新型强化融合蛋白Ec-LDM-TF体外抗乳腺癌细胞活性研究  

Fusion protein Ec-LDM-TF shows high efficacy against breast cancer in vitro

作　　者：张玉萍 万海涛 梁华 刘文娟 ZHANG Yu-ping;WAN Hai-tao;LIANG Hua;LIU Wen-juan(Qingdao Central Hospital^Qingdao 266000,P.R.China;Shandong Cancer Hospital and Institutey Shandong First Medical University and Shandong Academy of Medical Sciences,Jinan 250117,P.R.China)

机构地区：[1]青岛市中心医院放疗二科,山东青岛266000 [2]青岛市中心医院内一科,山东青岛266000 [3]山东省肿瘤防治研究院(山东省肿瘤医院基础实验室),山东第一医科大学(山东省医学科学院),山东济南250117

出　　处：《中华肿瘤防治杂志》2019年第18期1348-1354,1360,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81502691)

摘　　要：目的乳腺癌在我国发病率呈上升趋势,严重危害女性身体健康。本研究探讨新型融合蛋白Ec-LDM-TF的抗乳腺癌活性及其作用机制。方法采用酶联免疫吸附测定法(enzyme-linked immuno sorbent assay,ELISA)和细胞免疫荧光法检测Ec-LDM-TF与人乳腺癌肿瘤细胞MCF-7和SK-BR-3表面表皮生长因子受体(epidermal growth factor receptor,EGFR)的亲和活性;采用噻唑蓝(MTT)法测定Ec-LDM-TF对人纤维肉瘤细胞HT1180、人皮肤癌细胞A431、人乳腺癌细胞SK-BR-3和MCF-7的体外杀伤活性;流式细胞术检测Ec-LDM-TF作用后肿瘤细胞的凋亡和细胞周期变化;蛋白质印迹法检测Ec-LDM-TF对细胞凋亡通路中相关蛋白的影响。结果 Ec-LDM-TF可与高表达EGFR的MCF-7和SK-BR-3细胞表面结合。Ec-LDM-TF对人乳腺癌细胞MCF-7和SK-BR-3,以及人纤维素肉瘤细胞HT1080均有杀伤效果,其中对细胞SK-BR-3的杀伤效果最强,其IC50为13.93×10-12 mol/L。流式细胞术检测结果显示,MCF-7细胞在Ec-LDM-TF孵育24h后,晚期凋亡的细胞0.5、1nmol/L Ec-LDM-TF占比分别为(4.653±1.128)%和(21.233±0.900)%,与对照组的(1.86±1.3)%相比,差异有统计学意义,均P<0.05;而SK-BR-3细胞晚期凋亡的细胞为0.5、1nmol/L Ec-LDM-TF占比分别为(5.125±1.318)%和(33.055±2.312)%,与对照组(2.418±1.376)%相比,差异有统计学意义,均P<0.05。提示Ec-LDM-TF促使MCF-7和SK-BR-3细胞产生凋亡。在24h后SK-BR-3细胞Ec-LDM-TF给药组1nmol/L时G2期为(76.84±1.29)%,与对照组(5.52±0.95)%相比,差异有统计学意义,t=44.62,P<0.001;Ec-LDM-TF促使SK-BR-3肿瘤细胞G2/M期阻滞。蛋白质印迹法检测结果显示,在Ec-LDM-TF作用下,SK-BR-3细胞内Cl-Caspase-3和Cl-PARP均出现裂解带,Bax表达上调,Bcl-2表达下调。结论本研究制备的具有EGFR靶向的新型融合蛋白Ec-LDM-TF对人乳腺癌细胞具有杀伤活性,其作用机制为促使细胞凋亡。OBJECTIVE The incidence of breast cancer is first among female malignant tumors in China,which seriously endangers women’s health.This study detected the anti-breast cancer activity and its mechanism of action of a novel fusion protein,Ec-LDM-TF.METHODS Immunofluorescence assay and ELISA assay were used to investigate the binding activity of Ec-LDP-TF to EGFR overexpressed breast cancer cells.The cytotoxicity of Ec-LDM-TF was measured by MTT assay.The cell apoptosis was tested by flowcytometry.PI staining with flow cytometry was used to observe cell cycle arrest.Key of proteins in the cell apoptosis pathways were detected by Western blotting.RESULTS Ec-LDP-TF showed strong binding activity to breast cancer cells highly expressing EGFR.MTT assay showed that the Ec-LDPM-TF had better cytotoxicity in EGFR-MCF-7 and SK-BR-3 expression breast cancer cells,and the killing effect on cell SK-BR-3 is the strongest.The IC50 of it was 13.93×10-12 mol/L.The results of flow cytometry showed that the percentage of MCF-7 cells in late apoptotic cells after Ec-LDM-TF incubation for 24 h was respectively(4.653±1.128)% Ec-LDM-TF(0.5 nmol/L),(21.233±0.900)% Ec-LDM-TF(1 nmol/L),and the control group was(1.86±1.3)%.The difference was statistically significant,P <0.005.The percentage of SK-BR-3 cells in late apoptotic cells for Ec-LDM-TF(0.5 nmol/L)was(5.125±1.318)%and for Ec-LDM-TF(1 nmol/L)was(33.055±2.312)%.Compared with the control group(2.418±1.376)%(P<0.005),indicating that Ec-LDM-TF promoted apoptosis of MCF-7 and SK-BR-3 cells.We examined the effect of Ec-LDM-TF on SK-BR-3 cell cycle after 24 h.The flow cytometry cell cycle analysis showed that 1 nmol/L Ec-LDM-TF induced cell cycle arrested at G2 phase(76.84±1.29)%,compared with the control group(5.52±0.95)%(t=44.62,P<0.001).Therefore,Ec-LDM-TF induced cell cycle arrest at G2/M phase in SK-BR-3 cells.The results of Western blotting showed that pretreatment with Ec-LDM-TF caused CL-Caspase-3 and CL-PARP cleavage.The proapoptotic protein Bax was up-regulated,and the a

关 键 词：力达霉素 烯二炔融合蛋白 乳腺癌 细胞凋亡 细胞周期阻滞 

分 类 号：R737.9[医药卫生—肿瘤]