WO2005053671A1

WO2005053671A1 - Mmp-9 inhibitor containing caffeic acid or caffeic acid phenethyl ester

Info

Publication number: WO2005053671A1
Application number: PCT/KR2004/000308
Authority: WO
Inventors: Cheorl-Ho Kim; Tae-Wook Chung; Young-Choon Lee
Original assignee: Cheorl-Ho Kim; Tae-Wook Chung; Young-Choon Lee
Priority date: 2003-12-08
Filing date: 2004-02-16
Publication date: 2005-06-16
Also published as: KR20050055479A

Abstract

Disclosed is an MMP-9 inhibitor which contains caffeic acid of caffeic acid phenethyl ester.

Description

MM P -9 INHIBITOR CONTAINING CAFFEIC ACID OR CAFFEIC ACID PHENETHYL ESTER

Technical Field The present invention relates to an MMP-9 inhibitor which contains caffeic acid or caffeic acid phenethyl ester.

Background Arts MMPs play an important role in degradation of various components in extracellular matrix (ECM). Examples of MMPs-mediated diseases include arteriosclerosis, restenosis, MMP -mediated osteopenia, inflammatory disease in central nerve system, Alzheimer's disease, skin aging, rheumatoid arthritis, osteoarthritis, septic arthritis, keratohelcosis, malunion of wound, bone disease, proteinuria, abdominal aortic aneurysms, cartilage-degenerative loss owing to traumatic joint damages, demyelinative diseases in nerve system, hepatic cirrhosis, glomerulus disorders, premature rupture of fetal membrane, inflammatory bowel diseases, periodontal disease, aging-related macular degeneration, diabetic retinopathy, prohferative vitreoretinopathy, immature retinopathy, opthalmia, conical cornea, Sjogren syndrome, short sightedness, ophthalmic tumor, corneal transplant rejection, vascularization, invasion and metastasis of carcinoma and the like.. Particularly, MMP-2 and MMP-9 among MMPs are widely known to have specific activities on type IV collagen, thereby playing a key role in diseases such as inflammation, stroke, tumor growth and metastasis. It has been reported that MMP-9 level in cerebrospinal fluid is related to multiple sclerosis and other neurotic diseases [Beeley, N.R.A. et al, supra.; Miyazaki, K. et al.,

Nature 362, 839~841(1993)], and contributes to degradation and accumulation of amyloid β-protein [Backstrom JR, et al, J neurosci 16(24), 7910-9 (1996)]. Further, it is also known that MMP-9 has an important role in invasion and metastasis of hepatocarcinoma. The metastasis and invasion of tumor are fundamental characteristic feature of malignant tumor cells. In the initial stage of cancer, occurred is the invasion of a tumor to blood vessel as well as its metastasis. Tumor cells go through several important steps during the invasion and metastasis process. The tumor cells are surrounded by extracellular matrix and basement membrane which act as a barrier against the invasion of tumor cells, accordingly, the substantial initial step in those processes is degradation of the extracellular matrix which constitutes the basement membrane. In the degradation process, various proteinases are involved, and then the external barrier, i.e. the extracellular matrix(ECM) and basement membrane, is finally degraded (Woessner, J. F. Jr. FASEB J., 5: 2145-2154, 1991). During the process, secreted from cells is an inactive proMMP-9, which is activated by a series of enzymatic activation processes including the generation of an active type of plasminogen (plasmin) by a urokinase-type plasminogen activator (uPA). The activated MMP-9 promotes the invasion potential of tumor cells (registered Korean Patent No. 2002-71674). The relation between the invasion and metastasis of tumor and the expression of

MMP-9 which has great effects thereon was investigated by using several analytic methods such as zymography, Northern blot and Western blot with MMP-9 being expressed on tumor cells. The MMP-9 expression on tumor cells was very high. The test on invasion using a matrigel showed plasmin-dependent increase in the degree of invasion. The results suggest that the transcription activity of MMP-9 plays important role in the invasion and metastasis of tumor cells. The MMP-9 expression has also important role in the invasion and metastasis of brain tumor, tumors of the nervous system and breast cancer (Rao et al., 1993; Rao et al., 1996 Scorilas et al., 2001). Further, the promoter region of MMP-9 has binding sites for AP-1 and NF-κB (Sato, H., and Seiki, M. Oncogene, 8: 395-405, 1993), therefore stimulating elements such as tumor cells, cytokines thereof or PMA (phorbol 12-myristate 13-acetate) control the MMP-9 expression by regulating the activation of transcription factors like AP-1 and NF-κB, etc. through Ras/Raf/ERK, JNK and PI-3K/AKT signal transduction pathways (Arai, K. et al., Glia. 43: 254-264, 2003; Sato, H., and Seiki, M.. Oncogene, 8: 395-405, 1993; Gum, R et al, Oncogene, 14: 1481-1493, 1997; Eberhardt, W., et al., J. Immunol., 165: 5788-5797,

2000; Abiru, S. et al., Hepatology, 35: 1117-1124, 2002; Kim, D et al, FASEB J., 15: 1953-1962, 2001 ; Sato, T. et al, Cancer Res., 62: 1025-1029, 2002). NF-κB controls the expression of various genes which are directly involved in the onset of cancer (Pahl H. L., Oncogene, 18: 6853-6866, 1999; Garg A., Aggarwal B. B., Leukemia (Baltimore), 16: 1053-1068, 2002). Examples of the various genes include antiapoptosis genes, etc. such as survivin, TRAF, bcl-2 and the like. Besides, NF-κB is a key transcription factor which is involved in the activation of inflammatory-cytokine genes such as tTNF-α or IL-lβ. The NF-κB also induces the activation of MMP-9, COX-2 and iNOS genes. Therefore, several substances which inhibit the activation of NF-κB are suggested to be potent to inhibit the onset and metastasis of cancer, and researches thereon for developing them as a therapeutic agent have been actively conducted. Caffeic acid (CA) is a phenolic compound which wildly presents in nature including fruits, vegetables, wine, vegetable oil, coffee and the like (Shahidi, F., and Naczk. M. Food phenolics. Sources, chemistry, effects, applications, Technomic Publishing Company, Inc, Lancaster, PA (1995). Formula 1

Caffeic acid phenethyl ester (CAPE) may be extracted mainly from bee propolis (Grunberger, D. et al., Experientia. 44: 230-232, 1988) and can be synthesized by esterification of CA (Nagaoka, T. et al., Bioorg Med Chem. 10: 3351-3359, 2002). Formula 2

It is well known that CA and CAPE have antioxidation activity (Grunberger, D. et al., Experientia. 44: 230-232, 1988; Vieira, O. et al., Biochem. Pharmacol. 55: 333-340,1998), and also an activity of inhibiting enzymes such as lipoxygenases, cyclooxygenase, glutathione S-transferase, xanthine oxidase and the like [Chan, W.S. et al, Anticancer Res. 15: 703-707, 1995; Schefferlie, J.G., and van Bladeren, P.J. Food Chem. Toxicol. 37: 475-482, 1993; Koshihara, Y. et al., Biochim. Biophys. Acta. 792: 92-97, 1984; Mirzoeva, O. K. et al, FEBS Lett. 396: 266-270, 1996]. Further, it has been reported that CA and CAPE have antitumor activity [Tanaka, T. et al., Carcinogenesis. 14: 1321-1325, 1993; Frenkel, K. et al, Cancer Res., 53: 1255-1261, 1993], anti-inflammatory activity [Michaluart, P. et al, Cancer Res. 59: 2347-2352, 1999; Fernandez, M. A. et al, J. Pharm. Pharmacol. 50: 1183-1186, 1998], HIV replication inhibition [Kashiwada, Y. et al., J. Nat. Prod. 58: 392-400, 1995; Fesen, M. R. et al, Proc.

Natl. Acad. Sci. USA 90: 2399-2403, 1993]. Further, CA effectively inhibits the binding activity of a ceramide-induced NF-κB (Nardini, M. et al, Free Radic Biol Med. 30: 722-733, 2001), and CAPE inhibits the activity of a transcription factor NF-κB in a specific and effective way (Natarajan, K. et al, Proc Natl Acad Sci USA 93: 9090-9095, 1996). However, although the anti-tumor metastatic activity of CAPE is known

(Nagaoka, T. et al, Biol. Pharm. Bull. 26: 487-491, 2003), neither a direct inhibition of MMP-9 activity by CA and CAPE nor an inhibitory activity on the invasion and metastasis of cancer through the direct inhibition of MMP-9 gene expression have been reported.

Summary of Invention Therefore, an object of the present invention is to provide a novel use of CA and CAPE as a direct inhibitor to the enzymatic activity of MMP-9. In order to achieve the above object, the inventors of the present invention have intensively studied and developed an MMP-9 inhibitor which contains caffeic acid (CA) and caffeic acid phenethyl ester (CAPE). The present invention relates to an MMP-9 inhibitor having CA or CAPE as an active ingredient. The present invention is further characterized in that the inhibitor suppresses the generation of MMP-9 enzyme. The present invention is also characterized in that the inhibitor suppresses the transcription for MMP-9 expression. The present invention is further characterized in that the inhibitor suppresses the activity of an MMP-9 promoter. Additionally, the present invention is characterized in that the inhibitor is used to rheumatoid arthritis, osteoarthritis, osteoporosis and diseases related with the metastasis, invasion or development of tumor. Further, the present invention relates to a use of CA or CAPE as an MMP-9 inhibitor.

Brief description of Drawings Fig. 1 is a plot showing the results of XTT assay for estimating the inhibitory effect of CA and CAPE on the proliferation of a hepatocarcinoma cell, HepG2, wherein (A) is CA and (B) is CAPE. Fig. 2 is a plot showing the results of zymography assay for estimating the inhibitory effect of CA and CAPE on an MMP-9 enzyme, wherein (A) is CA and (B) is CAPE. Fig. 3 is the results of Western blot assay for demonstrating the inhibitory effect of CA and CAPE on MMP-2 generation in hepatocarcinoma cell. Fig. 4 shows the inhibitory effect of CA and CAPE on transcription and promoter activity in PMA-induced MMP-9 gene expression, wherein (A) is a genetic map regarding cis-regulatory elements of an MMP-9 promoter region; (B) is the results of a zymography assay on the inhibitory effect on the transcription of MMP-9 gene; and (C) is the results of

RT-PCR and Northern blot assay. Fig. 5 is a plot showing the inhibitory effect of the NF-κB binding site in CA and

CAPE on the promoter activity of MMP-9 gene, wherein (A) shows the structure of an MMP-9 promoter for testing the luciferase activity and its luciferase activity, and (B) the luciferase activity of various mutated promoter regions: Mut-NF-κB, Mut-AP-1-1 and

Mut-AP-1-2. Fig. 6 is a photo showing electrophoretic mobility shift assay for determining the inhibitory effect of CA and CAPE on the PMA-induced NF-κB activation, wherein (A) represents M4 reaction specific to NF-κB, AP-1-1, AP-1 -2 specific ( $ } !:-§- ^όl 2]--ϊ-. °

i ); (B) is the results of Western blot assay of p65 antibody, with GAPDH as a control group. Fig. 7 is a plot showing the inhibitory effect of CA and CAPE on the metastasis and invasion of hepatocarinoma cell, HepG2.

Embodiments for Carrying Out the Invention Hereinafter, the present invention is further described in detail with referencing the examples provided below. However, those examples are only provided for illustrative purposes, by no means limiting the scope of the present invention.

Example 1: Assay on the inhibitory effect of CA and CAPE on the cell proliferation of HepG2 Human hepatocarcinoma cell line HepG2 was cultured in Dulbecco's modified Eagle medium (DMEM, Gibco-BRL, USA) containing a heat-treated 10% fetal bovine serum (FBS, Gibco-BRL, USA), penicillin (lOOunitsM), streptomycin (lOOβg/ml) and sodium bicarbonate (2.2g/£) in a 5% CO₂-incubator at 37 ^°C . An XTT kit-II (commercially available from Boehringer Mannheim, Germany) was used to determine cell proliferation. Briefly, the HepG2 cells were plated and cultured in a 96-well plate for 24 hours till obtaining the cell concentration of 10³ cells per each 100 μJl of DMEM, and then the media of the 96-well plate were replaced with 100/z£ fresh media each containing various concentrations of CA or CAPE. The plate was incubated at 37^°C for 24 hours under constant 5% CO atmosphere in a humidity-controlled incubator. Completing the cell cultivation, the medium was discarded and the cells were washed with a PBS solution. Then, 50μA of an XTT reagent prepared by mixing 5uji of an XTT-indicator reagent and 100/z# of an electron coupling reagent was added to each well of the plate. The plate was again incubated at 37^°C for 4 hours in 5% CO₂-incubator, and light absorbance was measured at 490nm with an ELISA kit (Molecular Devices, USA). In order to determine the effects of CA and CAPE on the proliferation of the hepatocarcinoma cell, HepG2, HepG2 cells were added to various concentrations of CA and CAPE and maintained for 24 hours for reaction, and the inhibitory activity on cell proliferation was measured with XTT assay. Light absorbance was measured at 490nm with an ELISA kit (Molecular Devices, USA). The results are shown in Fig.l. In Fig. 1, (A) and (B) represent the individual effect of CA and CAPE, respectively. The plots of Fig. 1 were expressed with the percentage of cell proliferation compared to a control group without containing CA or CAPE, and each experimental data was obtained by running 3 times of independent test and represented as the average±SD (standard deviation). As seen from the results, it was demonstrated that both of CA and CAPE had an inhibitory activity on the proliferation of hepatocarcinoma cell line. Example 2; Effects of CA and CAPE on the enzymatic activity of MMP-9 In order to determine the inhibitory effects of CA and CAPE on the gelatinolyic enzymatic activity of MMP-9, a conditioned medium obtained from a HepG2 culture broth treated with PMA was again diluted with the same buffer solution containing 62.5mM of Tris-HCl (pH 6.8), 10% glycerol, 2% SDS and 0.00625%(w/v) bromophenol blue. The resulted solution was subjected to electrophoresis in 10% polyacrylamide gel containing 0.1%(w/v) gelatin, without heating. After the electrophoresis, the gel was immersed into 2.5% Triton X-100 (2X30 min) at room temperature to wet, and washed with distilled water, NanoPure. Then, the gel was cut into slices by each electrophoresis lane, and the slices were immersed into an individual tank containing a reaction buffer solution that contains a series of concentrations of CA or CAPE [50mM Tris-HCl(pH 7.5), 200mM NaCl, 2.5mM CaCl₂]. Thus prepared gels treated with CA and CAPE were incubated at 37^°C for 24 hours. Dyeing with the coomassie brilliant blue R-250 showed a white band which indicates an enzymatic activity (Fig. 2). Addtionally, in order to determine the inhibitory effects of CA and CAPE on the

PMA-induced MMP-9 expression, HepG2 cells were treated with CA or CAPE in the presence of 200nM of PMA, and the degree of MMP-9 expression was assayed by using zymography. HepG2 cells were cultured in 10% FBS/DMEM, washed with a PBS solution, and then cultured in a serum-free DMEM together with CA or CAPE in the presence of PMA for 24 hours. Then, the conditioned medium was collected and mixed with 10% acrylamide gel containing 0.1 % gelatin for electrophoresis. After the electrophresis, the gel was washed with 2.5%(v/v) Triton X-100 for 1 hour to remove SDS, and was maintained at a constant temperature in a reaction buffer solution for 24 hours to decompose the gelatin substrate. A white-colored band that indicates an enzymatic activity was developed by dyeing with the coomassie brilliant blue R-250 (Bio-Rad, USA) and the molecular weight was estimated by comparing with a pre-dyed SDS-PAGE marker. In order to determine the effects of CA and CAPE on MMP-9 activity, gelatin zymography assay was used by culturing the PMA-induced hematocarcinoma HepG2 in a conditioned medium with increasing the concentration of CA and CAPE. As seen from

Fig. 2, the strength of bands obtained from gelatin zymography was reduced as the addition of CA and CAPE when measuring with a densitometry. Specifically, the IC₅₀ value (the average 50% inhibitory concentration) of CA was 8.2/zg , and the IC₅₀ of CAPE was 2.lμg/ml, accordingly CAPE showed approximately 4 times stronger activity than C A. The resulted values were obtained by running 3 times of independent test, and represented as the average value ±SD.

Example 3; Effects of CA and CAPE on MMP-9 gene expression in hepatocarcinoma cell In order to determine the effects of CA and CAPE on the expression of MMP-9, a hepatocarcinoma HepG2 was treated with CA and CAPE at various concentrations in the presence of 200nM of PMA. In 24 hours after the treatment of the conditioned medium, it was collected to perform gelatin-zymography. HepG2 cell extracts treated with CA and CAPE at various concentrations were prepared in the presence of 200nM of PMA, and the degree of expression was determined on a protein basis by Western blot assay. In the assay, GAPDH(glyceraldehyde-3 -phosphate dehydrogenase) was used as an internal control group. The cells were crushed in the same buffer solution containing 50mM Tris-HCl(pH 8.0), 150mM NaCl, 0.02% NaN₃, 100/zg PMSF, l//g/πtf aprotinin and 1% Triton X-100. Protein quantification of the cell extracts were carried out by using a Bio-Rad protein quantification kit (Bio-Rad, USA). In order to measure the degree of NF- B activity, the cell extracts were separated according to EMSA method. 20 zg of samples from the total cell extracts and the cell nucleus extracts were subjected to SDS-PAGE electrophoresis and electrically transferred to a nitrocellulose membrane by using a Hoefer electrotransfer (Amersharm Biosciences, UK). Protein detection for MMP-9, p65 and GAPDH was performed with commercially obtained antibodies for each MMP-9 (Serotec, UK), p65 (SantaCruz Biotechnology, USA) and GAPDH (Chemicon, USA). Horseradish peroxidase-binding anti-mouse antibody was used for the antigen-antibody reaction, and ECL chemiluminescence kit (Amersham, USA) was used for the protein detection. The results were represented in Fig. 3. From the results of the Western blot assay in Fig. 3, it is recognized that the protein amount was decreased as the addition of CA and CAPE. Particularly, it was found that CA at the concentration of 100 zg suppressed most of MMP-9 generation, and CAPE at 5.0/zg eliminated most of the enzyme generation. The same results were obtained from the enzyme zymography and the immunological Western blot assay. The resulted data were obtained by running 3 times of independent test, and represented as the average value ±SD.

Example 4; Inhibition of the transcription and promoter activity of a PMA-induced MMP-9 by using CA and CAPE Promoter activity was determined by cloning 710 bps of a human MMP-9 gene in 5'-promoter region of the MMP-9 gene (Accession No. D10051) with the primers of: 5'-ACATTTGCCCGAGCTCCTGAAG-3 '(forward/Sαci) and

5'-AGGGGCTGCCAGAAGCTTATGGT - 3' (reverse/Hind III). pGL2-Basic vector was used as a vector, which has a polyadenylation signal upstream region of a luciferase gene. To the vector, a MMP-9 promoter was inserted for cloning to a pGL2-Basic vector (WT-MMP9pro) at SacIIHindlll site. The size of PCR product (MMP-9 promoter part) was determined by electrophoresis and then its base sequence was determined. Further, mutants ofAP-l-l, AP-l-2 and NF-κB (Mut-AP- 1 - 1 , Mut-AP- 1-2 and Mut-NF-κB, respectively) were constructed in WT-MMP-9 promoter by using a quick Change Site-Directed Mutagenesis Kit (Stratagene, USA). The cells were spread to a 6-well plate at the density of 10⁵ cells/well and co-transduced with \βg of a constructed body of MMP-9 promoter-luciferase reporter and \βg of β-galactosidase reporter plasmid by a LipofecAMINE method (Invitrogen, USA). The cells were cultured in 10% FBS medium and selected for 24 hours. The activity of each luciferase and β-galactosidase was determined by using a luciferase and β-galactosidase measuring kit (Promega, USA), and the luciferase activity in the cell extracts was corrected as the β-galactosidase activity and the average value was used after running 3 times of independent test. HepG2 cells were temporarily transduced to the WT-MMP-9 promoter (Fig. 4A) where 710 bps of 5 '-promoter region in the MMP-9 genes have been constructed, and divided into two groups by the presence or absence of 200nM PMA added thereto. Then, the two groups were again divided into two groups by a CA (100/tg/M) or CAPE

treated group and a non-treated group and measured the luciferase activity of each group. The measurement of the luciferase activity was performed independently to each cell extract. The resulted data were obtained by running 3 times of independent test, and represented as the average value ±SD (Fig. 4B). In order to determine the effects of CA and CAPE on the expression of PMA-induced MMP-9, the amount of MMP-9 mRNA contained in the total RNA separated from each cell was measured by RT-PCR method. In the RT-PCR, beta-actin was used as an internal control group. The expression of MMP-9 genes was detected by using RT-PCR and Northern blot assay. In RT-PCR, the total RNA was separated from

HepG2 with RNAzol B reagent (Tel-test, USA) according to the instruction contained in the kit. With 1/tg of the total RNA as a substrate, cDNA was synthesized by using an AMV RNA PCR kit (Takara, Japan), and then the cDNA was amplified with the two primers below: MMP-9(537 bps): 5'-CGGAGCACGGAGACGGGTAT-3' (sense) and

5*-TGAAGGGGAAGACGCACAGC-3'(antisense); Beta-actin(247bps): 5'-CAAGAGATGGCCACGGCTGCT -3'(sense) and 5'-TCCTTCTGCATCCTGTCGGCA-3'(antisense). The PCR products were assayed by agarose gel electrophoresis and dyed with ethidium bromide. In the results using the luciferase activity as an indicating activity as above, the concentration for decreasing the transcription activity by 50% was approximately

100/zg in CA and 4.0/zgM in CAPE, therefore CAPE showed approximately 25 times stronger activity. In addition, the result thereof was again demonstrated by the result of RT-PCR (Fig. 4C).

Example 5: Inhibiting effects of the NF-κB binding site in CA and CAPE on the activity of an MMP-9 gene promoter The structure of the MMP-9 promoter constructed for determining the luciferase activity, has one NF-κB binding site and two AP-1 binding sites per 710 bps MMP-9 promoter region. Therefore, mutation to the corresponding 2 bps was induced and introduced into the NF-κB or AP-1 binding site in WT-MMP-9. HepG2 cells were temporarily transduced by the WT or Mut-MMP-9 promoter and conditioned in the presence or absence of 200nM PMA added thereto for 24 hours, and the luciferase activity thereof was measured (Fig. 5A). HepG2 cells were temporarily transduced into each Mut-NF-κB, Mut-AP-1-1 and Mut-AP- 1-2 plasmid and treated in the presence or absence of 200nM PMA added thereto for 24 hours. Then the luciferase activity thereof was measured and represented in Fig. 5B. The resulted data were obtained by running 3 times of independent test, and represented as the average value ±SD. From the results, it has been found that CA and CAPE strongly inhibit the activity of the promoter that induces the expression of MMP-9 gene through the NF-κB binding site. Therefore, no activity was detected in the PMA-added group, due to complete inhibition on the promoter activity.

Example 6; The inhibiting effect of CA and CAPE on the activation of the PMA-induced NF-κB For the electrophoretic mobility shift assay(EMSA), cell nucleus extracts were prepared as mentioned below. Cells were cooled, washed with a physiological saline solution-phosphate buffer solution and then suspended to a buffer solution containing 0.4m^ of lOmM HEPES, lOmM KC1, O.lmM EDTA, O.lmM EGTA, ImM DTT, 0.5mM PMSF, l.Oβg/n of leupeptin and 2.0#g/m# of aprotinin dissolved therein. After maintaining the suspension still on the ice for 15 minutes, it was strongly shaken for 10 seconds with 25/z# of 10% Nonidet P-40 added thereto, and the resulted solution was centrifuged with 13,000rpm, at 4^°C for 2 minutes. The cell nucleus extract was diluted with a nucleus extract containing 50 ^ of 20mM HEPES, 0.4M NaCl (pH 7.9), ImM EDTA, ImM EGTA, ImM DTT, ImM PMSF, 2.0βgM of leupeptin and 2.0/tg/ of aprotinin. The diluted solution was allowed to stand for 15 minutes and then mildly mixed, and the nucleus extract was centrifuged with 13,000rpm, at 4^°C for 5 minutes. The supernatant fluid was immediately used and remained fraction was stored at -70 J . With a Bio-Rad protein quantification kit (Bio-Rad, USA), the protein was quantified and EMSA was performed with a gel shift measurement kit (Promega, USA). Double stranded oligoneucleotides having AP-l-l(5'-TGACCCCTGAGTCAGCACTT-3'),

AP-l-2(5'-AGGAAGCTGAGTCAAAGAAG-3') and

NF- B(5'-CCAGTGGAATTCCCCAG-3') as a conserved region were labeled with [γ-³²P] adenosine triphosphate (3000Ci/mmol; Amersham Pharmacia Biotech, UK) by T4 polynucleotide kinase and used to the EMSA. Competitive binding reaction was measured with wild-type oligomers of AP-1 -1, AP-1 -2, NF-κB which are not labeled with radioisotope or mutant type oligomers (AP-1-1; 5'-TGACCCCTGAGTTGGCACTT-3', AP-1-2; 5'-AGGAAGCTGAGTTGAAGAAG-3', NF-KB;

5'-CCAGTGGAATTGGCCAGCCT-3') and the like. The nucleus extract (2 zg) was incubated at warm temperature(with a gel-shift binding buffer solution (4% glycerol, ImM MgCl₂, 0.5mM EDTA, 0.5mM dithiothreitol, 50mM NaCl and lOmM Tris-HCl(pH 7.5)) and 0.05 zg/m^β of poly (deoxyinosine-deoxycytosine for 10 minutes for reaction, and then again incubated at room temperature with a radio-labeled probe for 20 minutes. Then, the sample was subjected to electrophoresis using a modified polyacrylamide gel, 0.5xTBE buffer solution at 250V for 20 minutes and developed. In order to assay the inhibiting effects of CA and CAPE on the activation of the PMA-induced NF-κB, HepG2 cells were treated with CA(100/zg/mfc) or

in the presence of 200nM PMA. After preparing the cell nucleus extract, the NF-κB activation degree thereof were measured with EMSA method. The cell nucleus extract used was separated from cells each incubated with [γ-³²ρ] ATP-labeled NF-κB, AP-1-1 and AP-1 -2 double stranded oligonucleotides at warm temperature. Competitive reaction was carried out with non-labeled wild type or mutant type oligonucleotide, and each sample was was subjected to electrophoresis using 4% nondenaturing polyacrylamide gel and 0.5X TBE buffer solution at 250V for 20 minutes. The resulted gel was developed by autoradiography (Fig. 6A). Nucleus extracts were separated from each cell and measured by Western blot assay, using an antibody to p65. As an internal control group, GAPDH was used. From the result, it was demonstrated that CA and CAPE strongly inhibit the specific binding of two transcription factors to the corresponding NF-κB and AP-1 binding site in the transcription control region of the promoter in MMP-9 gene, accordingly inhibiting the expression of MMP-9 gene (Fig. 6B).

Example 7: Inhibiting effects of CA and CAPE≤I on the metastasis and invasion of cancer in Matrigel of hepatocarcinoma HepG2 An assay for determine the inhibiting effects of CA and CAPE on the metastasis and invasion of a hepatocarcinoma HepG2 was carried out as follows. In the assay, a filter (8/tm, Becton Dickinson, USA) coated with Matrigel being adaptable to a 24-well invasion chamber was used. For investigating the invasion activity, cells which was obtained by culturing hepatocytes and detaching the cultured cells from a plate, were again diluted with a conditioned medium (5X10⁴ cells/200/ti). The resulted cells were divided by the presence or absence of the addition of drugs (PMA, CA and CAPE) and the drug-free and drug-containing group were poured into the upper part of an invasion chamber, respectively. The conditioned medium (500βl) was poured into the lower part of the invasion chamber. The Matrigel invasion chamber was cultured at 37°C for 24 hours with 5% CO₂, and then thereto a filter was inserted and removed from the well. Cells present on the upper part of the filter were swabbed off. The filter was fixed, erected and dyed (Becton Dickinson). Cells invaded into the Matrigel set to the lower part of the filter. 3-5 numbers of chambers were normally used for each experiment being suitable for respective condition, and the resulted values were obtained by averaging the total cell number from 3 filters. For a drug treatment, the hepatocarcinoma HepG2 was rediluted with a conditioned medium at the concentration of 5X10⁴ cells/200/^, and added to the upper part of the Matrigel invasion chamber supplemented with CA or CAPE in the presence of PMA. After an 24 hour-incubated reaction at worm temperature, the total number of cells invaded into the lower part of the filter was calculated. The total cell number from each 3 filter were averaged to obtain the result. The final result data was obtained by running 3 times of independent test and represented as the average±SD. From the result that the metastasis and invasion activity of the hepatocarcinoma HepG2 in Matrigel were inhibited, it was demonstrated that CA and CAPE have an inhibiting effect on the metastasis and invasion of cancer (Fig. 7).

Example 8: Inhibiting effects of CA and CAPE on the proliferation of cancer cell HepG2 grafted in a nude mice of a cancer cell grafted mice model In order to determine the antitumor activity of CA and CAPE in a cancer cell grafted mice model, a hepatocarcinoma HepG2 was harvested from a tissue culture treated with tripsin, washed with a serum-free medium and diluted to a concentration of lX10⁷cells/m^. 0.1 ml of the diluted cell solution containing 10⁶ numbers of cells was subcutaneously injected to the right chest of a 8-week old nude mice. To the animals (n=5 per each experimental group), a tumor cell was grafted, and together with it, the oral intake of lOOmg/kg of CA and lOOmg/kg of CAPE and the subcutaneous injection of 50 tg/kg of CA or 50/zg/kg of CAPE were made 3 times a week. In a control group, normal physiological saline solution(0.9%>) was only provided to the mouse. On 10^th day after the subcutaneous injection and oral intake of CA and CAPE, the size of tumor was measured with a caliper. The difference in the size of tumor between the control group and the test groups was analyzed by using Students t-test. The result of the subcutaneous injection was represented in Table 1, and the result of the oral intake was represented in Table 2. CA and CAPE significantly inhibited the HepG2 cancer cell in the nude mouse grafted with a cancer cell, and specifically, in the subcutaneous injection, the degree of inhibiting cancer cell proliferation of CA was 61.0% and CAPE was 56.7% which is over the half. In the oral intake, CA showed 53.6% of inhibition and CAPE showed 47.1% of inhibition.

Table 1

Table 2 Inhibition of tumor growth (%) Oral intake Volume of tumor(mιif) Weight Saline solution (0.9%, NaCl) 3306±15.6 27.7±3.5 CA 1533±24.6*(53.6) 26.7±3.3 CAPE 1750±44.3*(47.1) 27.3±2.1

Industiral Applicability The present invention provides an inhibitor which can directly inhibit the MMP-9, by using caffeic acid or caffecic acid phenethyl ester as active ingredients. Therefore, the inhibitor of the present invention can be suitably used as a therapeutic agent of various MMP-mediated diseases such as MMP-9 dependant tumors or rheumatoid arthritis, etc.

Claims

What is Claimed is:

1. An MMP-9 inhibitor which contains caffeic acid or caffeic acid phenethyl ester as an active ingredient.

2. The MMP-9 inhibitor according to claim 1, characterized by suppressing the generation of MMP-9 enzyme.

3. The MMP-9 inhibitor according to claim 1, characterized by suppressing the transcription for MMP-9 expression.

4. The MMP-9 inhibitor according to claim 1, characterized by suppressing the activity of the MMP-9 promoter.

5. The MMP-9 inhibitor according to any one of claims 1 to 4, characterized by being used in rheumatoid arthritis, osteoarthritis, osteoporosis, periodontitis, gingivitis, or diseases related with metastasis, invasion or growth of a tumor.

6. Use of caffeic acid or caffeic acid phenethyl ester as an MMP-9 inhibitor.