School of Paramedical, Gerash University of Medical Sciences, Gerash, Iran.
Department of Medical Biotechnology, Kermanshah University of Medical Science, Kermanshah, Iran.
Life Sci. 2021 Jan 15;265:118866. doi: 10.1016/j.lfs.2020.118866. Epub 2020 Dec 7.
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.
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.
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.
Fusion construct could be a prospective candidate for targeted therapy of cancers upregulating GnRH receptor.
GnRH-DFF40（促性腺激素释放激素-DNA 片段因子 40）人源化重组免疫毒素可作为治疗过度表达促性腺激素释放激素受体（GnRHR）的恶性肿瘤的靶向治疗的候选药物。在这项研究中，我们试图构建一种由人源 DFF40 与编码凋亡核酸酶的 GnRH 融合而成的基于 GnRH 的嵌合蛋白，该蛋白特异性靶向表达 GnRH 受体的癌细胞。
构建了在 pET28a 表达载体中的 GnRH-DFF40 融合基因及其单链（DFF40）的密码子优化合成基因。在三种乳腺癌细胞系（MCF7、MDA-MB231 和 SKBR3）上评估这些表达蛋白的细胞毒性。在处理的细胞系和无细胞系统中研究了重组蛋白的稳定性和生物学活性。此外，通过流式细胞术、迁移试验和划痕愈合分析分别评估了该融合蛋白及其单一形式诱导凋亡、抑制转移和迁移的能力。还进行了计算机模拟分析，以了解 GnRH 与其受体之间的特异性相互作用。
成功表达了 GnRH-DFF40 融合蛋白和 DFF40。纯化的嵌合蛋白对所有三种细胞系均表现出剂量依赖性的细胞毒性。重组融合蛋白具有生物活性，具有核酶功能和诱导凋亡的能力。此外，融合蛋白可抑制 MDA-MB-231 细胞的侵袭特性。计算机模拟分析还表明，GnRH 结构域的四个残基和 GnRHR 的 11 个残基具有最多的相互作用位点，可特异性靶向递送至癌细胞中的免疫毒素。
融合构建物可能是治疗过度表达 GnRH 受体的癌症的有前途的候选药物。
