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

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 V β 3 receptor. The aim of this study was to produce the recombinan...

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Bibliographic Details
Published in:Biotechnology letters 2021-10, Vol.43 (10), p.1967-1976
Main Authors: Amrollahi-nia, Raheleh, Akbari, Vajihe, Shafiee, Fatemeh
Format: Article
Language:English
Subjects:
Antineoplastic Agents - pharmacology
Apoptosis
Apoptosis - drug effects
Applied Microbiology
Biochemistry
Biological effects
Biomedical and Life Sciences
Biotechnology
Breast cancer
Cell death
Cell Line, Tumor
Cell survival
Cell Survival - drug effects
Cell viability
Chitin
Cytotoxicity
Deoxyribonucleases - genetics
Deoxyribonucleic acid
DNA
E coli
Endonuclease
Female
Flow cytometry
Fusion protein
Humans
Life Sciences
Microbiology
Molecular docking
Molecular Docking Simulation
Oligopeptides - genetics
Original Research Paper
Poly-ADP-Ribose Binding Proteins - genetics
Proteins
Receptors
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - pharmacology
Toxicity
Triple Negative Breast Neoplasms - metabolism
Tumor cells
Tumors
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Summary: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 V β 3 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 V β 3 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 V β 3 positive) and MCF-7 (α V β 3 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 IC 50 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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-021-03178-y