Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins.
Growing endothelial cells at the sites of angiogenesis express high numbers of VEGF receptors and therefore may be particularly sensitive to VEGF-mediated drug delivery. To test this hypothesis we have constructed a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF121 (SLT-VEGF/L). Wild-type A-subunit is a site-specific N-glycosidase of 28S rRNA that inhibits protein synthesis after being delivered into cells by separate cell-binding B-subunits. SLT-VEGF/L retains functional activities of both SLT and VEGF121 moieties, since it inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2.5 x 10(5) VEGFR-2/cell with an IC50 of 0.2 nM and rapidly induces apoptosis at concentrations >1 nM. We found that sensitivity of VEGFR-2 transfected PAE cells to SLT-VEGF/L declined as the cellular VEGFR-2 density decreased; PAE cells expressing 25000 VEGFR-2/cell were as sensitive as parental cells lacking the receptor. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but take place without significant inhibition of protein synthesis. Selective cytotoxicity of SLT-VEGF/L against growing endothelial cells overexpressing VEGFR-2 suggests that it may be useful in targeting similar cells at the sites of angiogenesis.
Backer
M V
MV
SibTech, Inc., Newington, Connecticut 06111, USA. mbacker@sibtech.com
Backer
J M
JM
D020533:Angiogenesis Inhibitors / Q000493:pharmacokinetics / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016503:Drug Delivery Systems; D016228:Endothelial Growth Factors / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D008222:Lymphokines / Q000378:metabolism; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000096:biosynthesis / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000633:toxicity*; D022622:Shiga Toxin 1 / Q000633:toxicity; D013552:Swine; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020533:Angiogenesis Inhibitors; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011500:Protein Synthesis Inhibitors; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor