title: Cytotoxic Effect of Immunotoxin Containing The Truncated Form of Pseudomonas Exotoxin A and Anti-VEGFR2 on HUVEC and MCF-7 Cell Lines.
abstract: OBJECTIVE
  Immunotoxins (ITs) have been developed for the treatment of cancer, and comprise of antibodies linked to toxins. Also vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and the blockade of VEGF receptor-2 (VEGFR2) inhibits angiogenesis and tumor growth. The aim of this study was to produce anti-VEGFR2/rPE (Pseudomonas exotoxin) 38 IT to test its cytotoxic activity and mechanism of action.
  
  
  METHODS
  In this basic research and experimental study, at first, DNA that encodes recombinant PE38 protein was inductively expressed in Escherichia coli (E.coli) and purified by nickel-sepharose chromatography and further analyzed by western blot. Then, for production of IT, rPE38 was chemically conjugated to anti- VEGFR2. The cytotoxicity response of IT treatment was evaluated by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test in Human Umbilical Vein Endothelial Cell (HUVEC) and Michigan Cancer Foundation-7 (MCF-7) (VEGFR2+) cell lines. The mechanism of IT cytotoxicity was observed by Annexin V staining and flow cytometry. Continuous variables were compared with the analysis of variance (ANOVA; for all groups). P values less than 0.05 were considered statistically significant.
  
  
  RESULTS
  SDS-PAGE showed 98% purity of rPE38 and IT. In vitro dose-dependent cytotoxicity assay demonstrated that anti-VEGFR2/PE38 is toxic to VEGFR2-positive cells. IT treatment significantly inhibited proliferation of HUVEC and MCF-7 in a VEGFR2-specific manner as compared with the control groups (p<0.05). Flow cytometry showed that the mechanism of IT induced cell death is mediated by apoptosis.
  
  
  CONCLUSIONS
  IT treatment also caused remarkable synergistic cytotoxicity characterized by decreased cell viability, and an increased apoptotic index by both anti-VEGFR2 and PE38. Thus these results raise the possibility of using anti-VEGFR2/PE38 IT for cancer therapy because nearly all tumors induce local angiogenesis with high VEGFR expression.
authors.affiliation: Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Elahe
authors.initials: E
authors.lastname: Safari
authors.affiliation: Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Zavaran Hosseini
authors.affiliation: Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Zuhair
authors.initials: Z
authors.lastname: Hassan
authors.affiliation: Department of Biochemistry, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.affiliation: Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mehdi Shafiee
authors.initials: MS
authors.lastname: Ardestani
authors.affiliation: Immunology Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Behzad
authors.initials: B
authors.lastname: Baradaran
keywords: Immunotoxin; Pseudomonas Exotoxin; VEGFR2