title: Preparation and functional characterization of human vascular endothelial growth factor-melittin fusion protein with analysis of the antitumor activity in vitro and in vivo.
abstract: Vascular endothelial growth factor and its tyrosine kinase receptors have been identified as key mediators of the regulation of pathologic blood vessel growth and maintenance in the promotion of angiogenesis and tumor growth. Therefore, an alternative approach to destroying tumor endothelium would be to make this tissue particularly sensitive to VEGF-mediated drug delivery. To verify this hypothesis, we generated a protein containing VEGF165 fused to melittin. Melittin is a small linear peptide composed of 26 amino acid residues that can exert toxic or inhibitory effects on many types of tumor cells. This protein is a cytolytic peptide that attacks lipid membranes, leading to significant toxicity. In the present study, the Pichia pastoris expression system was used to express the fusion protein. Under optimal conditions, stable VEGF165-melittin production was achieved using a series of purification steps. The activity of VEGF165-melittin fusion protein was compared with melittin for its ability to suppress the growth of tumor cell line in vitro. The fusion toxin selectively inhibited growth of human hepatocellular carcinoma HepG-2 cell line with high expression of VEGFR-2. We found that sensitivity of VEGFR-2 transfected 293 cells to VEGF165-melittin enhanced as the cellular VEGFR-2 density increased. In an in vivo initial experiment, the fusion protein inhibited tumor growth in xenografts assays. Furthermore, successful expression and characterization of the fusion protein demonstrated its efficacy for use as a novel treatment strategy for cancer.
authors.affiliation: Department of Biotechnology, College of Life Science and Bio-pharmaceuticals, Guangdong Pharmaceutical University, Guangzhou, Guangdong, P.R. China.
authors.forename: Dingding
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Biotechnology, College of Life Science and Bio-pharmaceuticals, Guangdong Pharmaceutical University, Guangzhou, Guangdong, P.R. China.
authors.forename: Lili
authors.initials: L
authors.lastname: Hu
authors.affiliation: Department of Regenerative Medicine, College of Pharmacy, Jilin University, Changchun, Jilin, P.R. China.
authors.forename: Manman
authors.initials: M
authors.lastname: Su
authors.affiliation: Guangdong Provincial Key Laboratory of Bio-engineering Medicine (National Engineering Research Centre of Genetic Medicine), Guangzhou, Guangdong, P.R. China.
authors.forename: Ju
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, The Second Clinical Hospital, Jilin University, Changchun, Jilin, P.R. China.
authors.forename: Tianmin
authors.initials: T
authors.lastname: Xu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D008555:Melitten / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D008555:Melitten; D040301:Vascular Endothelial Growth Factor Receptor-2