111In- or 99mTc-labeled recombinant VEGF bioconjugates: in vitro evaluation of their cytotoxicity on porcine aortic endothelial cells overexpressing Flt-1 receptors.
BACKGROUND
The aims of this study were to (a) synthesize and characterize a novel vascular endothelial growth factor (VEGF-2K) recombinant protein expressed in Pichia pastoris and (b) compare its cytotoxicity when labeled with the Auger electron emitter (111)In or (99m)Tc, both of which are in the nanometer-micrometer range, toward porcine aortic endothelial (PAE) cells transfected with the flt-1 gene to overexpress Flt-1 receptors (PAE-Flt-1).


METHODS
The gene for the VEGF(165) isoform was fused to a sequence encoding an extended flexible peptide (KGGGGSK) with two accessible lysines for preferential derivatization with diethylenetriaminepentaacetic acid (DTPA) for complexing (111)In and a sequence for a His(6) affinity tag that bound the [(99m)Tc(CO)(3)(H(2)O)(3)](+) tricarbonyl complex. P. pastoris strain KM71H was transfected with the recombinant gene, the VEGF-2K protein expressed with methanol induction, and then purified by metal-affinity chromatography. VEGF-2K was modified with 13-mer peptides [CGYGPKKKRKVGG] containing the nuclear localization sequence (NLS) of SV-40 large T-antigen (underlined) to promote nuclear uptake following its receptor-mediated internalization.


RESULTS
(99m)Tc-DTPA-VEGF-2K bound strongly and preferentially to PAE-Flt-1 cells compared with non-transfected PAE cells, but NLS modification diminished the ratio of PAE-Flt-1 to PAE binding to 2.3-fold. Nuclear accumulation of (99m)Tc-labeled DTPA-VEGF-2K was not enhanced by NLS modification but was enhanced by 1.5-fold for (111)In-DTPA-VEGF-2K-NLS. However, confocal microscopy revealed intranuclear distribution of DTPA-VEGF-2K-NLS, whereas DTPA-VEGF-2K distribution was mainly perinuclear. (111)In-DTPA-VEGF-2K-NLS was the most cytotoxic to PAE-Flt-1 cells, reducing their clonogenic survival by 4-fold. (111)In-DTPA-VEGF-2K, (99m)Tc-DTPA-VEGF-2K or (99m)Tc-DTPA-VEGF-2K-NLS had less effect on the clonogenic survival of PAE-Flt-1 or PAE cells. The strong cytotoxicity of (111)In-DTPA-VEGF-2K-NLS toward PAE-Flt-1 cells was associated with a 27-fold increase in nuclear foci of immunofluorescence for phosphorylated histone-2AX corresponding to sites of unrepaired DNA double-strand breaks. Monte Carlo modeling revealed that radionuclide decay in the nucleus would provide a 5-fold higher radiation absorbed dose for (111)In than for (99m)Tc, explaining their differential cytotoxicity, and intranuclear localization would amplify the radiation dose delivered by (111)In by 3-fold, explaining the greater potency of (111)In-DTPA-VEGF-2K-NLS compared with (111)In-DTPA-VEGF-2K.


CONCLUSIONS
We conclude that targeted Auger electron radiotherapy aimed at Flt-1 receptors is a promising strategy that should be explored further for treatment of tumors in which this angiogenic pathway is up-regulated. (111)In is a more cytotoxic radionuclide than (99m)Tc, unless DNA delivery can be achieved, due to the short range of the electrons emitted.
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
Conrad
C
Chan
Zhongli
Z
Cai
Ruifen
R
Su
Raymond M
RM
Reilly
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D015870:Gene Expression; D006801:Humans; D007205:Indium Radioisotopes / Q000737:chemistry*; D008954:Models, Biological; D009010:Monte Carlo Method; D004369:Pentetic Acid / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000235:genetics; D011829:Radiation Dosage; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D013552:Swine; D013667:Technetium / Q000737:chemistry*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics* / Q000378:metabolism
D007205:Indium Radioisotopes; D010455:Peptides; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D013667:Technetium; D004369:Pentetic Acid; D040281:Vascular Endothelial Growth Factor Receptor-1