Preferences help
enabled [disable] Abstract
Number of results
2009 | 56 | 1 | 115-124
Article title

Chimeric protein ABRaA-VEGF121 is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B16-F10 melanoma growth

Title variants
Languages of publication
It has been known that VEGF121 isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF121. Expression of the ABRaA-VEGF121 chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF121 preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (about 84 kDa homodimer and about 42 kDa monomer). ABRaA-VEGF121 is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF121 inhibited protein biosynthesis in a cell-free translation system. Preincubation of ABRaA-VEGF121 with antibody neutralizing the biological activity of human VEGF abolished the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF121 inhibits growth of B16-F10 murine melanoma tumors.
Physical description
  • Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
  • Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
  • Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
  • Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
  • Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
  • Arora N, Masood R, Zheng T, Cai J, Smith DL, Gill PS (1999) Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells. Cancer Res 59: 183-188.
  • Backer MV, Budker VG, Backer JM (2001) Shiga-like toxin-VEGF fusion proteins are selectively cytotoxic to endothelial cells overexpressing VEGFR-2. J Control Release 74: 349-355.
  • Brogi E, Schatteman G, Wu T, Kim EA, Varticovski L, Keyt B, Isner JM (1996) Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. J Clin Invest 97: 469-476.
  • Brown LF, Guidi AJ, Tognazzi K, Dvorak HF (1998) Vascular permeability factor/vascular endothelial growth factor and vascular stroma formation in neoplasia: insights from in situ hybridization studies. J Histochem Cytochem 46: 569-575.
  • Chow LP, Chou MH, Ho CY, Chuang CC, Pan FM, Wu SH, Lin JY (1999) Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina. Biochem J 338: 211-219.
  • Dickers KJ, Bradberry SM, Rice P, Griffiths GD, Vale JA (2003) Abrin poisoning. Toxicol Rev 22: 137-142.
  • Endo Y, Mitsui K, Motizuki M, Tsurugi K (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. J Biol Chem 262: 5908-5912.
  • Ferrara N, Kerbel RS (2005) Angiogenesis as a therapeutic target. Nature 438: 967-974.
  • Fodstad O, Olsnes S, Pihl A (1977) Inhibitory effect of abrin and ricin on the growth of transplantable murine tumors and of abrin on human cancers in nude mice. Cancer Res 37: 4559-4567.
  • Griffiths GD, Leek MD, Gee DJ (1987) The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissues and intestine. J Pathol 151: 221-229.
  • Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22: 346-353.
  • Hoeben A, Landuyt B, Highley MS, Wildiers H, van Oosterom AT, de Bruijn EA (2004) Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 56: 549-580.
  • Hwang KM, Foon KA, Cheung PH, Pearson JW, Oldham R (1984) Selective antitumor effect on L10 hepatocarcinoma cells of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen. Cancer Res 44: 4578-4586.
  • Kowanetz M, Ferrara N (2006) Vascular endothelial growth factor signaling pathways: therapeutic perspective. Clin Cancer Res 12: 5018-5022.
  • Liu Y, Cheung LH, Thorpe P, Rosenblum MG (2003) Mechanistic studies of a novel human fusion toxin composed of vascular endothelial growth factor (VEGF)121 and the serine protease granzyme B: Directed apoptotic events in vascular endothelial cells. Mol Cancer Ther 2: 949-959.
  • Mitrus I, Missol-Kolka E, Plucienniczak A, Szala S (2005) Tumour therapy with genes encoding apoptin and E4orf4. Anticancer Res 25: 1087-1090.
  • Narayanan S, Surolia A, Karande AA (2004) Ribosome-inactivating protein and apoptosis: abrin causes cell death via mitochondrial pathway in Jurkat cells. Biochem J 377: 233-240.
  • Ohba H, Moriwaki S, Bakalowa R, Yasuda S, Yamasaki N (2004) Plant-derived abrin-a induces apoptosis in cultured leukemic cell lines by different mechanisms. Toxicol Appl Pharmacol 195: 182-193.
  • Olsnes S, Pihl A (1973) Isolation and properties of abrin: a toxic protein inhibiting protein synthesis. Evidence for different biological functions of its two constituent-peptide chains. Eur J Biochem 35: 179-185.
  • Olsnes S, Fernandez-Puentes C, Carrasco L, Vazquez D (1975) Ribosome inactivation by the toxic lectins abrin and ricin. Kinetic of the enzymatic activity of the toxin A-chains. Eur J Biochem 60: 281-288.
  • Pan Q, Chatery Y, Wu Y, Rathore N, Tong RK, Peale F, Bagri A, Tessier-Lavigne M, Koch AW, Watts RJ (2007) Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting. J Biol Chem 282: 24049-24056.
  • Pastan I, Hassan RH, FitzGerald DJ, Kreitman RJ (2007) Immunotoxin treatment of cancer. Annu Rev Med 58: 221-237.
  • Qu X, Qing L (2004) Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation. J Biochem Mol Biol 37: 445-453.
  • Ran S, Huang X, Downes A, Thorpe PE (2003) Evaluation of novel antimouse VEGFR2 antibodies as potential antiangiogenic or vascular targeting agents for tumor therapy. Neoplasia 5: 297-307.
  • Shibuya M (2006) Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol 39: 469-478.
  • Shih S-F, Wu Y-H, Hung C-H, Yang H-Y, Lin J-Y (2001) Abrin triggers cell death by inactivation a thiol-specific antioxidant protein. J Biol Chem 276: 21870-21877.
  • Szala S (2004) Two-domain vascular disruptive agents in cancer therapy. Curr Cancer Drug Targets 4: 501-509.
  • Thorpe PE (2004) Vascular targeting agents as cancer therapeutics. Clin Cancer Res 10: 415-427.
  • Veenendaal LM, Jin H, Ran S, Cheung L, Navone N, Marks JW, Waltenberger J, Thorpe P, Rosenblum MG (2002) In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc Natl Acad Sci USA 99: 7866-7871.
  • Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K (2000) Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60: 203-212.
  • Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin C (1994) Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem 269: 26988-26995.
  • Wang L, Kang L, Hu T, Wang J (2004) Abrin-a A chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active. Biochimie 86: 327-333.
  • Wawrzynczak EJ, Cumber AJ, Henry RV, May J, Newell DR, Parnell GD, Worrell NR, Forrester JA (1990) Pharmacokinetics in the rat of a panel of immunotoxins made with abrin a chain, ricin a chain, gelonin, and momordin. Cancer Res 50: 7519-7526.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.