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-(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...

Cyclo19,31[D-Cys19]-uPA19-31 Is a Potent Competitive Antagonist of the Interaction of Urokinase-Type Plasminogen Activator with Its Receptor (CD87)

“…Similarly, reduction of growth of primary human breast carcinoma in immunodeficient mice was observed by administration of a fusion protein consisting of human or murine uPA fused to the Fc portion of human IgG (Tressler et al, 1999). A bifunctional inhibitor, consisting of uPA fused to domain II of the urinary trypsin inhibitor (UTI), suppressed experimental tumor invasion and metastasis by blocking uPA/uPAR-interaction via the amino-terminal fragment of uPA within this chimeric molecule and by inhibition of plasmin activity via UTI (Kobayashi et al, 1998). The application of large molecules to treat cancer patients appears rather difficult and depends on sophisticated, e. g. viral, delivery systems.…”

“…Recently, a non-competitive, allosteric antagonist of prouPA/uPAR-interaction (Å6-peptide) derived from a non-receptor binding region of uPA ) has been identified (Guo et al, 2000). As demonstrated in various experimental in vivo model systems for competitive inhibitors of uPA/uPAR-interaction (Crowley et al, 1993;Min et al, 1996;Kobayashi et al, 1998;Li et al, 1998;Tressler et al, 1999;Krüger et al, 2000;Lutz et al, 2001), administration of this non-competitive antagonist to tumor-bearing animals did also inhibited tumor growth and metastasis.…”

Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

“…TGF-␤-stimulated uPA protein expression was not impaired by S ODNs (lanes 4 and 7) or iAS ODNs (lanes 5 and 8). appropriate candidate, because AP-1 is activated by PI3K and MAPK and has also been involved in the induction of uPA (25). Further, Akt has been shown to up-regulate NF-B transcriptional activity in a variety of cells (26); we suspected that the up-regulation of uPA mRNA in HRA cells by Akt might occur via increased NF-B transcriptional activity.…”

A Novel Type of Bifunctional Inhibitor Directed against Proteolytic Activity and Receptor/Ligand Interaction

“…The arrow indicates injection of suPAR, the arrowhead, the end of injection. C, injection (arrow) of different concentrations of recombinant suPAR (10,20,30, and 40 g/ml, curves 1-4, respectively) to react with immobilized papain-chCys-uPA- (19 -31). From these curves, the binding constant for suPAR binding to chCys-uPA-(19 -31) was calculated using the BIAcore evaluation software.…”

“…Following intensive washing without decrease in the resonance signal, the ternary papain-cystatin (variant)-uPAR complex was formed by injecting 260 l of different concentrations of recombinant suPAR from Chinese hamster ovary cells (10,20,30, and 40 g/ml, respectively) (21). Regeneration conditions were not found, because it was not possible to dissociate uPAR and to restore the papain-cystatin variant complexes without loss of their uPAR binding capacity.…”