新型肿瘤导向肽融合抗菌肽对肿瘤细胞的杀伤效应及机制

Cytotocixity of antimicrobial peptide in tumor cells by conjugation to tumor homing peptide TMTP1 and mechanisms

目的 将新型肿瘤导向肽TMTP1与抗菌肽相融合,以增强对肿瘤细胞的杀伤效应.方法 验证新型多肽对肿瘤细胞的亲和能力,经融合多肽作用后,噻唑蓝（MTT）、流式细胞仪实验检测细胞的增殖与凋亡,Boyden小室分析对细胞侵袭转移能力的影响,Western blot检测细胞中活化的半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3、-8、-9的表达水平.结果 薪型融合多肽具有对肿瘤细胞的亲和能力,20 μmol/L的融合多肽引起近100％ PE-3M-1 E8和95％ MKN-45细胞死亡,10 μmol/L的融合多肽诱导（82.15±1.20）％ PC-3M-1E8细胞及（84.27 ±3.20）％的MKN-45细胞发生凋亡,而其侵袭转移能力下降为（52.38±3.30）％、（46.16±2.70）％,其机制和细胞中的Caspase-3、-8、-9活化有关.结论 新型肿瘤导向肽TMTP1与抗菌肽融合后显示强烈的肿瘤细胞杀伤效应.

Objective To enhance the cytotoxicity of antimicrobial peptide in tumor cells by conjugation to a tumor homing peptide （TMTP1）.Methods The fusion peptide binding to human prostate cancer cell line PC-3M-1 E8 and gastric cancer cell line MKN-45 was identified in vitro.Cytotoxicity exerted by the fusion peptide on the two tumor cell lines was measured by using methyl thiazol tetrazolium （MTT） assay and the induced apoptosis was examined by using flow cytometry.The invasive activity in the two cell lines was measured by uisng a modified Boyden chamber assay.The protein expression of Caspase-3,Caspase-8 and Caspase-9 was detected by using Western blotting.Results The fusion peptide expressed special high binding activty to PC-3M-1E8 and MKN-45 cells.Treatment of PC-3M-1E8 and MKN-45 cells with 20 μmol/L fusion peptide showed higher cytotoxicity by 100％ and 95％ respectively than control groups （P＜ 0.05）.The apoptosis rate of PC-3M-1E8 and MKN-45 cells was obviosuly increased to （82.15 ± 1.20）％ and （84.27 ± 3.20）％ respectively after treatment with 10 20 μmol/L fusion peptide.Invasive activity induced by fusion peptide was decreased by （52.38 ± 3.30）％ and （46.16 ± 2.70）％,respectively.The mechanism might be related to the up-regulation of the protein expression levels of Caspase-3,Caspase-8 and Caspase-9.Conclusion These results demonstrated that the fusion peptide showed high cytotoxicity to the target tumor cells after antimicriobial peptide conjugated to TMTP1.