结论 ANTP-SmacN7融合肽可明显提高H460细胞的辐射敏感性，作为一种新的Smac蛋白类似物有望用于肿瘤的辐射增敏治疗。

Radiosensitization Induced by ANTP-SmacN7 Fusion Peptide in H460 Cell Line

Background and objective hTe curative effect of radiotherapy may be limited by the radioresistance of tumor. Mimetic compounds of Second mitochondria-derived activator of caspase (Smac) were hopeful to become new drugs of radiosensitization for tumor because they can increase radiation induced apoptosis in tumor cells. hTe aim of present study is to observe the radiosensitization effect of a new Smac mimetic ANTP-SmacN7 fusion peptide in H460 cell line.Methods In order to observe if the fusion peptide can enter into tumor cell, ANTP-SmacN7 fusion peptide was synthesized and linked by FITC. H460 cell was divided into control, radiation only, ANTP-SmacN7 only and ANTP-SmacN7 combined with radia-tion group. hTe cells were exposed by 0, 2, 4 and 6 Gy and the concentration of ANTP-SmacN7 was 20 μmol/L. Proliferation of H460 tumor cell was detected by WST-1 assay. hTere are four groups in the present study: control group, radiation group, ANTP-SmacN7 group and ANTP-SmacN7 combined with radiation group. Apoptosis was detected by lfow cytometry at 24 and 48 hours atfer the treatment of all the groups. hTe level of caspase3 and cleaved caspase3 were detected by Western blot as-say.Results ANTP-SmacN7 can enter into cells and promote the radiosensitization of H460 cell obviously (F=25.1,P<0.01, sensitivity enhancement ratio was 1.86). The treatment of ANTP-SmacN7 combined with radiation decreased the cloning forming effciency (χ2=45.2,P<0.01; χ2=40.3,P<0.01), activated caspase3 by promoting the expression of cleaved caspase3 and increased the apoptosis of H460 cell line.ConclusionANTP-SmacN7 fusion peptide had remarkably radiosensitization effect on H460 cell line. ANTP-SmacN7 fusion peptide might be hopeful to be applied in radiosensitization therapy as a new Smac mimetic polypeptide in the future.