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.
Chan
Conrad
C
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
Cai
Zhongli
Z
Su
Ruifen
R
Reilly
Raymond M
RM
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