抗肿瘤活性的新型高效细胞穿膜肽[Cys-CPT(2,9)] penetratin的设计及活性评价

Development of novel cell-penetrating peptide [Cys-CPT^(2,9)] penetratin with high translocation ability and antitumor activity

本文将半胱氨酸-喜树碱(Cys-camptothecin,Cys-CPT)引进penetratin第2、9位,合成新型穿膜肽类似物[Cys-CPT^(2,9)]penetratin,旨在探索一条具有强穿膜活性且具有抗肿瘤活性的新型细胞穿膜肽。通过激光共聚焦实验和流式细胞术观察[Cys-CPT^(2,9)]penetratin在He La细胞中荧光摄取情况,使用不同内吞抑制剂研究[Cys-CPT^(2,9)]penetratin的细胞摄取机制,最后通过MTT实验测试[Cys-CPT^(2,9)]penetratin抗肿瘤活性。在激光共聚焦及流式细胞术实验中显示[Cys-CPT^(2,9)]penetratin的穿膜活性显著增强,它的胞内荧光强度比penetratin明显提高5倍。而对于[Cys-CPT^(2,9)]penetratin的细胞摄取机制,它主要通过网格蛋白和巨胞饮内吞途径进入细胞内。[Cys-CPT^(2,9)]penetratin对He La细胞具有抗肿瘤活性,并强于抗肿瘤药物喜树碱。结果说明,[Cys-CPT^(2,9)]penetratin是一个新的高膜转导活性的细胞穿膜肽,同时对HeLa细胞具有抗肿瘤活性。

To develop a cell-penetrating peptide with high membrane penetrating ability and effective antitumor activity, we designed and synthesized an analogue of penetratin, [Cys-CPT 2,9] penetratin, by substitution of Gln2 and Asn9 with Cys-CPT. We investigated the transmembrane activity and antitumor activity of [Cys-CPT2,9] penetratin. The fluorescence intensity of [Cys-CPT2,9] penetratin in HeLa cells was observed by laser confocal microscopy and flow cytometry, and the cell uptake mechanism of [Cys-CPT2,9] penetratin was evaluated by using different endocytic inhibitors, finally the anti-tumor activity of [Cys-CPT 2,9] penetratin was tested by MTT assay. The results showed that the membrane activity of [Cys-CPT2＇9] penetratin was significantly enhanced in laser confocal microscopy and flow cytometry assay, and the intracellular fluorescence intensity was 5 times higher than penetratin. The cell uptake mechanism study of [Cys-CPT2,9] penetratin indicated that it mainly entered the cell through the clathrin and endocytosis. Moreover, [Cys-CPT 2,9] penetratin exhibited anti-tumor activity against HeLa cells, and its inhibitory effect on cancer cells was stronger than CPT. [Cys-CPT 2,9] penetratin is a new cell-penetrating peptide with high translocation ability, and has anti-tumor activity against HeLa cells.