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Carcinogenesis Advance Access originally published online on October 27, 2006
Carcinogenesis 2007 28(4):837-847; doi:10.1093/carcin/bgl203
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Methylseleninic acid inhibits PMA-stimulated pro-MMP-2 activation mediated by MT1-MMP expression and further tumor invasion through suppression of NF-{kappa}B activation

Jong-Min Park, Aeyung Kim, Jang-Hee Oh and An-Sik Chung*

Department of Biological Science, Biochemical Toxicology Lab, Korea Advanced Institute of Science and Technology 373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701, South Korea

*To whom correspondence should be addressed. Email: aschung{at}kaist.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Selenium, an essential biological trace element, reduces the incidence of cancer. Our previous studies show that selenite inhibits tumor invasion by suppressing the expression of matrix metalloproteinases (MMP) -2 and -9.Methylseleninic acid (MSeA), an immediate precursor of methylselenol, inhibits tumor cell growth in vitro and mammary carcinogenesis in vivo. In this study, we demonstrate that MSeA suppresses pro-MMP-2 activation in a dose-dependent manner induced by 12-O-tetradecanoylphorbol-13-acetate (PMA), and further decreases the invasiveness of HT1080 tumor cells. Membrane type-1-MMP (MT1-MMP) is a crucial element in the process of pro-MMP-2 activation. Pro-MMP-2 binds MT1-MMP, using tissue inhibitor of metalloproteinase-2 (TIMP-2) as an adaptor, by forming a trimolecular complex on the cell surface. MSeA blocked MT1-MMP in a dose-dependent manner, but not TIMP-2 expression. MMP-9 and TIMP-1 levels were not affected by MSeA. Selenite induced a decrease in protein levels of both pro-MMPs -9 and -2, but not active forms of pro-MMP-2. MT1-MMP expression is regulated by NF-{kappa}B. Our data show that the effect of MSeA on MT1-MMP expression is mediated through suppression of NF-{kappa}B activity. Methylselenol generated by selenomethionine (SeMet) and methioninase (METase) inhibited pro-MMP-2 activation induced by PMA, confirming the effect of MSeA on pro-MMP-2 activity. Moreover, ROS production induced by PMA was partly decreased in the presence of MSeA. This suppression of ROS production may be related to diminished NF-{kappa}B activity. Thus, our results suggest that MSeA blocks tumor invasion in vitro via inhibiting pro-MMP-2 activation mediated by suppression of MT1-MMP expression, which is regulated by the NF-{kappa}B signal pathway.

Abbreviations: DCFH-DA, dichlorofluorescin diacetate; DDC, diethyldithiocarbamic acid; METase, Methioninase; MMP, matrix metalloproteinase; MSeA, Methylseleninic acid; MT1-MMP, membrane-type 1-matrix metalloproteinase; PMA, 12-O-tetradecanoyl-phorbol-13-acetate; ROS, reactive oxygen species; SeMet, selenomethionine; TIMP, tissue inhibitor of metalloproteinase


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tumor invasion and metastasis are a major cause of cancer-related death, and involve several biological processes. Cell–Extracellular matrix (ECM) interactions, disconnection of intercellular adhesion, degradation of ECM and invasion of lymph and blood vessels are critical steps for cancer invasion and metastasis (1). A number of proteolytic enzymes participate in the degradation of environmental barriers, such as the ECM and basement membrane (2). Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, play an important role in the proteolysis of various ECM components, and are involved in the metastasis and angiogenesis of cancer cells (3). In particular, MMPs are crucial in the proteolysis of ECM proteins, collagen and fibronectin (4). MMPs are synthesized as preproenzymes, and secreted from cells as proenzymes. Among the human MMPs reported to date, MMPs -2 and -9 are the key enzymes involved in degrading Type I and IV collagen, and ECM (3,4). Both MMPs -2 and -9, which are abundantly expressed in various malignant tumors (5), contribute to cancer invasion and metastasis (6). MMP-2 is constitutively expressed and secreted as a latent zymogen, pro-MMP-2. Its main activation occurs on the cell surface, and is mediated by membrane-type matrix metalloproteinases (MT-MMPs), such as MT1-MMP (7,8). The concerted action of highly expressed MT1-MMP and adequate expression of tissue inhibitor of metalloproteinase (TIMP)-2 leads to activation of pro-MMP-2 (9–11). Following activation, MMP-2 digests components of the basement membrane and ECM, such as Type IV collagen and fibronectin (12).

Extensive animal and human data show that selenium is a chemopreventive agent with a protective role against cancer (13,14). Results obtained from the ‘nutritional prevention of cancer’ clinical trials indicate that dietary supplementation with 200 µg/day selenium reduces the incidence of prostate and colon cancer (13). Studies using animal carcinogenesis models strongly implicate a monomethyl selenium metabolite pool, possibly methylselenol, as the active in vivo selenium species responsible for chemopreventive activity (15–17). Methylseleninic acid (CH3SeOOH, MSeA), the immediate precursor of methylselenol, is a monomethylated selenium compound, and an excellent tool for molecular mechanism studies on selenium in cell culture, since it mimics the generation of methylselenol from monomethylated selenoamino acids (18). In mice, dietary supplementation with selenite and selenomethionine reduces lung metastasis of melanoma cells (19,20), and Se yeast inhibits the spread of Lewis lung carcinoma cells (21). To date, functional studies on selenium compounds have mainly focused on their chemopreventive effects, whereas the relationship between methylselenol and tumor metastasis remains to be established. To further clarify the possible role of selenium in pro-MMP-2 activation, we used HT1080 cells, a type of fibrosarcoma. Similar to various malignant tumors, HT1080 cells constitutively express MT1-MMP, which can activate pro-MMP-2 (23), and high levels of MMPs -2 and -9 (22).

In the present study, we examined the effects of MSeA on pro-MMP-2 activation and tumor invasion induced by treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (PMA), in HT1080 cells. MT1-MMP and TIMP-2 expression in relation to pro-MMP2 activity were additionally analyzed in the presence of MSeA. To further elucidate the molecular mechanisms by which MSeA regulates pro-MMP-2 activation and MT1-MMP expression, we investigated the transcription factor, NF-{kappa}B, in HT1080 cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture and materials
HT1080 (fibrosarcoma) cells were grown in DMEM (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10 mM HEPES, 50 mg/l gentamycin (Life Technologies, Rockville, MD), and 10% heat-inactivated fetal bovine serum (FBS; Gibco/Invitrogen, Carlsbad, CA) in a humidified 5% CO2 incubator. MSeA was provided by Dr Julian Spallholz (PharmaSe, Lubbock, TX). L-methionine {gamma}-lyase (designated ‘METase’) was purchased from Wako Pure Chemical Industries, Ltd (Richmond, VA). 1-Pyrrolidinecarbodithioic Acid (PDTC) was obtained from Calbiochem (La Jolla, CA). Anti-MT1-MMP and Anti-MMP-9 antibodies, selenite, seleno-L-methionine (SeMet), PMA, 2',7'-dichlorofluorescin diacetate (DCFH-DA), and diethyldithiocarbamic acid (DDC) were from Sigma Chemical Co. (St Louis, MO). Anti-MMP-2 and anti-TIMP-2 antibodies were acquired from Chemicon International (Temecula, CA). The anti-phospho-p65 antibody was obtained from Cell Signaling Technology (Beverly, MA). Anti-NF-{kappa}B p65, anti-phospho-I{kappa}B-{alpha}, and anti-I{kappa}B-{alpha} antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). NF-{kappa}B reporter vectors were purchased from CLONTECH (Palo Alto, CA).

Cell viability
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Roche Molecular Biochemicals, Germany) assays were performed, as described in the supplier's protocol, to evaluate cytotoxicity. To confirm MTT assay results, we additionally performed a trypan blue dye exclusion assay.

Zymography and reverse zymography
All experiments, including zymography, were performed in the absence of serum. Enzymatic activities of MMPs -2 and -9 were assayed by gelatin zymography (24). Samples were electrophoresed on gelatin-containing 10% SDS-polyacrylamide gels. The gel was washed twice with washing buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl and 2.5% Triton X-100), followed by brief rinsing in washing buffer without Triton X-100. This was followed by treatment with incubation buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.02% NaN3 and 1 µM ZnCl2) at 37°C. Next, the gel was stained with Coomassie brilliant blue R-250 (Sigma, St Louis, MO), and destained. A clear zone appearing on the gel signified the presence of MMP. Reverse zymography was employed to detect TIMPs -1 and -2 activity in conditioned media, as described previously (25).

Western blot analysis
Western blot analysis was performed for MMPs -2, -9 and TIMP-2. Conditioned media were collected and concentrated using a Centricon filter (Millipore, Bedford, MA). The MT1-MMP protein was identified using cell membrane proteins, which were prepared using the Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce Biotechnology, Rockford, IL). Whole-cell lysates were generated with M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL). Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL), according to the manufacturer's instructions. Briefly, samples were resuspended in reducing 5x Sample buffer (60 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol and 0.1% bromophenol blue), boiled for 5 min, and subjected to SDS–PAGE. Proteins were transferred to Hybond-ECL (Amersham Biosciences, Little Chalfont, Buckshire, UK). Blots were blocked with TTBS (25 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.05% Tween-20) containing 1% bovine serum albumin, and probed with primary and secondary antibodies coupled to peroxidase. Blots were developed using an enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, Buckshire, UK).

Transient transfection and reporter gene assay
HT1080 cells were plated in six-well plates, and incubated at 37°C. At 70–80% confluence, cells were washed with DMEM, and incubated with DMEM without serum or antibiotics for 5 h. The NF-{kappa}B reporter vector (2 µg) and ß-galactosidase vector (0.5 µg) were transfected using LipofectAMINE 2000 reagent (Invitrogen, San Diego, CA, USA). After incubation, cells were lysed, and luciferase activity measured using a luminometer (Microlumat LB 96P luminometer, EG&G Berthold, Gaithersburg, MD). ß-Galactosidase activity was measured using O-nitrophenyl ß-galactopyranoside as a substrate.

RNA isolation and northern blot analysis
Total cellular RNA was purified from cultured cells, using TRI reagent (Gibco/Invitrogen, Carlsbad, CA). For northern blot analysis, 15 µg of RNA was electrophoresed on 1% agarose gels containing 37% formaldehyde, and transferred to Hybond-N membrane (Amersham Biosciences, Little Chalfont, Buckshire, UK) by capillary transfer. The membrane was fixed using an optimized UV cross-linking procedure. Prehybridization and hybridization were performed at 68°C in ExpressHyb hybridization solution (CLONTECH, Palo Alto, CA). cDNA probes for MT1-MMP and glyceraldehyde-3-phosphate dehydrogenase were labeled with 3000 Ci/mmol [32P]dCTP (Amersham Biosciences, Little Chalfont, Buckshire, UK) using a random primer kit (Takara, Japan). The blot was washed twice with 2x SSC (300 mM NaCl and 30 mM sodium citrate, pH 7.0) containing 0.05% SDS at 25°C, and 0.1x SSC containing 0.1% SDS at 55°C, and autoradiographed at –70°C.

RT–PCR
Total RNA from cultured cells was isolated using TRI reagent (Gibco/Invitrogen, Carlsbad, CA) and reverse-transcribed to cDNA using reverse transcriptase (Promega) and oligo(dT). cDNA aliquots corresponding to 1 µg RNA were semi-quantitatively analyzed by PCR. The following primers were employed for amplification: TIMP-2, 5'- GAG ACA AAG AGG AGA GAA AGT TTG C-3' and 5'- TTT ATC TGC TTG ATC TCA TAC TGG A-3'; and GAPDH, 5'- CCA TGG AGA AGG CTG GGG -3' and 5'- CAA AGT TGT CAT GGA TGA CC -3'. PCR products were resolved on 1% agarose gels, and visualized by ethidium bromide staining.

Electrophoretic mobility shift assay (EMSA)
Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL), according to the manufacturer's instructions. Two double-stranded deoxyoligonucleotides corresponding to the NF-{kappa}B sequence (Promega) were end-labeled with 32P[{gamma}]ATP using T4 kinase (TaKaRa), and nuclear extracts incubated with 1 µg/µl of poly (dI-dC) and 32P-labeled DNA probe in binding buffer (25 mM HEPES, 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol and 50 mM NaCl). The DNA–protein complex was resolved on a 6% polyacrylamide gel and analyzed by autoradiography.

Measurement of ROS formation
Flow cytometry was performed to measure intracellular H2O2. Briefly, cells were incubated with DCFH-DA (5 µM) for 30 min at 37°C. Cells were washed with PBS and trypsinized. Next, 10 µl of propidium iodide (2.5 mg/ml) was added, and the amount of H2O2 measured with a flow cytometer (FACSCalibur, Becton Dickinson, NJ).

Cell invasion assay
An in vitro invasion assay was performed using a 24-well Transwell unit with polycarbonate filters having a diameter of 6.5 mm and a pore size of 8.0 µm (Corning Costar, Cambridge, MA). A fixed number of cells (5 x 104/chamber) were used for the invasion assays. The lower part of Transwell was coated with 10 µl of Type I collagen (0.5 mg/ml), and the upper part of Transwell was coated with 20 µl of 1:2 mixture of Matrigel:DMEM (Matrigel; BD Biosciences, Bedford, MA). Cells were plated on the Matrigel-coated Transwell. The medium compartments of the lower chambers contained 0.1 mg/ml of bovine serum albumin. Inserts were incubated for 12–18 h at 37°C. Cells invading the lower surface of the membrane were fixed with methanol, and stained with hematoxylin and eosin (H&E stain). Random fields were counted under a light microscope.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effects of MSeA on pro-MMP-2 activation
HT1080 cells were employed as a model to study the effects of MSeA on pericellular gelatin lytic activity. PMA enhanced MMP-9 cell expression and partially converted/proteolytically processed the MMP-2 proenzyme to its active 62 and 64 kDa forms in a dose-dependent manner at 24 h (Figure 1A). Pro-MMP-2 activation peaked at 4–5 nM PMA (non-cytotoxic concentrations), and 5 nM PMA was therefore used for all subsequent experiments. In the presence of PMA, MSeA at a concentration of 3 µM did not have any toxic effects on cell viability in the absence of serum (Figure 1B). Thus, non-cytotoxic concentrations of MSeA (1–3 µM) were used for subsequent experiments. MSeA dramatically suppressed the proteolytic activation of pro-MMP-2 induced by PMA, but increased the pro-MMP-2 level. Moreover, MSeA did not affect PMA-induced MMP-9 expression (Figure 1C). However, MMP activity was not directly affected by MSeA alone. Direct incubation of the conditioned medium with MSeA in a test tube did not affect MMP-2 activation (Data not shown). No direct interactions were evident between MSeA and MMP proteins. Thus, inhibition of PMA-stimulated pro-MMP-2 activation by MSeA is a cell-dependent process.


Figure 1
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  		Fig. 1 Effects of MSeA on PMA-induced upregulation of MMP activity. (A) HT1080 cells were treated with various concentrations of PMA without serum. Conditioned media were prepared 24 h after treatment, and used for zymography. Cell viability was tested by the MTT assay, as described in Materials and methods. Results are expressed as a percentage of cell proliferation of the control in the absence of MSeA. Data are the presented as means ± SD of three independent experiments. (B) HT1080 cells were treated with various concentrations of MSeA and 5 nM PMA, and 24 h thereafter, cell viability was tested using the MTT assay, as described in Materials and methods. Results are expressed as a percentage of cell proliferation of the control in the absence of PMA treatment. Data are presented as means ± SD of three independent experiments. (C) HT1080 cells were preincubated with various concentrations of MSeA for 6 h, and treated with 5 nM PMA. Conditioned media were prepared 24 h after treatment, and used for zymography. Differences were considered statistically significant at P < 0.01 (values are compared with control). Data are a representative of three independent experiments.

 
Effects of MSeA and selenite on MMP activity
Previous experiments by our group demonstrate that selenite inhibits tumor invasion by blocking expression of MMPs -2 and -9 (26). Pretreatment with MSeA dramatically suppressed pro-MMP-2 activation by PMA, but not MMP-9 expression (Figure 2A). Following pretreatment with selenite, expression of MMPs -2 and -9 induced by PMA was decreased, but the effect on pro-MMP-2 activation was not as significant as that of MSeA. Western blot data disclosed that MSeA decreases the level of active MMP-2, while selenite suppresses expression levels of pro-MMPs -2 and -9 (Figure 2B). HT1080 cells were incubated with MSeA or selenite for 3 days. The expression of MMPs -2 and -9 was not affected by MSeA, while selenite decreased the levels of these proteins (Figure 2C). Moreover, MSeA did not affect the levels of pro-MMPs -2 or -9 secreted from cells. In contrast, selenite inhibited the secretion of pro-MMPs -2 and -9.


Figure 2
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  		Fig. 2 Effects of selenium on MMP activity. (A) HT1080 cells were preincubated with various concentrations of MSeA and selenite for 6 h, and treated with 5 nM PMA. The conditioned media were prepared 24 h after treatment, and used for zymography and (B) western blot analysis. Data are a representative of three independent experiments. (C) To examine the activities of MMPs -2 and -9, HT1080 cells were treated with various concentrations of MSeA and selenite. At 3 days after treatment, conditioned media were collected, and gelatin zymography was performed. HT1080 cells were treated with MSeA and selenite, and 3 days thereafter, cell viability was tested using the MTT assay, as described in Materials and methods.

 
Effects of MSeA on transcription and translation of MT1-MMP
Since MT1-MMP plays a critical role in regulating pro-MMP-2 activation, we determined whether MSeA suppresses its expression in HT1080 cells stimulated with PMA. Upon application of PMA to HT1080 cells, MT1-MMP mRNA levels increased in a time-dependent manner, with maximal expression at 12 h (Figure 3A). Northern blot data revealed that MSeA suppressed MT1-MMP mRNA levels induced by PMA (Figure 3B). In the presence of MSeA, the MT1-MMP mRNA levels were decreased at 6 h. However, no effect was observed with selenite (Figure 3C). Moreover, MSeA treatment led to a dose-dependent decrease in the MT1-MMP mRNA level at 12 h (Figure 3D). As MSeA suppresses the transcriptional level of MT1-MMP stimulated by PMA, protein expression was examined at the membrane site under similar experimental conditions. The levels of 63, 60 and 43 kDa MT1-MMP proteins were decreased upon treatment with MseA (Figure 3E). Our results clearly demonstrate that MSeA suppresses both MT1-MMP mRNA and protein induced by PMA.


Figure 3
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  		Fig. 3 Effects of MSeA on MT1-MMP expression. (A) After treatment with 5 nM PMA, RNA was extracted at the indicated times. (B) HT1080 cells were preincubated with 2.5 µM MSeA for 6 h, and treated with 5 nM PMA. RNA was extracted at the indicated times. (C) HT1080 cells were preincubated with various concentrations of MSeA and selenite for 6 h. RNA was extracted at 6 h (C) and 12 h (D). RNA was loaded on 1% agarose gels, and northern blots were performed, as described in Materials and methods. RNA loading was normalized using the signal obtained with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. Band intensities were quantitated by densitometry analysis using TINA 2.0 software, and data are expressed as a ratio relative to control. (E) HT1080 cells were preincubated with various concentrations of MSeA for 6 h, and treated with 5 nM PMA. After 18 h, membrane proteins were collected after cell lysis, and protein levels detected by western blot analysis. Data are a representative of three independent experiments.

 
Effect of MSeA on TIMP-2 activity
Significantly, pro-MMP-2 activation is mediated by highly expressed MT1-MMP and the adequate expression of TIMP-2 (9–11). MSeA induced a significant increase in TIMP-2 activity in a dose-dependent manner that was inhibited by PMA, but did not affect TIMP-1 activity (Figure 4A). Moreover, the level of TIMP-2 protein in the medium was significantly increased in the presence of MSeA (Figure 4B). However, the TIMP-2 mRNA level was not affected by PMA and MSeA (Figure 4C). In the absence of PMA, MSeA did not affect TIMP-2 activity (Figure 4D). Pro-MMP-2 binds MT1-MMP using TIMP-2 as an adaptor by forming a trimolecular complex on the cell surface. Based on the data, we suggest that TIMP-2 activity is modulated by MT1-MMP on the cell surface through binding. This finding is consistent with the observed regulation of MT1-MMP levels in membrane extracts upon similar treatment (Figure 3E).


Figure 4
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  		Fig. 4 Effects of MSeA on TIMP activity. (A) HT1080 cells were preincubated with various concentrations of MSeA and selenite for 6 h, and treated with 5 nM PMA. The conditioned media were prepared 24 h after treatment, and used for reverse zymography and (B) western blot analysis. Data were considered statistically significant at P < 0.01 (values are compared with ‘PMA alone’). (C) TIMP-2 mRNA levels in total RNA were assayed by RT–PCR. GAPDH was included as an internal control. (D) To analyze the activity of TIMP-2, HT1080 cells were treated with various concentrations of MSeA. At 3 days after treatment, conditioned media were collected, and reverse zymography performed. Data are a representative of three independent experiments.

 
Effects of MSeA on NF-{kappa}B activity
Treatment with PMA led to a pronounced increase in DNA binding, nuclear translocation, and transactivation of NF-{kappa}B (Figure 5). Our results indicate that the nuclear NF-{kappa}B complex by detection of EMSA was increased after 30–180 min of PMA treatment (Figure 5A) and MSeA diminished nuclear localization of the NF-{kappa}B complex (Figure 5B). One of the most critical steps in NF-{kappa}B activation is the dissociation of I{kappa}B, which is mediated through phosphorylation and subsequent proteolytic degradation of this inhibitory subunit. We measured the levels of I{kappa}B-{alpha} and phospho- I{kappa}B-{alpha} in whole-cell extracts, and the nuclear translocation of p65, the functionally active subunit of NF-{kappa}B, in the nucleus. Further analyses disclosed that the nuclear fraction of p65 is decreased in the presence of MSeA, while the cytosolic fraction is increased (Figure 5C). Application of PMA resulted in I{kappa}B-{alpha} phosphorylation, which was significantly repressed upon MSeA pretreatment at 10 min (Figure 5D). Phosphorylation of the p65 subunit by a variety of kinases induces modifications in NF-{kappa}B transcriptional activity. In our experiments, MSeA completely blocked PMA-mediated p65 phosphorylation in whole-cell extracts (Figure 5E). Our results indicate that MSeA inhibits PMA-induced translocation of p65 to the nucleus through blockade of I{kappa}B-{alpha} and p65 phosphorylation. To determine NF-{kappa}B transcriptional activity, a NF-{kappa}B reporter vector was transfected into HT1080 cells. Treatment with 1 µM MSeA reduced NF-{kappa}B activity induced by PMA and 2–3 µM MSeA further decreased NF-{kappa}B to the basal level (Figure 5F). Next, we investigated the functional significance of NF-{kappa}B transactivation in pro-MMP-2 activation in HT1080 cells. Treatment with PDTC (a general inhibitor of NF-{kappa}B) reduced pro-MMP-2 activation and MT1-MMP expression induced by PMA (Figure 5G). The above findings collectively suggest that MSeA inhibits PMA-induced activation of pro-MMP-2 by suppressing NF-{kappa}B activation in HT1080 cells.


Figure 5
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  		Fig. 5 Effects of MSeA on NF-{kappa}B activity. (A) EMSA for NF-{kappa}B DNA-binding activity. Nuclear extracts were prepared from HT1080 cells after treatment with 5 nM PMA for the indicated time-periods. (B) HT1080 cells were preincubated with various concentrations of MSeA for 6 h, and treated with 5 nM PMA. Nuclear extracts were prepared 1 h after treatment, and used for EMSA. Nuclear extracts were incubated with an isotope-labeled oligo NF-{kappa}B probe. The NF-{kappa}B–DNA complex is indicated with an arrowhead. An EMSA was performed, as described in Materials and methods. Data are a representative of three independent experiments. (C) p65 nuclear translocation was investigated. HT1080 cells were preincubated with 2.5 µM MSeA for 6 h, and treated with 5 nM PMA for the indicated time intervals. Nuclear and cytoplasmic extracts were prepared, and the p65 protein levels measured by western blot analysis. (D) HT1080 cells were preincubated with 2.5 µM MSeA for 6 h, and treated with 5 nM PMA for the indicated time intervals. Whole-cell extracts were prepared, and assayed by western blot analysis. Phosphorylation of I{kappa}B-{alpha} (D) and p65 (E) were investigated at 10, 30 and 60 min after PMA treatment. The degree of phosphorylation was quantitated as the ratio of band intensity of the phospho-form/total form. Band intensities were quantitated by densitometry analysis using TINA 2.0 software. (F) The NF-{kappa}B reporter vector was transfected, as described in Materials and methods. After pretreatment with various concentrations of MSeA for 6 h, cells were treated with 5 nM and 10 nM PMA. After 24 h, luciferase activity was measured using a luminometer. Data are presented as means ± SD of three independent experiments. Differences were considered statistically significant at P < 0.05 (values are compared with ‘PMA alone’). (G) After pretreatment with the NF-{kappa}B inhibitor for 3 h, 5 nM PMA was added. At 1 day after treatment, conditioned media were collected, and gelatin zymography was performed. Membrane proteins were collected after cell lysis, and protein levels were detected by western blot analysis. Data are a representative of three independent experiments.

 
Effects of MSeA on pro-MMP-2 activation by other reagents
Previous experiments show that phenazine methosulfate (PMS) induces pro-MMP-2 activation through the receptor tyrosine kinase/phosphatidylinositol 3-kinase/NF-{kappa}B pathway by the production of reactive oxygen species (ROS) (27). Moreover, ciglitazone, a PPAR-{gamma} agonist, triggers pro-MMP-2 activation through the ERK/NF-{kappa}B pathway via ROS production (unpublished data). Pretreatment with MSeA blocked the activation of pro-MMP-2 and NF-{kappa}B induced by PMS and ciglitazone (Figure 6A and B). The data indicate that MSeA decreases pro-MMP-2 activation by PMA and other reagents through blocking NF-{kappa}B activation. Pretreatment with MSeA led to the blockage of pro-MMP-2 activation induced by PMS, ciglitazone and PMA, which produce ROS in cells. Flow cytometry analyses disclosed PMA concentration- and time-dependent increase in intracellular H2O2 (Figure 6C), whereas pretreatment with MSeA partly blocked the production of H2O2 at 3–6 h (Figure 6D). Diethyldithiocarbamic acid, a potent superoxide dismutase inhibitor, and the antioxidant, N-acetylcysteine, were effective in inhibiting pro-MMP-2 activation by PMA (Figure 6E). These results suggest that MSeA partly blocks pro-MMP-2 activation by scavenging ROS produced by these reagents.


Figure 6
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  		Fig. 6 Effects of MSeA on other pro-MMP-2 activation reagents. (A) After treatment of HT1080 cells with various concentrations of MSeA for 6 h, PMS and ciglitazone were added for pro-MMP-2 activation. At 2 days after treatment, conditioned media were collected and gelatin zymography analysis performed. (B) After HT1080 cells were treated with various concentrations of MSeA for 6 h, PMS and ciglitazone were added for pro-MMP-2 activation. At 1 day after treatment, nuclear extracts were collected, and assayed for NF-{kappa}B binding by EMSA. (C) Following treatment with various concentrations of PMA at the indicated times, DCFH-DA was added, cells were subjected to flow cytometric analysis for ROS detection. (D) Pretreatment with 2.5 µM MSeA for 6 h was followed by the addition of 5 nM PMA for 3–6 h and cytometric analysis. (E) Following pretreatment with the ROS inhibitor for 3 h, 5 nM PMA was added. At 1 day after treatment, conditioned media were collected, and gelatin zymography was performed.

 
Effects of MSeA on invasion of PMA-induced HT1080 cells in vitro
Pro-MMP-2 activation due to upregulation of MT1-MMP expression induces an invasive cell phenotype. We examined whether the tumor invasion properties of PMA-induced HT1080 cells were affected by MSeA. The invasiveness of PMA-induced HT1080 cells was significantly increased, compared to untreated cells, as determined with the matrigel invasion assay. Treatment with 1 µM MSeA reduced the invasiveness of cells induced by PMA, while 2–3 µM MSeA significantly blocked tumor invasion (Figure 7). These results suggest that MSeA may be used for suppressing tumor invasion of HT1080 cells, and further tumor metastasis via modulation of MMP-2 activity induced by PMA.


Figure 7
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  		Fig. 7 Effects of MSeA on in vitro tumor invasion of HT1080 cells. For the invasion assay, the lower and upper parts of transwells were coated with collagen and matrigel, respectively. HT1080 cells and various concentrations of MseA, along with 5 nM PMA, were added. At 12–18 h incubation, cells on the filter were fixed, stained and counted, as described in Materials and methods. Data are presented as means ± SD of at least three independent experiments. Differences were considered statistically significant at P < 0.05 (values are compared with ‘PMA alone’).

 
Effects of Methylselenol on pro-MMP-2 activation
MSeA is an immediate precursor of methylselenol. To establish whether methylselenol is directly responsible for inducing this cellular effect, we provide experimental evidence that this compound is generated in cell culture by L-methionine-{alpha}-deamino-{gamma}-mercaptomethane lyase (METase) and seleno-L-methionine (SeMet). Specifically, the enzyme and substrate act in concert to promote these cellular and biochemical effects (28). As shown in Figure 8, both SeMet and METase decreased pro-MMP-2 activation induced by PMA, while METase or SeMet alone did not exhibit any effects on activation. Our results imply that methylselenol generated from organic selenium compounds, such as SeMet, inhibits pro-MMP-2 activation.


Figure 8
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  		Fig. 8 Effects of methylselenol on pro-MMP-2 activation. HT1080 cells were preincubated with SeMet and METase (0.02 U/ml) for 6 h, and treated with 5 nM PMA. At 1 day after treatment, conditioned media were collected, and gelatin zymography analysis was performed. Differences were considered statistically significant at P < 0.05 (values are compared with ‘PMA alone’).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Two selenium metabolites are critical for chemoprevention and tumor invasion. One is methylselenol generated from organic selenium compounds, such as SeMet and Se-methylselenocysteine (SeMSC), and the other is hydrogen selenide from inorganic selenium compounds, such as selenite and selenate (29,30). Methylselenol, an active Se metabolite in vivo, has anticancer properties, including chemopreventive and anti-angiogenic activity (31,32). However, the relationship between methylselenol and tumor metastasis has yet to be firmly established. Here we use MSeA, an immediate precursor of methylselenol, for MMP inhibition. MMPs play a major role in promoting angiogenesis and tumor metastasis (33). MMPs -2 and -9 are crucial enzymes in the process of tumor metastasis derived from ECM degradation (34). There are limited reports on the role of MSeA in MMP inhibition and regulation of MT1-MMP expression. Our findings demonstrate that MSeA is a strong inhibitor of pro-MMP-2 activation induced by PMA, and significantly suppresses tumor invasion of HT1080 cells. In this study, we sought to elucidate the mechanism by which MSeA regulates pro-MMP-2 activation via MT1-MMP expression.

PMA was applied to induce pro-MMP-2 activation. PMA, TNF-{alpha}, and concanavalin A up-regulate MMP-9 expression and convert pro-MMP-2 to active MMP-2 (35–37). MSeA is water-soluble, non-volatile, and ideal for cell culture analysis of the carcinostatic mechanisms of selenium. Cell exposure to MSeA triggered a dose-dependent reduction in pro-MMP-2 activation induced by PMA, while selenite treatment led to a dose-dependent reduction in expression of MMPs -2 and -9 (Figure 2). The data reveal distinct patterns of modulation of MMP expression by MSeA versus selenite exposure. We recently reported that selenite inhibits the invasion of HT1080 cells by suppressing expression of MMP -2 and -9 (26). In this study, treatment with MSeA blocked tumor invasion and pro-MMP-2 activation induced by PMA, which is consistent with earlier results obtained using methylselenol (38). Both TIMPs -1 and -2 protein levels were increased in the presence of methylselenol, while TIMP-2 expression was not altered by MSeA in our system, possibly due to the different systems and concentrations.

A recent study showed that enhanced production of MT1-MMP correlates with pro-MMP-2 activation (9). Here, we demonstrate that MSeA markedly decreases the MT1-MMP protein level at the membrane site and the MT1-MMP transcriptional activity induced by PMA (Figure 3). The inhibitory effect of MSeA on MT1-MMP expression provides a reasonable explanation for the observed decrease in pro-MMP-2 activation. For activation, pro-MMP-2 binds MT1-MMP using TIMP-2 as an adaptor, by forming a trimolecular complex on the cell surface. In this complex, the N-terminal domain of TIMP-2 binds the catalytic domain of MT1-MMP for inhibition, and its C-terminal domain binds the hemopexin-like domain of pro-MMP-2. Another MT1-MMP protein near the complex cleaves the propeptide bond of pro-MMP-2, and generates an intermediate, which is converted to the fully activated enzyme by an auto-proteolytic mechanism (39). Thus, the balance between MT1-MMP and TIMP-2 is important for pro-MMP-2 activation. In our system, MSeA does not appear to exert its action through transcriptional regulation of TIMP-2 (Figure 4C). Interestingly, TIMP-2 activity and protein levels suppressed by PMA were increased in the presence of MSeA (Figure 4). The reduction in pro-MMP-2 activity may be explained by a decrease in both the protein level and activity of MT1-MMP. Subsequently, both TIMP-2 expression and activity were increased (Figure 3E).

We are further interested in the transcriptional mechanism of MT1-MMP expression by MSeA. PMA enhances MMP production through the activation of transcription factors, such as NF-{kappa}B. The promoter region of the MT1-MMP gene contains binding sites for NF-{kappa}B (40,41). NF-{kappa}B activation by PMA was confirmed by EMSA and nuclear localization of the p65 subunit, similar to earlier observations (42). Nuclear translocation and phosphorylation of the p65 subunit, and phosphorylation of I{kappa}B-{alpha} occurring immediately after treatment with PMA were inhibited by MSeA (Figure 5). Mechanistically, MSeA inhibited I{kappa}B-{alpha} phosphorylation and degradation, and p65 phosphorylation and translocation. PDTC, an inhibitor of NF-{kappa}B, also suppressed pro-MMP-2 activation induced by PMA (Figure 5G). These results clearly indicate that MSeA inhibits pro-MMP-2 activation via reducing the transcription factor, NF-{kappa}B. In particular, MSeA inhibited PMS and ciglitazone-stimulated activation of pro-MMP-2 and activation of NF-{kappa}B (Figure 6A and B). Thus, it is evident that NF-{kappa}B is an important transcriptional factor of MT1-MMP expression, which in turn promotes pro-MMP-2 activation and MSeA is likely a potent universal inhibitor for NF-{kappa}B activation. Results obtained with HT1080 cells are consistent with previous experimental findings that MSeA and selenite inhibit I{kappa}B-{alpha} phosphorylation and NF-{kappa}B transcriptional activity in prostate cancer cells (43), and that selenite suppresses NF-{kappa}B translocation and binding to DNA (44). Earlier studies by our group also show that selenite inhibits MMPs -2 and -9 activation via NF-{kappa}B and AP-1 (26), and PMS induces MMP-2 activity through induction of MT1-MMP (27).

Selenium is an essential component of several enzymes, such as glutathione peroxidase (45), thioredoxin reductase (46), and selenoprotein P and W (47–49). These proteins have critical antioxidant functions in several biological processes. Recent studies show that antioxidant selenoproteins block the cellular proliferation induced by excess ROS, which are produced by cancer cells (50–52). In our experiments, treatment with MSeA blocked ROS production induced by PMA. One of the anticarcinogenic mechanisms of Se may involve the antioxidant function of these selenoproteins. Another anticarcinogenesis mechanism of Se is related to its pro-oxidant effect at high dosages (53). SeMSC at a concentration of 50 µM induces apoptosis via ROS production (54), while at non-toxic levels, selenite (2–3 µM) inhibits tumor invasion by inhibiting expression of MMPs -2 and -9 (26). Selenite and methylselenol oxidize a critical thiol- containing cellular substrate, which alters the intracellular redox state and results in oxidative modification of proteins. Micromolecular levels of selenite and methylselenol facilitate intramolecular S-S bond formation in the cysteine-containing catalytic subunit of protein kinase C, leading to its inactivation. On the other hand, thioredoxin reductase reverses Se-induced inactivation of protein kinase C, which may facilitate the development of advanced malignant cells (55,56). The molecular mechanisms of Se in anticarcinogenesis have been a topic of debate, and can be explained by the ‘double-edged sword’ hypothesis.

While the results obtained with various methylselenium compounds implicate methylselenol as the common carcinostatic metabolite, past studies have not been able to establish whether methylselenol is directly responsible for inducing these cellular effects. Here, direct experimental evidence demonstrates that methylselenol generated in cell cultures from SeMet and METase promotes these cellular and biochemical effects (28). In our experiments, methylselenol produced in this way inhibited pro-MMP-2 activation (Figure 8). It is proposed that MSeA is a useful agent for analyzing the anticarcinogenic effects of methylselenol. Several studies have found that increased activation of pro-MMP-2 by stimulators triggers the invasiveness of some cancer cells, and that MT1-MMP is overexpressed in specific types of malignant tumor cells (57). Our data demonstrate that MSeA inhibits the PMA-induced invasive properties of cells, as evident from the matrigel invasion assay (Figure 7). MSeA may contribute to the suppression of tumor invasion by inhibiting pro-MMP-2 activation.

Further animal and human intervention studies are required to establish whether selenium compounds, such as SeMSC or SeMet, can be effectively used for chemoprevention and blocking tumor metastasis.

In conclusion, our data strongly imply that MSeA inhibits pro-MMP-2 activation. First, MSeA suppresses PMA-induced activation of pro-MMP-2 and tumor invasion. Second, MSeA regulates MT1-MMP involved in pro-MMP-2 activity. And third, this regulation is mediated by NF-{kappa}B activity, both directly and indirectly. We propose that the anti-tumorigenic effects of MSeA on pro-MMP-2 activation are mediated by the suppression of MT1-MMP gene expression via inhibition of NF-{kappa}B activity. MSeA is a useful model selenium compound for studying the chemopreventive effects of methylselenol derived from organoselenium compounds.


   Acknowledgments
 
We thank Dr Julian Spallholz (Texas Tech University, Lubbock, TX) for providing the methylseleninic acid used in these experiments. This work was supported by the Brain Korea 21 project.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 17, 2006; revised September 14, 2006; accepted October 16, 2006.


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