Inhibitory effect of a conjugate between human urokinase and urinary trypsin inhibitor on tumor cell invasion in vitro.
Proteolytic enzymes such as urokinase-type plasminogen activator (uPA), plasmin, and collagenase mediate proteolysis by a variety of tumor cells. uPA secreted by tumor cells can be bound to a cell surface receptor via a growth factor-like domain within the amino-terminal fragment (ATF) of the uPA molecule with high affinity. Urinary trypsin inhibitor (UTI) efficiently inhibits the soluble and the tumor cell-surface receptor-bound plasmin and subsequently reduces tumor cell invasion and the formation of metastasis. The anti-invasive effect is dependent on the anti-plasmin activity of the UTI molecule, domain II in particular. We synthesized a conjugate between ATF of human uPA and a native UTI molecule or domain II of UTI (HI-8). The effect of the conjugates (ATF.UTI or ATF.HI-8) on tumor cell invasion in vitro was investigated. ATF.UTI and ATF.HI-8 bound to U937 cells in a rapid, saturable, dose-dependent, and reversible manner. A large part of receptor-bound ATF-UTI and ATF.HI-8 remains on the cell surface for at least 5 h at 37 degrees C. Inhibition of tumor cell-surface receptor-bound plasmin by ATF.UTI and ATF.HI-8 was markedly enhanced when compared with tumor cells treated either with ATF, UTI, or HI-8. Results of a cell invasion assay showed that ATF.UTI and ATF.HI-8 is very effective at targeting HI-8 specifically to uPA receptor-expressing tumor cells, whereas tumor cells devoid of uPA receptor may be less affected by the conjugates. Our results indicate that cell surface uPA and plasmin activity is essential to the invasive process and that the conjugates exhibit plasmin inhibition to the close environment of the cell surface and subsequently inhibit the tumor cell invasion through Matrigel in an in vitro invasion assay.
Kobayashi
H
H
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan.
Gotoh
J
J
Hirashima
Y
Y
Fujie
M
M
Sugino
D
D
Terao
T
T
D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D005341:Fibrinolysin / Q000037:antagonists & inhibitors; D006023:Glycoproteins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D011956:Receptors, Cell Surface / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology*
D006023:Glycoproteins; D011956:Receptors, Cell Surface; D014361:Trypsin Inhibitors; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; C028665:urinastatin