From the Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka, 431-3192, Japan
Received for publication, October 10, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Bikunin is a Kunitz-type protease
inhibitor, acting at the level of tumor invasion and metastasis. The
goal of this study was to investigate the effect of
bikunin-dependent signal transduction involved in the
expression of a plasminogen activator (PA) system and invasion. We
report here the following. 1) The human ovarian cancer cell line HRA
produced secreted and cell-associated urokinase-type PA (uPA) and PA
inhibitor type 1 (PAI-1). The plasma membrane of the cells showed
enzymatically active uPA even in the presence of high level of PAI-1,
as measured by zymography, Western blot, chromogenic assay,
enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells
leading to invasion are induced through up-regulation of uPA
expression. 3) HRA cells specifically released transforming growth
factor- Tumor cell invasion is dependent on finely regulated
extracellular proteolytic activity, which allows tumor cells to invade the extracellular matrix (1). Among the proteolytic enzymes involved in
this process are PAs,1 whose
expression in cultured cells is regulated by several types of growth
factors and cytokines (2). Invasive tumor cells not only express
cell-associated proteases but also secrete anti-proteases, preventing
the overdigestion of the extracellular matrix, which leads to a loss of
cell attachment. The balance between proteolytic activity and
inhibition is crucial in the invasive and metastatic event (3). Indeed,
the increment of a specific PA inhibitor, PAI-1, could have an
important regulatory role on the extracellular proteolysis and might
explain the decrease of net PA and gelatinolytic activities measured in
the medium (3, 4). Even in the high levels of PAI-1 in the medium,
however, several growth factors, including TGF- One prototype signal transduction therapy agent is bikunin,
which is a Kunitz-type protease inhibitor with
tumor-suppressive potential in several malignant cell types, acting at
the level of tumor invasion and metastasis (5-7). It was subsequently
demonstrated that bikunin inhibits tumor invasion, at least in part, by
a direct inhibition of plasmin activity as well as by inhibiting uPA
(5, 6) and uPAR (7) expression at the gene and protein levels. Interestingly, in cell-free solutions, bikunin does not inhibit uPA
activity effectively. Mechanistic studies in several cell types
demonstrated that bikunin interferes with an upstream target(s) of
selected MAP kinase signaling processes such as phosphorylation of MEK
and ERK, leading to overexpression of uPA (6). A recent study in our
laboratory demonstrated that bikunin could interfere with selected
calcium-sensitive signaling processes such as agonist-induced cytokine
expression in several types of cells, including human umbilical vein
endothelial cells, uterine myometrial cells, vascular endothelial
cells, neutrophils, HL60 leukemia cells, and tumor cells (8, 9). It has
been established that calcium mobilization is a common signaling
response in malignant and nonmalignant cells treated with a variety of
growth factors, including TGF- We have been reported that in preclinical studies with
several types of rodent and human malignant cells, bikunin has proven effective as an anti-invasive and anti-metastatic agent (5-7). In
recently completed clinical studies, bikunin has shown promise at
stabilizing disease progression in patients with advanced ovarian cancer (11). Despite the recent biochemical evidence that bikunin specifically inhibits expression of uPA and uPAR mRNA and proteins, the molecular mechanism underlying the tumor-suppressive effect of
bikunin remains elusive.
A growing body of evidence has recently implicated that members of
TGF- In our experimental system, we chose the human ovarian cancer cell line
HRA because these cells mediate plasminogen activation primarily by uPA
but produce low amounts of matrix metalloproteases (MMPs), and the cell
invasion is a uPA-dependent. We examined whether bikunin
can reduce cell invasion by modulating cell surface expression of PA
system through suppression of tumor-associated production of cytokines,
including TGF- Cell Culture--
The human ovarian cancer cell line HRA was
grown and cultured as described previously (6). The HRA cells were
maintained in RPMI 1640 medium (Invitrogen) supplemented with 10%
fetal bovine serum in a cell culture incubator (constantly set at
37 °C with 5% CO2).
Chemicals and Reagents--
Bikunin was purified from human
urine as described previously (5-7). Reagents of molecular biology
grade for protein gel electrophoresis, protein staining, and protein
concentration analysis were purchased from Bio-Rad. Proteolytic
enzymes, protease substrates, and specific protease inhibitors were
obtained from American Diagnostica (Greenwich, CT) and include
recombinant single-chain tPA, high molecular weight recombinant uPA,
synthetic chromogenic substrate of uPA (Spectrozyme UK), synthetic
chromogenic substrate of tPA (Spectrozyme TPA), Glu-type plasminogen,
purified plasmin, chromogenic plasmin substrate (Spectrozyme PL),
purified tPA-neutralizing monoclonal antibody (374B), purified
uPA-neutralizing monoclonal antibody (394), and anti-PAI-1 antibody
(379). Monoclonal antibodies against MMP-2 and MMP-9 were from Oncogene
Science (Cambridge, MA) and R & D Systems (Minneapolis, MN). Rabbit
polyclonal antibodies against the total and phosphorylated ERK were
from Zymed Laboratories Inc. (San Francisco, CA) and
Calbiochem, respectively. Polyclonal antibodies that recognize the
total and phosphorylated (activated) forms of JNK and p38 were from
Santa Cruz Biotechnology. Ultrapure natural human TGF- Preparation of Conditioned Medium and Cell Lysate--
Cells
were seeded in 35-mm dishes and basically 24 h later washed twice
with phosphate-buffered saline and then treated with several reagents,
including bikunin or TGF- Quantitation of uPA, PAI-1, and TGF- Detection of Cell Surface-associated PAs by Colorimetric
Assay--
HRA cells were seeded at 10,000 cells/well in 96-well
plates in the maintenance medium and allowed to reach ~90%
confluence. The cells were washed once with phosphate-buffered saline
and analyzed in a reaction buffer (100 µl/well) containing 0.8 mM MgCl2 and 0.2 µg/ml leupeptin. The
reactions were initiated by the addition of the chromogenic amidolytic
substrate specific for either uPA (Spectrozyme UK) or tPA (Spectrozyme
TPA) to a final concentration of 0.2 mM in the absence or
presence of 1 mM amiloride or neutralizing antibodies to 10 µg/ml uPA or 10 µg/ml tPA. No amidolytic substrate was added in the
blank reactions. The photometric absorbance of the reaction mixtures at
405 nm was monitored at 37 °C over the next 30 min. The trypan blue
cell staining procedure was performed as described by Broman et
al. (18).
Plasmin activities in cell-conditioned medium and cell lysate were
determined spectrophotometrically using a Spectrozyme PL as described
(12).
[3H]Thymidine Incorporation--
Cells were seeded
in 24-well plates (Corning Costar Corp.). After 2 days, cells were
washed with phosphate-buffered saline and treated with the indicated
concentrations of TGF- Cell Growth Assay--
HRA cells were grown on a flat bottomed
96-well plate at 10,000 cells/well containing the complete medium
including 10% fetal bovine serum. 24 h later, cells were grown in
RPMI 1640 medium with or without several reagents. After 1, 3, and 5 days of culture, cell proliferation was assayed by the addition of 20 µg of the vital dye MTT (1 mg/ml; Sigma). The blue dye taken up by
cells during a 4-h culture was dissolved in 100 µl/well dimethyl
sulfoxide, and its A490 nm was read on an
automated microplate reader. A preliminary study by MTT assay showed
that absorbance was directly proportional to the number of cells.
Substrate Zymography for Protease
Activities--
Confluent monolayers of HRA cells in 35-mm tissue
culture dishes were washed twice with serum-free medium and incubated
for 12 h in the presence of serum-free medium, the indicated
concentrations of TGF- Effects of Inhibitors on uPA Expression--
HRA cells were
cultured and serum starved. Treatment was achieved by adding 0.5-50
µM SK&F 96365 to the medium for 20 h. In addition,
cells were incubated for 12 h with one of the following agents: 50 µM MEK inhibitor PD98059, 50 µM
L-type calcium channel blocker nifedipine or verapamil, 25 µM general tyrosine kinase inhibitor genistein, or 10 µM selective Src tyrosine kinase inhibitor PP2. Control
cells received just vehicle (0.1-0.5% dimethyl sulfoxide for PD98059,
SK&F 96365, genistein, and PP2; 0.1% ethanol for nifedipine and verapamil).
Isolation of Cytoplasmic RNA and Northern Blot Analysis--
The
HRA cells were plated at a density of 106 cells/100-mm
dish. When cells grown in monolayer reached an early phase of
confluence, total cellular RNA samples were isolated from cells using a
Qiagen RNeasy kit (Qiagen Ltd.) according to the manufacturer's
protocol. For Northern blot analysis, 20 µg of cytoplasmic RNA was
electrophoresed onto a formaldehyde and 1.0% agarose gel and blotted
onto a nylon filter. After transfer, RNA was cross-linked onto the
membranes by UV irradiation. The nylon filter was hybridized with a
32P-labeled cDNA probe in 50% formamide, 5 × saline-sodium phosphate, EDTA, 0.1% SDS, 5 × Denhardt's
solution, and 100 µg/ml salmon sperm DNA at 42 °C for 20 h.
Quantitative analysis was performed with a Bio-Rad model 620 video
densitometer with a one-dimensional Analyst software package for Macintosh.
The uPA cDNA probe was prepared as described previously (6). The
Western Blotting--
Confirmation of the presence of uPA,
PAI-1, and MMP expression was obtained by Western blot using each
monoclonal antibody. Briefly, samples were electrophoresed under
nonreducing (for uPA and PAI-1) and reducing (for MMPs) conditions and
transferred to polyvinylidene difluoride membrane using a semidry
transfer system. Nonspecific binding sites were blocked for 1 h in
Tris-buffered saline, pH 7.6, containing 2% bovine serum albumin.
Blots were incubated with 0.5 µg/ml primary antibody for 1 h and
with secondary antibody (goat anti-mouse horseradish
peroxidase-conjugated IgG diluted 1: 5,000 in blocking solution) for
1 h. After several washes, reactive bands were visualized by a
chemiluminescence detection kit (Amersham Biosciences).
Invasion Assay--
The ability of cells to migrate across a
Matrigel barrier (invasion) was determined by the modified Boyden
chamber method (25). Briefly, HRA cells (105/chamber) were
added to polyvinylpyrrolidone-free, 8-µm polycarbonate filters coated
with 50 µl of 50 µg/ml Matrigel and incubated with complete medium
containing 0.1% bovine serum albumin for 36 h at 37 °C. NIH3T3
fibroblast-conditioned medium was used as a chemoattractant, and
serum-free medium containing 0.1% bovine serum albumin was used as a
negative control. Filters were removed from the chambers and stained
with hematoxylin. Cells were counted at a 100× magnification, and the
mean numbers of cells/field in five random fields were recorded.
Duplicate filters were used, and the experiments were repeated three times.
Statistics--
Data are expressed as the mean ± S.D. of
at least three independent triplicate experiments. Statistical analysis
was performed by one-way analysis of variance followed by Student's
t test. p < 0.05 was considered
statistically significant.
HRA Cells Produce Primarily uPA, but Not tPA or MMPs--
The
human ovarian cancer cell line HRA was chosen for our study because we
have shown previously that HRA cells exhibited a
uPA-dependent invasion and metastasis (6). In this study, HRA cells were evaluated for their capability to secrete and activate PAs and MMPs in great detail. Fig. 1,
A and B, shows the zymographic profiles of the
plasminogen-dependent (for PA (A)) and
plasminogen-independent (for MMP (B)) gelatinolytic
activities of both the cell lysate and the conditioned medium (10×
concentrated) of HRA cells. Compared with the purified tPA, uPA, MMP-2,
and MMP-9 standards, both the lysate and the conditioned medium of HRA
cells contained primarily uPA, although a trace amount of tPA was also
detected (Fig. 1A). Gelatin zymography (Fig. 1B)
and Western blotting (not shown) indicate that HRA cells basally
secreted detectable but low amounts of 92- and 65-kDa gelatinases whose
positions identify the produced molecules as pro-MMP-9 and pro-MMP-2
(Fig. 1B). As shown in Fig. 1, C and
D, analysis of the uPA and PAI-1 levels using Western blotting showed a band of 33 kDa (for low molecular mass uPA), 50 kDa (for high molecular mass uPA), 60 kDa (for PAI-1), 110 kDa (for
uPA·PAI-1 complex), and 130 kDa (for tPA·PAI-1 complex) in the
conditioned medium and the cell lysate. Moreover, HRA cells produced
extremely low amounts of PAI-2 as revealed by ELISA (data not shown).
ELISA data showed that the basal cell-associated uPA protein level
measured in HRA cells was 14.8 ± 2.1 ng/mg cell lysate protein
(mean ± S.D.), whereas the basal level of uPA released in the
medium by the cells during a 24-h period was 12.0 ± 3.8 ng/ml/106 cells/24 h. Furthermore, the cells also produced
cell-associated PAI-1 and secreted PAI-1 (5.2 ± 1.7 ng/mg protein
for cell-associated PAI-1; 30.2 ± 8.7 ng/ml/106
cells/24 h for secreted PAI-1).
The conditioned medium of HRA cells was not very active in converting
Glu-plasminogen to plasmin, which in turn did not lead to the cleavage
of Spectrozyme PL. In contrast, when Spectrozyme UK or Spectrozyme TPA
was added to the monolayer of HRA cells, the Spectrozyme UK was
specifically cleaved (Fig. 1E). 1 mM amiloride (a catalytic inhibitor of uPA) and 10 µg/ml uPA-neutralizing
antibody, but not 10 µg/ml tPA-neutralizing antibody, blocked the
cleavage of Spectrozyme UK on the plasma membrane of the HRA cells. It is worth noting that throughout this biochemical assay, cells remained
intact, showing little or no plasma membrane permeability by trypan
blue and a full recovery of proliferation in the subsequent cell growth
assay (data not shown).
HRA Cells Invade through a uPA-dependent
Mechanism--
Effects of bikunin at concentrations from 1 to 1,000 nM on the cell proliferation were tested in dose-response
and time course experiments over a 14-day period. Bikunin
concentrations varying from 1 to 1,000 nM did not show any
significant effect on growth (data not shown). Thus, the invasion assay
could be performed at a time (36 h) when there was no growth
inhibition. To test whether the HRA cells had an ability to invade by a
PA system-dependent manner, cells were preincubated with
E64 and amiloride as well as specific neutralizing antibodies to uPA,
tPA, MMP-2, or MMP-9 and analyzed using a chemoinvasion assay. As shown
in Fig. 2, the uPA-neutralizing antibody
specifically inhibited HRA cell invasion in a
dose-dependent manner. Antibody against uPA A-chain 3471 (containing the domain interacting with uPA receptor) also strongly blocked the invasion. We have shown previously that bikunin inhibits the in vitro invasion of several types of cancer
cells, including HRA cells. Cells preincubated with the
uPA-neutralizing antibody were not inhibited further by bikunin in the
Matrigel invasion assay. Neutralizing antibodies to tPA, MMP-2, and
MMP-9 neither inhibited cell invasion nor altered the
invasion-inhibitory effect of bikunin (data not shown). In addition,
enhanced invasion of HRA cells was inhibited markedly by the
uPA-specific inhibitor, amiloride. E64, which totally inhibits soluble
cathepsins and uPA, had a weak inhibitory effect.
We next examined whether the uPA level is modulated by bikunin in the
medium collected from the upper chamber during the invasion experiment.
The presence of bikunin caused a dramatic reduction of the uPA level
(107 ± 21.5 [+bikunin] versus 283 ± 43.6 pg/ml/105 cells/36 h [ Neutralization of Endogenous TGF-
Confluent cultures of HRA cells were incubated in the presence of
different concentrations of neutralizing anti-TGF-
ELISA data revealed that in the conditioned medium, the
TGF- Inhibition of Invasion of HRA Cells through TGF- Effect of Exogenously Added TGF- Effect of TGF- Bikunin Specifically Suppresses Invasion through Down-regulation of
TGF- Analysis of the Signals Regulating Bikunin-induced Suppression of
TGF-
In addition, the constitutive expression of uPA observed in HRA cells
was inhibited by preinoculation of the cells with bikunin, SK&F 96365, genistein, PP2, or PD98059, but not with nifedipine, verapamil, and
sodium vanadate. Again, bikunin showed no additive effect on SK&F
96365-, genistein-, or PP2-mediated suppression of uPA expression.
Suppression by Bikunin of TGF-
We hypothesized that bikunin-mediated suppression of calcium influx
could inhibit the constitutive expression of TGF- The results of this study extend earlier observations about the
inhibitory effect of bikunin on tumor cell invasion (5-7). Our
previous data provide evidence that bikunin not only has the capacity
of functioning as a protease inhibitor to suppress directly cell
surface-associated plasmin activity but it can also reduce cell
invasion by modulating uPA mRNA and protein expression
through inhibition of an upstream target(s) of protein kinase C- and/or MAP kinase-dependent signaling cascade (6). More recent
data (7) demonstrate that uPAR expression is also down-regulated by
bikunin in human chondrosarcoma HCS-2/8 cells and two human ovarian
cancer cell lines, HRA and HOC-I.
The novel findings of the present study are as follows. First, HRA
cells have an ability to invade through a PA
system-dependent manner. This is an extension of our
previous findings that HRA cells exhibited a uPA-mediated fibrinolytic
activity (6). Even in the high levels of PAI-1 in the medium, HRA cells
exhibited an increase of net PA activity on the plasma membrane, which
results in strong proteolytic activity in some specific sites, such as areas of cell-to-cell or cell-to-extracellular matrix contacts (26,
27). Therefore, up-regulation of net PA activity on the plasma membrane
is important for the invasive process of HRA cells. Secreted PAI-1
inhibited activation of plasminogen to plasmin in the growth medium,
thereby preventing plasmin-induced detachment of cells (28).
Second, we found that expression of the PA system in HRA cells is
dependent on endogenously secreted TGF- type 1 (TGF-
1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-
1 could induce change in uPA/PAI-1 expression, which could in turn modify
the invasive behavior of the cells. 5) The constitutive expression of
TGF-
1 as well as up-regulation of the PA system observed in HRA
cells was inhibited by preinoculation of the cells with bikunin or
calcium channel blocker SK&F 96365 but not with nifedipine or
verapamil, with an IC50 of ~100 nM for
bikunin or ~30 µM for SK&F 96365, respectively, as
measured by enzyme-linked immunosorbent assay. Bikunin showed no
additive effect on SK&F 96365-mediated suppression of TGF-
1
expression. 6) The ability of TGF-
1 to elevate free intracellular
Ca2+, followed by activation of Src and ERK, was reduced by
preincubation of the cells with bikunin. In conclusion, bikunin could
inhibit the constitutive expression of TGF-
1 and TGF-
1-mediated,
Src- and ERK-dependent, PA system signaling cascade, at
least in part, through inhibition of a non-voltage-sensitive calcium channel.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1, induced the
increase of net PA and gelatinolytic activities on the plasma membrane,
which results in strong proteolytic activity in some specific sites,
such as areas of cell-to-cell or cell-to-extracellular matrix contacts.
For this reason, drugs that manipulate or suppress signal transduction
on the PA system in malignant cells offer a potentially new approach to
anti-cancer therapy.
and TNF-
, which are associated
with tumor progression (10). These findings underscore the importance
of calcium in the biology of malignant cells.
family are potent regulators of multiple cellular functions,
including cell proliferation, differentiation, migration, organization,
and death (4, 12, 13). TGF-
1 is produced by host cells and/or tumor
cells in a latent form and is activated by proteases in a
cell-dependent manner. TGF-
1 produced by tumor cells
including prostate cancer (4) and breast cancer (12-14) is a candidate
responsible for the induction of PA system expression. Thus, TGF-
1
had a positive effect on cell proliferation and increased uPA or uPAR
expression (15). The previous results (14) also indicated that the
effects of TGF-
1 on cell growth can be dissociated from its effects
on the uPA system and that TGF-
1 may promote invasion of tumor cells
by increasing uPA activity and PAI-1 levels. In contrast, the TGF-
signaling pathway is considered to be one of the most important
mechanisms for tumor suppression (16). Therefore, the role of TGF-
1
on tumor cell invasion is controversial.
1. We also investigated whether bikunin can inhibit
tumor-associated production of TGF-
1 and the TGF-
1-stimulated PA
system through suppression of the calcium-dependent signaling.
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 was from
Genzyme (Cambridge, MA) and R & D Systems. Neutralizing antibody
against TGF-
1 was purchased from R & D Systems and Sigma.
Neutralizing mouse monoclonal IgG antibodies to TNF-
or IL-1 were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY). MEK
inhibitor PD98059 was from New England BioLabs (Beverly, MA). Matrigel
was purchased from Collaborative Research (Bedford, MA). PP2
(Calbiochem), genistein, nifedipine, sodium vanadate, and verapamil
were from Sigma. SK&F 96365 was obtained from BioMol (Plymouth Meeting,
PA) and dissolved in dimethyl sulfoxide. The final dimethyl sulfoxide
concentration was held constant throughout each experiment at either
0.5% or 1%. Control experiments indicated that neither of these
dimethyl sulfoxide concentrations affected gene expression or cell
viability. Unless otherwise specified, all other chemicals and reagents
were of the highest purity and obtained from Sigma.
1 in serum-free medium. The conditioned
medium was produced by incubating the cells (~90% confluent) for
24 h in a serum-free medium. All conditioned medium thus obtained
was concentrated ~10-fold in Centricon-10 units (Amicon). The cell
monolayers were extracted with 0.1 M Tris-HCl, pH 7.5, 2 mM EDTA, 0.5% Triton X-100 (v/v) in the presence of inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) at 4 °C for 15 min and scraped with a rubber
policeman. Cell extracts were then centrifuged at 3,000 × g to remove cell debris. All samples were stored at
20 °C until use. In parallel, cells treated in the same condition
in different dishes were harvested and counted using a hemocytometer.
1 Levels--
The amount
of uPA was measured in the cell-conditioned medium and in the cell
lysate using an Imubind uPA ELISA kit from American Diagnostica. PAI-1
levels in the cell-conditioned medium were determined using an Imubind
PAI-1 ELISA kit from the same vendor. Lower detection levels were set
at 10 pg/ml for the uPA kit and below 1 ng/ml for the PAI-1 kit. The
active and total (active plus latent) TGF-
1 were assayed with a
human TGF-
1 ELISA kit (BIOSOURCE International
Company, Tokyo) according to the manufacturer's instructions. To
measure total TGF-
1, samples were acidified with acetic acid and
then neutralized as described previously (17).
1 for 24 h in serum-free medium. Four h
before the end of culture, cells were incubated with 2 µCi/ml
[3H]thymidine (ICN, specific activity 2 Ci/mM). Cells were then washed with 10% trichloroacetic
acid at 4 °C followed by four washes with water and solubilized with
0.5 M sodium hydroxide; the radioactivity was measured with
a liquid scintillation counter (LKB).
1, neutralizing antibodies to TGF-
1,
TNF-
, IL-1
, or preimmune IgG. At the end of the incubation, cell
extracts and supernatants were analyzed by zymography as described
(19). Conditioned medium (10 µl of 10 × concentrated) and 20 µg of cell lysate were electrophoresed in nonreducing SDS-PAGE
conditions in gel copolymerized with 0.1% gelatin for gelatinase
activities and with 0.1% casein plus 15 µg/ml Glu-plasminogen for PA
activities. Plasminogen-dependent gelatinolytic
zymogram was performed as described previously (20).
-actin probe was an XhoI-XhoI fragment of
pHF
A-1 for (ATCC, Rockville, MD). For pSP64-hPAI-1, a 1.4-kbp
EcoRI-BglII fragment (position 54-1480 (Ref.
21)) isolated from pPAI1-C1, a plasmid containing a 2.2-kbp human PAI-1
cDNA insert (22), was subcloned between the BamHI and
EcoRI sites of pSP64 (23). For pSP65-hTA, a 614-bp
BglII-EcoRI fragment (position 188-801) isolated
from pW349F, a plasmid containing a 2.6-kbp human tPA cDNA insert
(24), was subcloned between the EcoRI and BamHI sites of pSP65 (23). The SmaI-NcoI fragment pf a
PCR product, which encompasses 634-886 bp of MMP-2 cDNA, was
subcloned into the pGem5 vector (Promega, Madison, WI). A PCR fragment,
which encompasses 151-442 bp of MMP-9 cDNA, was subcloned into the
pGem-T vector (Promega).
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Fig. 1.
Plasminogen activator system in the cell
lysate and the conditioned medium of HRA cells. A and
B, zymographic analysis. Gelatinolytic zymographic profiles
of plasminogen activators (A) and matrix metalloproteases
(B) in the cell lysate (CL) and conditioned
medium (CM) of HRA cells are shown. A total of 20 µg of
the lysate protein or 10 µl of the 10× concentrated conditioned
medium was loaded on the zymogram gels. Purified high molecular weight
uPA (0.1 NIH unit) and tPA (0.2 NIH unit) were used as standards. The
detected signals were identified on the basis of their relative
molecular masses (arrows). C and D,
Western blot analysis. The cell lysate and conditioned medium protein
of HRA cells were analyzed by Western blotting using anti-uPA antibody
(C) or anti-PAI-1 antibody (D), respectively.
E, colorimetric analysis. The monolayer of HRA cells was
analyzed by colorimetric assay in the presence of the Spectrozyme UK or
Spectrozyme TPA in the presence or absence of amiloride, neutralizing
antibodies to uPA, or tPA. Open circles, Spectrozyme UK
alone; open squares, Spectrozyme TPA alone; open
triangles, Spectrozyme UK plus 1 mM amiloride;
filled squares, Spectrozyme UK plus 10 µg/ml neutralizing
anti-uPA antibody; filled circles, Spectrozyme TPA plus 10 µg/ml neutralizing anti-tPA antibody; and filled
triangles, Spectrozyme UK plus 10 µg/ml neutralizing anti-tPA
antibody. The data represent an average result of triplicate repeats,
and the error bars indicate the S.D. values, which are less
than 15%.
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Fig. 2.
HRA cell invasion through cell
surface-mediated uPA. The cell invasion assay was performed in the
presence of no additional factor; 2.5, 5, and 10 µg/ml
uPA-neutralizing antibody; 10 µg/ml tPA-neutralizing antibody; 10 µg/ml MMP-2-neutralizing antibody; 10 µg/ml MMP-9-neutralizing
antibody; 10 µg/ml preimmune IgG; 1 mM amiloride; 10 µg/ml E64; 1 µM bikunin; and 1 µM bikunin
plus 10 µg/ml uPA-neutralizing antibody. Results are expressed as the
average number of cells present in five fields/membrane and are the
mean ± S.D. of three different determinations; unlike
letters (a-e) stand for statistical differences
(p < 0.05).
bikunin]). At a concentration of
1 µM, the uPA level is inhibited by 62%. These data
support that the HRA cells leading to invasion are induced through
up-regulation of uPA expression and a specific inhibitory activity of
bikunin toward the endogenous expression of uPA.
Specifically Suppresses uPA
and PAI-1 mRNA Expression--
To investigate whether HRA cells
released several types of cytokines participating in an
autocrine/paracrine regulation of HRA cell invasion, the conditioned
medium of HRA cells was tested by ELISA for TGF-
1, TNF-
, and
IL-1
. ELISA data revealed that TGF-
is significantly produced by
HRA cells (TGF-
, 396 ± 29.1; TNF-
, 63.1 ± 10.5; and
IL-1
, <20 pg/ml/106 cells/24 h). 24-h, serum-free
conditioned medium from HRA cells contained both latent and activated
TGF-
1 (latent TGF-
1, 128 ± 25.1; active TGF-
1, 180 ± 32.5 pg/ml/106 cells/24 h).
antibody or other
neutralizing antibodies. As shown in Fig.
3A, Northern blot analysis of
HRA cells incubated for 6 h in the presence of 10 µg/ml
anti-TGF-
1 antibody revealed a significant decrease in
uPA and PAI-1 mRNA, but no significant
modulation of tPA, MMP-2, and MMP-9
mRNA levels. In contrast, antibodies to other cytokines (TNF-
and IL-1
) or nonimmune IgG had no significant effect on expression
of uPA and PAI-1 mRNA. No morphologically
identifiable signs of cytotoxicity were observed in cells incubated
with these antibodies.
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Fig. 3.
A neutralizing
anti-TGF- 1 monoclonal antibody specifically
inhibits expression of uPA and PAI-1
mRNA and protein as well as the HRA cell invasion. Five × 105 cells were plated in 100-mm dishes and cultured for
48 h, then washed three times with phosphate-buffered saline.
Confluent monolayers of HRA cells were incubated for 6 h with the
indicated concentrations of neutralizing monoclonal antibodies to
TGF-
1, TNF-
, IL-1
, or nonimmune IgG. A, total RNAs
were extracted and hybridized with uPA, PAI-1,
tPA, MMP-2, MMP-9, and
GAPDH cDNA. Each filter was loaded with 20 µg of total
RNAs. Filters were exposed for 5 h (PAI-1 and
-actin), 24 h (uPA, MMP-2, and MMP-9), or 3 days (tPA). The uPA (B) and
PAI-1 (C) levels in the conditioned medium were analyzed by
ELISA. Results are from at least three separate experiments. *,
p < 0.05 compared with the control. D, a
cell suspension, 200 µl (500,000 cells/ml) in RPMI 1640 containing
1% fetal bovine serum, was placed on the Matrigel-coated surface of
the upper chamber. The indicated concentrations of neutralizing
monoclonal antibodies to TGF-
1, TNF-
, IL-1
, or nonimmune
rabbit IgG were added to the upper compartment. After a 36-h
incubation, cells that invaded through the Matrigel-coated membrane
were stained and counted under the microscope. All experiments were
performed three times, and typical data are shown. Results are the
mean ± S.D. of three different determinations; unlike
letters (a-d) stand for statistical differences
(p < 0.05).
1-neutralizing antibody inhibited up-regulation of uPA (Fig. 3B) and PAI-1 (Fig. 3C) protein expression in a
dose-dependent manner, with a 50% inhibition occurring at
5.0-7.0 µg/ml for uPA and PAI-1 of the antibody. Zymographic
analysis of the cell extracts revealed a dose-dependent
decrease in cell-associated uPA activity (data not shown). This result
suggests a major role for TGF-
1 endogenously produced by HRA cells.
1-mediated
Modulation of the Plasminogen Activator System--
Treatment of the
HRA cells with the neutralizing anti-TGF-
1 antibody reduced cell
invasion (Fig. 3D). The responses were found to be
concentration-dependent. Antibodies against TNF-
and
IL-1
reduced HRA cell invasion to a lesser degree, demonstrating that both TNF-
and IL-1
were less potent than TGF-
1. These data allow us to hypothesize that neutralization of endogenous TGF-
1
by the anti-TGF-
1 antibody could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of HRA cells.
1 on Expression of uPA and PAI-1
by HRA Cells and Tumor-promoted Plasmin Activity--
We next
investigated whether exogenously applied TGF-
1 could enhance
expression of cell surface and secreted uPA and PAI-1 by colorimetric,
zymographic, ELISA, and Western blot analyses (Fig.
4). In the cell lysate and
cell-conditioned medium, we found that TGF-
1 induced a significant
increase in the amount of cell-associated and secreted uPA and PAI-1
compared with untreated cultures. TGF-
1 stimulated uPA production in
a dose-dependent manner. The incubation with 10 ng/ml
TGF-
1 resulted in about a 5-fold increased cell-associated uPA
expression (51.8 ± 7.0 versus 10.8 ± 1.5 ng/mg
protein) (Fig. 4A) and in about 2.5-fold increased
expression of uPA in the conditioned medium (30.5 ± 5.3 ng/ml/106 cells/24 h versus 12.1 ± 1.1 ng/ml/106 cells/24 h) (Fig. 4B). Furthermore,
TGF-
1 increased cell-associated PAI-1 about 2-fold and cell-secreted
PAI-1 4-fold in HRA cells. Zymography (Fig. 4C) and Western
blot (Fig. 4, D and E) analyses revealed that
exogenously added TGF-
1 could enhance expression of cell surface
(data not shown) and secreted uPA and PAI-1 levels in a
dose-dependent manner. Anti-TGF-
1 antibody abrogated the TGF-
1-mediated expression of uPA (Fig. 4F).
Concomitantly, plasmin activity decreased to 30% of untreated cells in
the cell-conditioned medium and increased 3-fold on the plasma membrane
for the TGF-
1-treated cultures by colorimetric assay (data not
shown). In contrast, TGF-
1-treated HRA cells secreted low amounts of
tPA (data not shown).
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Fig. 4.
Effect of TGF- 1 on
expression of uPA and PAI-1 by HRA cells. Cells were seeded in
35-mm dishes and then incubated for 24 h with either serum-free
medium or serum-free medium containing 0.1, 1, or 10 ng/ml TGF-
1.
The uPA (open bars) and PAI-1 (filled bars)
contents of cell lysate (A) and cell-conditioned medium
(B) were measured by ELISA. The data represent mean
values ± S.D. of three experiments and triplicate determinations.
C, the secreted PA activities were measured by zymography.
First lane, purified uPA (0.1 NIH unit/lane). Cells
were treated with TGF-
1 (0, second lane; 0.1, third
lane; 1.0, fourth lane; and 10 ng/ml, fifth
lane). The secreted uPA (D) and PAI-1 (E)
levels were measured by Western blotting. F, cells were
treated with 10 ng/ml TGF-
in the presence of the indicated
concentrations of neutralizing anti-TGF-
1 antibody for 24 h,
and the uPA level in the cell lysates was analyzed by Western blotting
using anti-uPA antibody. 20 µg of the cell lysate protein and 10 µl
of the 10× concentrated conditioned medium were loaded on the gels.
Results are the mean ± S.D. of three different determinations;
unlike letters (a, b, c,
a', b', and c') stand for statistical
differences (p < 0.05).
1 on Cell Proliferation--
To examine the
sensitivity of ovarian cancer cells to TGF-
1, we measured the
anchorage-dependent growth of HRA cells for a 24 h-period
of treatment with the drug. Different concentrations of TGF-
1
(0.1-10 ng/ml) were tested for their effect on cell proliferation. At
any of the doses tested, TGF-
1 had no effect on cell proliferation
during the incubation period because no change in cell growth could be
detected for either untreated or TGF-
1-treated samples as assessed
with the [3H]thymidine incorporation assay and the MTT
cell viability assay (data not shown). Also, no significant change in
cell growth could be detected by direct cell enumeration measurements
(data not shown). Confluent monolayers of HRA cells treated with
TGF-
1 showed no evidence of cytotoxicity as measured by the trypan
blue exclusion test. These data indicate that TGF-
1 has no immediate effects on cell proliferation.
1--
To assess the specificity of the effect of bikunin, the
conditioned medium of HRA cells was tested by ELISA for uPA, PAI-1, and
TGF-
1. ELISA data revealed that bikunin specifically suppresses uPA
and PAI-1 secretion into the conditioned medium in a
dose-dependent manner (Fig.
5A). We showed that treatment
of HRA cells with bikunin induced a suppression of endogenously
secreted TGF-
1 with doses as low as 30 nM, with an IC50
of ~300 nM (Fig. 5A), which is associated with
suppression of uPA/PAI-1 secretion. Confluent monolayers of HRA cells
treated with bikunin showed no evidence of cytotoxicity as measured by
the trypan blue exclusion test or by the MTT assay. We also found that
treatment of cells with bikunin induced a suppression of the
TGF-
1-stimulated secretion of uPA and PAI-1 in a
dose-dependent manner (Fig. 5B). In addition, we
found that bikunin could reduce the invasion of the HRA cells treated
with or without TGF-
1 (Fig. 5C). Thus, bikunin
specifically inhibited tumor cell invasion through suppression of
endogenous and exogenous TGF-
1-mediated secretion of uPA/PAI-1.
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Fig. 5.
Effects of bikunin on secretion of uPA,
PAI-1, and TGF- 1 as well as on cell
invasion. HRA cells were seeded in 35-mm dishes and then treated
with the indicated concentrations of bikunin in the absence
(A) or presence (B) of 10 ng/ml TGF-
1 for
24 h. The uPA (open circles), PAI-1 (filled
circles), and TGF-
1 (open squares) contents of
cell-conditioned medium were measured by ELISA. Results represent mean
values ± S.D. of three experiments and triplicate determination.
*, p < 0.05 compared with the control. C,
the indicated concentrations of bikunin and 10 ng/ml TGF-
1 were
added to the upper compartment. After a 36-h incubation, cells that
invaded through the Matrigel-coated membrane were stained and counted
under the microscope (×100). Results are expressed as the average
number of cells present in five fields/membrane; unlike
letters (a-e) stand for statistical differences
(p < 0.05).
1 and uPA Expression--
To analyze the signals regulating
bikunin-mediated suppression of TGF-
1, cells were treated with
calcium channel blockers (the L-type voltage-sensitive calcium channel
blockers nifedipine (50 µM) and verapamil (50 µM) and the non-voltage-sensitive calcium channel blocker
SK&F 96365 (0.5-50 µM)), protein tyrosine kinase inhibitor (genistein (25 µM), PP2 (10 µM)),
MAP kinase inhibitor (PD98059 (50 µM)), and protein
phosphatase (sodium vanadate (0.1 mM)), or bikunin
(1-1,000 nM), aspirate cell-conditioned medium, and
assayed for TGF-
1 (Fig. 6A)
and uPA (Fig. 6B) levels by ELISA. The constitutive
expression of TGF-
1 observed in HRA cells was inhibited by
preinoculation of the cells with bikunin or SK&F 96365 but not with
nifedipine, verapamil, genistein, PP2, PD98059, or sodium vanadate. The
addition of increasing doses of bikunin and SK&F 96365 strongly
decreased TGF-
1 secretion in a dose-dependent manner,
with an IC50 of ~30 µM for SK&F 96365. Further, we
tried to establish whether bikunin causes any modulation of SK&F
96365-dependent down-regulation of TGF-
1. However,
bikunin showed no additive effect on SK&F 96365-mediated suppression of
expression of TGF-
1.
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Fig. 6.
Effects of calcium channel blockers, protein
tyrosine kinase inhibitor, MAP kinase inhibitor, and protein
phosphatase on the secretion of TGF- 1 and
uPA. HRA cells were seeded in 35-mm dishes and then treated with
the indicated concentrations of drugs in serum-free medium for 24 h. The TGF-
1 (A) and uPA (B) contents of
cell-conditioned medium were measured by ELISA. Results are the
mean ± S.D. of three different determinations; unlike
letters (a-e) stand for statistical differences
(p < 0.05).
1-induced Calcium Influx Leading
to Inhibition of ERK Activation--
To investigate TGF-
1-induced
signaling in the HRA cells, we examined the Src and MAP kinase family
using HRA cells. Among MAP kinase family members, expression of ERK,
JNK, and p38 proteins was determined by Western blotting (Fig.
7A). When HRA cells were exposed to TGF-
1 for various periods, the phosphorylation of ERK was
enhanced from 5 min after addition of 10 ng/ml TGF-
1 and peaked at
15 min. In contrast, JNK and p38 did not appear to be activated by
TGF-
1. Thus, activation of ERK was most relevant among three subsets
of MAP kinase family members in response to TGF-
1. Previous reports
have demonstrated that an increase of intracellular calcium is
important for activation of ERK (26). Therefore, we examined the
contribution of extracellular calcium to the TGF-
1-stimulated ERK
activation using EGTA and SK&F 96365. When HRA cells were incubated in
the presence of EGTA (data not shown) or SK&F 96365, TGF-
1-stimulated phosphorylation of ERK was abrogated (Fig.
7B). In addition, the TGF-
1-stimulated ERK activation was
also abrogated when HRA cells were treated with bikunin, PP2, or
PD98059. Again, bikunin showed no additive effect on SK&F 96365-, PP2-,
or PD98059-mediated suppression of the TGF-
1-stimulated activation
of ERK. These results suggested that an increase of intracellular
calcium via non-voltage-gated calcium channels is required for the
TGF-
1-stimulated ERK activation in HRA cells.
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Fig. 7.
Effects of bikunin on the
TGF- 1-stimulated activation of the MAP kinase
family members. A, time-dependent MAP kinase
activation by TGF-
1 in HRA cells. HRA cells were exposed to 10 ng/ml
TGF-
1 for the periods indicated. Cell lysates were prepared; and
phosphorylation of ERK, JNK, and p38 was measured by Western blotting
analysis using specific antibodies against the phosphorylated proteins.
The same filters were immunoblotted with each of the specific
antibodies to demonstrate total amounts of ERK, JNK, and p38.
B, HRA cells were exposed to 10 ng/ml TGF-
1 in the
absence or presence of bikunin, SK&F, PP2, PD98059, SK&F plus bikunin,
PP2 plus bikunin, or PD98059 plus bikunin for 15 min. Total and
phosphorylated ERK were measured by Western blotting. Results are from
at least two separate experiments with similar results.
1 by HRA cells. We
examined the influence of bikunin on calcium mobilization in response
to TGF-
1 and TNF-
, two agonists previously shown to elevate
cellular calcium in ovarian cancer cells rapidly (10) and stimulate
expression of uPA. Using suspension of HRA cells loaded with the
calcium fluoroprobe Fura-2, we observed that the ability of TGF-
1 to
elevate free intracellular Ca2+ was reduced ~50% by a
preincubation of the cells with 1 µM bikunin (Fig.
8). Similar attenuations of calcium
signaling were observed in bikunin-treated HOC-I cells (human ovarian
cancer cell line) (data not shown). SK&F 96365 also inhibited
TGF-
1-stimulated elevation of intracellular Ca2+, with
10 µM SK&F 96365 causing significant inhibition. Higher concentration of SK&F 96365 produced further inhibition, with 50 µM agent causing around 85% inhibition. In contrast,
nifedipine did not suppress TGF-
1-stimulated intracellular
Ca2+ elevation. These results suggest that the bikunin
inhibitory effect on TGF-
1 signaling may act, at least in part,
through a non-voltage-sensitive calcium-dependent
pathway.
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Fig. 8.
Effects of bikunin and SK&F 96365 on
TGF- 1-induced elevation of
[Ca2+] in HRA cells. HRA cells were loaded with
Fura-2 and preincubated with the 0 nM bikunin (trace
1), 100 nM bikunin (trace 2), 500 nM bikunin (trace 3), or 1,000 nM
bikunin (trace 4) or 50 µM SK&F (trace
5) for 30 min before the addition of 10 ng/ml TGF-
1, indicated
by the dip in the traces between 0 and 1 min. The results shown are representative of three experiments.
Variability between replicates generally was less than 20% in a given
experiment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1, that exogenously added
TGF-
1 can specifically induce uPA and PAI-1 secretion and promotes
binding of uPA at the external plasma membrane with increased membrane-associated uPA/plasmin activity even in the presence of high
level of PAI-1 in the conditioned medium, and that exogenously added
TGF-
1 stimulated activation of MAP kinase (i.e.
phosphorylation of ERK), which resulted in up-regulation of the PA
system leading to enhancement of HRA cell invasion. In addition,
expression of the PA system was inhibited by a protein tyrosine kinase
inhibitor, genistein (25 µM), and a selective inhibitor
of Src tyrosine kinase, PP2 (10 µM). PP2 also prevented
the activation of ERK. Our results are supported by the facts that uPA
gene transcription is increased in certain cells after transformation
by the Src protein tyrosine kinase (29, 30); that Src kinases have been
shown to be involved in Gi protein-mediated ERK activation
(31), suggesting that Src kinase could be an upstream component of the
ERK cascade involved in uPA expression; that Src kinase plays an
important role in relaying signals from TGF-
1-coupled receptors to
MAP kinase (31); that selected mediators, including TGF-
, TNF-
,
lipopolysaccharide and phorbol ester, enhance uPAR expression in
certain tumor cell lines (32); and that the uPA interacts with uPAR to
promote proteolysis, and uPAR mediates cell signaling for migration.
uPAR appears to modulate phosphorylation of MAP kinase cascade (33), suggesting that the MAP kinase is a downstream target of uPAR. Thus,
TGF-
1 produced by HRA cells is a candidate responsible for the
regulation of the uPA/uPAR system and cell invasive process. Therefore,
we hypothesize that TGF-
1 can stimulate uPA expression through the
Src-dependent, ERK-specific signaling cascade. Further, TGF-
1-induced overexpression of uPAR (32) may lead to activation of
MAP kinase components, demonstrating a cross-talk between
TGF-
1-mediated MAP kinase cascade and the uPA/uPAR system (Fig.
9).
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Fig. 9.
Putative mechanism of bikunin-mediated
suppression of tumor invasion capacity through the
TGF- 1-dependent signal
transduction cascade. Bikunin can directly inhibit production of
TGF-
1 and TGF-
1-mediated MAP kinase activation through
suppression of calcium influx. Reduction of
TGF-
1-dependent signals by bikunin might cause
inhibition of the PA system, leading to suppression of cell
invasion.
Third, SK&F 96365 is an agent known to inhibit non-voltage-gated
calcium influx (34). The suppression by bikunin of TGF-1 and uPA
expression could be mimicked by the addition of exogenous SK&F 96365. Thus, bikunin could inhibit the constitutive expression of TGF-
1 and
TGF-
1-mediated PA system signaling cascade possibly through
suppression of the intracellular calcium ion influx. Removal of
extracellular calcium and the non-voltage-sensitive calcium channel
blocker SK&F 96365 but not the L-type voltage-sensitive calcium channel
blockers nifedipine and verapamil, inhibited both the constitutive
expression of TGF-
1 and TGF-
1-stimulated phosphorylation of ERK
and uPA/PAI-1 expression, suggesting that the non-voltage-sensitive calcium channel is specifically involved in these processes.
Finally, TGF-1-stimulated phosphorylation of ERK and uPA/PAI-1
expression was significantly inhibited by bikunin, SK&F 96365, PP2, and
by PD98059. Bikunin showed no additive effect on SK&F 96365-, PP2-, or
PD98059-mediated suppression of uPA expression, suggesting that bikunin
may function at the upstream target of Src tyrosine kinase and MAP
kinase cascade. A part of this is in agreement with previous data that
the ERK cascade, often an event downstream of Src tyrosine kinase
activation, has been shown to mediate uPA expression to a variety of
agents, including phorbol ester (29, 30) and that bikunin could
inhibit an upstream component of the ERK cascade involved in phorbol
ester-mediated uPA expression in human chondrosarcoma cell line HCS-2/8
(6).
SK&F 96365 and another non-voltage-sensitive calcium channel blocker
carboxyamidotriazol were shown previously to have anti-metastatic properties at low micromolar concentrations. Above 10 µM,
carboxyamidotriazol inhibited all second messenger pathways (36).
Therefore, the influx of calcium seems to be an event upstream of the
activation of Src and ERK, followed by uPA/PAI-1 expression. In
addition, we found that TGF-1 activity needs active uPA because
inhibitors of uPA/plasmin activity (p-aminobenzamidine and
anti-catalytic antibodies) block the activation of latent TGF-
1
present in the conditioned medium (data not shown). This suggests that
TGF-
1 is secreted by HRA cells as a latent form and is activated
successively by tumor cell-secreted uPA/plasmin. Taken together, we
conclude that expression of uPA/PAI-1 in HRA cells is dependent upon
continuous activation of the ERK signal transduction cascade, which is
downstream of an influx of extracellular calcium, and activation of Src
tyrosine kinases. Thus, bikunin may function as a voltage-independent
calcium influx antagonist.
The differences in bikunin sensitivity could be the result of variability in drug metabolism, a higher dependence on calcium-sensitive functions for cell invasiveness in certain types of cells, or a variability of the amounts of bikunin receptors on tumor cell membrane (37, 38). There appeared to be a specific interaction between bikunin and the tumor cell surface. HRA cells do not produce endogenous bikunin, but they do exhibit two types of bikunin receptors. Bikunin at low concentrations (<200 nM) bound to HRA cells in a dose-dependent manner. As the concentration of bikunin exceeded 200 nM, the amount of cell surface-bound bikunin was not further increased, suggesting that the binding of bikunin had reached a level of saturation (data not shown).
Because calcium is a pleiotropic second messenger in cell regulation and function, it is likely that bikunin will affect several cellular events simultaneously. A recent study of our laboratory demonstrated that bikunin has a specific inhibitory effect on calcium influx (8, 9). The inhibition of the uPA expression seen in the presence of bikunin could be caused by efficient inhibition of calcium influx, rather than direct inhibition of protein tyrosine kinase or phosphorylation of MAP kinase cascade, which was confirmed by the in-gel kinase assay.2 It is strongly supported that bikunin interferes with selected calcium-sensitive transmembrane signal transduction events.
Proteins of the Kunitz family may act as a calcium channel blocker. Bovine pancreatic trypsin inhibitor is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. Bovine pancreatic trypsin inhibitor is also homologous to dendrotoxin peptides from mamba snake venom, which has been characterized as an inhibitor of various types of voltage-dependent K+ channels (39). Bovine pancreatic trypsin inhibitor interferes directly with cation entry to the channel and is found to inhibit a large conductance Ca2+-activated K+ channel. The trypsin inhibitory loop of bovine pancreatic trypsin inhibitor recognizes a specific site on the channel protein. Calcicludine isolated from the green mamba venom has been identified recently as blocking a large set (i.e. L-, N-, and P-type) of calcium channels. Calcicludine also adopts the Kunitz-type protease inhibitor fold. Its three-dimensional structure is similar to that of dendrotoxins (35, 40). The C-terminal region of the large conductance Ca2+-activated K+ channels contains a domain homologous to serine proteases. These results support our present results, in which bikunin may function as an inhibitor of certain types of calcium channels, in particular, non-voltage-gated calcium channel.
We conclude that up-regulation of the PA system in HRA cells is
dependent upon activation of the TGF-1-mediated ERK signal transduction cascade, which is downstream from an influx of
extracellular calcium, and activation of Src tyrosine kinases and that
bikunin can function as a non-voltage-sensitive calcium channel
blocker. These findings point to a novel role for bikunin in regulating the PA system and TGF-
1-mediated invasion. Treatment with bikunin may be beneficial as an adjuvant therapy to delay the appearance of
metastatic disease and/or combined with cytotoxic agents to improve
treatment efficacy in advanced ovarian cancers.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Fujie, K. Shibata, T. Noguchi, and A. Suzuki (Equipment Center and Photo Center, Hamamatsu University School of Medicine) for helping with the biochemical analysis. We are also thankful to Drs. H. Morishita, Y. Kato, K. Kato, and H. Sato (BioResearch Institute, Mochida Pharmaceutical Co., Gotenba, Shizuoka), Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co. Ltd., Tokyo), and Drs. S. Miyauchi and M. Ikeda (Seikagaku Kogyo Co. Ltd., Tokyo) for continuous and generous support of our work.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (to H. K.) and by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-53-435-2309; Fax: 81-53-435-2308; E-mail:
hirokoba@hama-med.ac.jp.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M210407200
2 H. Kobayashi, M. Suzuki, Y. Tanaka, N. Kanayama, and T. Terao, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
PA(s), plasminogen activator(s);
ELISA, enzyme-linked immunosorbent assay;
ERK, extracellular signal-regulated kinase;
IL-1, interleukin-1;
JNK, c-Jun N-terminal kinase;
MAP, mitogen-activated protein;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
MMP, matrix metalloproteinase;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
PAI-1, plasminogen activator inhibitor type 1;
PP2, 4- amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
TGF- , transforming growth factor-
;
TNF-
, tumor necrosis
factor-
;
tPA, tissue-type plasminogen activator;
uPA, urokinase-type
plasminogen activator;
uPAR, urokinase-type plasminogen activator
receptor.
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REFERENCES |
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