Purpose

DNA fragmenting factor (DFF40), an endonuclease inducing irreversible apoptosis protein, is down-regulated in many types of tumor cells. iRGD is a tumor-penetrating peptide with high affinity to cancer cells overexpressing α_Vβ₃ receptor. The aim of this study was to produce the recombinant DFF40-iRGD protein as a new molecule to selectively induce cytotoxicity in cancer cells and evaluate its biological effects.

Methods

The three-dimensional structure of DFF40-iRGD was predicted using Modeller software and its interaction with α_Vβ₃ receptor was evaluated by HADDOCK web-server. Recombinant DFF40 and DFF40-iRGD proteins were produced using intein fusion system in Escherichia coli BL21 (DE3). To improve the soluble expression, the inducer concentration, temperature and incubation time were optimized. After purification of DFF40 and DFF40-iRGD using chitin column, the cytotoxic and apoptotic effects of the proteins against MDA-MB-231 (α_Vβ₃ positive) and MCF-7 (α_Vβ₃ negative) cell lines were evaluated using cell viability assay and flow cytometric analysis.

Results

The results of molecular docking indicated the proper interaction of DFF40-iRGD with the integrin receptor comparable to iRGD. The optimum conditions of soluble expression of proteins were the induction by 0.5 mM and 0.1 mM of IPTG for DFF40 and DFF40-iRGD, respectively, at 7 °C for 24 h. After 48 h of incubation, DFF40-iRGD exhibited significantly higher cytotoxic effect against MDA-MB-231 cells than MCF-7 cells as IC₅₀ values of 19.25 and 41 nM were found for MDA-MB-231 and MCF-7 cells, respectively. However, DFF40 cytotoxicity was not significantly different in two cell lines. Furthermore, Flow cytometry results showed that the fusion protein can induce remarkably apoptotic cell death in cancer cells.

Conclusion

In this study, DFF40-iRGD protein was produced in soluble form and its inhibitory effects on cancer cell survival and induction of apoptosis were established; therefore, it has the potential to be used as a drug candidate for targeted treatment of breast cancer, especially Triple Negative Breast Cancer Cells.

中文翻译：

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DFF40-iRGD，一种新型嵌合蛋白，对三阴性乳腺癌细胞具有有效的细胞毒性和凋亡作用

目的

DNA 片段化因子 (DFF40) 是一种诱导不可逆细胞凋亡蛋白的核酸内切酶，在许多类型的肿瘤细胞中被下调。iRGD 是一种肿瘤穿透肽，对过度表达 α _V β ₃受体的癌细胞具有高亲和力。本研究的目的是生产重组 DFF40-iRGD 蛋白作为一种新分子，以选择性地诱导癌细胞的细胞毒性并评估其生物学效应。

方法

使用 Modeller 软件预测 DFF40-iRGD 的三维结构，并通过 HADDOCK 网络服务器评估其与 α _V β ₃受体的相互作用。重组 DFF40 和 DFF40-iRGD 蛋白是在大肠杆菌BL21 (DE3) 中使用内含肽融合系统产生的。为了提高可溶性表达，优化了诱导剂浓度、温度和孵育时间。使用几丁质柱纯化 DFF40 和 DFF40-iRGD 后，使用细胞评估蛋白质对 MDA-MB-231（α _V β ₃阳性）和 MCF-7（α _V β ₃阴性）细胞系的细胞毒性和凋亡作用。活力测定和流式细胞术分析。

结果

分子对接的结果表明 DFF40-iRGD 与与 iRGD 相当的整联蛋白受体的适当相互作用。蛋白质可溶性表达的最佳条件是分别用 0.5 mM 和 0.1 mM IPTG 诱导 DFF40 和 DFF40-iRGD，7°C 24 小时。孵育 48 小时后，DFF40-iRGD 对 MDA-MB-231 细胞的细胞毒作用显着高于 MCF-7 细胞，因为发现 MDA-MB-231 和 MCF-7 细胞的IC ₅₀值分别为 19.25 和 41 nM . 然而，DFF40 细胞毒性在两种细胞系中没有显着差异。此外，流式细胞术结果显示融合蛋白可以显着诱导癌细胞的凋亡细胞死亡。

结论

在本研究中，DFF40-iRGD 蛋白以可溶性形式产生，并确定了其对癌细胞存活和诱导细胞凋亡的抑制作用；因此，它有可能作为靶向治疗乳腺癌，尤其是三阴性乳腺癌细胞的候选药物。