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INTRODUCTION |
Metastasis is the major cause of death among cancer patients. The
cancer cells metastasis requires several sequential steps, such as
changes in cell-ECM1
interaction, disconnection of intercellular adhesions, and separation of single cells from solid tumor tissue, degradation of ECM, locomotion of tumor cells into the extracellular matrix, invasion of lymph and
blood vessels, immunologic escape in the circulatory system, adhesion
to endothelial cells, extravasation from lymph and blood vessels,
proliferation of cells, and induction of angiogenesis (1).
Attachment of cells to ECM molecules is mediated by the integrin family
of extracellular matrix receptors. Integrins are a large family of
heterodimeric proteins that transduce a variety of signals from the
ECM. Through integrin and matrix interactions, many of the genes, which
are critical for cell migration, survival, proliferation,
differentiation (2), and ECM degradation, are activated (3, 4). In the
majority of metastasizing tumors, cellular interactions with the ECM,
which promote adhesion and migration, are thought to be required for
primary tumor invasion, migration, and metastasis.
The main groups of proteolytic enzymes involved in the tumor invasion
are matrix metalloproteinases (MMPs) and serine proteases. The MMPs, a
family of zinc-dependent endopeptidases, are involved in
tumor invasion, metastasis, and angiogenesis in cancer (5). MMPs are
important enzymes for the proteolysis of extracellular matrix proteins
such as collagen, proteoglycan, elastin, laminin, and fibronectin (6).
MMPs are synthesized as preproenzymes, and most of them are secreted
from the cells as proenzymes. Among human MMPs reported previously,
MMP-2 (gelatinase A/Mr 72,000 type IV
collagenase) and MMP-9 (gelatinase B/Mr 92,000 type IV collagenase) are thought to be key enzymes for degrading type IV collagen, which is a major component of the basement membrane (5).
Both MMP-2 and MMP-9 are abundantly expressed in various malignant
tumors (7) and contribute to invasion and metastasis documented in many
reports (8). The serine proteases, urokinase-type plasminogen activator
(uPA), can convert plasminogen to plasmin, which is capable of
degrading extracellular matrix proteins like fibrin, fibronectin,
vitronectin (9), and type IV collagen (10) as well as activating latent
forms of MMPs (11). Therefore, uPA leads to a synergistic effect with
MMPs.
Selenium, an essential trace element for animals, has been shown to
prevent cancer in numerous animal model systems (12) and cancer
chemopreventive efficacy in humans (13). The known functions of
selenium as an essential element in animals are attributed to ~12
known mammalian selenoproteins, glutathione peroxidase, thioredoxin
reductase, phospholipid hydroperoxide, etc., that contain
selenocysteine, specifically incorporated through a unique co-translational mechanism (14). However, the studies of the functions
of selenium mainly have been focused on the chemopreventive effects,
whereas the relationship between selenium and metastasis of cancer
cells has not been firmly established. Here we demonstrate that
selenite decreases the invasiveness of tumor cells in vitro, which is derived from inhibition of cell-matrix interaction,
suppression of the MMPs and uPA expressions, and up-regulation of
tissue inhibitor of metalloproteinase-1 (TIMP-1). These results suggest
that selenite can contribute to the reduction of invasion and
metastasis in tumors.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Materials--
HT1080 (fibrosarcoma), 293 (embryonic kidney), and MDA-MB-231 (breast adenocarcinoma) cells were
grown in DMEM supplemented with 10 mM HEPES, 50 mg/liter
gentamicin (Life Technologies, Inc.), and 10% heat-inactivated fetal
bovine serum. T98G (glioblastoma) and NUGC-3 (gastric adenocarcinoma)
cells were grown in RPMI 1640 supplemented with 10 mM
HEPES, 50 mg/liter gentamicin, and 10% heat-inactivated fetal bovine
serum. Human type I and IV collagens, bovine serum albumin, gelatin,
sodium selenite, sodium selenate, plasminogen, fibrinogen, and thrombin
were purchased from Sigma. Anti-MMP-9, anti-TIMP-1, anti-uPA, and
anti-uPA inhibitor-1 (uPAI-1) were obtained from Chemicon
International, Inc. (Temecula, CA). Matrigel was purchased from Becton
Dickinson (Bedford, MA).
Cytotoxicity
Assay--
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) (Roche Molecular Biochemicals) assays were performed as described in the supplier's protocol to evaluate the cytotoxicity of
selenite and selenate. To confirm the MTT assay results, we also tested
trypan blue dye exclusion assay.
Cell Invasion and Motility Assays--
5 × 104
cells/chamber were used for each invasion assay. The lower and upper
parts of Transwell (Corning Costar, Cambridge, MA) were coated with 10 µl of type I collagen (0.5 mg/ml) and 20 µl of 1:2 mixture of
Matrigel:DMEM, respectively. Cells were plated on the Matrigel-coated
Transwell in the presence of various concentrations of selenite and
selenate. The medium in the lower chambers also contained 0.1 mg/ml
bovine serum albumin. The inserts were incubated for 18 h at
37 °C. The cells that had invaded to the lower surface of the
membrane were fixed with methanol and stained with hematoxylin and
eosin. Random fields were counted under a light microscope.
To determine the effect of the selenite and selenate on cell motility,
cells were seeded into Transwell on membrane filters coated with 10 µl of type I collagen (0.5 mg/ml) at the bottom of the membrane.
Migration in the absence or presence of selenite and selenate was
measured as described in the invasion assay.
Zymography--
MMP-2 and MMP-9 enzymatic activities were
assayed by gelatin zymography (15). uPA activity was assayed as
described previously (16) with some modification. For receptor-bound
uPA, cells were incubated for 3 min with 50 mM glycine HCl
(pH 3.0) containing 0.1 M NaCl. Samples of serum-free
conditioned medium and buffers containing receptor-bound uPA were
electrophoresed on a 10% SDS-polyacrylamide gel. After
electrophoresis, the gel was washed twice with washing buffer, followed
by a brief rinsing in washing buffer without Triton X-100. The gel was
placed on a 0.5% agarose gel containing 0.3% (w/v) fibrinogen, 0.1 unit/ml thrombin, 0.2 unit/ml human plasminogen and incubated at
37 °C. After incubation, the gel was stained and destained. In this
gel, a clear zone of fibrin digestion that appeared indicated the
presence of uPA.
Plasmids--
MMP-9 promoter region (
670 to +3) was
PCR-amplified and inserted upstream of the pGL3 luciferase reporter
vector (Promega, Madison, WI). uPA promoter containing pGL2 luciferase
reporter vector was provided by Dr. F. Blasi (Universita Vita-Salute
San Raffaele, Milan, Italy). NF-
B and AP-1 reporter constructs were purchased from CLONTECH (Palo Alto, CA).
Transient Transfection and Reporter Gene Assay--
HT1080 cells
were plated in 6 wells and incubated at 37 °C. At 70-80%
confluency, cells were washed with DMEM and incubated with DMEM without
serum and antibiotics for 5 h. 2 µg of MMP-9 promoter containing
pGL3 vector and 0.5 µg of
-galactosidase vector were transfected
using LipofectAMINE 2000 reagent (Life Technologies, Inc.). After
24 h, various concentrations of selenite and selenate were
treated. After 24 h of incubation, cells were lysed, and
luciferase activity was measured using a luminometer.
-Galactosidase
activity was measured using o-nitrophenyl
-galactopyranoside as a substrate. The same method was used for the
measurement of uPA promoter, NF-
B, and AP-1 activities.
Cell-Cell Adhesion Assay--
HT1080 cells were plated in 24 wells and incubated at 37 °C to 100% confluency. Along with this,
other HT1080 cells were radiolabeled with [3H]thymidine
overnight and trypsinized. Radiolabeled cells were resuspended in DMEM
with 10% fetal bovine serum and added to the unlabeled attached 100%
confluent 24 wells. After 2-3 h of incubation, nonadherent cells were
collected. Then plates were rinsed with PBS (137 mM NaCl,
2.7 mM KCl, 4.3 mM
Na2HPO4 7H2O, and 1.4 mM KH2PO4), and this was collected
in the same container. Following this procedure, bound cells were
trypsinized completely and collected in other containers. Radioactivity
was measured by liquid scintillation counter, and the percentage of
adherent cells was calculated.
Cell-Matrix Adhesion Assay--
24-Well plates were coated with
10 µg/ml type I collagen or type IV collagen. Nonspecific binding was
blocked by PBS containing 2% bovine serum albumin for 2 h at room
temperature. Cells were radiolabeled with [3H]thymidine
overnight and trypsinized. Cells were then plated on coated culture
plates and incubated for 30 min. Nonadherent and adherent cells were
collected and counted, and the percentage of adherent cells was
calculated as described in cell-cell adhesion assay method.
RNA Isolation and Northern Blot Analysis--
Total cellular RNA
was purified from cultured cells using TRIZOL reagent (Life
Technologies, Inc.). For Northern blot analysis, 15 µg of RNA were
electrophoresed on 1% agarose gels containing 37% formaldehyde and
transferred to Hybond-N membranes (Amersham Pharmacia Biotech) by
capillary transfer. Membrane was fixed using an optimized UV
cross-linking procedure. Prehybridization and hybridization were
performed at 68 °C in ExpressHyb hybridization solution
(CLONTECH). cDNA probes for MMP-2, MMP-9,
TIMP-1, TIMP-2, uPA, uPAI-1, and glyceraldehyde-3-phosphate
dehydrogenase were labeled with [32P]dCTP (3000 Ci/mmol,
Amersham Pharmacia Biotech) using a random primer kit (Takara, Japan).
The blot was then washed twice with 2× SSC (300 mM NaCl,
30 mM sodium citrate, pH 7.0) containing 0.05% SDS at
25 °C, 0.1× SSC containing 0.1% SDS at 55 °C, and autoradiographed at
70 °C.
Western Blot Analysis--
Conditioned media were collected and
concentrated using Centricon (Millipore, Bedford, MA). Western blot
analysis for secreted MMP-9, uPA, TIMP-1, and uPAI-1 was performed
according to Burnette's method (17).
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RESULTS |
Effect of Selenite and Selenate on Proliferation of HT1080
Cells--
Selenite and selenate are inorganic selenium compounds
dissolved in water. Because they have rather different chemical and biological characteristics (18), these compounds were used throughout the experiments for comparison of antitumor activities. We first tested
the cytotoxic effects of selenite and selenate on HT1080, human
fibrosarcoma cells, by MTT assay in serum-free and 10%
serum-containing media. Selenite showed higher toxicity than selenate
in both conditions, with and without serum (Fig.
1). Treatment with 3 µM
selenite increased cell viability, but a further increase in
concentration of selenite decreased cell viability. At the serum-free
conditions, 5 µM selenite began to show toxic effects to
cells, and at media containing 10% fetal bovine serum, 10 µM selenite showed cytotoxicity (Fig. 1A).
Selenate did not show any cytotoxic effect below 50 µM in
the presence or absence of serum (Fig. 1B). To confirm MTT assay, we performed trypan blue dye exclusion assay, and the results were similar to those of MTT assay, as expected. Noncytotoxic concentration of selenite and selenate was used for additional experiments.

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Fig. 1.
Effects of selenite and selenate on viability
of HT1080 cells. HT1080 cells were treated with selenite and
selenate, and after 3 days the viability was tested by MTT assay as
described under "Experimental Procedures." HT1080 cells were
treated with selenite (A) and selenate (B)
varying the concentrations with (open circle) or without
(closed circle) serum.
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Effects of Selenite and Selenate on Invasion and Motility of HT1080
Cells--
As shown in Fig.
2A, invasion of HT1080 cells
was significantly reduced by treatment with 1 µM selenite
and further inhibited with increased concentrations of selenite.
Selenate did not exhibit a significant effect on the invasion at the
concentrations used (Fig. 2B). However, neither selenite nor
selenate showed dramatic changes of motility (Fig. 2, C and
D, respectively).

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Fig. 2.
Effects of selenite and selenate on in
vitro invasion and motility of HT1080 cells. For
invasion assay, the lower and upper parts of Transwells were coated
with collagen and Matrigel, respectively. HT1080 cells and various
concentrations of selenite (A) and selenate (B)
were added. After 16-18 h, cells on the bottom side of the filter were
fixed, stained, and counted as described under "Experimental
Procedures." For motility assay, the lower parts of filters were
coated with collagen. HT1080 cells and various concentrations of
selenite (C) and selenate (D) were added and
assayed as described under "Experimental Procedures." Data
represent the mean ± S.E. of at least three independent
experiments. Results were statistically significant (*,
p < 0.05) using Student's t test.
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Effects of Selenite and Selenate on Adhesion of HT1080 cells
to ECM Proteins and Cells--
Cell-matrix interaction is important
for cancer cell invasion because this interaction affects protease
expression, tumor cell locomotion, and survival. So, we tested whether
selenite and selenate affect cell-matrix interaction. When HT1080 cells were preincubated for 6 h with selenium compounds, selenite
markedly reduced the cells attachment to type I and type IV collagen in a dose-dependent manner (Fig.
3A). The attachment of cells
to type I collagen was much more affected by selenite than type IV collagen. Unlike selenite, selenate did not significantly affect cell-collagen interactions (Fig. 3B).

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Fig. 3.
Effects of selenite and selenate on
cell-matrix and cell-cell attachments. For cell-matrix attachment
assay, radiolabeled HT1080 cells, which were preincubated with selenite
(A) and selenate (B) for 6 h, were seeded
onto the collagen-coated wells. After 30 min, unattached and attached
cells were collected and counted. For cell-cell interaction assay,
radiolabeled HT1080 cells were preincubated with selenite
(C) and selenate (D) for 6 h and seeded
onto the 100% HT1080 confluent wells. 2-3 h after treatment,
unattached and attached cells were collected and counted. Data
represent the mean ± S.E. of at least three independent
experiments. Results were statistically significant (*,
p < 0.05; **, p < 0.01) using
Student's t test.
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To invade extracellular matrix, tumor cells must dissociate from
solid tumors. At this time, cell-cell interaction is loosened. To
examine the effects of selenite and selenate on HT1080 cells adhesion
to HT1080 cells themselves, a monolayer cell adhesion assay was carried
out. Adhesion of HT1080 cells to HT1080 was not changed even after
6 h (Fig. 3, C and D) and even longer
treatment with selenite and selenate (data not shown).
Effects of Selenite and Selenate on MMPs and uPA
Activities--
After the tumor cell became detached from the
neighboring cells by loosening its intercellular junctions, the
extracellular matrix was proteolytically degraded in order to allow
migration and invasion of the cell. Extracellular matrix breakdown is
vital to cellular invasion, indicating that matrix-degrading
proteinases are essential for tumor cell metastasis. HT1080 cells
constitutively secreted high levels of the MMP-2, MMP-9, and uPA. To
clarify whether activities of MMPs and uPA are involved in inhibition of invasion by selenite, we evaluated the effects of selenite and
selenate on MMPs and uPA activities with the use of gelatin and fibrin
zymography, respectively. MMPs and uPA bands were confirmed by size
markers. When HT1080 cells were incubated with selenite and selenate
for 3 days, selenite markedly reduced MMP-9 (Fig. 4A) and uPA (Fig.
4C) activities in a concentration-dependent manner but less effectively decreased MMP-2 activity (Fig.
4A). Membrane-bound uPA activity was also decreased by
selenite (data not shown). But selenate did not show any inhibitory
effect on MMP-2, -9, and uPA activities (Fig. 4, B and
D). To confirm that the inhibitory effect of selenite on
MMPs in HT1080 cells is general phenomena, several cell lines such as
MDA-MB-231, NUGC-3, T98G, and 293 cells were further tested. The
activities of MMP-9 were dramatically decreased by selenite in these
cells, but those of MMP-2 were less dramatic (Fig.
5).

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Fig. 4.
Effects of selenite and selenate on the
activities of MMP-2, MMP-9, and uPA. To test of activities of
MMP-2 and MMP-9, HT1080 cells were treated with various concentrations
of selenite (A) and selenate (B). 3 days after
the treatment, conditioned media were collected, and gelatin zymography
was performed as described under "Experimental Procedures." For the
uPA activity test, various concentrations of selenite (C)
and selenate (D) were treated for 3 days, and fibrin
zymography was performed.
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Fig. 5.
Effects of selenite on the activities of
MMP-2 and MMP-9 in various cell lines. T98G, HEK 293, MDA-MB-231,
and NUGC-3 cells were treated with various concentrations of selenite
for 3 days, and gelatin zymography was performed as described under
"Experimental Procedures."
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Effect of Selenite on Stimulatory or Inhibitory Chemicals of MMP-9
Expression--
We attempted to detect whether selenite is also
effective in the presence of TPA and TNF-
, which are known to
up-regulate MMP-9 expression and activate pro-MMP-2 to active MMP-2
(19, 20). Treatment of HT1080 cells with TPA and TNF-
resulted in increased levels of MMP-9 expression, but pretreatment with selenite also decreased level of MMP-9 (Fig. 6,
A and B). The expression and the activation of
MMP-2 were much less affected by selenite, as expected. We next treated
dexamethasone, which is known to decrease MMP-9 expression (21). When
selenite and dexamethasone were treated in cells simultaneously, MMP-9
activity was much more decreased than that of selenite or
dexamethasone-treated cells only (Fig. 6C). Unlike selenite,
selenate showed no marked effects on MMP-9 activity in all cases (data
not shown).

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Fig. 6.
Effects of selenite on the activities of
MMP-2 and MMP-9 in the presence of TPA, TNF- ,
and dexamethasone. HT1080 cells were preincubated with selenite
for 6 h and treated with TPA (50 nM) (A) or
TNF- (10 ng/ml) (B). After 24 h, conditioned media
were collected and analyzed by gelatin zymography. To find the effects
of dexamethasone and/or selenite on MMPs activities, 50 nM
dexamethasone and/or 3 µM selenite were treated, and
gelatin zymography was performed (C).
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Effects of Selenite and Selenate on Transcription of Proteases and
Their Inhibitors--
We further tested whether the strong inhibitory
effects of selenite on MMP-9 and uPA are by direct association between
selenite and proteases. We performed two experiments. First, we
incubated various concentrations of selenite with conditioned media
without any HT1080 cells. Direct exposure of collected conditioned
medium of HT1080 cells to selenite had no effect on MMPs and uPA
gelano- and fibrolytic activities (data not shown), indicating that
inhibition of MMPs and uPA was not attributable to a direct effect of
the selenite on secreted MMPs and uPA. Second, we performed Western blotting to identify the protein amount. The results of Western blotting showed that selenite decreased MMP-9 and uPA, but increased or
didn't change TIMP-1 and uPAI-1 levels (Fig.
7), respectively.

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Fig. 7.
Effects of selenite on the protein levels of
MMP-9, TIMP-1, uPA, and uPAI-1. HT1080 cells were treated with
various concentrations of selenite for 3 days and lysed, and 80 µg of
each sample was subjected to SDS-polyacrylamide gel electrophoresis,
and Western blot analysis was performed.
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These two experiments indicate that selenite may exert its actions
through the transcriptional regulation of these proteins. Further
examination showed that the reduced activities of proteases are related
to their mRNA levels. In addition, mRNA levels of their
inhibitors were also studied. As shown in Northern blotting results
(Fig. 8), selenite decreased MMP-2,
MMP-9, and uPA transcriptional levels, and MMP-9 mRNA was
dramatically decreased at 3 µM selenite. Interestingly,
selenite increased TIMP-1 mRNA in a dose-dependent manner but decreased TIMP-2 mRNA levels. This Northern blotting pattern was similar to the results of zymography (Fig. 4, A
and C) and Western blotting (Fig. 7). However, selenate did
not produce much of an effect on MMPs and uPA mRNA levels, as of
zymographic analysis, and rather decreased TIMP-1 and short transcript
of uPAI-1. Because the mRNA levels of MMP-9 and uPA were markedly decreased by selenite, we further examined them using MMP-9 and uPA
promoter vectors. In this experiment, selenite reduced the activities
of both promoters in a dose-dependent manner (Fig. 9), which was a similar trend of Northern
blotting. All of these results suggest that proteases and their
inhibitors are transcriptionally regulated by selenium compounds,
especially selenite.

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Fig. 8.
Effects of selenite and selenate on the
transcriptional levels of proteases and their inhibitors. HT1080
cells were treated with various concentrations of selenite
(A) and selenate (B), and RNA was extracted. RNA
were loaded on 1% agarose gels, and Northern blotting was carried out
as described under "Experimental Procedures." RNA loading was
normalized using the signal obtained with a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe.
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Fig. 9.
Effects of selenite on the promoter
activities of MMP-9 and uPA. MMP-9 (A) or uPA
(B) promoter-containing reporter vectors were transfected,
and various concentrations of selenite were treated, and luciferase
activity was measured. Data represent the mean ± S.E. of at least
three independent experiments. Results were statistically significant
(*, p < 0.05) using Student's t
test.
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Effects of Selenite and Selenate on NF-
B and AP-1
Activities--
Several transcriptional factors regulate the
expressions of MMPs, uPA, and their inhibitors. AP-1 is a major
transcription factor, which regulates MMP-9, uPA, TIMPs, and uPAI-1
expression, and NF-
B is known to regulate MMP-9 and uPA expressions.
We then tested the activities of these transcription factors regulated by selenite and selenate using AP-1 and NF-
B reporter vectors, which
have binding sites to those factors. AP-1 and NF-
B activities were
significantly decreased by treatment with selenite in the presence as
well as in the absence of TPA (Fig. 10,
A and C, respectively), but selenate slightly
increased AP-1 and NF-
B activities (Fig. 10, B and
D, respectively).

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Fig. 10.
Effects of selenite and selenate on the
activities of AP-1 and NF- B. To elucidate
the effects of selenite and selenate on AP-1 activity, a report vector
that has AP-1-binding sites was transfected, and various concentrations
of selenite (A) and selenate (B) were treated.
After 6 h, 50 nM TPA was untreated (black
bar) or treated (white bar) and incubated for 24 h. The cells were then lysed, and luciferase activity was measured. The
same method was used for the test of effects of selenite (C)
and selenate (D) on NF- B activity. Data represent the
mean ± S.E. of at least three independent experiments. Results
were statistically significant (*, p < 0.05) using
Student's t test.
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DISCUSSION |
It was demonstrated that selenite exerted stepwise suppression of
cancer cell metastasis within the nontoxic range. Selenite significantly inhibited the invasion of HT1080 human fibrosarcoma cells. This research was to determine which steps were regulated by
selenite. Nontoxic levels of selenite markedly reduced cell-collagen attachment but had no effect on adhesion of the cell-cell and cell
motility. Many reports show the importance of cancer cell-matrix interaction. Cell and matrix interactions promote cell migration, proliferation, and ECM degradation (2-4). Also, it has been shown that
prevention of tumor cell adhesion and migration is related to
inhibition of tumor cell invasion into the basement membrane (22), and
agents that inhibit cell attachment in vitro decrease the
invasiveness and/or metastatic potential of tumor cells in vivo (23-26). Therefore, cellular interactions with the ECM,
which promote adhesion and migration, are thought to be required for primary tumor invasion, migration, and metastasis (27). It is reported
that selenite inhibits HeLa cells attachment to the ECM (28). We also
demonstrated here that attachment of HT1080 cells to the type I and IV
collagen was significantly decreased after 6 h of pretreatment
with 2 µM selenite and further dramatically decreased
with 3 µM. But selenate did not show any inhibitory effect on attachment. The cell-cell adhesion and cell motility are
closely associated with tumor cell invasion and metastasis (1, 29, 30),
but selenite had no marked inhibitory or stimulatory effects on them in
our experiments. Therefore, the inhibition of attachment of HT1080
cells to the matrix by selenite is a crucial mechanism for the
inhibition of invasion.
After the tumor cell has become detached from neighboring cells by
loosening its intercellular junctions, the ECM has to be proteolytically degraded in order to allow migration and invasion of
the cells. So, matrix-degrading proteinases are essential for tumor
cell metastasis. Many studies reveal that enhanced production of MMPs
and uPA correlates with the invasion, metastasis, and angiogenesis of
the tumors (10). Moreover, the relationship between the suppression of
MMP-9 and the inhibition of invasion and metastasis has been explored
with the use of anti-MMP-9 ribozyme (31), ursolic acid (32), and
aspirin (33). Down-regulation of uPA levels using monoclonal antibody
against urokinase or antisense oligonucleotides also showed reduced
invasion and metastasis of tumor cells in mice (34). Here it was
demonstrated that selenite decreased markedly MMP-9, uPA, and less
dramatically MMP-2 expressions. The inhibitory effect of selenite on
proteinases expression gives reasonable explanation for the inhibition
of invasion.
Moreover, selenite exerts its action through regulation of inhibitor of
MMP-9, TIMP-1. Interestingly, selenite increased the TIMP-1 mRNA
and protein level but decreased the TIMP-2 level. Although TIMP-1 and
TIMP-2 are inhibitors of MMP-9 and MMP-2, respectively, expressions of
these inhibitors are differentially regulated in vivo as
well as in the cell culture system (35, 36). As TIMP-1 is a natural
inhibitor of MMP-9, the increase in its mRNA and protein can
inhibit tumor cell invasion (37, 38). These kinds of effects on MMPs
and TIMPs are not confined to selenite. Genistein (39), ursolic acid
(32), and 1
,25-dihydroxyvitamin D3 and its analogues
(40) show similar results with selenite. Genistein decreases MMP-9 and
MMP-2 mRNA levels, whereas it increases TIMP-1 mRNA levels in
MDA-MB-231 and MCF-7 cells, and ursolic acid decreases MMP-9 but
increases TIMP-1 mRNA in HT1080 cells, but the MMP-2 level is not
significantly affected. 1
,25-Dihydroxyvitamin D3 and its
analogues also down-regulates MMP-9 and uPA, whereas it up-regulates
TIMP-1 and uPAI-1 levels in MDA-MB-231 cells. Selenate showed different
patterns compared with selenite in all cases. It did not change MMPs,
uPA and TIMP-2, significantly but decreased TIMP-1 and the short
transcript of uPAI-1.
We were further interested in the inhibitory mechanism of MMPs and uPA
expressions by selenite. The role of MAPKs in regulation of MMP-9 and
uPA expressions in malignant cells has been well understood. At least
two (extracellular signal-regulated kinase and c-Jun
NH2-terminal kinase/stress-activated protein kinase) of the
three so-called mitogenic pathways known so far in mammalian cells
induce up-regulation of MMP-9 and uPA (41, 42). Recently it has been
shown that inhibition of p38 leads to reduced MMP-9 expression and
invasion by tumor cells (43), and p38 stabilizes uPA mRNA and
invasiveness in MDA-MB-231 cells (44). To link MAPK and protease
expressions, we examined the effects of selenite and selenate to
transcription factors. We tested AP-1 activity change by selenite and
selenate. MMP-9, uPA, and TIMP-2 promoter contain AP-1-binding sites
(5, 36, 45) so that MAPKs pathways are important. Selenite is shown to
inhibit specifically AP-1 DNA binding in vitro through
conserved cysteine residues in the DNA-binding domains of Jun and Fos
(46, 47). Selenite also inactivates AP-1 via inhibition of MAPKs
pathways (48). In our experiments, selenite also suppressed AP-1
activity in the absence and in the presence of TPA, but selenate
slightly increased AP-1 activity. Interestingly, although AP-1 also
affects the basal transcription of TIMP-1 (49), this inhibitor
expression pattern was quite different from the MMPs, uPA and TIMP-2,
by selenite treatment. However, these patterns are not
selenite-specific. Genistein, ursolic acid, and
1
,25-dihydroxyvitamin D3 and the analogues of the latter
show similar patterns (39, 32, 40) as described above. The exact
mechanisms have not been elucidated up to now, but several reports
demonstrate that TIMP-1 and TIMP-2 expressions are differentially
regulated in vivo as well as in cell culture (35, 36). In
hepatic stellate cells, induction of c-Fos and c-Jun is unlikely to
result in transactivation of the TIMP-1 promoter (50). Therefore,
researchers suggest that unidentified factors may be involved in the
regulation of TIMP-1 expression (50-52). It is possible that selenite
may function with other factors that regulate TIMP-1 expression.
We then tested whether selenite and selenate affect NF-
B activity.
The MMP-9 and uPA expressions require NF-
B (53, 54) as well as AP-1.
Although MMP-2 promoter does not contain NF-
B-binding site, it has
been reported recently (55) that MMP-2 activation occurs in endothelial
cells through an NF-
B-dependent pathway. Selenite has
been shown to inhibit the binding of NF-
B to DNA (56) by oxidizing
the critical cysteine Iresidues in their DNA-binding domains like AP-1.
Our results also showed that NF-
B activity was inhibited by selenite
in HT1080 cells. The inhibitory effect of selenite on NF-
B activity
can explain the repression of MMP-9, uPA, and MMP-2 in concert with
AP-1 inhibition. Selenite not only directly affects AP-1 and NF-
B
transcription factors but also affects signal molecules such as c-Jun
NH2-terminal kinase, p38, and protein kinase C (48, 57). As
a result, AP-1 and NF-
B activities could be more strongly inhibited
by selenite. From these observations, it can be suggested that selenite
inhibits protease expressions through repression of AP-1 and NF-
B,
both directly and indirectly.
AP-1 and NF-
B affect each other (58-60), and it is known that these
factors are involved in inflammation, cell adhesion, cell invasion,
metastasis, and angiogenesis (5, 61, 62). From these observations, it
has been suggested that suppression of the AP-1 and/or NF-
B
activities give potential in blocking tumor initiation, promotion, and
metastasis (63), but there have been few reports on the relationship
between selenium and anti-metastasis. Only recently has it been
reported that selenite and methylselenocysteine inhibit angiogenesis by
reduction of intra-tumoral microvessel density and vascular endothelial
growth factor expression in mammary carcinomas (64). Genistein inhibits
cell-matrix attachment (65), down-regulates MMPs and uPA, but
up-regulates TIMP-1, inhibits cell proliferation, angiogenesis, and
invasion, and induces apoptosis (39, 66, 67). Therefore, from our
results and other reports mentioned previously, we can suggest that the
function of selenite is similar to that of genistein. Tumor cells
appear to be more sensitive than normal cells by the treatment with
selenium compounds (68, 69). Therefore, treatment with selenium
compounds can be utilized to prevent cancer incidence and further to
reduce tumor properties, especially metastasis. Further studies of
anti-metastatic properties of selenite are required.
In conclusion, we have shown that selenite inhibited several essential
steps of metastasis. First, selenite inhibited cell-matrix interaction.
Second, selenite regulated the activities of invasion-associated proteases and their inhibitors. Third, this regulation is mediated by
the regulation of transcription factors such as AP-1 and NF-
B, directly and indirectly. Unlike selenite, selenate had no marked effect
on cell invasion, cell matrix interaction, and expression of proteases
but rather increased mRNA levels of proteases through the partial
activation of AP-1 and NF-
B.