©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibitory Effect of a Conjugate between Human Urokinase and Urinary Trypsin Inhibitor on Tumor Cell Invasion in Vitro(*)

(Received for publication, December 12, 1994; and in revised form, January 25, 1995)

Hiroshi Kobayashi (1) Junko Gotoh (1) Yasuyuki Hirashima (1) Michio Fujie (2) Dan Sugino (3) Toshihiko Terao (1)

From the  (1)Department of Obstetrics and Gynecology and the (2)Equipment Center, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-31, Japan and the (3)Nissin Central Research Institute, Nissin Food Products Company, Ltd. Kusatsu, Shiga, 525, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (ATFbulletUTI or ATFbulletHI-8) on tumor cell invasion in vitro was investigated. ATFbulletUTI and ATFbulletHI-8 bound to U937 cells in a rapid, saturable, dose-dependent, and reversible manner. A large part of receptor-bound ATFbulletUTI and ATFbulletHI-8 remains on the cell surface for at least 5 h at 37 °C. Inhibition of tumor cell-surface receptor-bound plasmin by ATFbulletUTI and ATFbulletHI-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 ATFbulletUTI and ATFbulletHI-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.


INTRODUCTION

There are several reports that cell surface proteolytic enzymes (urokinase-type plasminogen activator (uPA), (^1)plasmin, certain cathepsins, or matrix metalloproteinases) are essential to the metastatic process of tumor cells(1, 2, 3, 4, 5) . Treatments with antibodies or specific inhibitors of uPA or plasmin have shown promise of inhibiting tumor cell invasion and metastasis(6, 7, 8, 9, 10) . In addition, competitive displacement of uPA from cellular uPA receptor (uPAR) decreases plasminogen-dependent degradation of extracellular matrix and basement membrane proteins by tumor cells, suggesting the prevention of metastasis by inhibition of the uPAR(11) . The number of lung tumor colonies following subcutaneous injection of tumor cells was decreased by preincubation of the tumor cells with anti-uPA antibody(12) . However, difficulties in clinical use of antibodies have been considered because antibodies induce severe complications that limit prolonged administration.

The receptor binding domain within the uPA molecule has been localized to amino acid residues 17-34 of the amino-terminal fragment (ATF) of uPA(13, 14) . ATF (residues 1-135 of the A-chain of uPA) comprising the so-called growth factor-like domain and the kringle, have been shown to bind to uPA receptors on normal and neoplastic cells with the same affinity as HMW-uPA. LMW-uPA, lacking ATF, does not bind.

In addition, occupation of uPA receptors on human ovarian cancer HOC-I cells or mouse Lewis lung carcinoma 3LL cells by enzymatically inactive human ATF or mouse peptide 17-34 specifically reduced tumor cell invasion, suggesting that prevention of rebinding of uPA synthesized by tumor cells to the receptor inhibits tumor cell metastasis(15) .

Recently we have reported that a highly purified urinary trypsin inhibitor (UTI) efficiently inhibits soluble and tumor cell-surface receptor-bound plasmin(16) . UTI inhibits not only tumor cell invasion in an in vitro assay but also production of experimental and spontaneous lung metastasis in an in vivo mouse model(16, 17) . The anti-invasive effect is dependent on the anti-plasmin activity of UTI. UTI peptide, which inhibits plasmin activity, synthesized by an automated peptide synthesizer showed mouse 3LL cell invasion inhibitory activity. UTI and the effective peptide inhibited tumor cell invasion through Matrigel. UTI did not inhibit tumor cell proliferation or the binding of the cells to Matrigel. Also, UTI did not inhibit chemotactic migration to fibronectin. It is likely that UTI acts as a protease inhibitor. Thus, conjugating a physiological plasmin inhibitor to ATF might target it to reduce cell-associated proteolytic activity to the close environment of the uPARexpressing tumor cell surface and subsequently may effectively inhibit tumor cell invasion in vitro.

In the present study, we show that ATFbulletUTI or ATFbulletHI-8 conjugates have high and specific ability to inhibit tumor cell invasion toward uPAR-bearing cells.


MATERIALS AND METHODS

Purification of the ATF Fraction of uPA

Crude ATF-fraction of uPA was first obtained as a side fraction by the preparation of LMW-uPA. Recombinant pro-uPA (10 mg, donated by Green Cross Ltd.) was incubated (6 h, 37 °C) with plasmin (10 µg) in 50 mM sodium phosphate buffer, 0.1 M NaCl, pH 6.0. Subsequently this mixture was adjusted to pH 8.0 and then further incubated for 12 h at 37 °C with 50 mM PBS, pH 8.0. The formed ATF was separated from LMW-uPA by ion-exchange column chromatography. 2 mg of crude ATF was subjected to cation-exchange chromatography (Mono S HR 5/5 column) applying in a HPLC system (Pep-S/C-18 Pharmacia Biotech Inc.) equilibrated with 50 mM sodium acetate buffer, pH 5.0. After washing with equilibrating buffer, bound proteins were eluted with a linear salt gradient (0.15-0.5 M NaCl) in 50 mM sodium acetate buffer, pH 5.0, for 30 min at 23 °C. Further purification was achieved by reverse-phase HPLC applying a C-18 column. Polypeptides were eluted with a linear gradient from 0 to 70% acetonitrile in 0.1% trifluoroacetic acid/H(2)O (Fig. 1, lane 1).


Figure 1: SDS-PAGE of the conjugates. LaneM contains molecular mass standards (106, 80, 50, 33, 28, and 19 kDa), with their approximate masses shown to the left. The purified proteins after SDS-PAGE in an 18% polyacrylamide gel are shown. Lane1, ATF; lane2, ATFbulletUTI; lane3, ATFbulletHI-8; lane4, UTI; lane5, HI-8.



Cells and Cultures

Promyeloid leukemia U937 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum. Cells were differentiated to macrophage-like cells by incubation in the above medium containing 1.0 µM phorbol 12-myristate 13-acetate (PMA, Sigma). Adherent cells, which were detached by 1 mM EDTA in PBS (5 min, 23 °C), were harvested by centrifugation, resuspended in PBS, and then used for the experiments(18) .

The choriocarcinoma cell line SMT-cc1 was established from a pulmonary metastatic region of human gestational choriocarcinoma(19) . The SMT-cc1 cells were maintained under an atmosphere of 5% CO(2) in RPMI 1640 (Nissui, Tokyo) medium supplemented with 10% heat-inactivated fetal calf serum. When the cells were confluent, they were removed from the flask by adding 4 mM EDTA and 0.01% DNase solution for 2 min at 23 °C, followed by gentle tapping of the tray against the bench. Complete detachment was confirmed by direct visualization. Single-cell suspensions were made by repeated pipetting through a 0.4-mm diameter canula. After this, the cells were kept in suspension at 4 °C by shaking. The cell viability was determined by trypan blue dye exclusion prior to use. SMT-cc1 cells were also used for binding studies.

Preparation of UTI and HI-8

A highly purified preparation of human UTI with a specific activity of 2,330 units/mg of protein and a molecular mass of 40 kDa (by SDS-PAGE) was kindly supplied by Mochida Pharmaceutical Co., Tokyo, Japan (Fig. 1, lane 4). The covalent structure of the polypeptide chain of the physiological inhibitor UTI has been already determined by Wachter E et al.(20) . A trypsin-Sepharose affinity column was used in order to obtain purified HI-8 (carboxyl terminus (domain II) of UTI). UTI bound to a trypsin-Sepharose column was treated with 80 mg/ml trypsin (37 °C, 16 h). After washing the column with PBS, trypsin-digested UTI bound to the column was eluted with 10 mM HCl containing 0.2 M NaCl. The major active fractions from a trypsin-Sepharose column were applied to a Sephadex G-100 gel chromatography. Each fraction was pooled, and amino-terminal amino acid sequencing was carried out to obtain purified HI-8 (Fig. 1, lane 5).

Preparation of the Conjugates

UTI (40 kDa) or HI-8 (8 kDa) was coupled to ATF by disulfide bonds introduced in both ATF and UTI or HI-8 by incubating with a 1.25-fold molar excess of N-succinimidyl-3-(2-pyridyldithio)propionate as described previously(21) . Derivatized UTI or HI-8 was then incubated with excess dithiothreitol (for 45 min at 23 °C), to reduce its disulfide groups, and was purified by gel filtration. Finally, a mixture of 10 mg of reduced UTI (or 2 mg of reduced HI-8) and 2 mg of N-succinimidyl-3-(2-pyridyldithio)propionate-derivatized ATF was incubated at 23 °C for 90 min to allow intermolecular conjugation. The molecular ratio between UTI and ATF (HI-8 and ATF) at the end of the reaction was approximately equal to 1. The mixture was then applied to a Mono S column preequilibrated with 25 mM sodium citrate, pH 5.5, and eluted with a gradient of 0-0.5 M NaCl in the same buffer. The fractions were analyzed by SDS-PAGE. The bands shown in Fig. 1(lanes 2 and 3) correspond to ATFbulletUTI and ATFbulletHI-8.

Specific Binding of the Conjugates to Tumor Cells

The conjugates (ATFbulletUTI and ATFbulletHI-8) were dialyzed against 0.1 M NaHCO(3), pH 9.0, for at least 4 h. The indicated amount of fluorescein isothiocyanate (FITC) in dimethyl sulfoxide was added in small aliquots to the conjugate under continuous rolling. The conjugate/FITC mixture was left for 3 h at 23 °C. A PD-10 column (Pharmacia Biotech Inc.) was used to purify FITC-conjugated ATFbulletUTI (FITCbulletATFbulletUTI) or FITC-conjugated ATFbulletHI-8 (FITCbulletATFbulletHI-8). U937 and SMT-cc1 cells were collected by centrifugation and suspended in PBS, 0.1% BSA. PMA-stimulated U937 cells and SMT-cc1 cells were treated with 50 mM glycine-HCl, 0.5 M NaCl, pH 3.0, to dissociate surface receptor-bound uPA, and then resuspended in HEPES buffer (0.5 M HEPES buffer, 0.1 M NaCl, pH 7.5). After centrifugation (1,200 rpm, 10 min, 4 °C), the cell pellet was adjusted to a density of 10^6 cells/ml in PBS, 0.1% BSA. The cells were suspended in 1.0 ml of PBS, 0.1% BSA containing FITC-conjugated ligands and rocked gently for 1 h at 4 °C. Cell-associated fluorescence (argon laser excitation at 488 nm) at real time (no wash) was immediately analyzed by EPICS PROFILE flow cytometer.

In addition, inhibition of binding of FITCbulletpro-uPA to U937 cells with the conjugates (ATFbulletUTI and ATFbulletHI-8) was tested. 10 nM FITCbulletpro-uPA in the presence of competitors (0-1,000 nM) was added to the acid-treated, PMA-stimulated cells (30 min, 4 °C). Nonspecific binding was determined in the presence of excess parent pro-uPA (500 nM). Cell-associated fluorescence was determined.

Phosphatidylinositol-specific Phospholipase C Treatment of Intact Tumor Cells

Incubation of the cells with 5 units/ml phosphatidylinositol specific-phospholipase C (Sigma) in culture medium without fetal calf serum was performed at 37 °C for 2 h with gentle shaking(22) . The cells were pelleted by centrifugation and resuspended in PBS, 0.1% BSA containing FITC-conjugated ligands (1 h, 4 °C). Cell-associated fluorescence was measured.

Inhibition of Proteolytic Activity

The inhibiting effects of UTI, HI-8, ATF, ATFbulletUTI, and ATFbulletHI-8 on several proteases (trypsin, plasmin, and human leukocyte elastase) were investigated. These agents (0-100 µM) were preincubated (5 min, 23 °C) with 2.0 µM plasmin (trypsin, elastase) in a 96-well microtiter plate, followed by the addition of chromogenic substrate S-2251 (S-2222, S-2484) in 50 mM Tris-HCl, 12 mM NaCl, and 0.1% BSA, pH 7.4 (5-30 min, 23 °C). Changes in absorbance at 405 nm were measured with an EIA reader.

In addition, monolayers of tumor cells were incubated with plasmin (10 µM, 4 h, 4 °C) and washed twice with cold PBS, 0.1% BSA. To investigate the inhibitory effects of UTI, HI-8, ATF, ATFbulletUTI, and ATFbulletHI-8 on the tumor cell surface receptor-bound plasmin, each agent (1.0 µM) was preincubated (0-60 min, 23 °C) with cell-associated plasmin in a 96-well microtiter plate. Then, they were incubated and washed twice with PBS, 0.1% BSA, followed by addition of S-2251 in 50 mM Tris-HCl, 12 mM NaCl, and 0.1% BSA, pH 7.4. The change in absorbance at 405 nm was measured in an EIA reader. When the receptor-bound plasmin was incubated with S-2251 in the absence of agents, the value at 405 nm was considered as 100%.

Internalization of the Conjugates in Tumor Cells

Active PAI-1 was purified from serum-free conditioned media of cultured human umbilical vein endothelial cells by affinity chromatography on Concanavalin A-Sepharose and immobilized anhydro-urokinase as described previously(23, 24) .

U937 cells were acid-washed and resuspended in RPMI 1640 containing 25 mM HEPES, pH 7.4, 2 mM glutamine, and 0.1% BSA. The cells were preincubated either with FITCbulletATFbulletUTI (20 nM), FITCbulletATFbulletHI-8 (20 nM), or FITCbulletHMW-uPA (10 nM) for 60 min at 4 °C. The cells were washed, resuspended, and then incubated in the presence or absence of active PAI-1 (0.5 µM) at 37 °C for different times. Free ligand was removed, and cells were washed. Immunofluorescence view of the internalization was analyzed by fluorescence microscopy.

Cell Invasion Assay

Polycarbonate filters (8-µm pore size; Costar) were coated with Matrigel and placed in a modified Boyden chamber. Blind well Boyden chambers were filled with 600 µl of RPMI 1640, 0.1% BSA in the lower compartment. Wells were pretreated with agents at various concentrations (100 µl, 1 h, 23 °C) in serum-free medium. Then, 100 µl of SMT-cc1 cell suspension (2 times 10^6 cells/ml) were placed in the upper compartment of the chamber. The plates were incubated at 37 °C in 5% CO(2) in air saturated with H(2)O for 12 h. Fibroblast-conditioned media or fibronectin were placed in the lower compartment as a source of chemoattractants. Under these conditions, very few cells died within the first 12 h, as measured by trypan blue dye exclusion in preliminary experiments. At the end of the incubation, the cells on the upper surface of the filter were completely removed with tissue paper, and the cells on the lower surface of the filter were stained and counted, as monitoredvisually under a light microscope. Each assay was carried out in triplicate(12, 15, 16) .

Cell attachment assay and the chemotactic assay were conducted as described previously(12, 15, 16) .


RESULTS

Dose-dependent Inhibition of Plasmin and Human Leukocyte Elastase by UTI, HI-8, ATF, and the Conjugates (ATFbulletUTI and ATFbulletHI-8)

Prior to examining the effects of the conjugate on cells, the ability of UTI, HI-8, ATF, ATFbulletUTI, and ATFbulletHI-8 to inhibit HMW-uPA was determined. HMW-uPA activity was not affected by increasing concentrations of these agents. To investigate the specificity of inhibition of plasmin and human leukocyte elastase by these agents, each protease was preincubated with different concentrations of the agents (Fig. 2). UTI is composed of two tandem Kunitz-type domains (amino terminus (domain I) and carboxyl terminus (domain II, HI-8) of UTI). UTI was found to be a strong inhibitor of plasmin and human leukocyte elastase. Efficient inhibition was observed at >1:1 molar ratio of UTI to proteases (20 min, 23 °C). HI-8 and the conjugate ATFbulletHI-8 efficiently inhibit plasmin activity, but they only inhibit human leukocyte elastase activity weakly. The anti-human leukocyte elastase activity of UTI, therefore, appears to be mainly confined to domain I. ATF showed essentially no inhibition. Comparing UTI (or HI-8) with the conjugate, free UTI (or HI-8) were at least 5-fold more effective in inhibiting plasmin, as assessed by the concentration giving 50% inhibition of the protease-inhibitor reaction using our colorimetric assay system.


Figure 2: Dose-dependent inhibition of plasmin (A) and human leukocyte elastase (B) by the conjugates. Plasmin (A) and human leukocyte elastase (B) (2.0 µM) were incubated with different concentrations of ATF (), UTI (circle), HI-8 (bullet), ATFbulletUTI (), or ATFbulletHI-8 (box). The remaining protease activity was assayed with the chromogenic substrates S-2251 and S-2484, respectively. Like anti-plasmin activity, antitrypsin activity of UTI is assigned to the domain II subunit (data not shown).



Despite the presence of plasmin inhibitors in the culture medium, cell-associated plasmin activity could subsequently be recovered from the cell layer by tranexamic acid eluates (data not shown). In the presence of UTI, HI-8, or the conjugates, however, plasmin activity could not be recovered, suggesting that cell-bound plasmin might be inhibited by UTI, HI-8, or the conjugates in a dose-dependent manner (data not shown). In the previous study, we confirmed that UTI has the ability to inhibit the tumor cell-associated plasmin(16, 17) . Titration of the SMT-cc1 cell-associated plasmin with each agent revealed that, comparing the conjugate with UTI, the conjugate gives rise to a more than 10-fold enhancement in the inhibiting action on cell-bound plasmin (Fig. 3). Also, compared with HI-8, ATFbulletHI-8 gives rise to a 3-fold enhancement.


Figure 3: Titration of the tumor cell surface receptor-bound plasmin with the conjugates. The hydrolysis of chromogenic substrate S-2251 catalyzed by receptor-bound plasmin was measured at 405 nm. The receptor-bound plasmin was preincubated with 0.2 µM UTI (circle), HI-8 (bullet), ATFbulletUTI (), or ATFbulletHI-8 (box) for various time intervals (0-60 min) at 23 °C. The value at 405 nm was defined as 100%, when the receptor-bound plasmin was incubated with S-2251 for 60 min at 23 °C in the absence of any agents. After incubation for 60 min, the cells were washed twice, and the amidolytic activity was determined.



Binding of the Conjugates to U937 Cell Receptors Assessed by FCM

Binding assays employing FCM were performed to examine the peptide sequence within ATF responsible for binding to uPAR on tumor cells. pro-uPA, HMW-uPA, ATF, UTI, HI-8, and the conjugates were tested for binding to uPAR on U937 and SMT-cc1 cells. The results of experiments using PMA-stimulated initially adherent U937 cells are shown in Fig. 4. 50% inhibition of FITCbulletpro-uPA binding to U937 cells was obtained with 1.5 nM pro-uPA, 3 nM HMW-uPA, 5 nM ATF, 10 nM ATFbulletHI-8, and 10 nM ATFbulletUTI, respectively. UTI and HI-8 do not bind to U937 cells within 30 min at 4 °C. Thus, the conjugate has an affinity for the uPAR of approximately 6-fold less than pro-uPA itself. Similar results were obtained in SMT-cc1 cells.


Figure 4: Effects of the conjugates on uPA binding to PMA-stimulated U937 cells. Acid-treated PMA-stimulated U937 cells were reacted with 10 nM FITCbulletpro-uPA (30 min, 4 °C). The dose-dependent quench of cell-associated fluorescence by the conjugates is shown. Competition between FITCbulletpro-uPA and pro-uPA (down triangle), HMW-uPA (up triangle), ATF (), UTI (circle) HI-8(bullet), ATFbulletUTI (), or ATFbulletHI-8 (box) for binding to the uPAR on U937 cells is shown. The fluorescence mean channels were calculated. The cell signal in the absence of competitors was set to 100%.



Competitive inhibition of binding of ATFbulletHI-8 to U937 cells was observed to various extents by preincubation of ATFbulletHI-8 with moAB 3471 (which reacts with amino acid sequence 17-34 of uPA; American Diagnostica) (data not shown). It is likely that a masking of the intact receptor binding sequence within the ATF molecule by moAB 3471 is the cause of the inhibition.

Apparent saturation of uPAR was achieved at approximately 10 nM FITCbulletATFbulletUTI, 10 nM FITCbulletATFbulletHI-8, and 3 nM FITCbulletHMW-uPA for PMA-stimulated U937 cells (4 °C, 30 min) (Fig. 5A). Binding of FITCbulletATFbulletUTI is inhibited by excess of pro-uPA and HMW-uPA, but not by LMW-uPA or UTI (4 °C, 30 min) (data not shown). 50% displacement is obtained at a molar ratio of 1:5 for pro-uPA and FITCbulletATFbulletUTI. This lower affinity of FITCbulletATFbulletUTI might be due to derivatization with FITC of the -amino group of one or several lysines present in the growth factor-like domain of uPA(21) .


Figure 5: Specific binding of the conjugates to U937 cells. A, acid-treated PMA-stimulated U937 cells were incubated with increasing concentrations of FITCbulletATFbulletHI-8 (box), FITCbulletATFbulletUTI (), or FITCbulletHMW-uPA (up triangle) (30 min, 23 C). B, in a parallel experiment, phosphatidylinositol specific-phospholipase C-treated cells were incubated with increasing concentrations of FITCbulletATFbulletHI-8 (box), FITCbulletATFbulletUTI (), or FITCbulletHMW-uPA (up triangle) (30 min, 23 °C).



Release of uPAR from the cell surface with phosphatidylinositol specific-phospholipase C incompletely prevented FITCbulletATFbulletUTI binding to the cells (Fig. 5B).

It has been reported that internalization of the uPA-PAI-1 complexes is mediated by uPAR, and ATF is neither internalized nor degraded despite its ability to bind uPAR(25) . The internalization of FITCbulletATFbulletUTI in the presence of active PAI-1 in U937 cells has been tested by immunofluorescence (Fig. 6). The fluorescent spots seen represent patches of FITCbulletATFbulletUTI bound to uPAR. Positive staining on the cell surface was observed even after incubation of the cells with active PAI-1 at 37 °C for 5 h, indicating that FITCbulletATFbulletUTI is neither internalized nor degraded.


Figure 6: Immunofluorescence view of ligand internalization in the presence of PAI-1. U937 cells, preincubated either with FITCbulletATFbulletUTI (1) or FITCbulletHMW-uPA (2), were incubated with active PAI-1, washed, and then shifted to 37 °C for 10 min (A, times 400), 1 h (B, times 400), 3 h (C, times 400), or 5 h (D, times 600). As a negative control (not shown), the cells were incubated with FITCbulletATFbulletUTI in the presence of 2 µM pro-uPA and showed no staining. FITCbulletATFbulletUTI is neither internalized nor degraded despite its ability to bind uPAR.



In contrast, the cells were preincubated with FITCbulletHMW-uPA in the presence of PAI-1, washed, and then shifted to 37 °C. 60 min after raising the temperature to 37 °C, the immunofluorescence clearly shifted from the cell surface to the cytosol. The internalization was specific for HMW-uPA (Fig. 6).

Effect of the Conjugates on Tumor Cell Invasion in Vitro

If a selective protease is indeed essential to the invasion process by cancer cells, tumor invasion would be suppressed after inhibition of membrane-associated protease activities by selective protease inhibitors. We first examined whether the conjugate had a toxic effect on cells in vitro at the doses tested. SMT-cc1 cells in logarithmic growth phase and at confluence were cultured in the presence or absence of the conjugate (5 µM) for 3 days. Under these conditions, very few cells died as measured by trypan blue dye exclusion in preliminary experiments. No remarkable morphological changes were observed.

To confirm the effect of the conjugates on tumor cell invasion using an in vitro model, we examined the effects of the conjugates, ATF, HI-8, and UTI on invasion through Matrigel using a modified Boyden chamber. When these agents were incubated with SMT-cc1 cells at various concentrations in the upper compartment of the chamber, invasion by tumor cells through Matrigel was inhibited in a dose-dependent manner, although ATF, HI-8, and UTI were less effective than the conjugates (Fig. 7). Treatment of SMT-cc1 cells with higher concentrations of ATF, HI-8, and UTI inhibited invasion as described previously(16, 26) . We have investigated whether this effect was attributable to the physicochemical properties of these agents, such as high viscosity at high concentration. Since high concentration of LMW-uPA showed no significant inhibitory effects, this was considered to be specific for ATF and HI-8. We confirmed that the conjugates exhibited better reproducibility in replicate experiments. Comparing ATFbulletUTI with UTI, ATFbulletUTI gave rise to an approximately 20-fold enhancement of the inhibitory effect on tumor cell invasion, as assessed by the concentration giving 50% inhibition of tumor cell invasion using our in vitro assay system.


Figure 7: Effect of the conjugates on tumor cell invasion in vitro. SMT-cc1 cells were tested in an in vitro invasion assay. The number of cells that had attached to the lower surface of the filter at 12 h was used to calculate cell invasion. Control experiments were carried out in the absence of agents. Either UTI (circle), HI-8 (bullet), ATF (), LMW-uPA (), ATFbulletUTI (), or ATFbulletHI-8 (box) was added to the upper chamber 60 min before the cells were plated on the Matrigel. Each experiment was performed at least in triplicate.



The cell chemotactic response was also tested to determine whether the inhibitory effect of the conjugates on cell invasion of basement membranes was due to an inhibition of chemotaxis. The cells tested showed good chemotactic migration in the presence of the conjugates (data not shown). The lack of negative effects on chemotaxis is consistent with an absence of toxicity of the conjugates. In addition, we examined the effects of the conjugates used in this study on cell attachment. No inhibition of attachment to Matrigel (or fibronectin) was seen with the conjugates (data not shown).


DISCUSSION

This paper describes a series of experiments designed to test directly the hypothesis that uPA and plasmin might be involved in tumor cell invasion and metastasis(12, 15, 16, 17, 18, 26) . In this study, we describe the production of novel plasmin inhibitors: chimeric proteins including the receptor binding domain of uPA and a plasmin inhibitory domain from UTI or the complete UTI. These chimeric proteins were shown to bind to cell surfaces, inhibit plasmin, and to block tumor cell invasion.

Our previous results indicate that occupation of uPAR on 3LL cells by the enzymatically inactive mouse peptide 17-34 (Gly-Val-Cys-Val-Ser-Tyr-Lys-Tyr-Phe-Ser-Arg-Ile-Arg-ArgCys-Ser-Cys-Pro) or prevention of rebinding of uPA synthesized by tumor cells to its receptor specifically reduced tumor cell invasion and the formation of metastasis(12, 15, 18) . Since the proteolytic enzyme plasmin might be involved in tumor cell invasion, the use of UTI (a physiological plasmin inhibitor) and an effective peptide that inhibits plasmin activity within UTI specifically reduces invasion by tumor cells(16, 17, 26) . In order to extend our idea, we attempted to synthesize conjugates between ATF and UTI. Conjugating a plasmin inhibitor to ATF might target it to the cell membrane of uPAR-expressing tumor cells because the cell surface uPAR might be a critical component of the metastatic machinery(11) . Now we have examined whether the conjugates have a strong ability to inhibit tumor cell invasion in an in vitro assay. We have found that (a) the conjugates (ATFbulletUTI and ATFbulletHI-8) efficiently compete for pro-uPA binding; (b) a large part of receptor-bound conjugates remains on the cell surface; and (c) the conjugates are very effective at targeting UTI (or HI-8) specifically to uPAR-expressing tumor cells. Our results indicate that cell surface uPA and plasmin activity is essential to the invasive process and that the conjugates exhibit plasmin inhibition in the close environment of the cell surface and subsequently inhibit the tumor cell invasion through Matrigel. The more dramatic effects obtained in this study may be related to more complete inhibition of cell surface proteolytic activity by the conjugates. Alternatively, since plasmin is a known activator of metalloproteinases, one might speculate that inhibition of plasmin generation will result in diminished metalloproteinase activation with decreased collagen digestion. We suggest that the conjugates between ATF and UTI (blockade of the uPAR) may hold therapeutic promise. No complex formation between the conjugates and plasminogen activator inhibitors (PAI-1 and PAI-2) has been shown (data not shown). In addition, we confirmed that the conjugates are not internalized into the cells. This may be the reason why the results obtained in an in vitro invasion assay are more dramatic than expected in inhibition of cell-surface receptor-bound plasmin activity by the conjugates.

In the previous study, certain tumor cells and human neutrophils were shown to express a UTI-like substance, which might be an endogenous regulator of the cell migration(27) . If the very high levels of plasmin-dependent proteolytic activity could cause uncontrolled degradation of extracellular matrix proteins, the interaction of cells and matrix may be interrupted. The inhibition of excessive plasmin activity may stabilize and increase cell matrix contacts(28) . Therefore, an increase in endogenous UTI expression may control proteolysis and contributes to prevent the excessive fibrinolysis in conditions such as tumor growth and invasion. A further reduction in plasmin activity by exogenously added ATFbulletUTI or ATFbulletHI-8 should result in a decrease of tumor cell invasion once the minimal requirement is passed.

We have speculated that conjugate binding to tumor cells via uPAR permits efficient inhibition of plasmin activity at discrete sites on the tumor cell membrane. Of interest is the recent observation that inhibition of uPAR in PC3 prostate carcinoma cells resulted in significant suppression of spontaneous metastasis in nude mice(11) . A better understanding of the potential role of cell surface-localized UTI in the regulation of inhibition of extracellular proteolysis will require in increased understanding of the binding, interaction, and the process of internalization(27) .

In an attempt to develop a strategy to kill selectively uPAR-bearing cells, a conjugate between uPA and saporin, a ribosome-inactivating protein, has been chemically synthesized by Cavallaro et al.(21) . The conjugate between uPA and toxins is able to kill target cells by blocking protein synthesis when carried to the cytosol. Various attempts have been made to regulate tumor cell invasion during the metastatic process. A high dose of the effective peptide (the synthetic peptides within the growth factor-like domain of uPA or peptides within domain II of UTI) was needed to obtain a sufficient effect probably because of rapid clearance of the synthetic peptide from the systemic circulation or lower affinity with their cell surface receptors(15) . Therefore, it seems important clinically to develop anti-invasive substances with high affinity to tumor cells. Thus, the conjugate between uPA and UTI or superior peptide analogues with higher affinity might provide a therapeutically promising basis for the prevention of tumor metastasis.

In conclusion, our data demonstrate that inhibition of tumor cell-derived receptor-bound uPA as well as suppression of cell surface receptor-bound plasmin activity by exogenously applied ATFbulletUTI or ATFbulletHI-8 efficiently prevents tissue invasion by tumor cells in vitro.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: uPA, urokinase-type plasminogen activator; ATF, amino-terminal fragment of uPA; ATFbulletHI-8, conjugate between ATF and HI-8; ATFbulletUTI, conjugate between ATF and UTI; FITC, fluorescein isothiocyanate; HI-8, a domain II of UTI molecule; HMW, high molecular weight; LMW, low molecular weight; PAI-1, plasminogen activator inhibitor type-1; PAGE, polyacrylamide gel electrophoresis; uPAR, uPA receptor; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin.


ACKNOWLEDGEMENTS

We thank Drs. T. Kobayashi and N. Kanayama for helpful discussion and steady encouragement during the course of this study.


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