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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-(78Ϫ136)-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-(1Ϫ134)-UTI-(78Ϫ136)] consisting of the uPAR-binding NH 2 -terminal fragment [UTI-(78Ϫ136)-peptide] of uPA at the NH 2 -terminus of UTI-(78Ϫ136)-peptide was produced in Escherichia coli by genetic engineering. The purified hybrid protein inhibited trypsin and plasmin 2Ϫ3-fold less effectively than UTI-(78Ϫ136)-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-(1Ϫ134)-UTI-(78Ϫ136) 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-(1Ϫ134)-peptide, UTI-(78Ϫ136)-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.Keywords : amino-terminal fragment; bikunin; hybrid protein; urinary trypsin inhibitor ; urokinase-type plasminogen activator.Tumor cells produce urokinase-type plasminogen activator cell surface is the preferred site for uPA-mediated protein degra-(uPA) in an enzymatically inactive proenzyme form (pro-uPA) dation [11]. Various different approaches to interfere with the [1, 2]. Secreted pro-uPA can immediately bind to the specific expression or reactivity of uPA or uPAR at the gene or protein uPA receptors (uPAR) on tumor cell surfaces with high affinity. level were tested successfully, including the use of antisense The uPAR specifically recognizes pro-uPA and active high-mo-oligonucleotides, antibodies, inhibitors and recombinant or synlecular-mass uPA via their growth-factor-like terminal domain. thetic uPA and uPAR analogues [12Ϫ15]. It has been reported uPAR is a glycoprotein of approximately 55 kDa, its affinity for that a soluble recombinant truncated form of the uPAR is able to uPA is high (0.2 nM), and the rate of dissociation is low [3, 4]. block binding of uPA to cell-surface-bound uPAR [12]. Another Receptor-bound uPA catalyzes the formation of plasmin on the feasible approach to be tested is the repression of uPA or uPAR cell surface to generate the proteolytic cascade that contributes synthesis by agents that block transcriptional and translational to the breakdown of basement membranes and the extracellular factors known to be involved in uPA/uPAR express...

Using the human ovarian cancer cell line HOC-1, we investigated the effects of urinary trypsin inhibitor (UTI) purified from human urine and its related synthetic peptides on the invasive potential of cancer cells in an in vitro assay. Invasiveness of tumor cells was determined using a modified Boyden chamber and a reconstituted basement membrane Matrigel. Three peptides (peptide 1, peptide 2, and peptide 3), representing sequences within UTI, were synthesized. HOC-1 cells showed detectable and reproducible levels of expression of surface urokinase-type plasminogen activator (uPA) and plasminogen/plasmin by cell ELISAs and enzyme assays. UTI was found to strongly inhibit plasmin and human leukocyte elastase (HLE). Peptide 2 and peptide 3 specifically inhibit HLE and plasmin activity respectively. Peptide 1 has essentially no inhibitory activity. Treatment with UTI and peptide 3 reduces the incidence of invasion, whereas peptide 1 and peptide 2 do not affect invasion. The inhibitory effect on cell invasion is dose-dependent. The proteolytic enzyme plasmin may be involved in human ovarian cancer invasion in extracellular matrix degradation, and the use of UTI and peptide 3 that inhibits plasmin specifically reduces invasion by tumor cells.

Urokinase‐type plasminogen activator (uPA), uPA receptors, and cathepsin B were quantitatcd by using an immunological method, enzyme‐linked immunosorbent assay, and amidolytic activity assays in 15 malignant and 10 benign epithelial ovarian tumors. The levels of uPA and uPA receptors, as well as cathepsin B, were found to be higher in membrane preparations obtained from malignant tumors than in those obtained from benign tumors. Acid‐treated membranes acquired the ability to bind uPA, indicating that uPA is bound to a specific surface receptor that is not completely saturated. Levels of single‐chain uPA (pro‐uPA) and high‐molecular‐weight uPA in membrane preparations were measured by immunoadsorbent‐amidolytic assay. The finding of a significant increase in amidolytic activity following activation of uPAs by plasmin suggested that less than half (30–40%) of all membrane immunoreactive uPAs is present in the enzymatically inactive pro‐uPA form. In the membranes of malignant tumors, levels of uPA receptor and cathepsin B did not vary with stage of disease. On the other hand, we found that the level of receptor‐bound uPA antigen/activity was significantly increased in advanced malignant tumors. Receptor‐bound uPA may play an important role in determining invasive potential of tumor cells. Since ovarian cancer cells produce both pro‐uPA and cathepsin B, the possibility of activation of tumor cell‐derived pro‐uPA by cellular protease cathepsin B must be considered.