TY - JOUR
AR - COR-2019-5-102
TI - P28-Fur-Grb, A Novel Fusion Protein with Enhanced Targeted Cytotoxicity Against Breast Cancer
AU - Aria, Momeni
AU - Azadeh, Reshadmanesh
AU - Azita, Fakhravar
AU - Nafiseh, Paydarnia
AU - Saeed, Ranjbar
JO - Clinical Oncology and Research
PY - 2019
DA - Mon 28, Oct 2019
SN - 2613-4942
DO - http://dx.doi.org/10.31487/j.COR.2019.05.02
UR - https://www.sciencerepository.org/p28-fur-grb-a-novel-fusion-protein-with-enhanced-targeted-cytotoxicity-against-breast-cancer_COR-2019-5-102 
KW - Granzyme B, p28, azurin, breast cancer, fusion protein, targeting
AB - 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 BT474, 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.