P28-Fur-Grb, A Novel Fusion Protein with Enhanced Targeted Cytotoxicity Against Breast Cancer

Abstract

Keywords: Granzyme B p28 azurin breast cancer fusion protein targeting A B S T R A C T Targeting tumor cells via multiple pathways promises the emergence of a new era in cancer therapy.Consisting of a cell-binding ligand and a cytotoxic moiety, cytolytic fusion proteins can selectively bind and kill target cells with minimal adverse effects. We designed a novel immunoproapoptotic fusion protein, p28-fur-GrB, composed of the cancer-specific azurin-derived cell penetrating peptide, p28, and a mutant version of human serine protease granzyme B. The two moieties were genetically fused by a furin sensitive linker, allowing in vivo cleavage and activation of the immunotoxin after cell entry. Synthesized coding gene of the recombinant protein was cloned and expressed in HEK293T cells, and nickel chromatography was applied for protein purification. After in vitro furin cleavage and primary analyses of SDS-PAGE, Western blotting, GrB activity and ELISA binding assay, the fusion protein was tested for its cytotoxicity on various breast cancer cell lines. Suppression of cell proliferation and viability was evaluated using the WST-1 assay. Furthermore, DNA fragmentation was measured as an indication of apoptotic effects of the fusion protein on treated cells. Based on our results, p28-fur-GrB was efficiently cleaved by furin and showed high GrB activity and binding affinity after cleavage. Following 72h of incubation with IC50 values of the fusion protein, significant cytotoxic effects of 80.6%, 77.1%, 74% and 69.6% were recorded for BT-474, MCF7, SK-BR-3 and MDA-MB-231 tumor cells, respectively. Proliferative potential of MCF 10A normal cells was not affected by the treatment. Analysis of the rate of apoptosis in treated cells confirmed our cytotoxicity results. We concluded that p28-fur-GrB is a potent anti-tumor agent with high cytotoxicity against breast cancer cells.