Shiga-like toxin-VEGF fusion proteins are selectively cytotoxic to endothelial cells overexpressing VEGFR-2.
Growing endothelial cells at sites of angiogenesis may be more sensitive than quiescent endothelial cells to toxin-VEGF fusion proteins, because they express higher numbers of VEGF receptors. We have constructed, expressed and purified a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF(121) (SLT-VEGF/L). SLT-VEGF/L inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation in cells overexpressing VEGFR-2 indicating that both SLT and VEGF moieties are properly folded in the fusion protein. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2-3x10(5) VEGFR-2/cell with an IC(50) of 0.1 nM, and rapidly induces apoptosis at concentrations >1 nM. Similar results are observed with human transformed embryonic kidney cells, 293, engineered to express 2.5x10(6) VEGFR-2/cell. In contrast, SLT-VEGF/L does not affect three different types of endothelial cells (PAE/KDR(low), HUVE, MS1) expressing between 5x10(3) and 5x10(4) VEGFR-2/cell, and quiescent endothelial cells overexpressing VEGFR-2. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but occur without significant inhibition of protein synthesis. The selective cytotoxicity of SLT-VEGF proteins against growing endothelial cells overexpressing VEGFR-2 suggests that they may be useful in targeting similar cells at sites of angiogenesis.
Backer
M V
MV
SibTech Inc., 705 North Mountain Road, Newington, CT 06111, USA. mbacker@sibtech.com
Budker
V G
VG
Backer
J M
JM
D000818:Animals; D015153:Blotting, Western; D053178:Caspase 6; D020169:Caspases / Q000096:biosynthesis; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D016228:Endothelial Growth Factors / Q000008:administration & dosage / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008222:Lymphokines / Q000008:administration & dosage / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis*; D017978:Receptors, Growth Factor / Q000096:biosynthesis*; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000494:pharmacology; D022621:Shiga Toxin / Q000008:administration & dosage / Q000494:pharmacology*; D013552:Swine; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D022621:Shiga Toxin; D004247:DNA; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; C505514:CASP6 protein, human; D053178:Caspase 6; D020169:Caspases