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Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A comparative in vitro study using EGFR-positive breast cancer cells.

Abstract：

:Epidermal growth factor (EGF) is a local growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. EGF and EGFR are involved in many aspects of the development of carcinomas. Because EGFR has been found to be over-expressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. In this study we designed a mutated form of hEGF (mEGF) with a deletion of four amino acids residues (Gln43, Tyr44, Arg45 and Asp46) in order to show importance of Leu spatial location for EGFR binding/activation. Expression vector pET32a+ and E. Coli, strain Rosetta-gami B (DE3) were used to enhance solubility of the recombinant protein with yielding approximately 10mg/l of cell culture. The purified cleaved hEGF as well as non-cleaved fusion protein were biologically active, which was confirmed by their equal ability to stimulate proliferation of MCF7 cells. The mEGF showed specificity and high affinity for EGFR binding, however binding affinity of mEGF for EGFR was reduced about 11.5 fold compared with that of hEGF. The mEGF effect on the MCF7 cell proliferation had a relatively different outcome; mEGF simulated differential cell growth in a dose dependent manner. On the other hand, in MDA-MB468 cells, hEGF and mEGF induced growth inhibition, which was much more severe for hEGF than that of mEGF. Also, hEGF strongly induced the phosphorylation of EGF receptor in MDA-MB468 cells while mEGF induced poor EGFR phosphorylation. The same observations were also made for migration of cancer cells, especially induction of MDA-MB468 migration by mEGF was significantly lower than that of hEGF, suggesting a connection between tyrosine phosphorylation of EGFR and cell migration. Docking analysis revealed that the binding affinity and the buried surface area of mEGF to EGFR complex are lower than those of hEGF/EGFR. Although theoretical studies confirmed reduction in mEGF-EGFR binding affinity, the data of the present study indicate that mEGF is a potential EGFR blocker but may highlight it as excellent delivery agent of protein/non-protein toxins as well as for α-, β-, γ-emitting radio-immunotherapy.

journal_name

Int J Biol Macromol

journal_title

International journal of biological macromolecules

authors

Mehrabi M,Mansouri K,Soleymani B,Hoseinkhani Z,Shahlaie M,Khodarahmi R

doi

10.1016/j.ijbiomac.2017.05.035

subject

Has Abstract

pub_date

2017-10-01 00:00:00

pages

275-285

eissn

0141-8130

issn

1879-0003

pii

S0141-8130(17)30899-1

journal_volume

103

pub_type

杂志文章

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Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A comparative in vitro study using EGFR-positive breast cancer cells.