DFF40-iRGD, a novel chimeric protein with efficient cytotoxic and apoptotic effects against triple-negative breast...

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.

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.

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.

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.