A bifunctional hybrid molecule of the amino-terminal fragment of urokinase and domain II of bikunin efficiently inhibits tumor cell invasion and metastasis.
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.
Kobayashi
H
H
Department of Obstetrics and Gynecology, Hamamatsu University, Shizuoka, Japan.
Sugino
D
D
She
M Y
MY
Ohi
H
H
Hirashima
Y
Y
Shinohara
H
H
Fujie
M
M
Shibata
K
K
Terao
T
T
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*
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