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National Research Laboratory for Glycobiology and Department of Biochemistry and Molecular Biology, Dongguk University College of Oriental Medicine, Kyungju, Kyungbuk, Korea;
* Department of Pathology, College of Medicine, Daegu Catholic University, Daegu, Korea;
Proteome Research, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea;
Faculty of Biotechnology, Dong-A University, Pusan, Korea;
Proteome Analysis Team, Korea Basic Science Research Institute, Daejon, Korea; and
|| Department of Microbiology, Kyungpook National University, Daegu, Korea
2Correspondence: National Research Laboratory for Glycobiology and Department of Biochemistry and Molecular Biology, Dongguk University and, Sukjang-Dong 707, Kyungju City, Kyungbuk 780-714, Korea. E-mail: chkimbio{at}dongguk.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, but not AP-1. We confirmed that CA and CAPE suppressed the growth of HepG2 tumor xenografts in nude mice in vivo. The subcutaneous and oral administrations of CA and CAPE significantly reduced the liver metastasis. These results confirm the therapeutic potential of the compounds and suggest that the anti-metastatic and anti-tumor effects of CA and CAPE are mediated through the selective suppression of MMP-9 enzyme activity and transcriptional down-regulation by the dual inhibition of NF-B as well as MMP-9 catalytic activity.—Chung, T.-W., Moon, S.-K., Chang, Y.-C., Ko, J.-H., Lee, Y.-C., Cho, G., Kim, S.-H., Kim, J.-G., Kim, C.-H. 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.
Key Words: hepatocellular carcinoma • tumor regression • Euonymus alatus • MMP-9 • NF-B • CAPE • xenograft
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The MMP family has been shown to play an important role in the proteolysis of various components of the ECM. MMP-2 and MMP-9 of the MMP family are known to degrade type IV collagen, a major constituent of the basement membrane during cancer invasion and metastasis, and are expressed in various tumor cells (2
3
4
5
6)
. It has been reported that the expression of MMP-9 is associated with the progression and invasion of cancer (7)
. These observations have led to the development of MMP inhibitors to prevent tumor metastasis. Several novel MMP inhibitors have been developed and are being investigated in clinical trials (8
, 9)
. Since some broad-spectrum MMP inhibitors (i.e., Batimastat/BB-94 and Marimastat/BB-2516) may have serious musculoskeletal side effects such as arthralgia, myalgia, stiffness, and edema (10)
, selective (narrow-spectrum) MMP inhibitors such as BAY12-9566 (11)
, KB-R7785 (12)
, N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA) (13
, 14)
, and MMI-166 (15)
have been developed to reduce the risk of such side effects.
On the other hand, stimulators, such as cytokines and PMA control the expression of MMP-9 by modulating the activation of transcription factors such as AP-1 and NF-kappaB (NF-B) through Ras/Raf/ERK, JNK, and PI-3K/AKT signaling pathways (16
17
18
19
20
21
22)
, since the promoter region of MMP-9 has AP-1 and NF-B binding sites (17)
. NF-B has been shown to regulate the expression of a number of genes, the products of which are involved in tumorigenesis (23
, 24)
. These include anti-apoptosis genes such as TRAF, bcl-2, cyclin D1, c-Myc, and cIAP (25
26
27
28)
. NF-B is a key transcription factor involved in the activation of genes that encode inflammatory cytokines such as the tumor necrosis factor-
(TNF-) and IL-1ß. NF-B can induce the activation of MMP-9 and COX-2 (23
, 24)
. Thus, several agents able to suppress NF-B activation have the potential to suppress tumorigenesis and metastasis, and show therapeutic potential.
Orally active and natural products-based anticancer drugs include green tea polyphenols, resveratrol, limonene, and organosulfur compounds (29)
. Plant polyphenols and one of its constituents (epigallocatechin gallate, EGCG) have been shown to cause strong inhibition of the gelatinolytic activities of MMP-2 and MMP-9 and of the elastinolytic activity of MMP-12 (30)
. We recently developed an MMP-9 inhibitor as an antitumor agent from methanol extracts of plant Euonymus alatus (Thunb.) Siebold, known as winged euonymus and long used in cancer treatment (31)
. The methanol extract of winged euonymus was not toxic to mammalian cells (31)
.
Caffeic acid (CA) (Fig. 1
A) is a widespread phenolic acid that occurs naturally in many agricultural products such as fruits, vegetables, wine, olive oil, and coffee (32)
. CA phenylester (CAPE) (Fig. 1B
) was extracted from honeybee propolis (33)
and has been synthesized by esterification of CA (34)
. Besides their well-known antioxidant activity (34
, 35)
, CA and CAPE inhibit certain enzyme activities such as lipoxygenases, cyclooxygenase, glutathione S-transferase, and xanthine oxidase (36
37
38
39
40)
. CA and CAPE have been reported to have antitumor activity (41
, 42)
and anti-inflammatory properties (38
, 43)
and to inhibit HIV replication (44
, 45)
. CA efficiently inhibits ceramide-induced NF-B binding activity (46)
, and CAPE is a potent and specific inhibitor of transcription factor NF-B activation (47)
. However, although CAPE has been reported to have anti-metastatic activity (48)
, inhibitory effects on MMP-9 gene expression and enzyme catalytic activity directly by CA and CAPE have not been reported to date. In this study, therefore, we for the first time demonstrate that CA and CAPE dually suppress gene expression and the enzymatic activity of MMP-9 that play an important role in metastasis and the invasion of cancer.
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Although many potent anti-cancer drugs can be clinically applicable, the above drugs are generally known to have strong cellular cytotoxicity and side effects. Thus, strong anti-cancer drugs without any cytotoxicity and side effects would be highly desirable. The specific aim of the present study was to develop powerful anti-cancer drugs that meet the above criteria, with specific tumor regression and anti-metastatic potential having dual inhibitory activities for the specific MMP-2 and -9 enzyme activities and gene transcription at the molecular level.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of the MMP-9-inhibiting compound caffeic acid from the stems of Euonymus alatus (Thunb.) Siebold and synthesis of CAPE
The plant was collected in Kyungju City, the Republic of Korea, and a sample and voucher specimen were deposited in the herbarium of the College of Oriental Medicine, Dongguk University. The plant samples were extracted three times with methanol at 70°C for 5 h. Extracts were filtered through a 0.45 µm filter and lyophilized. The w/w yield of extracts was 
2.25%. The methanol extract (45 g) was suspended in water (500 mL) and successively re-extracted with 500 mL each (3 times) of hexane (yield: 10.1 g), chloroform (yield: 11.6 g), ethyl acetate (yield: 13.4 g), and butanol (yield: 3.9 g). All fractions, including the final remaining water fraction (yield: 9.0 g), were concentrated under reduced pressure using a rotary evaporator, then freeze dried. Strong MMP-9-inhibitory activity was observed in the butanol fraction and was subjected to recycling HPLC to isolate the target compounds (31)
. The final yield of caffeic acid was 0.5 g. Caffeic acid phenyl ester was synthesized by the esterification of caffeic acid chloride with phenethyl alcohol in the presence of p-toluenesulfonic acid as detailed previously (33)
.
XTT proliferation assay
Cell proliferation was investigated using a commercially available proliferation kit II (XTT, Boehringer Mannheim, Mannheim, Germany). Briefly, HepG2 cells were subcultured into 96-well culture plates at a density of 103 cells/ well in 100 µL of DMEM culture medium. After 24 h of incubation, the medium, in a 96-well plate, was discarded and replaced with 100 µL of new medium containing various concentrations of CA or its derivative CAPE. The plates were incubated in a 37°C humidified incubator in an atmosphere of 5% CO2 for 24 h. At the end of the incubation, the medium was discarded and cells were washed with PBS. Fifty µL of XTT test solution prepared by mixing 5 mL of XTT labeling reagent and 100 µL of electron coupling reagent was then added to each well. After 4 h of incubation in a 37°C and 5% CO2 incubator, absorbance was measured on an ELISA reader (Molecular Devices, Palo Alto, CA, USA) at a test wavelength of 490 nm.
Enzyme assay for artificial substrate
For the MMP-1 (collagenase-1) assay, a commercial assay kit with a natural substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, was used (Yagai Co., Yamagata, Japan). MMP-3 (stromelysin-1) and MMP-7 (Matrilysin) were obtained from the Yagai Co. and enzyme activities were assayed in a reaction buffer [300 mM NaCl, 10 mM CaCl2, 0.005% Brij35, 0.01% NaN3, and 50 mM Tris-HCl (pH 7.5)] using 20 µM MOAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as the substrate. After a 90 min incubation at 25°C, the fluorescence was measured with excitation and emission at 320 and 405 nm, respectively, with a Fluoroscan Ascent (Labosystems, Franklin, MA, USA). For enzyme inhibition assays, the enzymes were preincubated with inhibitor for 60 min. MMP-2 and MMP-9 were purified from the culture supernatant of HepG2 cells with enzyme activities assayed in the linear range using the substrate peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 (Peptide International, Louisville, KY, USA). Recombinant cathepsin K (Cat K) as a cysteine protease was expressed in Escherichia coli, and a 32 kDa protein was purified by DEAE-Sepharose column chromatography, as described previously (49)
. Cat K activity was assayed using the substrate peptide, Z-Leu-Arg-4-MßNA obtained from Bachem Co. (King of Prussia, PA, USA). The optimal MMP concentration for each proximity-based substrate peptide assay was determined empirically under reducing conditions.
Zymography
To investigate the inhibitory effect of CA or its derivative CAPE on the gelatinolytic activity of MMP-9, conditioned medium obtained from PMA-induced HepG2 cells was resuspended in a sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.00625% (w/v) bromophenol blue and loaded without boiling in 10% polyacrylamide gel containing 0.1% (w/v) gelatin. After electrophoresis, gels were soaked in 2.5% Triton X-100 (2x30 min) at room temperature and rinsed in NanoPure water. The gel was cut into slices corresponding to the lanes, then put in different tanks containing incubation buffer [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2.5 mM CaCl2] with various concentrations of CA or CAPE. The gel treated with CA or CAPE was incubated at 37°C for 24 h. Bands corresponding to activity were visualized by negative staining using Coomassie Brilliant blue R-250. To determine the inhibitory effects of CA or CAPE on PMA-induced MMP-9 expression, HepG2 cells were treated with CA or CAPE in the presence of 200 nM PMA and MMP-9 expression was evaluated by zymography. HepG2 cells were grown in 10% FBS/DMEM and rinsed with PBS, then incubated in serum-free DMEM with or without drugs (CA or CAPE) in the presence of PMA for 24 h and the conditioned medium was collected. The conditioned medium was resolved in 10% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gels were washed for 1 h in 2.5% (v/v) Triton X-100 to remove SDS, then incubated for 24 h at 37°C in the incubation buffer to allow proteolysis of the gelatin substrate. Bands corresponding to activity were visualized by negative staining using Coomassie Brilliant blue R-250 (Bio-Rad, Richmond, CA, USA) and molecular weights were estimated by reference to prestained SDS-PAGE markers.
Reverse transcription-polymerase chain reaction (RT-PCR)
To detect the expression of MMP-9 using RT-PCR and Northern blot analysis, total RNA was prepared from HepG2 cells using the RNAzol B reagent (Tel-test, Friendswood, TX, USA) according to the manufacturer’s instructions. For RT-PCR, a cDNA was synthesized from 1 µg of total RNA using a AMV RNA PCR Kit (Takara, Japan) according to the manufacturer’s protocol. The cDNA was amplified by PCR with the following primers: MMP-9 (537 bp), 5'-CGGAGCACGGAGACGGGTAT-3' (sense) and 5'-TGAAGGGGAAGACGCACAGC-3' (antisense); ß-actin (247 bp), 5'-CAAGAGATGGCCACGGCTGCT-3' (sense) and 5'-TCCTTCTGCATCCTGTCGGCA-3' (antisense). PCR products were analyzed by agarose gel electrophoresis and visualized by treatment with ethidium bromide.
Western blot analysis
HepG2 cells were treated with various concentrations of CA or its derivative CAPE in the presence of 200 nM PMA. To determine MMP-9 expression using Western blot analysis, cells were homogenized in a sample buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% NaN3, 100 µg/mL PMSF, 1 µg/mL aprotinin, and 1% Triton X-100. Protein concentrations were measured using the Bio-Rad protein assay. To determine the activation of NF-B, nuclear extracts of cells were isolated by the protocol of EMSA. Twenty µg samples of total cell lysates and nuclear extracts were size fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes using the Hoefer electrotransfer system (Amersharm Biosciences, Amersham, UK). To detect MMP-9, p65, and GAPDH protein, membranes were incubated with the MMP-9 (Serotec, Kidlington, Oxford, UK), p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH antibodies (Chemicon, El Segundo, CA, USA). Detection was performed using a secondary horseradish peroxidase-linked anti-mouse antibody and the ECL chemiluminescence system (Amersham, Arlington Heights, IL, USA).
Electrophoretic mobility shift assay (EMSA)
The nuclear extract of each cell was prepared as described below. Cells were washed with cold PBS and suspended in 0.4 mL of lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2.0 µg/mL leupeptin, and 2.0 µg/mL aprotinin. The cells were allowed to swell on ice for 15 min, then 25 µL of 10% Nonidet P-40 was added. The tube was vigorously vortexed for 10 s, and the homogenate centrifuged at 4°C for 2 min at 13,000 rpm. The nuclear pellet was resuspended in 50 µL of ice-cold nuclear extraction buffer containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2.0 µg/mL leupeptin, and 2.0 µg/mL aprotinin. The tube was incubated on ice for 15 min with intermittent mixing. The nuclear extract was then centrifuged at 4°C for 5 min at 13,000 rpm and the supernatant was either used immediately or stored at –70°C for later use. The protein content was measured using the Bio-Rad protein assay. EMSA were performed using a gel shift assay system kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, double-stranded oligonucleotides containing the consensus sequences for AP-1-1 (5'-TGACCCCTGAGTCAGCACTT-3'), AP-1-2 (5'-AGGAAGCTGAGTCAAAGAAG-3') and NF-B (5' CCAGTGGAATTCCCCAG-3') were end-labeled with [
-32P] ATP (3000 Ci/mmol; Amersham Pharmacia Biotech) using T4 polynucleotide kinase and used as probes for EMSA. Competition was performed using either the unlabeled wild-type AP-1-1, AP-1-2, NF-B, or mutant oligomers (AP-1-1; 5'-TGACCCCTGAGTTGGCACTT-3', AP-1-2; 5'-AGGAAGCTGAGT TGAAGAAG-3', NF-B; 5'-CCAGTGGAATTGGCCAGCCT-3'). Nuclear extract proteins (2 µg) were preincubated with the gel shift binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.05 mg/mL poly (deoxyinosine-deoxycytosine) for 10 min, then incubated with the labeled probe for 20 min at room temperature. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5 x TBE buffer at 250 V for 20 min. The gel was dried and subjected to autoradiography.
Promoter assay
A 710 bp fragment from the 5'-promoter region of the MMP-9 gene was cloned. A 710 bp fragment at the 5'-flanking region of the human MMP-9 gene was amplified by PCR using specific primers from the human MMP-9 gene (accession no. D10051): 5'-ACATTTGCCCGAGCTCCTGAAG (forward/SacI) and 5'-AGGGGCTGCCAGAAGCTTATGGT (reverse/Hind III). The pGL2-Basic vector containing a polyadenylation signal upstream from the luciferase gene was used to construct the expression vectors by subcloning PCR-amplified DNA of the MMP-9 promoter into the SacI/HindIII site of the pGL2-Basic vector (WT-MMP9pro). PCR products (fragment of MMP-9 promoter) were confirmed by their size, as determined by electrophoresis, and by DNA sequencing. The AP-1-1, AP-1-2, and NF-B mutants (Mut-AP-1-1, Mut-AP-1-2, and Mut-NF-B) from WT-MMP9pro were generated using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA, USA). Cells were plated onto 6-well plates at a density of 105 cells/well and grown overnight. Cells were cotransfected with 1 µg of MMP-9 promoter-luciferase reporter constructs and 1 µg of ß-galactosidase reporter plasmid by the LipofecAMINE method (Invitrogen, San Diego, CA, USA). Cells were cultured in 10% FBS medium and incubated with drugs for 24 h. Luciferase activity and ß-galactosidase activity were assayed by using the luciferase and ß-galactosidase enzyme assay system (Promega). Luciferase activity was normalized with the ß-galactosidase activity in the cell lysate and calculated as an average of three independent experiments.
Invasion assays
Matrigel-coated filter inserts (8 µm pore size) that fit into 24-well invasion chambers were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). Liver cells to be tested for invasion were detached from the tissue culture plates, washed, resuspended in conditioned medium (5x104 cells/200 µL), then added to the upper compartment of the invasion chamber in the presence or absence of drugs (PMA, CA, CAPE, EGCG, and TIMP-1). Conditioned medium (500 µL) was added to the lower compartment of the invasion chamber. The Matrigel invasion chambers were incubated at 37°C for 24 h in 5% CO2. After incubation, filter inserts were removed from the wells and the cells on the upper side of the filter were removed using cotton swabs. The filters were fixed, mounted, and stained according to the manufacturer’s instructions (Becton Dickinson). The cells that invaded through the Matrigel and were located on the underside of the filter were counted. Three to five invasion chambers were used per condition. The values obtained were calculated by averaging the total number of cells from three filters.
Mouse model studies for tumor growth and liver metastasis
The antitumoral efficacy of CA and CAPE was tested in vivo. HepG2 cells were harvested from tissue culture flasks with trypsin treatment. The cells were then washed with serum-free medium and suspended at a concentration of 1 x 107/mL in serum-free medium. A 0.1 mL suspension containing 106 cells was injected subcutaneously into the right flank of nude mice (8 wk old).
To evaluate the effect of CA and CAPE on tumor growth and liver metastasis, treatment of the drugs was carried out by oral and subcutaneous administration. For subcutaneous administration of the drugs, experimental animals (n=7 for each group) were treated with CA or CAPE (5.0 mg/kg) three times/wk beginning on the day of tumor cell implantation. Control mice were treated with normal saline (0.9%). Ten days after injection of CA or CAPE, tumor volume was determined by measuring tumor size with calipers.
For oral administration of the drugs, 25 mice were randomly divided into two groups. Seven mice were given CA or CAPE at a daily dose of 20.0 mg/kg on 6 days/wk for 5 wk starting on the fifth day after orthotopic implantation (the CA or CAPE group). The same volume of vehicle was given to another 11 mice (the control group). All of the mice were killed 6 wk after tumor implantation.
Statistics
Statistical significance of difference in tumor volume and metastasis between control and treated mice was assessed by Student’s t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). CA inhibited the activities of MMP-2 and MMP-9, with an IC50 of 10–20 µM, but did not inhibit MMP-1, MMP-3, MMP-7, and Cat K (IC50S values were 234, 335, 433, and >500 µM, respectively). The CA derivative CAPE inhibited the activities of MMP-2 and MMP-9 more strongly than CA, with an IC50 of 2–5 µM, but weakly inhibited MMP-1, MMP-3, and MMP-7 (IC50S values were 187, 224, and 335 µM, respectively). In contrast, CAPE did not inhibit Cat K (serine protease) activity (IC50>500 µM). Neither CA nor CAPE inhibited typical serine proteases (neutrophil elastase, plasmin, trypsin, and chymotrypsin), cysteine proteases (cathepsins B and L), aspartic protease (HIV-1 protease), or metalloproteinase (aminopeptidase M) (data not shown).
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Effect of CA or its derivate CAPE on cell viability
To evaluate the viability of HepG2 cells after exposure to CA or CAPE, cell viability was measured using an XTT assay. As shown in Fig. 2
, the resulting survival curve shows that CA and CAPE had a dose-dependent effect on the proliferation of cells. The addition of 200 µg/mL of CA reduced the viability to 61% of the controls, and treatment with CAPE (20 µg/mL) in HepG2 cells reduced the viability to 72% of the controls.
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Inhibitory effect of CA or CAPE on MMP-9 enzyme activity
We examined the effect of CA or CAPE on MMP-9 activity, which is related to the invasion and metastasis of hepatocellular carcinoma (HCC) as evidenced by gelatin zymography. As shown in Fig. 3
, CA and CAPE dramatically inhibited the proteolytic activity of MMP-9 in a dose-dependent manner. A tissue inhibitor of metalloproteinase-1 (TIMP-1), an MMP-9-specific inhibitor, suppressed the enzymatic activity of MMP-9; (–)-epigallocatechin-3-gallate (EGCG), an MMP-9 inhibitor isolated from green tea, inhibited MMP-9 activity (Fig. 3C
). These results show that CA and CAPE inhibit the enzymatic activity of the MMP-9 protein secreted from HepG2 cells via induction by PMA. We further investigated whether CA and CAPE inhibit PMA-induced MMP-9 expression in HepG2 cells. Expression and secretion of MMP-9 was induced by PMA in HepG2 cells, as evidenced by gelatin zymography. On the other hand, when HepG2 cells were treated with CA or CAPE in the presence of PMA, CA and CAPE decreased the secretion of MMP-9 measured by zymography. CA and CAPE blocked PMA-induced MMP-9 expression in a dose-dependent manner as evidenced by Western blot analysis (Fig. 4
A, B). Moreover, as shown in Fig. 4C
, we observed inhibitory effects of CA and CAPE on PMA-induced MMP-9 expression in human breast cancer cells (MCF-7), human acute promyelocytic leukemia cells (HL-60), and human monocytic leukemia cells (U937). These results indicate that CA and CAPE decrease MMP-9 expression as well as MMP-9 enzyme activity in PMA-induced HepG2 cells and other cancer cells.
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Suppression of MMP-9 transcription and promoter activity by CA and CAPE
Our finding that CA and CAPE inhibited PMA-stimulated MMP-9 expression in HepG2 cells as measured by Western blot analysis prompted us to investigate the effect of CA or CAPE on MMP-9 promoter activity and levels of MMP-9 mRNA. As shown in Fig. 5
B, luciferase activity was increased up to 13-fold in HepG2 cells that had been treated with PMA compared with untreated cells. On the other hand, luciferase activity of cells treated with CA (100 µg/mL) and CAPE (5 µg/mL) in the presence of PMA was reduced by 2- or 2.5-fold, respectively, compared with that of PMA-stimulated HepG2 cells. To confirm the effect of CA or CAPE on the levels of MMP-9 mRNA in HepG2 cells stimulated by PMA, treatment of HepG2 cells with CA or CAPE in the presence of PMA induced a decrease in the levels of MMP-9 mRNA compared with cells not treated with CA or CAPE in the presence of PMA, as evidenced by RT-PCR (Fig. 5C
). These results show that CA and CAPE inhibit the transcriptional activity of MMP-9 in PMA-induced HepG2 cells.
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Inhibition of transcriptional activity of MMP-9 gene through suppression of PMA-stimulated NF-B activity by CA and CAPE
The AP-1 and the NF-B elements of MMP-9 promoter are centrally involved in the induction of the MMP-9 gene associated with the invasiveness of tumor cells by PMA and cytokines. CA modulates ceramide-induced NF-B activation (46)
. Natarajan et al. have reported that the effect of CAPE on the inhibition of NF-B binding to DNA was specific inasmuch as binding of other transcription factors (including AP-1, Oct-1, and TFIID) to their DNA was not affected (47)
. In previous data, CA and CAPE were found to inhibit the transcriptional activity of MMP-9 in PMA-induced HepG2 cells. Thus, to investigate whether CA and CAPE modulate MMP-9 expression through the inhibition of PMA-induced NF-B activity, we examined the promoter activity of the MMP-9 gene. As shown in Fig. 6
A, luciferase activity remained essentially unchanged when cells were transfected with the promoterless and enhancerless control vector (pGL2-basic) in the presence or absence of PMA and respectively transfected with WT-MMP9pro, Mut-AP-1-1, Mut-AP-1-2, and Mut-NF-B in the absence of PMA. The increase in luciferase activity of HepG2 cells transfected with WT-MMP9pro was observed in the presence of PMA vs. cells transfected with pGL2-basic in the presence of PMA. Promoter activities of Mut-AP-1-1, Mut-AP-1-2, and Mut-NF-B in PMA-induced HepG2 cells were decreased by up to 2-fold, respectively, compared with the promoter activity of WT-MMP9pro. As shown in Fig. 6B
, luciferase activity in the cells transfected with Mut-NF-B was slightly reduced by treatment with CA (100 µg/mL) and CAPE (5 µg/mL) in the presence of PMA whereas luciferase activity in cells transfected with Mut-AP-1-1 and Mut-AP-1-2, respectively, was significantly reduced by treatment with CA (100 µg/mL) and CAPE (5 µg/mL) in the presence of PMA.
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To further confirm that CA and CAPE are directly involved in the inhibition of NF-B-mediated transcriptional activation of MMP-9, we examined the inhibitory effect of CA and CAPE on the binding of AP-1 and NF-B isolated from PMA-stimulated HepG2 cells to wild-type oligonucleotides that contain the sequence for the AP-1 and NF-B binding sites from the MMP-9 promoter using EMSA. As shown in Fig. 7
A, the findings confirm that nuclear lysates isolated from HepG2 cells treated with PMA induced an electromobility shift compared with untreated cells when [-32P]-labeled AP-1-1, AP-1-2, and NF-B wild-type oligonucleotides were introduced. The formation of an electrophoretically retarded complex by PMA was inhibited when unlabeled AP-1-1, AP-1-2, and NF-B wild-type oligonucleotides were introduced. The formation of an electrophoretically retarded complex by PMA was maintained when unlabeled AP-1-1, AP-1-2, and NF-B mutant-type oligonucleotides were introduced. CA (100 µg/mL) and CAPE (5 µg/mL) blocked NF-B activation by treatment with PMA, but CA and CAPE had no effect on PMA-stimulated AP-1 activation, as evidenced by EMSA.
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Stimulation of cells with various agents including PMA and cytokines lead to NF-B activation, which results in phosphorylation of p65 and the translocation of NF-B into the nucleus. Thus, we investigated the effects of CA and CAPE on the PMA-stimulated nuclear translocation of the active NF-B complex composed of p65 and p50 subunits. As shown in Fig. 7B
, treatment with CA (100 µg/mL) and CAPE (5 µg/mL) of PMA-induced HepG2 cells inhibited the nuclear translocation of NF-B complex as measured by Western blot analysis. These results clearly show that CA and CAPE regulate the transcriptional activation of MMP-9 through the inhibition of PMA-stimulated NF-B activity.
Inhibitory effects of CA and CAPE on the invasion of PMA-induced liver cancer cells in vitro
We previously showed that CA and CAPE suppress NF-B-mediated MMP-9 expression in PMA-induced HepG2 cells. Because the up-regulation of MMP-9 expression might be expected to contribute to an invasive phenotype, we examined whether the invasiveness of PMA-stimulated liver cancer cells was decreased by CA (100 µg/mL) and CAPE (5 µg/mL). As shown in Fig. 8
, the invasiveness of PMA-induced HepG2 cells was increased when compared with the invasiveness of PMA-untreated HepG2 cells, as evidenced by a Matrigel invasion assay whereas CA and CAPE blocked the invasiveness of PMA-induced HepG2 cells. MMP-9 inhibitors TIMP-1 and EGCG suppressed the invasiveness of HepG2 cells stimulated by PMA. From these results, we conclude that with respect to liver cells, CA and CAPE induce a decrease in the potential for invasion.
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Inhibitory effect of CA and CAPE on tumor growth in mice
To investigate the effect of CA and CAPE on tumor growth in vivo, HepG2 cells were injected subcutaneously into the right flank of nude mice (8 wk old). Experimental animals (n=7 in each group) were treated with CA or CAPE (5.0 mg/kg) three times/wk beginning on the day of tumor cell implantation. As shown in Table 2
, tumor size showed a significant reduction in the CA-treated group (61% inhibition) compared with the normal saline-treated group. An 56.7% inhibition of tumor growth in the CAPE-treated group was observed compared with the normal saline-treated group. However, body weight in the CA- and CAPE-treated group was not reduced significantly compared with the normal saline-treated group. In the case of oral administration, the actual tumor weights at the end of the experiment were significantly reduced with 53.6% and 47.1% inhibition of tumor growths in the CA-treated and CAPE-treated groups, respectively.
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Inhibitory effect of CA and CAPE on liver metastasis
A suppressive effect of the subcutaneous administration of CA and CAPE on liver metastasis was significantly demonstrated when compared with the control group (Table 3
; P=0.0024 for CA and P=0.0035 for CAPE). Liver metastasis was observed in 10 mice (10/11, 90.1%) from the control group, whereas only 1 mouse (1/7, 14.3%) showed liver metastasis in the CA group and 2 (2/7, 28.3%) in the CAPE group. Subcutaneous administrations of CA and CAPE clearly decreased the number of metastatic foci in the liver (Table 3)
. In the case of oral administration, similar results were observed, with a significant reduction in liver metastasis in the CA-treated and CAPE-treated groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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51
52)
. MMPs play a major role in promoting angiogenesis and tumor metastasis (53)
. An enhanced expression of MMP-9 has been shown to be associated with the progression and invasion of tumors (5
6
7)
. Overexpression of MMP-9 associated with the capsular infiltration of HCC and growth of small HCC has been reported (54
, 55)
.
Some synthetic MMP inhibitors now undergoing clinical trials for cancer treatment carry undesirable side effects (56)
. To develop powerful anti-cancer drugs with the dual inhibitory activities of specific MMP-2 and -9 enzyme activities and gene transcription, we for the first time describe the isolation of a strong and selective MMP-2/9 inhibitory molecule from the winged euonymus. We determined its structure by conventional positive ion FAB mass spectrometry together with CID (data not shown). The isolated molecule was identified as CA. To examine the MMP-9 inhibitory activities of CA, its phenyl esteric derivative CAPE was synthesized (data not shown).
Because tumor-derived and host stroma-derived MMPs may play pivotal roles in tumor growth (57)
, the effect of MMP inhibitory activity is important for a therapeutic experimental model. We have confirmed the cross-inhibitory effects of CA and CAPE on MMP-2 and MMP-9 by the use of an artificial substrate and gelatin zymography. Since both compounds showed similar pharmacokinetic profiles, the difference found in the biological activities of these compounds is mainly due to the extent of their inhibition of MMP-2 and MMP-9. However, one difference between CA and CAPE in biological significance is that CAPE has higher inhibitory activities against MMP-2 and MMP-9. Structurally, CAPE is composed of two phenolic rings linked by an esteric bond whereas CA is a phenolic compound only. Therefore, the relationship between the structural and biological differences of CA and CAPE needs to be further elucidated. CA and CAPE selectively inhibit MMP-2 and MMP-9 activities, differentiating these compounds from other broad-spectrum MMP inhibitors such as BB-94 and BB-22516 (57
, 58)
. These findings have important implications for the therapeutic potential of CA and CAPE, which may reduce the incidence of adverse events such as pain and tenderness in the joints and pain affecting the shoulders and hands that appear in long-term treatment with broad-spectrum MMP inhibitors in clinical studies (58)
.
It has been shown that different agents including growth factors such as epidermal growth factor, platelet-derived growth factor, transforming growth factor-ß, and inflammatory cytokines such as TNF and IL-1 modulate the expression of the MMP-9 gene (59
, 60)
. PMA, a well-known selective activator of protein kinase C, has been shown to enhance the production of MMP-9 through the activation of transcription factors such as NF-B and AP-1 (61)
. The promoter region of the MMP-9 gene has binding sites for NF-B and AP-1. The MMP-9 promoter is unique in its requirement for the NF-B element for the induction by inflammatory cytokines that activates transcription factor NF-B via a multiprotein complex (62)
. The AP-1 DNA binding element plays an important role in regulating MMP expression by growth factors, cytokines, and oncogenes (63)
. To determine the inhibitory effect of CA and CAPE on MMP-9 gene transcription through suppression of transcription factor activity, we carried out an MMP-9 promoter luciferase assay and EMSA. Data show that luciferase activity is significantly increased in HepG2 cells that have been transiently transfected with the wild-type MMP-9 promoter by treatment with PMA, as evidenced by a luciferase promoter assay, whereas PMA-induced luciferase activity is significantly reduced in NF-B and AP-1 mutant MMP-9 promoters. CA and CAPE were shown to inhibit wild-type MMP-9 promoter activity stimulated by PMA in HepG2 cells. The activity of Mut-NF-B in HepG2 cells transfected with NF-B-mutated MMP-9 promoter, was not affected by treatment with CA and CAPE in the presence of PMA. CA and CAPE induced a drastic decrease in Mut-AP-1-1 and Mut-AP-1-2 activity in PMA-stimulated HepG2 cells. As measured by EMSA, MMP-9 promoter-derived oligonucleotides can bind to AP-1 and NF-B proteins that are isolated from HepG2 cells induced by PMA. However, CA and CAPE in PMA-stimulated HepG2 cells block NF-B activation by suppressing the interaction of NF-B proteins with oligonucleotides that contain the sequence for the NF-B binding site from the MMP-9 promoter. On the other hand, inhibition of binding AP-1 protein to oligonucleotides was not affected by CA and CAPE in HepG2 cells induced by PMA. Thus, CA and CAPE in PMA-stimulated HepG2 cells suppress NF-B-mediated MMP-9 gene transcriptional activity.
NF-B, a transcription factor, plays an important role in the expression of various target genes that control apoptosis, cell proliferation, differentiation, and immune and inflammatory responses. In resting cells, heterodimeric NF-B complexes are located in the cytoplasm of most cell types by inhibitory proteins of the IB family (64)
. These inhibitors block NF-B nuclear localization and inhibit its DNA binding activity. The IB inhibitor is rapidly phosphorylated and degraded in cells after stimulation by various reagents. This permits NF-B nuclear translocation, DNA binding to specific recognition sequences in promoters, and transcription of the target genes (65)
. In a previous study, we reported that CA and CAPE inhibit the binding of NF-B to oligoncleotides including the NF-B binding site in the MMP-9 promoter. PMA has been shown to induce the phosphorylation of NF-B p65 required for the translocation of p65 to the nucleus. Whereas CA and CAPE treatment inhibited p65 translocation induced by PMA, CA and CAPE had no effect on the suppression of PMA-activated PKC/ERK phosphorylation in HepG2 cells. These results clearly suggest that CA and CAPE suppress the function of NF-B through blocking the nuclear translocation of NF-B.
Several studies have reported that an increased expression of MMP-9 by stimulators induces the invasiveness of some cell lines (20
, 21)
. The present data show that PMA-induced MMP-9 expression leads to invasiveness for HCC cells. However, CA and CAPE inhibit the PMA-induced invasiveness potential as evidenced by a Matrigel invasion assay. We confirmed that the TIMP-1 protein, a known MMP-9-specific inhibitor, and EGCG, a known MMP-9 inhibitor, block the Matrigel invasion of PMA-induced HepG2 cells as well as PMA-stimulated MMP-9 activity. Tumors and stimulators have been shown to induce MMP-9 expression in multiple biological functions such as migration, adhesion, angiogenesis, and tumorigenicity. To investigate the effect of CA and CAPE on tumor growth in vivo, HepG2 cells were injected subcutaneously into the right flank of nude mice. Experimental animals were treated with CA or CAPE (5.0 mg/kg) by subcutaneous or oral administration (20.0 mg/kg each) three times/wk beginning on the day of tumor cell implantation. We confirmed that CA and CAPE suppressed the growth of HepG2 tumor xenografts in nude mice in vivo. Moreover, CA and CAPE reduced tumor invasion at a metastatic site in liver, so that it may inhibit secondary metastasis subsequent to liver metastasis, including intrahepatic metastasis.
In conclusion, as illustrated in Fig. 9
, we show that CA and its derivative CAPE 1) inhibit the enzymatic activity of MMP-9 that plays an important role in cancer invasion and metastasis, 2) block invasiveness potential through the suppression of MMP-9 gene transcription by inhibiting NF-B function in PMA-stimulated HepG2 cells, and 3) suppress the growth of HepG2 cell xenografts in nude mice. We propose that the anti-metastatic and anti-tumorigenic effects of CA and its derivate CAPE may be mediated through suppression of MMP-9 gene expression by the activation of NF-B and MMP-9 catalytic activity. Therefore, these two drugs are strong candidates for treatment of cancer and metastasis via dual mechanisms (dual inhibition of metastasis-specific enzyme activity and gene transcription). Based on the findings herein, the current state of the art for cancer treatment will progress with the novel mechanism for drug action on cancer treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication May 9, 2004. Accepted for publication July 14, 2004.
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