Inhibitory Effect of Selenite on Invasion of HT1080 Tumor Cells*

Sang-Oh Yoon, Moon-Moo Kim, and An-Sik ChungDagger

From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea

Received for publication, February 6, 2001, and in revised form, March 23, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 by selenite. Moreover, selenite reduced expression of matrix metalloproteinase-2 and -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 protease expressions was mediated by the suppression of transcription factors, NF-kappa B and AP-1. However, selenate showed no remarkable effect on all the steps of cancer cell invasion.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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 beta -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. beta -Galactosidase activity was measured using o-nitrophenyl beta -galactopyranoside as a substrate. The same method was used for the measurement of uPA promoter, NF-kappa 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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."

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-alpha , 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-alpha 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-alpha , and dexamethasone. HT1080 cells were preincubated with selenite for 6 h and treated with TPA (50 nM) (A) or TNF-alpha (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).

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.

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.

Effects of Selenite and Selenate on NF-kappa 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-kappa 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-kappa B reporter vectors, which have binding sites to those factors. AP-1 and NF-kappa 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-kappa 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-kappa 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-kappa 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 1alpha ,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. 1alpha ,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 1alpha ,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-kappa B activity. The MMP-9 and uPA expressions require NF-kappa B (53, 54) as well as AP-1. Although MMP-2 promoter does not contain NF-kappa B-binding site, it has been reported recently (55) that MMP-2 activation occurs in endothelial cells through an NF-kappa B-dependent pathway. Selenite has been shown to inhibit the binding of NF-kappa B to DNA (56) by oxidizing the critical cysteine Iresidues in their DNA-binding domains like AP-1. Our results also showed that NF-kappa B activity was inhibited by selenite in HT1080 cells. The inhibitory effect of selenite on NF-kappa 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-kappa 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-kappa 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-kappa B, both directly and indirectly.

AP-1 and NF-kappa 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-kappa 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-kappa 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-kappa B.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Dept. 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: aschung@mail.kaist.ac.kr.

Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M101143200

    ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; AP-1, activator protein-1; NF-kappa B, nuclear factor kappa B; MMPs, matrix metalloproteinases; uPA, urokinase-type plasminogen activator; uPAI-1, anti-uPA inhibitor-1; MTT, dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; MAPK, mitogen-activated protein kinase; TIMP-1, tissue inhibitor of metalloproteinase-1; TPA, 12-O-tetradecanoylphorbol-13-acetate; TNF-alpha , tumor necrosis factor alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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