A-NGR fusion protein induces apoptosis in human cancer cells

The NGR peptide is one of the well-known peptides for targeting tumor cells. It has the ability to target aminopeptidase N (CD13) on tumor cells or the tumor vascular endothelium. In this study, the NGR peptide was used for targeting A subunit of the Shiga toxin to cancer cells. The cytotoxic effect of the A-NGR fusion protein was assessed on HT1080, U937, HT29 cancer cells and MRC-5 normal cells. For this purpose, cells were treated with different concentrations of A-NGR (0.5-40 µg/ml). The evaluation of cell viability was achieved by MTT assay. Apoptosis was determined by annexin-V/PI double staining flow cytometry. Alterations in the mRNA expression of apoptosis - related genes were assessed by real time RT- PCR. The results showed that A-NGR fusion protein effectively inhibited the growth of HT1080 and U937 cancer cells in comparison to negative control (PBS) but for CD13-negative HT-29 cancer cells, only at high concentrations of fusion protein was inhibited growth recorded. On the other hand, A-NGR had little cytotoxic effect on MRC-5 normal cells. The flow cytometry results showed that A-NGR induces apoptosis. Furthermore, the results of real time RT-PCR revealed that A-NGR significantly increases the mRNA expression of caspase 3 and caspase 9. Conclusively, A-NGR fusion protein has the ability of targeting CD13-positive cancer cells, the cytotoxic effect on CD13-positive cancer cells as well as has low cytotoxic effect on normal cells.

[1]  M. Shokrgozar,et al.  Cloning, Expression, and Assessment of Cytotoxic Effects of A-NGR Fusion Protein , 2017, International Journal of Peptide Research and Therapeutics.

[2]  M. Shokrgozar,et al.  sIL-24 peptide, a human interleukin-24 isoform, induces mitochondrial-mediated apoptosis in human cancer cells , 2017, Cancer Chemotherapy and Pharmacology.

[3]  E. Pirogova,et al.  Evaluation of the use of therapeutic peptides for cancer treatment , 2017, Journal of Biomedical Science.

[4]  B. Bauvois,et al.  The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13 , 2015, Oncotarget.

[5]  A. Melton-Celsa.  Shiga Toxin (Stx) Classification, Structure, and Function. , 2014, Microbiology spectrum.

[6]  A. Corti,et al.  Peptide-Mediated Targeting of Cytokines to Tumor Vasculature: The NGR-hTNF Example , 2013, BioDrugs.

[7]  Jyothi Thundimadathil,et al.  Cancer Treatment Using Peptides: Current Therapies and Future Prospects , 2012, Journal of amino acids.

[8]  Zhi Jie Li,et al.  Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery , 2012, Journal of Translational Medicine.

[9]  A. Khaled,et al.  The use of therapeutic peptides to target and to kill cancer cells. , 2012, Current medicinal chemistry.

[10]  M. Shokrgozar,et al.  Assessment of selective toxicity of insect cell expressed recombinant A1-GMCSF protein toward GMCSF receptor bearing tumor cells , 2012, Research in pharmaceutical sciences.

[11]  K. Sandvig,et al.  Shiga toxins. , 2012, Toxicon : official journal of the International Society on Toxinology.

[12]  N. Engedal,et al.  Shiga toxin and its use in targeted cancer therapy and imaging , 2010, Microbial biotechnology.

[13]  Rongsheng E. Wang,et al.  Development of NGR peptide-based agents for tumor imaging. , 2011, American journal of nuclear medicine and molecular imaging.

[14]  H. Buhr,et al.  Specific targeting of tumor endothelial cells by a shiga-like toxin-vascular endothelial growth factor fusion protein as a novel treatment strategy for pancreatic cancer. , 2010, Neoplasia.

[15]  V. L. Tesh.  Induction of apoptosis by Shiga toxins. , 2010, Future microbiology.

[16]  W. Arap,et al.  The neovasculature homing motif NGR: more than meets the eye. , 2008, Blood.

[17]  S. Rabbani,et al.  Binding and internalization of NGR-peptide-targeted liposomal doxorubicin (TVT-DOX) in CD13-expressing cells and its antitumor effects , 2007, Anti-cancer drugs.

[18]  J. Meng,et al.  High-yield expression, purification and characterization of tumor-targeted IFN-alpha2a. , 2007, Cytotherapy.

[19]  S. Bouzari,et al.  Selective cytotoxicity of recombinant STXA1-GM-CSF protein in hematopoetic cancer cells , 2006, Cell Biology and Toxicology.

[20]  G. Colombo,et al.  Immunogenic and structural properties of the Asn-Gly-Arg (NGR) tumor neovasculature-homing motif. , 2006, Molecular immunology.

[21]  A. Corti,et al.  Synergistic Antitumor Activity of Cisplatin, Paclitaxel, and Gemcitabine with Tumor Vasculature-Targeted Tumor Necrosis Factor-α , 2006, Clinical Cancer Research.

[22]  R. Cherla,et al.  Shiga Toxin 1 Induces Apoptosis in the Human Myelogenous Leukemia Cell Line THP-1 by a Caspase-8-Dependent, Tumor Necrosis Factor Receptor-Independent Mechanism , 2005, Infection and Immunity.

[23]  A. Corti,et al.  Targeted delivery of IFNgamma to tumor vessels uncouples antitumor from counterregulatory mechanisms. , 2005, Cancer research.

[24]  A. Corti.  Strategies for improving the anti-neoplastic activity of TNF by tumor targeting. , 2004, Methods in molecular medicine.

[25]  Wadih Arap,et al.  Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia, and myeloid cells. , 2002, Cancer research.

[26]  Fulvio Magni,et al.  Enhancement of tumor necrosis factor α antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13) , 2000, Nature Biotechnology.

[27]  Erkki Ruoslahti,et al.  Anti-cancer activity of targeted pro-apoptotic peptides , 1999, Nature Medicine.