核糖核酸酶-表皮生长因子融合蛋白对表皮生长因子受体过表达细胞生长抑制作用的机制。

Mechanisms of the growth-inhibitory effect of the RNase-EGF fused protein against EGFR-overexpressing cells.

作者信息

Hoshimoto Sojun, Ueda Masakazu, Jinno Hiromitsu, Kitajima Masaki, Futami Junichiro, Seno Masaharu

机构信息

Department of Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan.

出版信息

Anticancer Res. 2006 Mar-Apr;26(2A):857-63.

PMID:16619480

Abstract

BACKGROUND

We previously showed the usefulness of a fused protein of human pancreatic ribonuclease1 (hRNase1) with human epidermal growth factor (hEGF) for molecular targeting of EGF receptor (EGFR)-overexpressing cells. In this study, the mechanisms underlying the inhibition of cell growth by RNase-EGF fused proteins was confirmed.

MATERIALS AND METHODS

Des.1-7 hRNase1 was genetically fused to hEGF. The fused proteins were expressed and isolated from Escherichia coli. The internalization of hRNase1-hEGF was confirmed by confocal fluorescence microscopy. The growth-inhibitory effect of the fused proteins was evaluated by MTT assay.

RESULTS

FITC-labelled hRNase1-hEGF was internalized into EGFR-overexpressing A431 cells. The internalization was not observed in A431 cells pre-treated with hEGF and EGFR-deficient H69 cells. The growth-inhibitory effect of des.1-7 hRNase1-hEGF against A431 cells was statistically significantly more pronounced than that of hRNase1-hEGF.

CONCLUSION

RNase-EGF fused proteins are internalized through EGFR and inhibit cell growth by exerting their ribonucleolytic activity in the cytosol.

摘要

背景

我们之前已证明人胰腺核糖核酸酶1（hRNase1）与人表皮生长因子（hEGF）的融合蛋白对表皮生长因子受体（EGFR）过表达细胞具有分子靶向作用。在本研究中，对核糖核酸酶 - EGF融合蛋白抑制细胞生长的潜在机制进行了确认。

材料与方法

将Des.1 - 7 hRNase1与hEGF进行基因融合。融合蛋白在大肠杆菌中表达并分离。通过共聚焦荧光显微镜确认hRNase1 - hEGF的内化。通过MTT法评估融合蛋白的生长抑制作用。

结果

异硫氰酸荧光素（FITC）标记的hRNase1 - hEGF被内化到EGFR过表达的A431细胞中。在用hEGF预处理的A431细胞和EGFR缺陷的H69细胞中未观察到内化现象。Des.1 - 7 hRNase1 - hEGF对A431细胞的生长抑制作用在统计学上比hRNase1 - hEGF更显著。