title: A bifunctional hybrid molecule of the amino-terminal fragment of urokinase and domain II of bikunin efficiently inhibits tumor cell invasion and metastasis.
abstract: Urinary trypsin inhibitor (UTI) inhibits efficiently tumor cell invasion and the formation of metastasis. The anti-metastatic effect is dependent on the COOH-terminal domain II of UTI [UTI-(78-136)-peptide]. To develop a molecule that binds with high affinity to the urokinase (uPA) receptor (uPAR) on tumor cell surfaces, a bifunctional hybrid molecule [uPA-(1-134)-UTI-(78-136)] consisting of the uPAR-binding NH2-terminal fragment [UTI-(78-136)-peptide] of uPA at the NH2-terminus of UTI-(78-136)-peptide was produced in Escherichia coli by genetic engineering. The purified hybrid protein inhibited trypsin and plasmin 2-3-fold less effectively than UTI-(78-136)-peptide and was found to bind to human tumor cells via uPAR, which was confirmed by cell binding and competition experiments. Using a modified Boyden chamber and an artificial basement membrane, Matrigel, it was found that the hybrid protein is very effective at inhibiting invasion by uPAR-expressing human tumor cells. Sensitivities of tumor cells towards the anti-invasive effect of uPA-(1-134)-UTI-(78-136) correlated with the density of uPAR on human tumor cells. Furthermore, in the spontaneous metastasis model, the hybrid protein inhibited the formation of lung and/or lymphatic metastasis by human ovarian carcinoma and choriocarcinoma cells. The hybrid protein was much more effective than uPA-(1-134)-peptide, UTI-(78-136)-peptide, or UTI. We conclude that this approach extends the possibility of applying recombinant protein for therapeutic use in inhibition of human tumor cell metastasis.
authors.affiliation: Department of Obstetrics and Gynecology, Hamamatsu University, Shizuoka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: D
authors.initials: D
authors.lastname: Sugino
authors.forename: M Y
authors.initials: MY
authors.lastname: She
authors.forename: H
authors.initials: H
authors.lastname: Ohi
authors.forename: Y
authors.initials: Y
authors.lastname: Hirashima
authors.forename: H
authors.initials: H
authors.lastname: Shinohara
authors.forename: M
authors.initials: M
authors.lastname: Fujie
authors.forename: K
authors.initials: K
authors.lastname: Shibata
authors.forename: T
authors.initials: T
authors.lastname: Terao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002822:Choriocarcinoma / Q000473:pathology; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000473:pathology / Q000517:prevention & control / Q000556:secondary*; D008562:Membrane Glycoproteins; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness / Q000517:prevention & control*; D009362:Neoplasm Metastasis / Q000517:prevention & control*; D010051:Ovarian Neoplasms / Q000473:pathology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C109148:SPINT2 protein, human; D015842:Serine Proteinase Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean; D014357:Trypsin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator