Yu Jiawen, Guo Meihua, Wang Ting, Li Xiang, Wang Dan, Wang Xinying, Zhang Qian, Wang Liang, Zhang Yang, Zhao Chunhui, Feng Bin
Department of Biotechnology, Dalian Medical University, Dalian, 116044, China.
Department of Hematology, The First Affiliated Hospital of Dalian Medical University, Dalian, 116011, China.
Cell Prolif. 2016 Feb;49(1):79-89. doi: 10.1111/cpr.12238. Epub 2016 Feb 3.
The aim of this study was to develop multifunctional fusion proteins for targeting and delivering therapy elements into glioma cells.
Multifunctional fusion proteins were expressed in Escherichia coli and purified using Ni-NTA resin affinity chromatography. Human glioma cells and primary astrocytes were used to analyse their functions. Targeting proteins location to glioma cells was observed by confocal microscopy. Effects of cell viability and proliferation were evaluated using the Cell Counting Kit 8 and colony formation assays. Glioma cell migration and invasion were assessed using transwell assays, and apoptosis was analysed by flow cytometry. In addition, changes in expression of proteins related to the cell cycle and apoptosis were determined by Western blotting.
The protein with highest bioactivity was GL1-riHA2-p53c+m-TAT (GHPc+mT), which combines glioma-targeting peptide GL1 (G), and C terminus (Pc) and mouse double minute domains (Pm) of p53, with the destabilizing lipid membrane peptide riHA2 (H) and cell-penetrating peptide TAT (T). The purified fusion protein was stable in cell culture medium and specifically targeted, and was internalized by, epidermal growth factor receptor (EGFR)-overexpressing glioma cells (U87ΔEGFR). It inhibited cell proliferation, migration and invasion, while flow cytometric analysis showed increased apoptosis. In addition, GHPc+mT caused significant changes in expression of proteins related to the cell cycle and apoptosis.
GHPc+mT is a multifunctional protein combining targeting, inhibition of glioma cell proliferation and induction of apoptosis, providing some potential to be developed into an effective protein drug delivery system for glioma therapy.
本研究旨在开发多功能融合蛋白，用于将治疗元件靶向递送至胶质瘤细胞。
多功能融合蛋白在大肠杆菌中表达，并用镍-亚氨基三乙酸树脂亲和层析法纯化。用人胶质瘤细胞和原代星形胶质细胞分析其功能。通过共聚焦显微镜观察靶向蛋白在胶质瘤细胞中的定位。使用细胞计数试剂盒8和集落形成试验评估细胞活力和增殖的影响。使用Transwell试验评估胶质瘤细胞的迁移和侵袭，并通过流式细胞术分析凋亡情况。此外，通过蛋白质印迹法测定与细胞周期和凋亡相关的蛋白质表达变化。
生物活性最高的蛋白是GL1-riHA2-p53c+m-TAT（GHPc+mT），它将胶质瘤靶向肽GL1（G）、p53的C末端（Pc）和小鼠双微体结构域（Pm）与去稳定化脂质膜肽riHA2（H）和细胞穿透肽TAT（T）结合。纯化的融合蛋白在细胞培养基中稳定且具有特异性靶向性，并被表皮生长因子受体（EGFR）过表达的胶质瘤细胞（U87ΔEGFR）内化。它抑制细胞增殖、迁移和侵袭，而流式细胞术分析显示凋亡增加。此外, GHPc+mT引起与细胞周期和凋亡相关的蛋白质表达发生显著变化。
GHPc+mT是一种兼具靶向性、抑制胶质瘤细胞增殖和诱导凋亡功能的多功能蛋白，具有开发成为有效治疗胶质瘤的蛋白质药物递送系统的潜力。
