title: Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator.
abstract: The aberrant signaling activation of vascular endothelial growth factor receptor (VEGFR) and urokinase plasminogen activator (uPA) is a common characteristic of many tumors, including lung cancer. Accordingly, VEGFR and uPA have emerged as attractive targets for tumor. KDR (Flk-1/VEGFR-2), a member of the VEGFR family, has been recognized as an important target for antiangiogenesis in tumor. In this study, a recombinant immunotoxin was produced to specifically target KDR-expressing tumor vascular endothelial cells and uPA-expressing tumor cells and mediate antitumor angiogenesis and antitumor effect. Based on its potent inhibitory effect on protein synthesis, Luffin-beta (Lβ) ribosome-inactivating protein was selected as part of a recombinant fusion protein, a single-chain variable fragment against KDR (KDRscFv)-uPA cleavage site (uPAcs)-Lβ-KDEL (named as KPLK). The KDRscFv-uPAcs-Lβ-KDEL (KPLK) contained a single-chain variable fragment (scFv) against KDR, uPAcs, Lβ, and the retention signal for endoplasmic reticulum proteins KDEL (Lys-Asp-Glu-Leu). The KPLK-expressing vector was expressed in Escherichia coli, and the KPLK protein was isolated with nickel affinity chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis test demonstrated KPLK was effectively expressed. Result of in vitro cell viability assay on non-small cell lung cancer (NSCLC) H460 cell line (uPA-positive cell) revealed that KPLK significantly inhibited cell proliferation, induced apoptosis, and accumulated cells in S and G2/M phases, but the normal cell line (human submandibular gland cell) was unaffected. These effects were enhanced when uPA was added to digest KPLK to release Lβ. For in vivo assay of KPLK, subcutaneous xenograft tumor model of nude mice were established with H460 cells. Growth of solid tumors was significantly inhibited in animals treated with KPLK up to 21 days, tumor weights were decreased, and the expression of angiogenesis marker CD31 was downregulated; meanwhile, the apoptosis-related protein casspase-3 was upregulated. These results suggested that the recombinant KPLK may have therapeutic applications on tumors, especially uPA-overexpressing ones.
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Qiying
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Dehong
authors.initials: D
authors.lastname: Huang
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Xianjun
authors.initials: X
authors.lastname: Tang
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.affiliation: Department of Biotherapy and Hemo-oncology, Chongqing Cancer Institute, Chongqing 400030, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Xiao
authors.affiliation: Center of Endoscopy Examination & Therapy, Chongqing Cancer Institute, Chongqing 400030, China win16@sina.com.
authors.forename: Jianghong
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics / Q000494:pharmacology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D000912:Antibodies, Neoplasm; D000970:Antineoplastic Agents; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D057127:Single-Chain Antibodies; C069038:luffin-b protein, Luffa cylindrica; D040301:Vascular Endothelial Growth Factor Receptor-2; D014568:Urokinase-Type Plasminogen Activator
keywords: A single-chain variable fragment against KDR; CD31; KDEL (Lys-Asp-Glu-Leu); KPLK protein; Luffin-beta; apoptosis; casspase-3; cytotoxicity; non-small cell lung cancer cell; solid tumor; urokinase plasminogen activator