In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells

Document Type : Original Article

Authors
Jahangir Langari 1
Morteza Karimipoor 1
Majid Golkar 2
Hossein Khanahmad 3
Sirous Zeinali 1
Skandar Omidinia 4
Reza Ahangari Cohan 5
Mahdi Behdani 1
Jalal Babaie 2
Roghaye Arezumand 1
Reza Moazami 1
1 Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
2 Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran
3 Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan; Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommunicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran
5 Department of Pilot Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran
Abstract
Background: Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. Materials and Methods: We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione. Results: The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC. Conclusion: The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

Keywords

1.
Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:10931-4.  Back to cited text no. 1
[PUBMED]    
2.
Vincenti V, Cassano C, Rocchi M, Persico G. Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3. Circulation 1996;93:1493-5.  Back to cited text no. 2
[PUBMED]    
3.
Safran M, Kaelin WG Jr. HIF hydroxylation and the mammalian oxygen-sensing pathway. J Clin Invest 2003;111:779-83.  Back to cited text no. 3
[PUBMED]    
4.
Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 2000;60:203-12.  Back to cited text no. 4
[PUBMED]    
5.
Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9-22.  Back to cited text no. 5
[PUBMED]    
6.
Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 1992;267:26031-7.  Back to cited text no. 6
[PUBMED]    
7.
Brown LF, Berse B, Jackman RW, Tognazzi K, Manseau EJ, Dvorak HF, et al. Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am J Pathol 1993;143:1255-62.  Back to cited text no. 7
[PUBMED]    
8.
Brown JM, Giaccia AJ. The unique physiology of solid tumors: Opportunities (and problems) for cancer therapy. Cancer Res 1998;58:1408-16.  Back to cited text no. 8
[PUBMED]    
9.
Wedge SR, Kendrew J, Hennequin LF, Valentine PJ, Barry ST, Brave SR, et al. AZD2171: A highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res 2005;65:4389-400.  Back to cited text no. 9
[PUBMED]    
10.
Kreitman RJ. Immunotoxins for targeted cancer therapy. AAPS J 2006;8:532-51.  Back to cited text no. 10
    
11.
Allured VS, Collier RJ, Carroll SF, McKay DB. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci U S A 1986;83:1320-4.  Back to cited text no. 11
[PUBMED]    
12.
Kreitman RJ, Pastan I. Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem J 1995;307:29-37.  Back to cited text no. 12
[PUBMED]    
13.
Kounnas MZ, Morris RE, Thompson MR, FitzGerald DJ, Strickland DK, Saelinger CB. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem 1992;267:12420-3.  Back to cited text no. 13
[PUBMED]    
14.
McKee ML, FitzGerald DJ. Reduction of furin-nicked Pseudomonas exotoxin A: An unfolding story. Biochemistry 1999;38:16507-13.  Back to cited text no. 14
[PUBMED]    
15.
Carroll SF, Collier RJ. Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J Biol Chem 1987;262:8707-11.  Back to cited text no. 15
[PUBMED]    
16.
Kondo T, FitzGerald D, Chaudhary VK, Adhya S, Pastan I. Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain. J Biol Chem 1988;263:9470-5.  Back to cited text no. 16
[PUBMED]    
17.
Pastan I. Immunotoxins containing Pseudomonas exotoxin A: A short history. Cancer Immunol Immunother 2003;52:338-41.  Back to cited text no. 17
[PUBMED]    
18.
Benedict WF, Baker MS, Haroun L, Choi E, Ames BN. Mutagenicity of cancer chemotherapeutic agents in the Salmonella/microsome test. Cancer Res 1977;37:2209-13.  Back to cited text no. 18
    
19.
Khandare JJ, Minko T. Antibodies and peptides in cancer therapy. Crit Rev Ther Drug Carrier Syst 2006;23:401-35.  Back to cited text no. 19
    
20.
Jain RK. Delivery of molecular and cellular medicine to solid tumors. J Control Release 1998;53:49-67.  Back to cited text no. 20
    
21.
Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994;367:576-9.  Back to cited text no. 21
    
22.
Wedge SR, Ogilvie DJ, Dukes M, Kendrew J, Curwen JO, Hennequin LF, et al. ZD4190: An orally active inhibitor of vascular endothelial growth factor signaling with broad-spectrum antitumor efficacy. Cancer Res 2000;60:970-5.  Back to cited text no. 22
    
23.
Oku T, Tjuvajev JG, Miyagawa T, Sasajima T, Joshi A, Joshi R, et al. Tumor growth modulation by sense and antisense vascular endothelial growth factor gene expression: Effects on angiogenesis, vascular permeability, blood volume, blood flow, fluorodeoxyglucose uptake, and proliferation of human melanoma intracerebral xenografts. Cancer Res 1998;58:4185-92.  Back to cited text no. 23
    
24.
Prewett M, Huber J, Li Y, Santiago A, O'Connor W, King K, et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res 1999;59:5209-18.  Back to cited text no. 24
    
25.
Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, et al. VEGF-Trap: A VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A 2002;99:11393-8.  Back to cited text no. 25
    
26.
Kreitman RJ. Immunotoxins for targeted cancer therapy. AAPS J 2006;8:E532-51.  Back to cited text no. 26
    
27.
Grünewald FS, Prota AE, Giese A, Ballmer-Hofer K. Structure-function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling. Biochim Biophys Acta 2010;1804:567-80.  Back to cited text no. 27
    
28.
Wild R, Dhanabal M, Olson TA, Ramakrishnan S. Inhibition of angiogenesis and tumour growth by VEGF121-toxin conjugate: Differential effect on proliferating endothelial cells. Br J Cancer 2000;83:1077-83.  Back to cited text no. 28
    
29.
Pastan II, Kreitman RJ. Immunotoxins for targeted cancer therapy. Adv Drug Deliv Rev 1998;31:53-88.  Back to cited text no. 29
    
30.
Frankel AE, Neville DM, Bugge TA, Kreitman RJ, Leppla SH. Immunotoxin therapy of hematologic malignancies. Semin Oncol 2003;30:545-57.  Back to cited text no. 30
    
31.
Siegall CB, Xu YH, Chaudhary VK, Adhya S, Fitzgerald D, Pastan I. Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin. FASEB J 1989;3:2647-52.  Back to cited text no. 31
    
32.
Pelham HR. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol 1989;5:1-23.  Back to cited text no. 32
    
33.
Seetharam S, Chaudhary VK, FitzGerald D, Pastan I. Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL. J Biol Chem 1991;266:17376-81.  Back to cited text no. 33
    
34.
Zhang J, Liu S, Shang Z, Shi L, Yun J. Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma. Theor Biol Med Model 2012;9:38.  Back to cited text no. 34
    
35.
Angov E. Codon usage: Nature's roadmap to expression and folding of proteins. Biotechnol J 2011;6:650-9.  Back to cited text no. 35
    
36.
Tegel H, Tourle S, Ottosson J, Persson A. Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta (DE3). Protein Expr Purif 2010;69:159-67.  Back to cited text no. 36
    
37.
Petsch D, Anspach FB. Endotoxin removal from protein solutions. J Biotechnol 2000;76:97-119.  Back to cited text no. 37
    
38.
Backer MV, Budker VG, Backer JM. Shiga-like toxin-VEGF fusion proteins are selectively cytotoxic to endothelial cells overexpressing VEGFR-2. J Control Release 2001;74:349-55.  Back to cited text no. 38
    
39.
Veenendaal LM, Jin H, Ran S, Cheung L, Navone N, Marks JW, et al.In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc Natl Acad Sci U S A 2002;99:7866-71.  Back to cited text no. 39
    
40.
Arora N, Masood R, Zheng T, Cai J, Smith DL, Gill PS. Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells. Cancer Res 1999;59:183-8.  Back to cited text no. 40
    
41.
Couffinhal T, Kearney M, Witzenbichler B, Chen D, Murohara T, Losordo DW, et al. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol 1997;150:1673-85.  Back to cited text no. 41