Mechanistic studies of a novel human fusion toxin composed of vascular endothelial growth factor (VEGF)121 and the serine protease granzyme B: directed apoptotic events in vascular endothelial cells.
The serine protease granzyme B (GrB; 25 kDa) is capable of inducing apoptosis through both caspase-dependent and caspase-independent mechanisms. We designed a novel vascular-targeting fusion construct designated as GrB/vascular endothelial growth factor (VEGF)121, which is composed of a non-heparin-binding isoform of VEGF and the proapoptotic pathway enzyme GrB fused via a short, flexible tether (G4S). The chimeric fusion gene was then cloned into a bacterial vector, and the protein was expressed in Escherichia coli and purified by nickel-NTA metal affinity chromatography. Western blotting confirmed incorporation of both VEGF121 and GrB proteins into the construct. GrB/VEGF121 specifically bound (ELISA) to porcine aortic endothelial (PAE)/FLK-1 cells overexpressing the FLK-1/KDR receptor but not to cells overexpressing the FLT-1 receptor. Immunofluoresence studies showed that the GrB moiety of GrB/VEGF121 was delivered efficiently and rapidly into the cytosol of PAE/FLK-1 cells but not into that of PAE/FLT-1 cells after 4 h treatment with GrB/VEGF121. Treatment of cells with GrB/VEGF121 showed that the IC50 was approximately 10 nM against PAE/FLK-1 cells; however, there were no cytotoxic effects observed on PAE/FLT-1 cells at doses up to 200 nM. GrB/VEGF121 induced apoptotic events specifically on PAE/FLK-1 as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, DNA laddering, and cytochrome c release from mitochondria. In addition, the fusion construct mediated the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase in target endothelial cells within 4 h after treatment. In conclusion, delivery of the human proapoptotic pathway enzyme GrB to tumor vascular endothelial cells or to tumor cells may have significant therapeutic potential and represents a potent new class of targeted therapeutic agents with a unique mechanism of action.
Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA.
Yuying
Y
Liu
Lawrence H
LH
Cheung
Philip
P
Thorpe
Michael G
MG
Rosenblum
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D053804:Granzymes; D006801:Humans; D020287:In Situ Nick-End Labeling; D020128:Inhibitory Concentration 50; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000737:chemistry*; D013552:Swine; D013997:Time Factors; D014118:Toxins, Biological / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry*; D051028:bcl-2-Associated X Protein
D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D014118:Toxins, Biological; D042461:Vascular Endothelial Growth Factor A; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases