(Received for publication, December 12, 1994; and in revised form, January 25, 1995)
From the
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 (ATFUTI 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
°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.
There are several reports that cell surface proteolytic enzymes
(urokinase-type plasminogen activator (uPA), ()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 ATFUTI or
ATF
HI-8 conjugates have high and specific ability to inhibit
tumor cell invasion toward uPAR-bearing cells.
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, ATFUTI; lane3, ATF
HI-8; lane4, UTI; lane5,
HI-8.
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 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.
In addition, inhibition of binding of FITCpro-uPA
to U937 cells with the conjugates (ATF
UTI and ATF
HI-8) was
tested. 10 nM FITC
pro-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.
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, ATFUTI, and ATF
HI-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%.
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 FITCATF
UTI (20
nM), FITC
ATF
HI-8 (20 nM), or
FITC
HMW-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 attachment assay and the chemotactic assay were conducted as described previously(12, 15, 16) .
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 (
), HI-8 (
),
ATF
UTI (
), or ATF
HI-8 (
). 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, ATFHI-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 (), HI-8 (
), ATF
UTI (
),
or ATF
HI-8 (
) 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.
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 FITCpro-uPA (30 min, 4 °C).
The dose-dependent quench of cell-associated fluorescence by the
conjugates is shown. Competition between FITC
pro-uPA and pro-uPA
(
), HMW-uPA (
), ATF (
), UTI (
) HI-8(
),
ATF
UTI (
), or ATF
HI-8 (
) 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 ATFHI-8 to U937 cells was observed to
various extents by preincubation of ATF
HI-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 FITCATF
UTI, 10
nM FITC
ATF
HI-8, and 3 nM FITC
HMW-uPA for PMA-stimulated U937 cells (4 °C, 30 min) (Fig. 5A). Binding of FITC
ATF
UTI 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 FITC
ATF
UTI. This lower
affinity of FITC
ATF
UTI 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 FITCATF
HI-8
(
), FITC
ATF
UTI (
), or FITC
HMW-uPA
(
) (30 min, 23 C). B, in a parallel experiment,
phosphatidylinositol specific-phospholipase C-treated cells were
incubated with increasing concentrations of FITC
ATF
HI-8
(
), FITC
ATF
UTI (
), or FITC
HMW-uPA
(
) (30 min, 23 °C).
Release of uPAR from
the cell surface with phosphatidylinositol specific-phospholipase C
incompletely prevented FITCATF
UTI 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 FITCATF
UTI in the
presence of active PAI-1 in U937 cells has been tested by
immunofluorescence (Fig. 6). The fluorescent spots seen
represent patches of FITC
ATF
UTI 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
FITC
ATF
UTI is neither internalized nor degraded.
Figure 6:
Immunofluorescence view of ligand
internalization in the presence of PAI-1. U937 cells, preincubated
either with FITCATF
UTI (1) or FITC
HMW-uPA (2), were incubated with active PAI-1, washed, and then
shifted to 37 °C for 10 min (A,
400), 1 h (B,
400), 3 h (C,
400), or 5 h (D,
600). As a negative control (not shown), the cells
were incubated with FITC
ATF
UTI in the presence of 2
µM pro-uPA and showed no staining. FITC
ATF
UTI
is neither internalized nor degraded despite its ability to bind
uPAR.
In
contrast, the cells were preincubated with FITCHMW-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).
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 ATFUTI with UTI, ATF
UTI 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 (), HI-8 (
), ATF
(
), LMW-uPA (
), ATF
UTI (
), or ATF
HI-8
(
) 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).
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
(ATFUTI and ATF
HI-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
ATFUTI or ATF
HI-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 ATFUTI or ATF
HI-8 efficiently prevents
tissue invasion by tumor cells in vitro.