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Carcinogenesis Advance Access originally published online on November 17, 2006
Carcinogenesis 2007 28(5):1104-1110; doi:10.1093/carcin/bgl217
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Ascofuranone suppresses PMA-mediated matrix metalloproteinase-9 gene activation through the Ras/Raf/MEK/ERK- and Ap1-dependent mechanisms

Hyun-Ji Cho, Jeong Han Kang, Jong-Young Kwak1, Tae-Sung Lee2, In-Seon Lee3, Nam Gyu Park4, Hiroo Nakajima5, Junji Magae6 and Young-Chae Chang*

Department of Pathology, Catholic University of Daegu School of Medicine, 3056-6, Daemyung-4-Dong, Nam-gu, Daegu 705-718, Korea
1 Medical Research Center for Cancer Molecular Therapy, Dong-A University, Busan 602-714, Korea
2 Department of Obstetrics and Gynecology, Catholic University of Daegu School of Medicine, Daegu 705-718, Korea
3 The Center for Traditional Microorganism Resources, Keimyung University, Daegu 704-701, Korea
4 Department of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, Korea
5 Department of Endocrine, Breast Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan
6 Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan

* To whom correspondence should be addressed. Tel: +82 53 650 4848; Fax: +82 53 650 4834; Email: ycchang{at}cu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of cancer cells. Here, we found that an antitumor antibiotic, ascofuranone, inhibits invasion and MMP-9 induction induced by phorbol myristate acetate (PMA) in human cell lines. Ascofuranone also inhibits the protein expression and transcription of MMP-9 induced by tumor necrosis factor-{alpha}. The inhibition of MMP-9 induction was observed in human cancer cell lines as well as primary rat mesangial cells. Furthermore, as evidenced by MMP-9 promoter and electrophoretic mobility shift assays, ascofuranone specifically inhibited MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, ascofuranone suppressed PMA-induced phosphorylation of Ras, Raf, MEK and extracellular signal-regulated kinase (ERK), upstream factors involved in AP-1activation, whereas the phosphorylation of p38 and JNK/mitogen-activated protein kinase was not affected by ascofuranone, suggesting that the primary target of ascofuranone for suppression of the AP-1 induction is present in upstream of ERK signaling pathway. These results suggest that the suppression of MMP-9 expression, at least in part, contributes to the antitumor activity of ascofuranone.

Abbreviations: AP-1, activator protein-1; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; NF-{kappa}B, NF-kappaB; PMA, phorbol myristate acetate; TIMPs, tissue inhibitors of metalloproteinases; TNF-{alpha}, tumor necrosis factor-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Ascofuranone (Figure 1A), an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of a phytopathogenic fungus, Ascochyta viciae (1,2). Ascofuranone has also been shown to have significant antitumor activity against various transplantable tumors, and to suppress the metastasis of a melanoma and a lung carcinoma in murine experimental models (3,4). It is generally assumed that the antitumor activity of ascofuranone is mediated by the activation of a host-defense mechanism against tumors, since pretreatment with ascofuranone prior to the tumor implantation is as effective as the treatment after the tumor inoculation. Ascofuranone activates macrophages and enhances IL-1 production and cytotoxic activity against syngeneic tumor cells, whereas it suppresses lymphocyte functions including IL-2 production and natural killer activity in vitro (5). The i.p. injection of ascofuranone activates phagocytic activity as evidenced by carbon clearance tests, natural killer activity in the spleen and the tumoricidal activity of macrophages and neutrophils induced in the peritoneal cavity (4,6). The inactivation of peritoneal phagocytes by the silica injection almost completely abolishes its prophylactic antitumor activity (4).


Figure 1
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  		Fig. 1. Ascofuranone prevents the invasion of U2OS cells in vitro. (A) Structure of ascofuranone (AF). (B) U2OS cells (left panel) or rat mesangial cells (right panel) were treated with ascofuranone for 24 h in medium containing 10% serum (closed circles) or serum-free medium (open circles) and viability was determined by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide assay. (C) U2OS cells and various concentrations of ascofuranone were cultured in the upper parts of Transwells coated with Matrigel. After 24 h, cells on the bottom side of the filter were counted. Data represent the mean ± standard error of three independent experiments. Statistically significant (*P < 0.05, **P < 0.01).

 
An inability to control metastasis and invasion is the leading cause of death in patients with cancer. The control of metastasis and invasion, therefore, represents an important therapeutic target. Matrix metalloproteinases (MMPs) play the greatest role in the process of metastasis and invasion. MMPs are a family of zinc-dependent endoproteinases that are capable of degrading all the components of the extracellular matrix proteins, including collagen (7–12). Among the previously reported human MMPs, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are the key enzymes that participate in the degradation of type IV collagen. These two gelatinases share structural and catalytic similarities, but their gene expression is regulated in a different manner, partly due to the distinct regulatory elements in the promoter region of their genes. MMP-9 is induced during the processes of renal development, macrophage differentiation, atherosclerosis, inflammation, rheumatoid arthritis and tumor invasion, whereas MMP-2 is usually expressed constitutively (10–12).

Furthermore, stimulators, such as cytokines and phorbol myristate acetate (PMA), control the expression of MMP-9 by modulating the activation of transcription factors such as activator protein-1 (AP-1) and NF-kappaB (NF-{kappa}B) through Ras/Raf/extracellular signal-regulated kinase (ERK), JNK and PI-3K/AKT signaling pathways (13–19), since the promoter region of MMP-9 has AP-1 and NF-{kappa}B binding sites (14). AP-1 has been shown to regulate the expression of a number of genes, the products of which are involved in tumorigenesis (20). AP-1 is a key transcription factor involved in the activation of genes that encode inflammatory cytokines such as the tumor necrosis factor-{alpha} (TNF-{alpha}) and IL-1{alpha} (21). Thus, several agents able to suppress AP-1 activation have the potential to suppress tumorigenesis and metastasis, and show therapeutic potential. In this study, we show that ascofuranone inhibits the invasion of osteosarcoma cells through suppression of MMP-9 expression by preventing induction of AP-1 transcription factor.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells and materials
Human U2OS, Caki-1 (renal carcinoma) and MDA-MD-231 (breast carcinoma cancer) cells were obtained from the American Type Culture Collection (Rockville, MD). Primary rat mesangial cells were isolated and cultured as described previously (22). In brief, glomeruli harvested from Sprague–Dawley rats by filtration with nylon meshes were collected, washed by centrifugation (4800 r.p.m.), and incubated with 250 U/ml collagenase (type I) for 30 min at 37°C. Rat mesangial cells were cultured in DMEM with 20% fetal bovine serum for 4 days. The medium was then changed every other day until confluence. All assays were performed on cells at passages 3–7. The culture medium used in the experiments was DMEM (Gibco, Grand Island, NY), containing 10% fetal bovine serum, 20 mM HEPES and 100 mg/ml gentamicin. Ascofuranone was purified from culture broth of A. viciae as described priviously (1). The lipofectamine reagent was obtained from Life Technologies (Rockville, MA). Luciferase assay and ß-galactosidase assay systems were from Promega (Madison, WI). Phorbol 12-Myristate 13-Acetate and TNF-{alpha} were purchased from Sigma Chemical (St Louis, MO).

Cytotoxicity assay
Ten thousand cells per well were incubated for 24 h, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) (Roche Molecular Biochemicals, Indianapolis, IN) was added 4 h prior to the termination of the culture. The amount of formazan deposits was quantified according to the supplier's protocol.

Cell invasion assay
Cell invasion assays were carried out as previously reported (23) with slight modifications, and 5 x 105 cells per chamber were seeded in each invasion assay. The upper chamber of a Transwell insert (Corning Costar, Cambridge, MA) was coated with 30 µl of a 1:2 mixture of Matrigel:PBS. The cells were plated on the Matrigel-coated upper chamber. The lower chamber was filled DMEM with various concentrations of ascofuranone. Cells in the chamber were incubated for 24 h at 37°C and cells that had invaded the lower surface of the membrane were fixed with methanol and stained with hematoxylin and eosin. Random fields were counted by light microscopy under a high power field.

Gelatin substrate gel zymography
Zymography was performed using the procedure described by Chung et al. (23) with minor modification. The U2OS cells were plated at 3 x 105 cells in 35 mm-diameter dishes and incubated until they reached 80% confluence. Fresh serum-free medium was added to each dish, followed by further culturing for 24 h. The resultant supernatant was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophroresis in 10% polyacrylamide gels that were copolymerized with 1 mg/ml of gelatin. After the electrophoresis runs, the gels were washed several times with 2.5% Triton X-100 for 1 h at room temperature to remove the sodium dodecyl sulfate and incubated for 24 h at 37°C in the buffer containing 5 mM CaCl2 and 1 µM ZnCl2. The gels were stained with Coomassie Brilliant Blue R250 (0.25%), (Bio-Rad, Hercules, CA) for 1 h, and then destained for 1 h in a solution of acetic acid and methanol. Proteolytic activity was evidenced as clear bands against the blue background of the stained gelatin.

Western blot analysis
Cell lysates were prepared by suspending 3 x 105 cells per 35 mm-diameter dish in 30 µl of lysis buffer [50 mM Tris, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.5% NP-40, 100 µM phenylmethylsulfonyl fluoride, 20 µM aprotinin and 20 µM leupeptin, adjusted to (pH 8.0)]. The cells were disrupted and extracted at 4°C for 30 min. The proteins were electrotransferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA). Detection of specific proteins was carried out with an enhanced chemiluminescence western blotting kit, following the manufacturer's instructions (Amersham-Pharmacia, NJ). The MMP-9- and phospho-ERK-specific antibodies were purchased from Santa Cruz (Santa Cruz, CA). Specific antibodies for phospho-Ras, phospho-Raf and phospho-MEK1/2 were purchased from Cell signaling (Beverly, MA).

Plasmids transfection and luciferase gene assays
MMP-9 wild-type (pGL2-MMP-9WT), AP-1 site-mutated MMP-9 (pGL2-MMP-9mAP-1-1 and pGL2-MMP-9mAP-1-2), NF-{kappa}B site-mutated MMP-9 luciferase promoter constructs (pGL2-MMP-9mNF-{kappa}B), SP-1 reporter constructs (23,24) were used in transient transfection assays as described previously. The AP-1 and NF-{kappa}B reporter constructs were purchased from Clontech (Palo Alto, CA). Cells were plated onto 35 mm dishes at a density of 3 x 105 cells and allowed to grow overnight. The cells were then cotransfected with 2 µg of various plasmid constructs and 1 µg of the pCMV-ß-galactosidase plasmid for 5 h by the Lipofectamine reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. After a 24 h incubation in fresh medium, the luciferase and ß-galactosidase activities were determined using commercial kits (Promega), according to manufacturer's protocols. Luciferase activity was calculated as luciferase activity normalized with ß-galactosidase activity in each cell lysate.

Northern blot analysis and RT–PCR
Radiolabeled probes used in the northern blot analyses were prepared by the random primer labeling method with [{alpha}-32P] dCTP using a random primer labeling kit (Promega). After the labeling reaction, the radiolabeled probes were purified on a micro Bio-Spin chromatography column (Bio-Rad). Total RNA for northern analysis was obtained by extraction with RNA ZolBee (Life Techology). Ten micrograms of total RNA was applied to a 1% formaldehyde-containing agarose gel and transferred to a Hybond-XL membrane (Amersham). The nylon membrane was hybridized at 55°C for 12 h with a radiolabeled DNA probe and washed according to the manufacturer's instructions. The membrane was then exposed to an X-ray film for 24 h. Loading differences were normalized using glyceraldehyde phosphate dehydrogenase expression levels. In the RT–PCR analysis, cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Promega). The PCR primers are described below. PCR products were analyzed by agarose gel electrophoresis and visualized by treatment with ethidium bromide. Used primers are as follows: tissue inhibitor of metalloproteinase (TIMP)-1 sense, 5-GGG GAC ACC AGA AGT CAA CCA GA-3; antisense, 5-CTT TTC AGA GCC TTG GAG GAG CT-3; TIMP-2 sense, 5-TGC AGC TGC TCC CCG GTG CAC-3; antisense, 5-TTA TGG GTC CTC GAT GTC GAG-3; ß-Actin sense, 5-GCC ATC GTC ACC AAC TGG GAC-3; antisense, 5-CGA TTT CCC GCT CGG CCG TGG-3.

Electophoretic mobility shift assay
Cultured cells were collected by centrifugation, washed and suspended in buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM PMSF]. After 15 min on ice, the cells were vortexed in the presence of 0.5% Nonidet P-40. The nuclear pellet was then collected by centrifugation and extracted with buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF] for 15 min at 4°C. The nuclear extracts (10 µg) were incubated at 4°C for 30 min in 25 mM HEPES buffer (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 0.05 M NaCl and 2.5% glycerol with 1 µg of poly dI/dC and 5 fmol (2 x 104 c.p.m.) of a probe end-labeled with {gamma}32P-ATP, and resolved by electrophoresis at 4°C in 6% polyacrylamide gels using a tris-borate (89 mM Tris, 89 mM boric acid and 1 mM EDTA) running buffer. Probes included 30mer oligonucleotides encompassing the consensus sequences for AP-1 and NF-{kappa}B (23). For competition assay to confirm the binding specificity, nuclear extracts were preincubated at 4°C for 30 min with a 100-fold excess of an unlabeled oligonucleotide. Gels were rinsed with water, dried and exposed to X-ray film overnight.

In vitro kinase assay of ERK and JNK
Cells (5–10 x 106 cells) detached from a dish were washed with 0.9% NaCl and centrifuged for 5 min at 3000 r.p.m. at 4°C, with a lysis buffer containing 50 mM Tris, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% Triton X-100, 0.5% NP40, 10% glycerol, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, 2 mM p-nitrophenylphosphate and 0.3 U/ml aprotinin. The cell lysate was centrifuged for 10 min at 10 000 g at 4°C and stored at –80°C. The immunoprecipitates were immobilized on protein G-Sepharose beads (Amersham-Pharmacia, Piscataway, NJ) by incubation for 4 h at 4°C overnight. The pellet was washed twice with the lysis buffer, suspended in kinase assay buffer [25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 20 mM ß-glycerophosphate, 0.1 mM Na3VO4, 2 µg/ml leupeptin, 1 mM phenylmethlsulfonyl fluoride, 0.3 U/ml aprotinin and 2 mM p-nitrophenylphosphate], mixed with either 20 µg c-Jun (for JNK assay) or 20 µg Elk-1 protein (for ERK-1 assay), 5 µCi of 32P-ATP, and incubated at 30°C for 30 min. The reaction was stopped by the addition of 2x laemmli buffer, and the sample was boiled, centrifuged and separated by electrophoresis. The gels were washed, dried and analyzed by a phosphorimage analyzer.

Statistical analysis
All in vitro results are representative of at least three independent experiments performed in triplicate; *P < 0.05, statistically significant between experimental and control values. Significance of differences between experimental and control values was calculated using analysis of variance with Newman–Keuls multicomparison test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Ascofuranone prevents invasion of human osteosarcma cells
Prior to the experiments for invasion in the medium containing 10% fetal bovine serum and for MMP activity in the serum-free medium, the cytotoxic effect of ascofuranone on a human osteosarcoma U2OS cell line was determined in the corresponding medium using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide assay. Treatment of cells with ascofuranone ranging in concentrations from 1 to 50 µM showed a 1–9% decrease in cell viability in serum-containing medium, and 3–15% decrease in cell viability in serum-free medium (Figure 1B). Ascofuranone did not show significant cytotoxicity to primary rat mesangial cells. These results show that ascofuranone has no significant cytotoxicity at these concentrations to tumor cells as well as normal cells. Cell invasion assays were carried out in Transwells coated with gels containing extracellular matrix proteins. As shown in Figure 1C, treatment of U2OS cells with PMA stimulated invasion about 3-fold. Ascofuranone inhibited PMA-dependent invasion in a dose-dependent manner, with inhibition in ascofuranone-treated cells reaching the levels of control cells by 1–50 µM. These results suggest that ascofuranone prevents invasion of human osteosarcoma at non-toxic concentrations.

Ascofuranone suppresses MMP-9 activity
The fact that ascofuranone had an inhibitory effect on invasion prompted us to examine the effect of ascofuranone on MMP-9 activity using gelatin zymography. The secretion of MMP-9 in the conditioned medium of U2OS cells was dramatically induced by PMA in a dose-dependent manner, when cultured in serum-free medium with various concentrations of PMA for 24 h (Figure 2A), whereas the level of MMP-2 expression was not detected by PMA as determined by gelatin zymography. As shown in Figure 2B, treatment of U2OS cells with ascofuranone, at doses above 10 µM, suppressed PMA-induced MMP-9 activity in a dose-dependent manner. Similar experiments were carried out with Caki-1, a human renal carcinoma, MDA-MB-231, a breast carcinoma cell, and primary rat mesagial cells. As evidenced by zymography, we were able to detect two bands in Caki-1 cells and rat mesangial cells, and a single weak band in MDA-MB-231 cells. Western blotting using specific antibodies indicated that they were MMP-9 and MMP-2 in Caki-1 cells and rat mesangial cells and MMP-9 in MDA-MB-231 cells (data not shown). The increased activities of MMP-9 induced by PMA were dramatically decreased by ascofuranone in these cell lines, but MMP-2 activity was not affected by the same treatment. TNF-{alpha} is one of the physiological inducers for MMP-9 (25). Ascofuranone significantly inhibited TNF-{alpha}-induced MMP-9 production in a dose-dependent manner, and MMP-9 expression returned to basal level in the presence of 30 µM ascofuranone (Figure 2C). These results indicate that ascofuranone inhibits MMP-9 induction in physiological relevance.


Figure 2
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  		Fig. 2. Inhibition of MMP-9 activity by ascofuranone. (A) Eighty percent confluent U2OS cells were treated with various concentrations of PMA in serum-free medium. Conditional media were collected after 24 h and gelatin zymography was performed. (B) U2OS, Caki-1, MDA-MB-231 cells and rat mesangial cells were incubated with varying concentrations of ascofuranone in the presence of PMA (50 nM) for 24 h. MMP activity in the medium was analyzed by gelatin zymography. (C) U2OS cells were incubated with varying concentrations of AF in the presence of TNF-{alpha}. MMP activity in the medium was analyzed by gelatin zymography.

 
Ascofuranone inhibits transcription of MMP-9
The results obtained on the zymography was further confirmed by western blot analysis (Figure 3A). The secretion of MMP-9 protein into the medium was gradually decreased in a dose-dependent manner, indicating that the reduced MMP-9 enzyme activity is the result of the decreased amounts of MMP-9 protein. In the northern blot analysis, the treatment of U2OS cells with ascofuranone decreased the levels of PMA-stimulated MMP-9 mRNA expression (Figure 3B), indicating that ascofuranone prevents the transcription of MMP-9 in response to PMA. Because activities of MMP-9 are tightly regulated by endogenous inhibitors, TIMPs (26), we further examined the expression level of TIMP-1 and -2 by RT–PCR, but their expression remained essentially unchanged by treatment with ascofuranone (Figure 3C).


Figure 3
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  		Fig. 3. Inhibition of PMA-induced MMP-9 expression by ascofuraone. (A) U2OS cells were incubated with ascofuranone and/or PMA (50 nM) for 24 h. MMP-9 expression in the medium was analyzed by western blotting. ß-actin in the cell lysate is shown as a control. (B) U2OS cells were incubated with ascofuranone and/or PMA (50 nM) for 24 h. mRNA expression of MMP-9 in the cells was analyzed by northern blotting. GAPDH expression was included as an internal control. (C) U2OS cells were incubated with ascofuranone and/or PMA (50 nM) for 24 h. The mRNA expression of TIMP-1 and 2 in the cells was analyzed by RT–PCR. ß-actin expression was included as an internal control.

 
Ascofuranone specifically suppresses AP-1 activity
The effect of ascofuranone on the activity of the MMP-9 promoter was investigated using U2OS cells that had been transiently transfected with a luciferase reporter gene linked to the MMP-9 promoter sequence. As shown in Figure 4A, luciferase gene expression was activated up to ~10-fold in cells that had been treated with PMA compared with untreated cells. Treatment of cells with ascofuranone decreased the PMA-mediated luciferase activity in a dose-dependent manner.


Figure 4
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  		Fig. 4. Inhibition of AP-1 activity in the MMP-9 promoter by ascofuranone. Mutations were introduced in the NF-{kappa}B or AP-1 binding sites of pGL2-MMP-9WT by 2 bp changes. U2OS cells were transfected with pGL2-MMP-9WT, pGL2-MMP-9mNF-{kappa}B, pGL2-MMP-9mAP-2 reporter plasmids (A), or with reporter plasmids containing tandem elements for AP-1, SP-1 or NF-{kappa}B binding sites (B). Cells were cultured with ascofuranone and/or PMA for 24 h, and the relative luciferase activity in the cell extract was determined. Each bar represents standard deviation of triplicate cultures. Statistically significant (*P < 0.05, **P < 0.01).

 
The MMP-9 promoter contains cis-acting regulatory elements for transcription factors that include two AP-1 sites (located at -79 bp and -533 bp) and an NF-{kappa}B site (located at -600 bp) (27–29). AP-1 had been described as a PMA-inducible transcription factor that addresses specific sequences in the enhancer of the metallothionein genes (30). To test which of these transcription factors may regulate the MMP-9 gene in U2OS cells, cells were transiently transfected with reporter genes that included the wild-type MMP-9 promoter, or the promoter with mutations in both AP-1 sites, or the NF-{kappa}B site (Figure 4A). The mutation of the AP-1 binding sites drastically decreased the response to PMA. The mutation in the NF-{kappa}B binding site also decreased PMA-induced MMP-9 reporter gene activity, but not to the extent achieved by mutation of the AP-1 sites. Treatment with ascofuranone in the presence of PMA decreased the transcription activity of the reporter with the NF-{kappa}B mutation, but was ineffective for the reporter with AP-1 mutations, suggesting that the target of ascofuranone is the AP-1 transcription factors.

In subsequent experiments, cells were transiently transfected with reporter vectors that included the tandem repeat of the AP-1, NF-{kappa}B or SP-1 binding sites, in order to confirm the specificity of ascofuranone-mediated inhibitory effect on AP-1. The luciferase activity in the cells transfected with the AP-1 reporter was significantly reduced by treatment with ascofuranone in the range of 1–50 µM in a dose-dependent manner, whereas ascofuranone showed no statistically significant effect on the luciferase activity of cells transfected with the NF-{kappa}B and Sp1 reporters (Figure 4B).

To confirm the results of reporter experiments, electrophoretic mobility shift assay was performed, using oligonucleotides containing the consensus sequences for AP-1 and NF-{kappa}B as probes. U2OS cells were incubated in the presence of PMA with different concentrations of ascofuranone for 24 h and nuclear extracts were prepared and analyzed for AP-1 and NF-{kappa}B DNA binding activity. As shown in Figure 5A, AP-1 induced by PMA dramatically decreased when cells were treated with ascofuranone. On the other hand, NF-{kappa}B binding activity induced by PMA was only negligibly affected by ascofuranone. These data were consistent with the reporter gene analysis and suggest that ascofuranone blocks MMP-9 expression, at least in part, by decreasing the expression or DNA binding activity of members of the AP-1 transcription factor family. Expression of AP-1 family was determined by western blotting (Figure 5B). PMA dramatically induced c-Fos expression, whereas it only minimally affected c-Jun expression. Ascofuranone significantly reduced c-Fos induction, suggesting that c-Fos reduction partly contributes to the decrease in AP-1 binding activity by ascofuranone-treatment in electrophoretic mobility shift assay.


Figure 5
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  		Fig. 5. Ascofurane specifically suppressed AP-1 binding activity induced by PMA. (A) Nuclear extract (10 µg) prepared from control or U2OS cells treated with 50 nM PMA in presence or absence of ascofuranone for 24 h was mixed with radioactive oligonucleotides containing AP-1 and NF-{kappa}B motif of MMP-9 promoter. Bound complexes were analyzed by electrophoresis. (B) U2OS cells were treated with 50 nM PMA in presence or absence of ascofuranone for 24 h, and the expression of c-Fos, c-Jun and ß-actin as an internal control were analyzed with western blotting.

 
Ascofuranone blocks ERK activation induced by PMA
It is known that activation of one or more mitogen-activated protein kinase (MAPK) pathways is important for the MMP-9 induction by PMA in various cell types (31). To evaluate the effects of ascofuranone on these signaling cascades, we used antibodies against the phosphorylated forms of the three MAPKs including ERK1/2, JNK/SAPK and p38. As shown in Figure 6A, their phosphorylations were increased by the stimulation with PMA. Ascofuranone specifically decreased ERK1/2 phosphorylation, whereas the levels of phosphorylated JNK and p38 remained unchanged. These results suggest that ascofuranone specifically suppresses ERK1/2 activity. This result was further confirmed by in vitro kinase assay for endogenous ERK-1 and JNK. ERK-1 and JNK were immunoprecipitated with specific antibodies, and incubated with and Elk-1 and c-Jun, specific substrates for ERK-1 and JNK, in the presence of [{gamma}-32P]ATP. Although PMA activated both ERK-1 and JNK activities, ascofuranone specifically decreased ERK-1 activity induced by PMA stimulation without affecting JNK activity (Figure 6B). Ascofuranone did not alter protein expression of ERK and JNK after PMA treatment. Taken together, these results indicate that ascofuranone has an inhibitory effect on PMA-stimulated ERK activation in U2OS cells. We also examined phosphorylation of upstream regulators in ERK signaling pathway by western blotting. PMA increases phosphorylations of ERK1/2, MEK and Raf, but not Ras phosphorylation (Figure 6C). Ascofuranone significantly inhibited phosphorylations of all these regulators in a dose-dependent manner from 1 to 30 µM without affecting protein expression levels. These results suggest that the primary target of ascofuranone for AP-1 inhibition is present in ERK signal transduction pathway upstream of Ras.


Figure 6
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  		Fig. 6. Ascofuranone specifically inhibits the activation of ERKs. (A) U2OS cells were incubated with ascofuranone and/or PMA as indicated, and the levels of phospho-JNK/SAPK, ERK1/2 and p38 were determined by western blotting with phospho-specific antibodies. Protein expression levels of ß-actin in cell lysates were used as a control. (B) U2OS cells were treated with ascofuranone and/or PMA for 24 h, and activities of ERK-1 and JNK immunoprecipitated with specific antibodies were determined by in vitro kinase assay using Elk-1 and c-Jun as specific substrate for ERK-1 and JNK, respectively. Total ERK1/2 and JNK in immunoprecipitates were also shown by western blotting. (C) U2OS cells were incubated with ascofuranone and/or PMA as indicated, and the levels of phospho-ERK1/2, phospho-Ras, phospho-Raf and phospho-MEK were determined by western blotting with phospho-specific antibodies. Values of a bar graph represent the mean ± standard error of at least three independent experiments. The densities of immunoreactive bands were measured by Quantity One 1-D analysis software program.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
A prenylphenol antibiotic, ascofuranone, has a broad spectrum of antitumor and antimetastatic activity, and we have demonstrated that the prophylactic antitumor activity is, at least in part, due to the activation of a host-defense mechanism (3,4,6). Our present results indicate that, in addition to the host-mediated mechanism, ascofuranone directly inhibits the invasive ability of tumor cells via the suppression of MMP-9 expression, a protease that is involved in tumor invasion and metastasis (7–12), in human tumor cell lines including an oseteosarcoma, a renal carcinoma and a breast cancer cell. Metastasis and invasion are major properties of malignant tumors that are associated with a poor prognosis. Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents. Osteosarcomas account for ~5% of the tumors in childhood and 80% of these tumors originate around the knee. Since ascofuranone prevents such invasion at non-toxic doses, it has the potential for clinical use in preventing the invasion and metastasis of human malignant tumors, including osteosarcoma.

Based on a sound understanding of the biochemistry of MMPs and the accumulated evidences that implicate MMPs in cancer dissemination, the pharmaceutical industry has invested heavily in developing effective MMP inhibitors, but none have been shown to be clinically effective in the treatment of advanced cancers (32). One of the reasons for the ineffectiveness is the redundancy and multiple functions of MMPs, and a new strategy other than the simple inhibition of the enzyme activity, which can specifically prevent MMPs involved in tumor invasion, will be required. Unlike these inhibitors, ascofuranone suppresses MMP-9-induction by the repression of transcription activation in the MMP-9 promoter. A mutation analysis of the promoter revealed that the major target of ascofuranone was AP-1, a finding that was further confirmed by the use of reporter plasmids containing synthetic elements that are specific for the transcription factors. Moreover, ascofuranone blocks the binding of AP-1 protein to oligonucleotides containing the consensus sequence for the AP-1 binding site from the MMP-9 promoter. Our results demonstrate that the suppression of AP-1 is another effective strategy for blocking MMP-9 induction, required for tumors to be malignant.

AP-1 is a sequence-specific transcriptional activator composed of members of the Jun and Fos families (33–35). These proteins, which belong to the bZIP group of DNA-binding proteins, associate to form a variety of homo- and heterodimers that bind to a common consensus sequence. Thus, AP-1 activity is regulated by the transcription of AP-1 family proteins, combination of members comprised of AP-1 heterodimers and modification of AP-1 family proteins. The phosphorylation of specific amino acid residues by protein kinases such as MAPKs modulates AP-1 activity including transcriptional activation, protein stability and the intracellular localization of AP-1 proteins. The ERK group of MAPKs phosphorylates Elk-1 that subsequently promotes the transcription of c-Fos by facilitating the formation of a ternary complex composed of itself, the serum response factor and the serum responsive elements present in the c-Fos promoter (36). The phosphorylation of Fra-1 by ERK protects the protein from proteasomal degradation (37,38). The intracellular localization and chromatin association of c-Fos family proteins in response to oxidative stress were reported to be regulated by ERK phosphorylation (39). Ascofuranone specifically suppressed PMA-mediated ERK activation without affecting pathways involving JNK and p38 and decreased expression of c-Fos. Phosphorylation of upstream signal transducers, Ras, Raf and MEK, was also suppressed by ascofuranone, suggesting that a primary target of ascofuranone for the suppression of AP-1 is present in upstream signal transduction pathway above Ras, although direct inhibition of kinase activity of protein kinase C is unlikely because ascofuranone has biological activities entirely different from potent protein kinase C inhibitors (data not shown).

On the basis of present results, we suggest that antitumor activity of ascofuranone is in part due to the inhibition of MMP-9 expression, which plays an important role in cancer invasion and metastasis, through the suppression of AP-1 activity. Beside antitumor activity, ascofuranone has variety of physiological activities, such as hypolipidemic activity and immunomodulation (2,4–6). Because MMPs play crucial roles in human diseases other than cancers such as atherosclerosis, aneurysms, nephritis, tissue ulcers, inflammatory disorders and fibrosis (40–42), inhibition of MMP might be involved in these physiological activities mediated by ascofuranone. In addition, MMP is an essential factor in normal physiological reactions such as embryonic development, morphogenesis, reproduction, tissue resorption and wound healing (43,44). Because ascofuranone suppressed MMP-9 induction in normal cells, side effects resulted from the inhibition of MMP-9 should be taken into account at the clinical application of ascofuranone. Nonetheless, considering the overwhelming evidence for a role of type IV collagenase in tumor cell invasion, inhibition of MMP-9 induction in tumor cells could be one of the most powerful strategies in cancer therapy. Present results demonstrate that ascofranone is a promising clinical drug in reducing malignant factors of cancers, invasion and metastasis.


   Acknowledgments
 
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-042-C00150).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Sasaki H, et al. (1973) Isolation and structure of ascofuranone and ascofranol, antibiotics with hypolipidemic activity. J. Antibiot. (Tokyo) 26:676–680.[Medline]
  2. Sawada M, et al. (1973) Hypolipidemic property of ascofuranone. J. Antibiot. (Tokyo) 26:681–686.[Medline]
  3. Magae J, et al. (1982) Antitumor protective property of an isoprenoid antibiotic, ascofuranone. J. Antibiot. (Tokyo) 35:1547–1552.[Medline]
  4. Magae J, et al. (1988) Antitumor and antimetastatic activity of an antibiotic, ascofuranone, and activation of phagocytes. J. Antibiot. (Tokyo) 41:959–965.[Medline]
  5. Magae J, et al. (1986) In vitro effects of an antitumor antibiotic, ascofuranone, on the murine immune system. Cancer Res 46:1073–1078.[Abstract/Free Full Text]
  6. Magae J, et al. (1986) Activation of natural cytotoxic activity and concomitant reduction of triglyceride content of murine spleen, treated with an antitumor antibiotic, ascofuranone. J. Antibiot. (Tokyo) 39:676–681.[Medline]
  7. Zucker S, et al. (1993) M(r) 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res 53:140–146.[Abstract/Free Full Text]
  8. Lakka SS, et al. (2004) Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis. Oncogene 23:4681–4689.[CrossRef][ISI][Medline]
  9. Shim YH, et al. (2003) p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis. Cancer Lett. 190:213–219.[CrossRef][ISI][Medline]
  10. Stetler-Stevenson WG, et al. (1996) Matrix metalloproteinases and tumor invasion: from correlation and causality to the clinic. Semin. Cancer Biol. 7:147–154.[CrossRef][ISI][Medline]
  11. Chambers AF, et al. (1997) Changing views of the role of matrix metalloproteinases in metastasis. J. Natl Cancer Inst. 89:260–270.[ISI][Medline]
  12. Nabeshima K, et al. (2002) Matrix metalloproteinases in tumor invasion: role for cell migration. Pathol. Int. 52:255–264.[CrossRef][ISI][Medline]
  13. Arai K, et al. (2003) Essential role for ERK mitogen-activated protein kinase in matrix metalloproteinase-9 regulation in rat cortical astrocytes. Glia 43:254–264.[CrossRef][ISI][Medline]
  14. Chung TW, et al. (2004) Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. FASEB J 18:1123–1125.[Abstract/Free Full Text]
  15. Gum R, et al. (1997) Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signalregulated kinase-dependent signaling cascades. Oncogene 14:1481–1493.[CrossRef][ISI][Medline]
  16. Eberhardt W, et al. (2000) Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J. Immunol. 165:5788–5797.[Abstract/Free Full Text]
  17. Abiru S, et al. (2002) Aspirin and NS-398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology 35:1117–1124.[CrossRef][ISI][Medline]
  18. Kim D, et al. (2001) Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J 15:1953–1962.[Abstract/Free Full Text]
  19. Sato T, et al. (2002) Inhibition of activator protein-1 binding activity and phosphatidylinositol 3-kinase pathway by nobiletin, a polymethoxy flavonoid, results in augmentation of tissue inhibitor of metalloproteinases-1 production and suppression of production of matrix metalloproteinases-1 and -9 in human fibrosarcoma HT-1080 cells. Cancer Res 62:1025–1029.[Abstract/Free Full Text]
  20. Eferl R, et al. (2003) AP-1: a double-edged sword in tumorigenesis. Nat. Rev. Cancer 3:859–868.[CrossRef][ISI][Medline]
  21. Pastore S, et al. (2005) ERK1/2 regulates epidermal chemokine expression and skin inflammation. J. Immunol. 174:5047–5056.[Abstract/Free Full Text]
  22. Park KK, et al. (2003) Inhibitory effects of novel E2F decoy oligodeoxynucleotides on mesangial cell proliferation by coexpression of E2F/DP. Biochem. Biophys. Res. Commun. 308:689–697.[CrossRef][ISI][Medline]
  23. Chung TW, et al. (2004) Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. FASEB J 18:1670–1681.[Abstract/Free Full Text]
  24. Chae YM, et al. (2004) Sp1-decoy oligodeoxynucleotide inhibits high glucose-induced mesangial cell proliferation. Biochem. Biophys. Res. Commun 319:550–555.[CrossRef][ISI][Medline]
  25. Mon NH, et al. (2006) A role for focal adhesion kinase signaling in tumor necrosis factor-alpha-dependent matrix metalloproteinase-9 production in a cholangiocarcinoma cell line, CCKS1. Cancer Res 66:6778–6784.[Abstract/Free Full Text]
  26. Kazes I, et al. (2000) Platelet release of trimolecular complex components MT1-MMP/TIMP2/MMP2: involvement in MMP2 activation and platelet aggregation. Blood 96:3064–3069.[Abstract/Free Full Text]
  27. Tsuchiya Y, et al. (1993) Tissue inhibitor of metalloproteinase 1 is a negative regulator of the metastatic ability of a human gastric cancer cell line, KKLS, in the chick embryo. Cancer Res 53:1397–1402.[Abstract/Free Full Text]
  28. Sato H, et al. (1993) v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements. A mechanism regulating gene expression independent of that by inflammatory cytokines. J. Biol. Chem. 268:23460–23468.[Abstract/Free Full Text]
  29. Woo JH, et al. (2003) Dykellic acid inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting nuclear factor kappa B transcriptional activity. Cancer Res 63:3430–3434.[Abstract/Free Full Text]
  30. Angel P, et al. (1987) Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729–739.[CrossRef][ISI][Medline]
  31. Woo JH, et al. (2004) Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta signal transduction. Oncogene 23:1845–1853.[CrossRef][ISI][Medline]
  32. Zucker S, et al. (2000) Critical appraisal of the use of matrix metallo-proteinase inhibitors in cancer treatment. Oncogene 19:6642–6650.[CrossRef][ISI][Medline]
  33. Angel P, et al. (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta. 1072:129–157.[Medline]
  34. Vogt PK. (2001) Jun, the oncoprotein. Oncogene 20:2365–2377.[CrossRef][ISI][Medline]
  35. Shaulian E, et al. (2001) AP-1 in cell proliferation and survival. Oncogene 20:2390–2400.[CrossRef][ISI][Medline]
  36. Karin M. (1995) The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270:16483–16486.[Free Full Text]
  37. Casalino L, et al. (2003) Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilization. Mol. Cell. Biol. 23:4401–4415.[Abstract/Free Full Text]
  38. Vial E, et al. (2003) Elevated ERK-MAP kinase activity protects the FOS family member FRA-1 against proteasomal degradation in colon carcinoma cells. J. Cell Sci. 116:4957–4963.[Abstract/Free Full Text]
  39. Burch PM, et al. (2004) An extracellular signal-regulated kinase 1- and 2-dependent program of chromatin trafficking of c-Fos and Fra-1 is required for cyclin D1 expression during cell cycle reentry. Mol. Cell. Biol. 24:4696–4709.[Abstract/Free Full Text]
  40. Caird,J J, et al. (2006) Matrix metalloproteinases 2 and 9 in human atherosclerotic and non-atherosclerotic cerebral aneurysms. Eur. J. Neurol. 13:1098–1105.[CrossRef][ISI][Medline]
  41. Deguchi JO, et al. (2006) Inflammation in atherosclerosis: visualizing matrix metalloproteinase action in macrophages in vivo. Circulation 114:55–62.[Abstract/Free Full Text]
  42. Henger A, et al. (2004) Gene expression fingerprints in human tubulointerstitial inflammation and fibrosis as prognostic markers of disease progression. Kidney Int. 65:904–917.[CrossRef][ISI][Medline]
  43. Philipp K, et al. (2004) Target TGF-beta in human keratinocytes and its potential role in wound healing. Int. J. Mol. Med. 14:589–593.[ISI][Medline]
  44. Ram M, et al. (2006) Matrix metalloproteinase-9 and autoimmune diseases. J. Clin. Immunol. 26:299–307.[CrossRef][ISI][Medline]
Received July 10, 2006; revised October 9, 2006; accepted November 2, 2006.


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