Synergistic effect of granzyme B-azurin fusion protein on breast cancer cells

Paydarnia, Nafiseh; Nikkhoi, Shahryar Khoshtinat; Fakhravar, Azita; Mehdiabdol, Mohsen; Heydarzadeh, Hedieh; Ranjbar, Saeed

doi:10.1007/s11033-019-04767-x

Cited by 12 publications

(7 citation statements)

References 38 publications

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“…Herein, we introduce the immunoproapoptotic fusion protein, p28-fur-GrB, as a novel anticancer agent with selective cytotoxicity against breast cancer cells. Previously, we designed a chimeric protein composed of GrB and azurin [16]. Our results showed selective cytotoxicity of the construct in breast cancer cells.…”

Section: Introductionmentioning

confidence: 77%

“…In a previous study, we evaluated the potential of GrB-azurin as a selective eradicator of cancer cells [16]. Given the positive yet improvable results obtained following bio-physico-chemical analyses, we decided to consider some adjustments in our original design.…”

Section: Resultsmentioning

confidence: 99%

“…The results showed binding of GrB and p28 to their respective ligands with relatively strong binding affinities ( Figure 5). reported for GrB-Azurin, highlighted a significant improvement in inhibitory effect and potency of the construct as a result of the abovementioned adjustments [16].…”

Section: Figurementioning

confidence: 86%

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

Momeni¹,

Reshadmanesh²,

Fakhravar³

et al. 2019

COR

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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.

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Section: Introductionmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

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

Momeni¹,

Reshadmanesh²,

Fakhravar³

et al. 2019

COR

Self Cite

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“…172 As azurin can be used to target P53 + tumour cells and to induce apoptosis and cell cycle arrest, a fusion protein in which GZMB has been fused to azurin using a (G4S)4 linker can be used to target and synergistically eliminate P53 + tumour cells. 173 GZMB fusion proteins have also been used to inhibit angiogenesis at tumour sites. Vascular endothelial growth factor receptors (VEGFRs) are considered the most important molecular markers of tumour angiogenesis.…”

Section: Related Fusion Proteinsmentioning

confidence: 99%

Dual roles of granzyme B

Wang

Huang

et al. 2021

Scand J Immunol

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Granzymes are important factors secreted by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The family of human granzymes consists of five members, namely granzyme A, granzyme B (GZMB), granzyme H, granzyme K and granzyme M. As GZMB exerts a robust killing effect on tumours, it has garnered considerable attention. Currently, other cells in addition to CTLs and NK cells are known to produce GZMB in the presence of inducing factors or in specific environments, including TIM-3 + regulatory T cells (Tregs), 1 B lymphocytes, 2-4 plasma cells, 5 myeloid-derived suppressor cells, 6 macrophages, 7 mast cells, 8-10 basophils, 11 chondrocytes, 12 polymorphonuclear neutrophils, 13 CD4 + helper T cells, 14 neutrophils, 15 monocytes, 16 retinal pigment epithelial cells, 10 keratinocytes 17 and mesenchymal-like androgen-repressed human prostate cancer cells. 18 The diversity of GZMB-secreting cells may contribute to the complexity of GZMB functions; therefore, a comprehensive understanding of GZMB is extremely important for conducting extensive research on the innovative application of GZMB. | RELATIONSHIP AMONG PERFORIN (PRF1), GRANULYSIN (GNLY) AND GZMBPRF1, GNLY and GZMB are important molecules secreted by CTLs and NK cells that function synergistically to exert a killing effect. Studies conducted by Dotiwala et al have revealed that PRF1, GNLY and GZMB gain entry into cells

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“…The significant cytotoxicity of azurin toward human carcinomas, such as melanoma, breast, liver, lung, prostate, ovarian, and colorectal sarcoma as well as fibrosarcoma are well-studied ( 23 , 24 ). The structure of azurin as a scaffold protein includes an invariant β-sheet sandwich formed by parallel and anti-parallel strands as well as an extended α-helix region situated outside the barrel ( Figure 1 ) that may help exert a cytostatic and cytotoxic effect ( 25 – 27 ).…”

Section: About Azurinmentioning

confidence: 99%

p28 Bacterial Peptide, as an Anticancer Agent

et al. 2020

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Cancer remains a major cause of morbidity and mortality irrespective of the type of conventional chemotherapy. Therefore, there is an urgent need for new and effective anticancer therapeutic agents. Bacterial proteins and their derivative peptides appear as a promising approach for cancer treatment. Several, including an amphipathic, α-helical, 28-amino acid peptide derived from azurin, a 128-amino acid copper-containing redox protein secreted from Pseudomonas aeruginosa, show clinical promise in the treatment of adult and pediatric solid tumors. The peptide, p28, is a post-translational, multi-target anticancer agent that preferentially enters a wide variety of solid tumor cells. Mechanistically, after entry, p28 has two major avenues of action. It binds to both wild-type and mutant p53 protein, inhibiting constitutional morphogenic protein 1 (Cop1)-mediated ubiquitination and proteasomal degradation of p53. This results in increased levels of p53, which induce cell-cycle arrest at G2/M and an eventual apoptosis that results in tumor cell shrinkage and death. In addition, p28 also preferentially enters nascent endothelial cells and decreases the phosphorylation of FAK and Akt inhibiting endothelial cell motility and migration. Here, we review the current basic and clinical evidence suggesting the potential of p28 as a cancer therapeutic peptide.

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Synergistic effect of granzyme B-azurin fusion protein on breast cancer cells

Cited by 12 publications

References 38 publications

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

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

Dual roles of granzyme B

p28 Bacterial Peptide, as an Anticancer Agent

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