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Four synthetic peptides (residues 20-30 and 17-34) within the growth factor-like domain (GFD) of murine and human urokinase-type plasminogen activator (uPA) were examined to determine whether they inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. In an in vivo experimental metastasis assay, which determines mainly the later steps of the metastatic migration process (extravasation from the bloodstream and then growth into pulmonary tumor), none of the peptides introduced by i.v. single co-injection into syngeneic C57B1/6 mice inhibited pulmonary metastasis, when 3LL cells were pre-incubated with the peptides followed by i.v. co-injection of the peptide and cells. In addition, none of the peptides, when injected i.p. daily for 7 days after i.v. tumor cell inoculation, reduced the number of lung tumor colonies. In a second in vivo assay that measures metastasis from a primary tumor (spontaneous metastasis model), multiple i.p. injections of the mouse peptide 17-34 for 7 days after s.c. tumor cell inoculation significantly inhibited metastatic lung tumor colonization in a dose-dependent manner, whereas human peptide 17-34 had no effect. Mouse and human peptide 20-30 had no effect either. The inhibition of lung metastasis was not due to direct antitumor effects of mouse peptide 17-34. Our results indicate that occupation of uPA receptors on 3LL cells by the enzymatically inactive mouse peptide 17-34 or prevention of rebinding of uPA synthesized by tumor cells to their receptor specifically reduced tumor cell invasion and formation of metastasis and that uPA may regulate more efficiently the mechanism involved in the entry of tumor cells into vascular circulation than extravasation during the metastatic process.

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...

A purified human urinary trypsin inhibitor (UTI) and its related synthetic peptides were examined to determine whether they could inhibit production of experimental and spontaneous lung metastases by murine Lewis lung carcinoma (3LL) cells. Three peptides, peptide I, peptide 2 and peptide 3, representing the amino acid sequences within the UTI molecule, were synthesized. UTI and peptide 2 inhibited human leukocyte elastase (HLE). UTI and peptide 3 specifically inhibited human and murine plasmin activity. Peptide I had essentially no inhibitory activity. In an in vivo spontaneous metastasis model, multiple s.c. injections of UTI or peptide 3 for 7 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. UTI reduced lung tumor colonization more effectively than peptide 3. Peptides 1 and 2, however, did not affect the formation of lung metastasis. Inhibition of lung metastasis was not due to direct anti-tumor effects of UTI and peptide 3. In an in vivo experimental metastasis assay, multiple s.c. injections of UTI for 7 days after i.v. tumor cell inoculation inhibited metastatic lung tumor colonization, while peptide 3 did not affect metastasis. Peptides 1 and 2 did not affect the formation of lung metastasis. When examined with an in vitro assay system using a modified Boyden chamber, UTI and peptide 3 suppressed the invasion of tumor cells through Matrigel. UTI and peptide 3 inhibited neither cell proliferation nor the binding of tumor cells to Matrigel and showed no significant suppression of chemotactic migration of tumor cells to fibronectin. Our results suggest that UTI efficiently regulates the mechanism involved in not only the entry into vascular circulation of tumor cells (intravasation, though, at least in part, inhibition of the proteolytic enzyme plasmin) but also the extravasation step of the metastatic process.

SummaryA selective inhibitory antibody, raised against human high molecular weight urokinase-type plasminogen activator (HMW-uPA), was examined to determine whether it would inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. Polyclonal antibody to human uPA cross-reads with the murine uPA and inhibits murine uPA activity. When examined with an in vitro assay system using a modified Boyden chamber, the anti-catalytic IgG to uPA suppressed the invasion of tumor cells through Matrigel. Anti-uPA IgG inhibited neither the cell proliferation nor the binding of tumor cells to Matrigel, and showed no significant suppression of chemotactic migration of tumor cells to tibronectin. In an in vivo spontaneous metastasis assay, multiple subcutaneous (s. c.) injections of anti-uPA IgG (up to a concentration of 200 pg [ 500 inhibitory unit/mousc/day]) for 7 days immediately after s. c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of anti-uPA IgG. In an in vivo experimental metastasis assay, multiple s. c. injections of anti-uPA IgG for 7 days after intravenous (i. v.) tumor cell inoculation did not reduce the number of lung tumor colonies. These results suggest that uPA more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation, during the metastatic process.