jbc papers in press. published on march 27, 2001 as ... such as collagen, proteoglycan, elastin,...

Inhibitory Effect of Selenite on Invasion of HT1080 Tumor Cells

Sang-Oh Yoon, Moon-Moo Kim, and An-Sik Chung*

Department of Biological Sciences, Korea Advanced Institute of Science and

Technology, Taejon 305-701, South Korea

Running Title: Selenite inhibits tumor cell invasion

*To whom correspondence should be addressed: Department of Biological Sciences,

Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea.

Tel: 82-42-869-2625; Fax: 82-42-869-2610; E-mail: [email protected]

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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 27, 2001 as Manuscript M101143200 by guest on June 7, 2018

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SUMMARY

Selenium, an essential biological trace element, has been shown to reduce and

prevent the incidence of cancer. Our previous studies have shown that selenite is

involved in the chemoprevention of cancer and induction of apoptosis of cancer cells. In

this study, we demonstrate that selenite also inhibits the invasion of tumor cells. Cancer

cell invasion requires coordinated processes, such as changes in cell-cell and cell-

matrix adhesion, degradation of the extracellular matrix, and cell migration. We found

that selenite inhibited invasion of HT1080 human fibrosarcoma cells. Adhesion of

HT1080 cells to the collagen matrix was also inhibited by treatment with selenite, but

cell-cell interaction and cell motility were not affected with selenite. Moreover, selenite

reduced expression of matrix metalloproteinase-2,-9 and urokinase type plasminogen

activator, which are involved in matrix degradation, but increased a tissue inhibitor of

metalloproteinase-1. This inhibitory effect of selenite on the proteases expressions was

mediated by the suppression of transcription factors, NF-κB and AP-1. However,

selenate showed no remarkable effects on above all the steps of cancer cell invasion.

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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-1ECM 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

which 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

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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 chemopreventvie 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., which 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,

while 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,

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suppression of the MMPs and uPA expressions, and upregulation of tissue inhibitor of

metalloproteinase-1 (TIMP-1). These results suggest that selenite can contribute to the

reduction of invasion and metastasis in tumors.

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/L gentamicin (Life Technologies, Inc., Rockville, MD),

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/L gentamicin, and 10% heat inactivated fetal bovine serum. Human type

I and IV collagens, bovine serum albumin, gelatine, sodium selenite, sodium selenate,

plasminogen, fibronogen and thrombin were purchased from Sigma (St. Louis, MO).

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, Germany) assays were performed as described in supplier’s

protocol to evaluate the cytotoxicity of selenite and selenate. To confirm this MTT

assay results, we also tested tryphan blue dye exclusion assay.

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Cell Invasion and Motility Assays- 5 ×10 4 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 of the

lower chambers also contained 0.1 mg/ml BSA. 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.

Zymograhpy-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, each. 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) fibronogen, 0.1

unit/ml thrombin, 0.2 unit/ml human plasminogen and incubated at 37°C. After

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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-κB and AP-1 reporter

constructs were purchased from Clontech (Palo Alto, CA).

Transient Transfection and Reporter Gene Assay- HT1080 cells were plated in 6

well 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 β-galactosidase vector were transfected

using LipofectAMINE 2000 reagent (Life Technologies). After 24 h, various

concentrations of selenite and selenate were treated. After 24 h incubation, cells were

lysed and luciferase activity was measured using luminometer. β-galactosidase activity

was measured using o-nitrophenyl β-galactopyranoside (ONPG) as a substrate. The

same method was used for the measurement of uPA promoter, NF-kB, and AP-1

activities.

Cell-Cell Adhesion Assay- HT1080 cells were plated in 24 well 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% FBS and added to the unlabeled attached 100% confluent 24 well. After 2-3

h incubation, nonadherent cells were collected. Then plates were rinsed with PBS (137

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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 of 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, counted and

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). 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, Arlington

Heights, IL) 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 GAPDH 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

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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).

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 characters (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

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with 3 µM selenite increased cell viability, but further increased 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% FBS, 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 tryphan

blue dye exclusion assay and the results were similar to those of MTT assay, as

expected. Noncytotoxic concentration of selenite and selenate was used to further

experiments.

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).

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

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interactions (Fig. 3B).

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

has become detached from the neighboring cells by loosening its intercellular junctions,

the extracellular matrix has to be 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 inhibiton 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 marker. 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

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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).

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-α, which are known to up-regulate MMP-9 expression, and activate proMMP-2

to active MMP-2 (19, 20). Treatment of HT1080 cells with TPA and TNF-α 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).

Effects of Selenite and Selenate on Transcription of Proteases and Their Inhibitors-

We further tested whether this strong inhibitory effects of selenite on MMP-9 and uPA

are through direct association between selenite and proteases. We performed two

experiments. Firstly, 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 inhibiton of MMPs and uPA was not

attributable to a direct effect of the selenite on secreted MMPs and uPA. Secondly, we

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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.

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 of selenite. Interestingly, selenite increased

TIMP-1 mRNA in a dose-dependent manner, but decreased TIMP-2 mRNA level.

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 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.

Effects of Selenite and Selenate on NF-κB 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

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expression, and NF-κB 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-κB reporter vectors, which have binding sites to those factors.

AP-1 and NF-κB 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-κB activities (Fig. 10, B and D, respectively).

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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 level 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 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

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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). And, the

relationship between the suppression of MMP-9 and the inhibition of invasion and

metastasis has been explored with the use of antiMMP-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 1α,25-dihydroxyvitamin D3 and its analogues (40) show similar

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results with selenite. Genistein decreases MMP-9 and MMP-2 mRNA levels while 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. 1α,25-dihydroxyvitamin D3 and its analogues also

down regulates MMP-9 and uPA, while it upregulates TIMP-1 and uPAI-1 levels in

MDA-MB-231 cells. Selenate showed different patterns compared to selenite in all

cases. It did not changed MMPs, uPA and TIMP-2 significantly, but decreased TIMP-

1 and 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 (ERK and

JNK/SAPK) 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 is 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 proteases expressions, we examined 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 specifically inhibit 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

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of TPA, but selenate slightly increased AP-1 activity. Interestingly, though AP-1 also

affects the basal transcription of TIMP-1 (49), this inhibitor expression pattern was

quite different from MMPs, uPA and TIMP-2 by selenite treatment. However, these

patterns are not selenite specific. Genistein, ursolic acid and 1α,25-dihydroxyvitamin

D3 and the latter’s analogues 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), and 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 to other factors

that regulate TIMP-1 expression.

We then tested whether selenite and selenate affect NF-κB activity. The MMP-9 and

uPA expressions require NF-κB (53, 54) as well as AP-1. Although MMP-2 promoter

does not contain NF-κB binding site, it is recently reported that MMP-2 activation

occurs in endothelial cells through an NF-κB-dependent pathway (55). Selenite has

been shown to inhibit the binding of NF-κB to DNA (56) by oxidizing the critical

cysteine residues in their DNA binding domains like AP-1. Our results also showed

that NF-κB activity was inhibited by selenite in HT1080 cells. The inhibitory effect of

selenite on NF-κB 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-κB

transcription factors but also affects signal molecules such as JNK, p38 and PKC (48,

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57). As a result, AP-1 and NF-κB activities could be more strongly inhibited by

selenite. From these observations, it can be suggested that selenite inhibits proteases

expressions through repression of AP-1 and NF-κB, both directly and indirectly.

AP-1 and NF-κB 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-κB activities give potential in blocking tumor initiation, promotion, and

metastasis (63), but there have been few reports on the relationship between selenium

and antimetastasis. Only recently, it has been reported that selenite and

methylselenocysteine inhibit angiogenesis by reduction of intra-tumoral microvessel

density and vascular endothelial growth factor (VEGF) expression in mammary

carcinomas (64). Genistein inhibits cell-matrix attachment (65), downregulates MMPs

and uPA, but upregulates TIMP-1, inhibits cell proliferation, angiogenesis and

invasion, and induces apoptosis (39, 66, 67). Therefore, we can suggest that the

function of selenite is similar to that of genistein, from our results and other reports

mentioned previously. 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 antimetastatic 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

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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-κB, directly

and indirectly. Unlike selenite, selenate had no marked effect on cell invasion, cell

matrix interaction, expression of proteases, but rather increased mRNA level of

proteases through the partial activation of AP-1 and NF-κB.

Acknowledgments-We thank Dr. Francesco Blasi (Universita Vita-Salute San

Raffaele, Milan, Italy) for the kind gift of the report vector containing uPA promoter,

and Dr. Seung-Taek Lee (Yonsei University, Seoul, Korea) for MMPs and TIMPs

vectors.

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FOOTNOTES

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1 The abbreviations used are: ECM, extracellular matrix; AP-1, activator protein-1;

NF-κB, nuclear factor κB; JNK, c-Jun NH2-terminal kinase; ERK, extracellular

signal-regulated kinase

FIGURE LEGENDS

Fig. 1. Effects of selenite and selenate on viability of HT1080 cells. HT1080 cells were

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treated with selenite and selenate, and 3 days after, viability was tested by MTT assay

as described in Materials and Method. HT1080 cells were treated with selenite (A) and

selenate (B) varying concentrations with (open circle) or without (closed circle) serum.

Fig. 2. Effects of selenite and selenate on in vitro invasion and motility of HT1080

cells. For invasion assay, 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 in Materials and Methods. For motility

assay, 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 in

Materials and Methods. Data represent the mean ± SE of at least three independent

experiments. Results were statistically significant (∗, P< 0.05) using Student’s t test.

Fig. 3. Effects of selenite and selenate on cell-matrix and cell-cell attachments. For

cell-matrix attachment assay, radiolabelled 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 ± SE of at least three independent experiments. Results were statistically

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significant (∗, P< 0.05; ∗(, P< 0.01) using Student’s t test.

Fig. 4. Effects of selenite and selenate on the activities of MMP-2, MMP-9 and uPA.

For the 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 zymograpy was performed as described in

Materials and Methods. For the uPA activity test, various concentrations of selenite (C)

and selenate (D) were treated for 3 days, and fibrin zymography was performed.

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 in Materials and Methods.

Fig. 6. Effects of selenite on the activities of MMP-2 and MMP-9 in the presence of

TPA, TNF- α, and dexamethasone. HT1080 cells were preincubated with selenite for 6

h and treated with TPA (50 nM) (A) or TNF-α (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).

Fig. 7. Effects of selenite on the protein levels of MMP-9, TIMP-1, uPA and uPAI-1.

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HT1080 cells were treated with various concentrations of selenite for 3 days, lysed, and

80 µg of each sample was subjected to SDS-PAGE, and Western blot analysis was

performed.

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 were extracted. RNA were loaded on 1% agarose gels, and

Northern blotting was carried out as described in Materials and Methods. RNA loading

was normalized using the signal obtained with a GAPDH cDNA probe.

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 ± SE of at least three independent experiments. Results were

statistically significant (∗, P< 0.05) using Student’s t test.

Fig. 10. Effects of selenite and selenate on the activities of AP-1 and NF-kB. To

elucidate the effects of selenite and selenate on AP-1 activity, a report vector which 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. Then, cells were lysed and luciferase activity was

measured. The same method was used for the test of effects of selenite (C) and selenate

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(D) on NF-kB activity. Data represent the mean ± SE of at least three independent

experiments. Results were statistically significant (∗, P< 0.05) using Student’s t test.

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Sang-Oh Yoon, Moon-Moo Kim and An-Sik ChungInhibitory effect of selenite on invasion of HT1080 tumor cells

published online March 27, 2001J. Biol. Chem.

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