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Design and characterization of a recombinant immunotoxin for targeted therapy of breast cancer cells: In vitro and in silico analyses.

Abstract：

AIMS:GnRH-DFF40 (gonadotropin releasing hormone-DNA fragmentation factor 40) humanized recombinant immunotoxin serves as a prospective candidate for targeted therapy of malignancies with over-expressed gonadotropin releasing hormone receptor (GnRHR). In this study, we attempted to generate a GnRH-based chimeric protein composed of human DFF40 fused with GnRH which encodes an apoptotic nuclease and specifically targets cancer cells displaying GnRH receptor overexpression. MATERIALS AND METHODS:A codon optimized, synthetic GnRH-DFF40 fusion gene and its single counterpart (DFF40) were constructed in pET28a expression vector. Cytotoxicity of these expressed proteins were evaluated on three breast cancer cell lines (MCF7, MDA-MB231, and SKBR3). The stability and biological activity of the recombinant proteins were investigated in the treated cell line and cell-free system. Also, the ability of this fusion and its single form in inducing apoptosis, and inhibiting metastasis and migration were evaluated by flow cytometry, migration assay and wound healing analysis, respectively. In silico analyses were also done to understand the specific interactions between GnRH and its receptor. KEY FINDINGS:GnRH-DFF40 fusion protein and DFF40 were successfully expressed. The purified chimeric protein showed dose-dependent cytotoxicity against all three cell lines. The recombinant fusion protein was biologically active with nucleolytic functionality and apoptosis induction ability. Moreover, the fusion could inhibit the invasion property of MDA-MB-231 cells. In silico analysis also showed that four residues from GnRH domain and 11 GnRHR residues had the most interaction sites for specific targeted delivery of the immunotoxin in cancer cells. SIGNIFICANCE:Fusion construct could be a prospective candidate for targeted therapy of cancers upregulating GnRH receptor.

journal_name

Life Sci

journal_title

Life sciences

authors

Barazesh M,Mohammadi S,Jalili S,Kavousipour S,Faraji SN,Mokarram P,Pirhadi S

doi

10.1016/j.lfs.2020.118866

subject

Has Abstract

pub_date

2021-01-15 00:00:00

pages

118866

eissn

0024-3205

issn

1879-0631

pii

S0024-3205(20)31619-2

journal_volume

265

pub_type

杂志文章

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