Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Biochemistry, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran.
Cell J. 2014 Summer;16(2):203-10. Epub 2014 May 25.
Immunotoxins (ITs) have been developed for the treatment of cancer, and comprise of antibodies linked to toxins. Also vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and the blockade of VEGF receptor-2 (VEGFR2) inhibits angiogenesis and tumor growth. The aim of this study was to produce anti-VEGFR2/rPE (Pseudomonas exotoxin) 38 IT to test its cytotoxic activity and mechanism of action.
In this basic research and experimental study, at first, DNA that encodes recombinant PE38 protein was inductively expressed in Escherichia coli (E.coli) and purified by nickel-sepharose chromatography and further analyzed by western blot. Then, for production of IT, rPE38 was chemically conjugated to anti- VEGFR2. The cytotoxicity response of IT treatment was evaluated by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test in Human Umbilical Vein Endothelial Cell (HUVEC) and Michigan Cancer Foundation-7 (MCF-7) (VEGFR2+) cell lines. The mechanism of IT cytotoxicity was observed by Annexin V staining and flow cytometry. Continuous variables were compared with the analysis of variance (ANOVA; for all groups). P values less than 0.05 were considered statistically significant.
SDS-PAGE showed 98% purity of rPE38 and IT. In vitro dose-dependent cytotoxicity assay demonstrated that anti-VEGFR2/PE38 is toxic to VEGFR2-positive cells. IT treatment significantly inhibited proliferation of HUVEC and MCF-7 in a VEGFR2-specific manner as compared with the control groups (p<0.05). Flow cytometry showed that the mechanism of IT induced cell death is mediated by apoptosis.
IT treatment also caused remarkable synergistic cytotoxicity characterized by decreased cell viability, and an increased apoptotic index by both anti-VEGFR2 and PE38. Thus these results raise the possibility of using anti-VEGFR2/PE38 IT for cancer therapy because nearly all tumors induce local angiogenesis with high VEGFR expression.
免疫毒素（ITs）已被开发用于癌症治疗，由与毒素连接的抗体组成。此外，血管内皮生长因子（VEGF）在肿瘤血管生成中起关键作用，阻断 VEGF 受体-2（VEGFR2）可抑制血管生成和肿瘤生长。本研究旨在生产抗-VEGFR2/rPE（绿脓杆菌外毒素）38 IT，以测试其细胞毒性活性和作用机制。
在这项基础研究和实验研究中，首先，在大肠杆菌（E.coli）中诱导表达编码重组 PE38 蛋白的 DNA，并通过镍琼脂糖色谱法进行纯化，然后通过 Western blot 进行进一步分析。然后，为了生产 IT，rPE38 通过化学方法与抗-VEGFR2 偶联。通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）试验评估 IT 处理对人脐静脉内皮细胞（HUVEC）和密歇根癌症基金会-7（MCF-7）（VEGFR2+）细胞系的细胞毒性反应。通过 Annexin V 染色和流式细胞术观察 IT 细胞毒性的机制。用方差分析（ANOVA；所有组）比较连续变量。P 值小于 0.05 被认为具有统计学意义。
SDS-PAGE 显示 rPE38 和 IT 的纯度为 98%。体外剂量依赖性细胞毒性试验表明，抗-VEGFR2/PE38 对 VEGFR2 阳性细胞有毒性。与对照组相比，IT 治疗以 VEGFR2 特异性方式显著抑制 HUVEC 和 MCF-7 的增殖（p<0.05）。流式细胞术显示 IT 诱导细胞死亡的机制是通过凋亡介导的。
IT 治疗还导致了明显的协同细胞毒性，表现为细胞活力降低，以及抗-VEGFR2 和 PE38 引起的凋亡指数增加。因此，这些结果提出了使用抗-VEGFR2/PE38 IT 进行癌症治疗的可能性，因为几乎所有肿瘤都会引起局部血管生成，伴有高 VEGFR 表达。
