2005;4:281-290. Published online February 15, 2005.
Mol Cancer Ther
Ching-Huai Ko, Shing-Chuan Shen, Tony J.F. Lee, et al.
cells
expression and enzyme activity in colorectal carcinoma
Myricetin inhibits matrix metalloproteinase 2 protein
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Myricetin inhibits matrix metalloproteinase 2 protein
expression and enzyme activity in colorectal
carcinoma cells
Ching-Huai Ko,1 Shing-Chuan Shen,3,4 Tony J.F. Lee,5,6and Yen-Chou Chen2
Graduate Institutes of1Pharmaceutical Sciences and 2
Pharmacognosy, School of Pharmacy and3Department of Dermatology, School of Medicine, Taipei Medical University, Taipei, Taiwan;4Department of Dermatology, Taipei Municipal Wan-Fang Hospital, Taipei, Taiwan;5Neuro-Medical Scientific Center, Tzu Chi Hospital, College of Life Sciences,
Tzu Chi University, Hualien, Taiwan; and6Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois
Abstract
Colorectal carcinoma is a leading cause of human mortality due to its high metastatic ability. Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, agents with the ability to inhibit MMP activity have potential in the treatment of colorectal carcinoma. In the present study, among 36 flavonoids examined, myricetin was found to be the most potent inhibitor of MMP-2 enzyme activity in COLO 205 cells (IC50=7.82 Mmol/L). Myricetin inhibition of MMP-2
en-zyme activity was also found in the human colorectal carcinoma cell lines COLO 320HSR, COLO 320DM, HT 29, and COLO 205-X (IC50=11.18, 11.56, 13.25, and 23.51
Mmol/L, respectively). In contrast, no inhibitory effect of MMP-2 protein expression or enzyme activity was ob-served in myricitrin (myricetin-3-rhamnoside)-treated cells. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated COLO 205 cells, an increase in MMP-2 protein expression and enzyme activity, as well as of protein kinase C (PKC)A protein translocation, extracellular signal-regulated kinase (ERK) 1/2 protein phosphorylation, and c-Jun protein expression was observed. ERK inhibitor (PD98059) and PKC inhibitors (GF-109203X and H-7), but not p38 inhibitor (SB203580) or c-jun-NH2-kinase inhibitor (SP600125),
significantly inhibited TPA-induced MMP-2 protein
expres-sion, with reduced ERK phosphorylation and c-Jun protein expression. Addition of myricetin but not myricitrin sup-pressed TPA-induced MMP-2 protein expression in COLO 205 cells by blocking the TPA-induced events, including translocation of PKCA from cytosol to membrane, phos-phorylation of ERK1/2 protein, and induction of c-Jun protein expression. Addition of PD98059 or GF-109203X significantly enhanced the inhibitory effect of myricetin on MMP-2 enzyme activity induced by TPA. Furthermore, myricetin, but not myricitrin, suppressed TPA-induced invasion of COLO 205 cells in an in vitro invasion assay using Engelbreth-Holm-Swarm sarcoma tumor extract Matrigel – coated Transwells. Results of the present study indicate that myricetin significantly blocked both endoge-nous and TPA-induced MMP-2 enzyme activity by inhibit-ing its protein expression and enzyme activity. The blockade involved suppression of PKC translocation, ERK phosphorylation, and c-Jun protein expression. [Mol Cancer Ther 2005;4(2):281 – 90]
Introduction
The extracellular matrix is a complex structure supporting cells in mammalian tissues, and there are several biomole-cules in the extracellular matrix including collage and casein involved in maintaining the three-dimensional structure of the body (1). During carcinogenesis, degradation of the extracellular matrix occurs in the process of metastasis of malignant tumors and is involved in the migration and invasion of malignant tumor cells (2, 3). Matrix metal-loproteinases (MMP) are proteins for extracellular matrix breakdown, and activation of these protein activities has been detected in malignant tumors including colon carcinoma, lung cancer, and hepatoma (4, 5). Both MMP-2 and MMP-9 have been shown to participate in the tumor metastatic process, and the more malignant the tumors the more potent MMP-2 and MMP-9 activity is in stimulating metastasis of tumors (6, 7). Therefore, an agent capable of inhibiting the activity of MMPs, especially MMP-2 and MMP-9, may potentially be an antitumor metastatic drug.
12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, induces a variety of cellular responses including differentiation, proliferation, and apoptosis. Pro-tein kinase C (PKC) activation by TPA via translocation of PKC proteins from cytoplasm to membrane has been shown to be an essential step in TPA-induced tumor promotion (8). At least five isoforms of PKC including a, h, g, y, and q have been isolated from different human tissues, and activation of PKCa is involved in the formation of colorectal carcinoma. After PKC activation, a series of downstream genes such as mitogen-activated protein kinases (MAPK),
Received 8/13/04; revised 11/9/04; accepted 12/8/04.
Grant support: National Science Council grants NSC93-2321-B-038-009, NSC92-2320-B-038-021, NSC92-2321-B-038-007, NSC-92-2320-B-320-025, and NCS93-2745-B-320-004-URD.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Yen-Chou Chen, Graduate Institute of
Pharmacognosy, School of Pharmacy, 250 Wu-Hsing Street, Taipei, Taiwan. Phone: 886-2-27361661 ext. 6152; Fax: 886-2-23787139.
E-mail: [email protected]
c-Jun, and c-Fos is stimulated. Our previous study indicated that induction of extracellular signal – regulated kinase (ERK) 1/2 phosphorylation, ornithine decarboxy-lasse, c-Jun, and cyclooxygenase 2 proteins is involved in TPA-induced transformation in NIH3T3 cells. Zeliadt et al. (9) indicated that TPA induced MMP-13 gene expression via an ERK-dependent pathway in mouse keratinocytes. In human cervical cancers, TPA induced MMP-9 activity via activation of PKCy and c-jun-NH2-kinase (JNK). Arnott
et al. (10) suggested that TPA was able to up-regulate MMP-9 expression that was closely correlated with tumor development. However, the exact mechanism of TPA regulation of MMPs in colorectal carcinoma is still unclear. Colorectal cancer is the third leading cause of cancer mortality in the Western world and increases strikingly. Early diagnosis and surgery to remove primary tumor have increased the survival rate of patients with colorectal cancer. Several studies indicated that metastasis is a major factor that causes death in patients with colorectal cancer (11). Therefore, investigators keep continuing to search for effective agents to inhibit the metastasis of colorectal cancer.
Flavonoids are benzo-g-pyrone derivatives with different numbers of hydroxyl substitutions in the structures. Beneficial effects of flavonoids include antioxidant, antitumor, and anti-inflammation effects (12, 13). Results of structure-activity relationship studies have indicated that OH substitutions may affect the biological functions of flavonoids, and increasing OH substitution seems to enhance the antioxidant activity of flavonoids. Our recent studies also showed that flavonoids such as quercetin and wogonin induced apoptosis of tumor cells, and addition of glycoside attenuated the apoptosis-inducing activity of flavonoids (14, 15). In addition, flavones with one or no OH substitution significantly inhibit epidermal growth factor – induced proliferation in A431 (16). These data suggest that chemical structure, especially OH substitution, plays important roles in the biological activities of flavonoids. However, the structure-activity relationship of flavonoids on inhibiting MMPs activity is still undelineated. In the present study, 36 structure-related flavonoids are used to investigate the inhibitory activities on MMP activity in colorectal carcinoma cells. The results suggest that myr-icetin is the most potent inhibitor on MMP-2 activity and TPA-induced cellular responses in COLO 205 cells. The inhibitory mechanism by myricetin via inhibiting MMP-2 gene expression and enzyme activity, and structure-activity relationship of flavonoids on MMP-2 inhibition were elucidated.
Materials and Methods
Cell Culture
The human colorectal carcinoma cells, including COLO 205, COLO 320HSR, COLO 320 DM, and HT 29 cells, were obtained from American Type Culture Collection (Rock-ville, MD). COLO 205-X cells were primary cultured from COLO 205 – xenografted nude mice. These cells were grown
in RPMI 1640 containing 10% heat-inactivated fetal bovine serum and maintained at 37jC in a humidified incubator containing 5% CO2. All culture reagents were purchased
from Life Technologies, Inc. (Gaithersburg, MD). Chemicals
Flavone, 3-OH flavone, 5-OH flavone, 6-OH flavone, 7-OH flavone, 2V-CH3O flavone, 3-CH3O flavone, 5-CH3O
flavone, 6-CH3O flavone, flavanone, 2V-OH flavanone,
4V-OH flavanone, 6-4V-OH flavanone, 7-4V-OH flavanone, 4V-CH3O
flavanone, 5-CH3O flavanone, 6-CH3O flavanone, 7-CH3O
flavanone, kaempferol, quercetin, quercitrin, myricetin, myricitrin, baicalein, baicalin, morin, wogonin, fisetin, luteolin, rutin, taxifoin, naringenin, naringin, hesperidin, hesperetin, (F)catechin, (+)catechin, ( )catechin, and H-7 (isoquinoline-5-sulfonic 2-methyl-1-piperazide) were pur-chased from Sigma Chemical Co. (St, Louis, MO). GF-109203X was purchased from Calbiochem Co. (La Jolla, CA). Gelatin from porcine skin and MMP-2 proteins from human fibroblast for direct enzyme inhibition assay were purchased from Sigma. Antibodies for MMP-1, MMP-2, MMP-3, and MMP-9 protein detection in Western blotting were obtained from Oncogene Research Products (Cambridge, MA). EHS-Matrigel for in vitro invasion assay was purchased from BD Biosciences (Bedford, MA).
Cell Viability
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining as described previously (15). Briefly, COLO 205 cells were plated at a density of 105cells per well into 24-well plates. After overnight growth, cells were treated with a different concentration of flavonoids described above for 12 hours. At the end of treatment, 30 AL tetrazolium compound MTT and 270 AL fresh RPMI medium were added. The supernatant was remove and formazon crystals were dissolved in DMSO. After incubation for 4 hours at 37jC, 200 AL of 0.1 N HCl in 2-propanol was replaced per well to dissolve the tetrazolium crystals. At the end, absorbance at wave-length 600 nm was recorded using an ELISA plate reader.
Western Blots
Total cellular extracts (30 Ag) were prepared, separated on 8% SDS-polyacrylamide minigels for poly(ADP-ribose) polymerase detection and on 12% SDS-polyacrylamide minigels for MMP-1, MMP-2, MMP-3, and MMP-9 detection, and transferred to Immobilon polyvinylidenedi-fluoride membranes (Millipore, Bedford, MA). The mem-brane was incubated overnight at 4jC with 1% bovine serum albumin and at room temperature for 1 hour and then incubated with indicated antibodies for a further 3 hours at room temperature followed by incubation with alkaline phosphatase – conjugated mouse IgG anti-body for 1 hour. Protein was visualized by incubating with the colorimetric substrate nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate as described in our previous article (14).
Gelatin Zymography in Gel
COLO 205 cells at logarithmic growth phase were treated with 100 or 200 Amol/L of myricetin and myricitrin in serum-free RPMI medium for 24 hours.
The conditioned media were collected and analyzed for MMPs using gelatin zymography (17). In brief, samples were mixed with SDS sample buffer without heating or reduction and applied to 10% polyacrylamide gels copoly-merized with 0.1% gelatin (from porcine skin). The loading volume of each conditioned medium sample was normal-ized according to the cell number. After electrophoresis, gels were washed for 1 hour at room temperature in buffer containing 2.5% (v/v) Triton X-100 in 50 mmol/L Tris-Cl (pH 7.4) to remove SDS. Gels were then incubated at 37jC in reaction buffer (50 mmol/L Tris-Cl, pH 7.4, with 5 mmol/L CaCl2, 1 Amol/L ZnCl2, and 0.02% NaN3) for 18
hours. Then the gels were stained with Coomassie brilliant blue R-250 (0.25%) in 10% acetic acid/20% ethanol and destained in the same solution without dye. The zones of gelatinolytic activity were detected as clear bands against a blue background and were quantified using a densitometer.
Direct MMP-2 Enzyme Activity Assay
MMP-2 (400 ng) from human fibroblasts were incubated with 0, 25, 50, 100, and 200 Amol/L of myricetin in 50 AL Tris buffer (50 mmol/L, pH 7.4) at 37jC for 30 minutes, respectively. Twenty microliters of incubated buffer were then analyzed for gelatinolytic activity as described above. The zones of gelatinolytic activity were detected as clear bands against a blue background and were quantified using a densitometer.
In vitro Invasion Assay
In vitro invasion assay was carried out by the method of Huang et al. (18). Briefly, 24-well Transwell units with 8 Amol/L porosity polycarbonate filters (Becton Dickinson, Franklin Lakes, NJ) were coated with 0.1 mL of 0.8 mg/mL Englebreth-Holm-Swarm sarcoma tumor extract (EHS Matrigel) at room temperature for 1 hour to form a genuine reconstituted basement membrane. COLO 205 cells (2 105 cells per 0.4 mL RPMI) were placed in the upper compartment, and TPA (25 ng per 0.4 mL RPMI) was added in the lower compartment. The indicated Transwell plates were then incubated at 37jC for 48 hours in a humidified atmosphere with 5% CO2. The cells migrated
on the lower surface were quantified by MTT assay and observed under light microscope by Giemsa staining. Each treatment was assayed in triplicate, and two independent experiments were done.
Statistics
Values are expressed as the mean F SE The significance of the difference from the respective controls for each experimental test condition was assayed by using Student’s t test for each paired experiment. A P value < 0.01 or < 0.05 was regarded as indicating a significant difference.
Results
Myricetin Suppresses MMP-2 Enzyme Activity in Hu-man Colorectal Carcinoma Cell Line COLO 205 among 36 Flavonoids
Thirty-six structure-related flavonoids including fla-vones, flavanones, and catechins were used to investigate the inhibitory effect on MMP-2 enzyme activity in human colorectal carcinoma cells COLO 205. These compounds
possess a basic benzo-g-pyrone in structure with dif-ferent substitutions such as OH, OCH3, and glycosides at different locations. Among them, flavones contain a double bond at C2-C3 of flavanones, and catechin possesses five OH substitutions at C3V, C4V, C3, C5, and C7 without an oxo group at C4 of flavanone. To investigate the inhibitory effect of tested compounds on MMP activity, release of MMPs in the medium was done by gelatin zymography. In COLO 205 cells, a detectable endogenous MMP-2, but not MMP-9, enzyme activity in the medium was examined in the absence of tested compounds. As elucidated in Table 1, myricetin at 200 Amol/L exhibited 90% inhibition of activity of MMP-2 enzyme released from COLO 205 cells. Kaempferol (200 Amol/L) also showed 47% inhibition of released MMP-2 enzyme activity in the medium. Other flavonoids examined in the present study exhibited <20% inhibition of released MMP-2 enzyme activity.
Results of MTT assay showed that flavone, 3-OH flavone, 2V-OCH3flavone, 2V-OH flavanone, 4V-OH flavanone, 6-OH
flavanone, quercetin, wogonin, luteolin, and baicalein, at 200 Amol/L, showed significant cytotoxicity (>50%) in COLO 205 cells. Myricetin and kaempferol at 200 Amol/L exhibited 20% and 30% reduction in the viability of COLO 205 cells, respectively. Morphologic observations and DNA integrity assay showed that myricetin did not change the morphology of COLO 205 cells and no DNA laddering effect was detected in myricetin-treated COLO 205 cells (data not shown). These data suggest that myricetin is the most effective inhibitor among 36 flavonoids examined on the activity of MMP-2 released from COLO 205 cells.
Myricetin Concentration-Dependently Inhibits MMP-2 Enzyme Activity in Colorectal Carcinoma Cell Lines COLO 205, COLO 320HSR, COLO 320DM, HT-29, and COLO 205-X
Previous data suggested that myricetin exhibited MMP-2 inhibitory effect without severe cytotoxicity in COLO 205 cells. Therefore, several colorectal carcinoma cells including COLO 320HSR, COLO 320DM, HT-29, and COLO 205-X cells were used to investigate the MMP-2 inhibition by myricetin. Figure 1A shows the structures of myricetin and its related glycosylated com-pound, myricitrin. Myricitrin contains a rhamnose at C3 of myricetin. Myricetin, but not myricitrin, dose-dependently inhibits MMP-2 enzyme activity released from COLO 205, COLO 320HSR, COLO 320DM, HT-29, and COLO 205-X cells (Fig. 1B). The IC50 values of myricetin on MMP-2
activity in COLO 205, COLO 320HSR, COLO 320DM, HT-29, and COLO 205-X cells are 7.82, 11.18, 11.56, 13.25, and 23.51 Amol/L, respectively. MTT assay showed that myricetin only at 200 Amol/L exhibited slight but signif-icant reduction on the viability of COLO 205 cells. These data suggest that myricetin inhibition of MMP-2 enzyme activity was not due to its cytotoxic effect on cells.
Myricetin Inhibits MMP-2 Released from COLO 205 by Decreasing MMP-2 Protein Expression and Inhibit-ing MMP-2 Enzyme Activity
Two possibilities might be involved in myricetin inhibition of activity of MMP-2 enzyme released from
COLO 205 cells: one is to block MMP-2 protein expression and the other is to directly inhibit MMP-2 enzyme. Results of Western blotting indicated that myricetin inhibited endogenous MMP-2 protein expression in COLO 205 cells without affecting that of MMP-1, MMP-3, and MMP-9 proteins. In contrast, myricitrin did not affect any MMP protein expression in COLO 205 cells (Fig. 2A).
Further-more, whether myricetin or myricitrin might directly affect MMP-2 enzyme was examined by direct incubation of different concentrations of myricetin or myricitrin with the medium taken from that culturing COLO 205 at 37jC for 30 minutes, and MMP-2 activity was examined by gelatin zymography. It indicated that myricetin, but not myricitrin, directly inhibited MMP-2 enzyme activity in vitro (Fig. 2B). These data suggested myricetin inhibition of activity of MMP-2 enzyme released from COLO 205 cells might be through both decreasing MMP-2 protein expression and/or suppressing MMP-2 enzyme activity.
Myricetin Directly Inhibited MMP-2 Activity Using Purified MMP-2 Protein
To elucidate if myricetin directly blocked MMP-2 activity, a purified MMP-2 protein isolated from human fibroblasts was incubated with or without indicated doses (25, 50, 100, 200 Amol/L) of myricetin or myricitrin at 37jC for 30 minutes, and MMP-2 enzyme activity was analyzed by gelatin zymography. Results shown in Fig. 2C and D indicated that purified MMP-2 protein was able to digest gelatin at three clear zones, and myricetin, but not myricitrin, concentration-dependently inhibited MMP-2 enzyme activity, as indicated by a reduction in gelatin digestion in the gel. Results from Western blot analysis using a specific MMP-2 antibody indicated that neither myricetin nor myricitrin caused a decrease in MMP-2 protein level. These data provide direct evidence suggesting that myricetin directly inhibits MMP-2 activity.
TPA Induction of MMP-2 Activity in COLO 205 Cells through PKC-A Translocation, ERK Phosphorylation, and c-Jun Expression
We further investigated if myricetin blocked TPA – induced MMP-2 enzyme activity. TPA is a potent tumor promoter in carcinogenesis. As shown in Fig. 3A, TPA time-dependently induced both MMP-2 protein expres-sion and enzyme activity released from COLO 205 cells by Western blotting and gelatin zymography. An increase in c-Jun, but not MMP-1, MMP-3, and MMP-9, protein was observed in a time-dependent manner under TPA treatment. Furthermore, PKCa translocation from cytosol to membrane and activation of ERK1/2, but not p38 and JNKs, were detected in TPA-treated COLO 205 cells by Western blotting. These data revealed events including induction of MMP-2 protein expression and enzyme activity and increases in phosphorylated ERK1/2 protein and c-Jun protein expression with translocation of PKCa from cytosol to membrane in TPA-treated COLO 205 cells.
Phosphorylation of ERK1/2 Proteins Located Down-stream of PKCA Translocation in TPA-Induced MMP-2 Activity
To elucidate if phosphorylation of ERK1/2, translocation of PKCa from cytosol to membrane, and induction of c-Jun protein are essential events in TPA-induced MMP-2 enzyme activity, pharmacologic studies using specific inhibitors including PD98059 (mitogen-activated protein/ERK kinase Table 1. MMP-2 inhibitory effects of flavonoids with their
cytotoxicy in human colorectal COLO 205 cells Compound Cytotoxicity (%)* MMP-2 inhibition (%)c Flavone 71 F 0.23 — 3-OH flavone 47 F 0.1 — 5-OH flavone 23 F 0.11 — 6-OH flavone 38 F 0.12 — 7-OH flavone 31 F 0.09 — 2V-CH3O flavone 47 F 0.06 — 3-CH3O flavone 33 F 0.001 11 5-CH3O flavone 30 F 0.012 — 6-CH3O flavone 25 F 0.12 — Kaempferol 35 F 0.13 47 Quercetin 59 F 0.12 — Quercitrin 9 F 0.18 — Myricetin 23 F 0.11 90 Myricitrin 9 F 0.08 — Baicalein 47 F 0.12 — Baicalin 20 F 0.15 — Morin 16 F 0.1 — Wogonin 71 F 0.1 — Luteolin 46 F 0.11 13 Rutin 13 F 0.11 — Flavanone 24 F 0.11 20 2V-OH flavanone 69 F 0.13 — 4V-OH flavanone 54 F 0.09 — 6-OH flavanone 55 F 0.14 11 7-OH flavanone 21 F 0.13 — 4V-CH3O flavanone 27 F 0.12 10 5-CH3O flavanone 25 F 0.11 15 6-CH3O flavanone 23 F 0.16 11 7-CH3O flavanone 18 F 0.24 12 Taxifoin 19 F 0.21 — Naringenin 21 F 0.17 — Naringin 20 F 0.13 — Hesperidin 23 F 0.07 — Hesperetin 21 F 0.01 — (F)Catechin 15 F 0.1 — (+)Catechin 17 F 0.11 11 ( )Catechin 45 F 0.07 —
*Human colorectal COLO 205 cells were treated with indicated compounds (200 Amol/L) for 24 hours and the cell viability was analyzed by MTT assay. The cytotoxicity was measured by the equation [(absorbance of control group) (absorbance of compound-treated group)]/(absorbance of control group) 100%.
cHuman colorectal COLO 205 cells were treated with indicated compounds (200 Amol/L) for 24 hours. The conditioned media were collected and normalized by cell numbers before gelatin zymography analysis. The density of each band was quantified by densitometry analysis. The inhibition percentage was measured by the equation [(density of control group) (density of compound-treated group)]/(density of control group) 100%.
inhibitor), SB203580 (p38 inhibitor), SP600120 (JNK inhibi-tor), H-7 (PKA and PKC inhibiinhibi-tor), and GF-109203X (specific PKC inhibitor) were done. The results indicated that TPA treatment induced significant morphologic changes in COLO 205 cells by microscopic observation with (Fig. 4, bottom) or without (Fig. 4, top) Giemsa staining. Addition of PD98059, H-7, and GF-109203X, but not SB203580 and SP600120, showed a significant inhibition of TPA-induced morphologic changes in COLO 205 cells (Fig. 4A). In addition, PD98059, GF-109203X, and H 7, but not SB203580 or SP600120, inhibit TPA-induced MMP-2 protein expression with a decrease in activity of MMP-2 enzyme released from COLO 205 cells (Fig. 4B). Moreover, PD98059, GF-109203X, and H 7 significantly blocked TPA-induced ERK1/2 protein phosphorylation and c-Jun protein expression (Fig. 4C). These data suggest that activation of ERK1/2 located downstream of PKC activa-tion was followed by inducing c-Jun protein expression, and these events occurred upstream of MMP-2 activation induced by TPA.
Myricetin Suppression of TPA-Induced MMP-2 Acti-vation Is Accompanied by Inhibition of PKCA Transloca-tion, ERK1/2 Protein PhosphorylaTransloca-tion, and c-Jun Protein E xpression in COLO 205 Cells
The possibility that myricetin inhibited TPA-induced MMP-2 activity was further examined. Results shown in Fig. 5A indicated that myricetin significantly inhibited TPA-induced activity of MMP-2 enzyme released from COLO 205 cells. In addition, TPA-induced PKCa translo-cation, ERK1/2 protein phosphorylation, and c-Jun and MMP-2 protein expression were significantly blocked by myricetin, but not by myricitrin (Fig. 5B). Addition of low concentrations of PD98059 (1, 2, and 4 Amol/L) or GF-109203X (0.5, 1, and 2 Amol/L) enhanced the
MMP-2 inhibition of myricetin (5 Amol/L) under TPA treatment (Fig. 5C). These data provided additional evidence that myricetin inhibited TPA-induced MMP-2 activity via blocking intracellular signaling transduction.
Anti-invasive Effect of Myricetin in COLO 205 Cells We further investigated if myricetin blocked TPA-induced invasion in COLO 205 cells. In vitro invasion assay using a Transwell chamber coated with a reconsti-tuted basement membrane (EHS Matrigel) was done to examine the anti-invasive effect of myricetin. In this assay system, COLO 205 cells were cultured on the upper chamber in the presence or absence of myricetin or myricitrin (100 and 200 Amol/L), and TPA (100 ng/mL) was added to the lower chamber. After 24 hours incuba-tion, COLO 205 cells in the lower chamber were observed microscopically with (Fig. 6A, bottom) or without (Fig. 6A, top) Giemsa staining, and the number of viable cells in the lower chamber was quantified by MTT assay (Fig. 6B). Results shown in Fig. 6A indicated that COLO 205 cells did not penetrate the EHS-coated filter in the absence of TPA in the lower chamber (control group). Addition of TPA induced invasion of COLO 205 cells from the upper to the lower chambers in the absence of myricetin or myricitrin. Addition of myricetin, but not myricitrin, significantly reduced TPA-induced invasion in COLO 205 cells. Results of quantification of cells in the lower chambers showed that myricetin but not myricitrin inhibited TPA-induced inva-sion in COLO 205 cells (Fig. 6B).
Discussion
Results of the present study show that myricetin signifi-cantly inhibits MMP-2 activity and tumor invasion in colorectal carcinoma cells. Myricetin inhibition of MMP-2 Figure 1. Differential inhibitory effect of myricetin (ME ) and its glycoside myricitrin (MI ) on MMP-2 enzyme activity in human colorectal carcinoma cells.
A,structures of myricetin and myricitrin. The latter contains a rhamnose at C3 of myricetin.B,myricetin but not myricitrin inhibits activity of MMP-2 enzyme released from indicated colorectal carcinoma cells. Cells were treated with different concentrations (6.25, 12.5, 25, 50, 100, and 200 Amol/L) of myricetin or myricitrin for 24 h, and 20 AL of medium were applied for gelatin zymography.C,cytotoxicity of myricetin and myricitrin in COLO 205 cells was evaluated by MTT assay as described in Materials and Methods.Columns, mean from three independent experiments; bars, SE. *, P < 0.05, significantly different from control group as analyzed by Student’st test. The intensity of each band was quantitated by densitometry analysis and expressed as folds of control. Gelatin zymography was done at least thrice, and the results are representative of all of the data.CON, control; O.D., optical density (absorbance).
activity was via a decrease in MMP-2 protein expression and a direct inhibition of MMP-2 enzyme activity. In the presence of TPA, myricetin suppressed PKC activation, ERK1/2 protein phosphorylation, and c-Jun protein ex-pression together with a reduction in MMP-2 protein expression and enzyme activity. Inhibition of TPA-induced tumor invasion by myricetin was shown by in vitro invasion assay in the present study. These results suggest that myricetin, a compound capable of blocking both MMP-2 protein expression and enzyme activity, is a potentially effective antitumor agent.
Flavonoids are known to possess several biological functions including antitumor, antioxidant, and anti-inflammation activities. Structure-activity analysis has
shown that the number and location of OH substitutions are essential for the biological effects of flavonoids (19, 20). Results of the present study indicated that myricetin significantly inhibited MMP-2 activity. However, quercetin, which is a potent antioxidant and an apoptotic inducer, did not suppress MMP-2 activity. In contrast to quercetin, myricetin contains an additional OH substitution at C4V of the quercetin structure. In addition, an OH group at C4V is also found in kaempferol and luteolin, and both showed about 50% and 13% inhibition of MMP-2 enzyme activity, respectively. These results suggest that OH at C4V of flavone is important for MMP-2 inhibition.
In addition, glycoside has been shown to affect the biological functions of flavonoids via increasing their Figure 2. Myricetin but not myricitrin inhibits both MMP-2 protein expression and enzyme activity.A,myricetin inhibits MMP-2 protein expression in COLO 205 cells. Cells were treated with myricetin or myricitrin (100 or 200 Amol/L) for 24 h, and expression of MMP-1, MMP-2, MMP-3, and MMP-9 protein in cells was detected by Western blotting using specific antibodies.B,myricetin inhibits MMP-2 enzyme activity in the medium. The conditioned medium after 24 h incubation of COLO 205 cells was incubated with different concentrations (6.25, 12.5, 25, 50, 100, and 200 Amol/L) of myricetin or myricitrin at 37jC for 30 min. Activity of MMP-2 enzyme in each sample was analyzed by gelatin zymography.CandD,myricetin inhibited MMP-2 enzyme activity using purified MMP-2 protein as a substrate. Purified MMP-2 protein (500 ng) from human fibroblast was incubated with different concentrations (25, 50, 100, and 200 Amol/L) of myricetin (C) or myricitrin (D) at 37jC for 30 min. Activity of MMP-2 enzyme in each sample was analyzed by gelatin zymography (top ). The amount of MMP-2 protein in each sample was detected by Western blotting (W.B. ) using a specific anti-MMP-2 antibody (middle ). Quantification of MMP-2 enzyme activity under different treatments was done by densitometry analysis and expressed as a fold of control (bottom ). Columns, mean from three independent experiments; bars, SE. **, P < 0.01, significantly different from control group as analyzed by Student’s t test. Gelatin zymography and Western blotting were done at least thrice, and the results are representative of all of the data.
hydrophilic properties. Our previous studies showed that rutinoside and rhamnoglucoside attenuated apoptosis-inducing activity and nitric oxide synthesis inhibition of flavonoids (14, 15, 21). In the present study, all the tested glycosylated compounds including quercitrin, myricitrin,
baicalin, rutin, naringin, and hesperedin did not inhibit activity of MMP-2 enzyme released from COLO 205 cells. A significant difference was found in myricetin and its glyco-side myricitrin. Myricitrin, which contains a rhamnoglyco-side at C3 of myricetin, did not inhibit endogenous and Figure 3. TPA time-dependently induced MMP-2 protein and enzyme activity, c-jun protein, ERK1/2 protein phosphorylation, and PKCa translocation in COLO 205 cells.A,COLO 205 cells were treated with TPA (50 ng/mL) for indicated time points, and MMP-2 activity in the medium and expression of indicated proteins including MMP-1, MMP-2, MMP-3, and MMP-9 proteins were analyzed by gelatin zymography and Western blotting, respectively.B,
TPA induced PKCa translocation from cytosol to membrane. Cells were treated with TPA for 30 min, and the fractions of membrane and cytosol were collected as described in Materials and Methods. The expression of PKCa protein was detected by Western blotting.C,TPA induced ERK1/2 but not p38 and JNK protein phosphorylation in COLO 205 cells. Cells were treated with TPA (50 ng/mL) for different times, and expression of indicated proteins was detected by Western blotting using specific antibodies.ERK , p38 , and JNK indicate the total amount of indicated proteins in cells, and p-ERK , p-p38 , and p-JNK represent the respective phosphorylated proteins. Western blotting was done at least thrice, and the results are representative of all of the data.
Figure 4. Induction of PKCa translocation, ERK1/2 protein phosphorylation, and c-Jun protein expression involved in TPA-induced transformation and MMP-2 enzyme activity.A,activation of PKCa and ERK1/2 protein participated in TPA-induced transformation in COLO 205 cells. Cells were treated with 10 Amol/L PD98059 (PD ), 10 Amol/L SB203580 (SB ), 10 Amol/L SP600125 (SP ), 10 Amol/L GF-109203X (GF ), or 10 Amol/L H 7 for 30 min followed by incubation with TPA (50 ng/mL) for an additional 24 h. Cell morphology was detected microscopically with (bottom ) or without (top ) Giemsa staining.B,
PD98059, GF-109203X, and H 7 inhibited TPA-induced MMP-2 protein expression and activity of enzyme released from COLO 205 cells. As described in
A, MMP-2 protein (bottom ) and enzyme activity (top ) in cells and medium were analyzed by Western blotting and gelatin zymography, respectively.C,
PD98059, GF-109203X, H 7 inhibited TPA-induced ERK1/2 protein phosphorylation and c-Jun protein expression in COLO 205 cells as described inA. Cells were pretreated with PD98059 (5, 10 Amol/L), GF-109203X (2.5, 5 Amol/L), or H 7 (5, 10 Amol/L) for 30 min followed by addition of TPA and incubation for an additional 30 min for p-ERK detection or 6 h for c-jun protein detection by Western blotting using specific antibodies. Western blotting was done at least thrice, and the results are representative of all of the data.
TPA-induced MMP-2 enzyme activity and invasion. These data support the notion that glycosides are negative moieties on MMP-2 inhibition and invasion in colorectal carcinoma cells.
It has been shown that flavonoids containing more hydroxyl substitutions exhibit stronger antioxidant activity (22, 23). Wang and Joseph (24) indicated that OH groups at C3V and C4V of the B ring and a 2,3 double bond in conjugation with a 4-oxo group in the C ring, along with the polyphenolic structures, were crucial for the protection from H2O2-induced oxidative stress. Cos et al. (25)
indicated the OH groups at C5 and C7 and a double bond between C2 and C3 were important for the inhibitory activity of flavonoids on xanthine oxidase activity. In contrast to OH substitutions, methyoxyl (OCH3) addition
inactivated both antioxidant and prooxidant activities of the flavonoids, and hydroxylation but not methyoxyla-tion at C2 of flavone showed high affinity on
benzodi-azepine receptors (26, 27). To elucidate the relationship between the antioxidant activity and MMP-2 inhibition of flavonoids, anti-1,1-diphenyl-2-picrylhydrazyl radical ac-tivity of indicated flavonoids was examined. It indicated that flavonoids with more than two OH substitutions, such as quercetin, kaempferol, baicalein, myricetin, myricitrin, and toxifolin, exhibited potent anti-1,1-diphenyl-2-picrylhydrazyl radical activity (data not shown). However, results of the present study indicated that myricetin but not others exhibited significant inhibition on MMP-2 enzyme activity. This suggests that myricetin inhibition of invasion and MMP-2 enzyme activity is not only due to its antioxidant activity but also to its suppression of MMP-2 protein expression and enzyme activity via blocking intracellular signaling transduction processes.
TPA, a tumor promoter, has been used extensively in induction of tumor progression in vitro and in vivo. Figure 5. Myricetin inhibits TPA-induced MMP-2 protein expression and enzyme activity by blocking PKCa translocation, ERK1/2 protein phosphorylation, and c-Jun protein expression in COLO 205 cells.A,myricetin inhibited TPA-induced MMP-2 protein expression and enzyme activity in COLO 205 cells. Cells were treated with TPA (50 or 100 ng/mL) for 24 h in the presence or absence of myricetin (100 or 200 Amol/L) pretreatment for 1 h. The MMP-2 enzyme activity in the medium was analyzed by gelatin zymography.B,myricetin inhibited TPA-induced PKCa translocation, ERK1/2 protein phosphorylation, and c-Jun protein expression in COLO 205 cells. Cells were pretreated with myricetin or myricitrin (100 or 200 Amol/L) for 1 h followed by addition of TPA (50 ng/mL). The expression of indicated proteins was detected by Western blotting using specific antibodies as described in Fig. 4.C,
addition of PD98059 or 109203X enhances the MMP-2 inhibition of myricetin. Cells were treated with or without PD98059 (1, 2, 4 Amol/L) or GF-109203X (0.5, 1, 2 Amol/L) for 30 min followed by addition of myricetin (5 Amol/L) and TPA for a further 24 h. MMP-2 enzyme activity in medium was examined by gelatin zymography. Gelatin zymography and Western blotting were done at least thrice, and the results are representative of all of the data.
Figure 6. Anti-invasion effect of myricetinin vitro .A,myricetin but not myricitrin inhibited invasion of COLO 205 cells.In vitro invasion assay using Transwells coated with EHS Matrigel was done. Cells in upper chambers were treated with or without myricetin or myricitrin (100 or 200 Amol/L), and TPA (100 ng/mL) was added into the lower chambers for 24 h. In the control group, no TPA added in the lower well.A,cells in the lower chambers were observed microscopically (top ) and cells in the lower chambers were detected by Giemsa staining and observed microscopically (bottom ).B,quantification of cells in the lower chambers, which was done by MTT assay.Columns, mean from three independent experiments; bars, SE. **, P < 0.01, significantly different from TPA-treated group as analyzed by Student’st test.
Activation of PKCs via translocation of PKCs from cytosol to membrane has been identified under TPA treatment (28). In addition to PKCs, phosphorylation of MAPKs, including ERK1/2, p38, and JNK, and induction of c-Jun gene expression were detected in TPA-induced prolifera-tion (8, 29). Hah and Lee (30) reported that TPA induced the invasion of the hepatocellular carcinoma cells through MMP-9 but not MMP-2 secretion. However, the effect of TPA on MMP-2 enzyme activity and invasion in colorectal carcinoma cells is still undefined. Results of the present study indicated that TPA induced invasion and MMP-2 protein expression in COLO 205 cells. Sequential induc-tion of PKCa translocainduc-tions, ERK1/2 protein phosphory-lation, and c-Jun protein expression were involved. Myricetin inhibited TPA-induced MMP-2 enzyme activity and invasion via blocking activated signaling cascades. These data suggested that addition of TPA was able to induce invasion via activation of MMP-2 enzyme activity in colorectal carcinoma cells, which was blocked by myricetin (Fig. 7).
Several natural products such as curcumin, epigalloca-techin, and resveratrol have been found to inhibit MMP activity and invasion in different assay systems (31 – 33). The mechanism of inhibition was attributed to blockade of MMP gene expression but not enzyme activity. In the present study, myricetin inhibited both endogenous and TPA-induced MMP-2 enzyme activity and invasion in colorectal carcinoma cells. The most important event was that myricetin inhibition of MMP-2 released from cells was through blocking both MMP-2 protein expression and enzyme activity. The inhibition by myricetin of MMP-2 protein expression may be due to interruption of signaling transduction cascades such as PKC translocations and ERK1/2 proteins phosphorylation. However, the reason for myricetin-inhibited MMP-2 enzyme activity is still unclear. Several previous studies showed the metal-chelating activity of flavonoids (34, 35). Therefore, it is suggested that a direct inhibitory effect of myricetin on MMP-2 enzyme activity may be through chelating metals such as zinc and calcium, which are essential for the activity of MMP-2.
In conclusion, we have provided evidence demonstrating that myricetin inhibits invasion and both MMP-2 protein expression and enzyme activity in colorectal carcinoma cells in both unstimulated and TPA-stimulated conditions. Therefore, myricetin is potentially a useful anti-invasive agent in the treatment of colorectal carcinoma.
References
1. Mercapide J, Lopez De Cicco R, Castresana JS, Klein-Szanto AJ. Stromelysin-1/matrix metalloproteinase-3 (MMP-3) expression accounts for invasive properties of human astrocytoma cell lines. Int J Cancer 2003; 106:676 – 82.
2. Bernardo MM, Fridman R. TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP. Biochem J 2003;374:739 – 45.
3. Hotary KB, Allen ED, Brooks PC, Datta NS, Long MW, Weiss SJ. Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix. Cell 2003;114: 33 – 45.
4. Maekawa R, Maki H, Yoshida H, et al. Correlation of antiangiogenic and antitumor efficacy ofN -biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor. Cancer Res 1999;59:1231 – 5.
5. Rosenblum G, Meroueh SO, Kleifeld O, et al. Structural basis for potent slow binding inhibition of human matrix metalloproteinase-2. J Biol Chem 2003;278:27009 – 15.
6. Shamamian P, Schwartz JD, Pocock BJ, et al. Activation of progelatinase A by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis. J Cell Physiol 2001;189:197 – 206.
7. Bourguignon LY, Gunja-Smith Z, Iida N, et al. CD44v (3, 8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metal-loproteinase (MMP-9) association in metastatic breast cancer cells. J Cell Physiol 1998;176:206 – 15.
8. Ko CH, Shen S-C, Lin H-Y, et al. Flavanones structure related inhibition on TPA-induced tumor promotion through suppression of extracellular signal-regulated protein kinases: involvement of prostaglandin E2 in anti-promotive process. J Cell Physiol 2002;193:93 – 102.
9. Zeliadt NA, Warmka JK, Winston SE, et al. Tumor promoter-induced MMP-13 gene expression in a model of initiated epidermis. Biochem Biophys Res Commun 2004;317:570 – 7.
10. Arnott CH, Scott KA, Moore RJ, et al. Tumor necrosis factor-a mediates tumor promotion via a PKCa- and AP-1-dependent pathway. Oncogene 2002;21:4728 – 38.
11. Hinoda Y, Okayama N, Takano N, et al. Association of functional polymorphisms of matrix metalloproteinase (MMP)-1 and MMP-3 genes with colorectal cancer. Int J Cancer 2002;102: 526 – 9.
12. Chen YC, Shen SC, Lee WR, et al. Wogonin and fisetin induction of apoptosis through activation of caspase 3 cascade and alternative expression of p21 protein in hepatocellular carcinoma cells SK-HEP-1. Arch Toxicol 2002;76:351 – 9.
13. Lee WR, Shen S-C, Lin H-Y, Hou WC, Yang LL, Chen Y-C. Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accom-panied by a decrease of reactive oxygen sepsis, and activation of caspase-3 and Ca2+-dependent endonuclease. Biochem Pharmacol 2002;
63:225 – 36.
14. Chen YC, Shen SC, Lin HY. Rutinoside at C7 attenuates the apoptosis-inducing activity of flavonoids. Biochem Pharmacol 2003;66: 1139 – 50.
15. Shen SC, Chen YC, Hsu FL, Lee WR. Differential apoptosis-inducing effect of quercetin and its glycosides in human promyeloleukemic HL-60 cells by alternative activation of the caspase 3 cascade. J Cell Biochem 2003;89:1044 – 55.
16. Shen SC, Ko CH, Hsu KC, Chen YC. 3-OH flavone inhibition of epidermal growth factor-induced proliferation through blocking prosta-glandin E2 production. Int J Cancer 2004;108:502 – 10.
17. Fishman DA, Liu Y, Ellerbroek SM, Stack MS. Lysophospha-tidic acid promotes matrix metalloproteinase (MMP) activation and
Figure 7. A proposed mechanism for myricetin in suppressing MMP-2 enzyme activity and that induced by TPA in colorectal carcinoma cells.
MMP-dependent invasion in ovarian cancer cells. Cancer Res 2001; 61:3194 – 9.
18. Huang YT, Hwang JJ, Lee LT, et al. Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated prop-erties in human epidermoid carcinoma A431 cells. Int J Cancer 2002;99: 505 – 13.
19. Shen SC, Ko CH, Tseng SW, Tsai SH, Chen YC. Structurally-related anti-tumor effects of flavanonesin vitro and in vivo : involvement of caspase 3 activation, p21 gene expression and reactive oxygen species production. Toxicol Appl Pharmacol 2004;197:84 – 95.
20. Ko CH, Shen SC, Chen YC. Hydroxylation at C4V or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase 3 cascade and production of reactive oxygen species. Free Radic Biol Med 2004;36:897 – 910.
21. Lin HY, Uan SH, Shen SC, Hsu FL, Chen YC. Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1. Biochem Pharma-col 2003;66:1821 – 32.
22. Arora A, Nair MG, Strasburg GM. Structure-activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radic Biol Med 1998;24:1355 – 63.
23. Fiorani M, De Sanctis R, De Bellis R, Dacha` M. Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes. Free Radic Biol Med 2002;32:64 – 72.
24. Wang H, Joseph JA. Structure-activity relationships of quercetin in antagonizing hydrogen peroxide-induced calcium dysregulation in PC12 cells. Free Radic Biol Med 1999;27:683 – 94.
25. Cos P, Ying L, Calomme M, et al. Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. J Nat Prod 1998;61:71 – 6.
26. Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of
flavonoids: structure-activity relationships. Free Radic Biol Med 1997; 22:749 – 60.
27. Huen Michael-SY, Hui KM, Leung Justin-WC, et al. Naturally occurring 20-hydroxyl-substituted flavonoids as high-affinity benzodiaze-pine site ligands. Biochem Pharmacol 2003;66:2397 – 407.
28. Fahrmann M, Kaufhold M, Rieg T, Seidler U. Different actions of protein kinase C isoformsa and epsilon on gastric acid secretion. Br J Pharmacol 2002;136:938 – 46.
29. Hess A, Wijayanti N, Neuschafer-Rube AP, Katz N, Kietzmann T, Immenschuh S. Phorbol ester-dependent activation of peroxiredoxin I gene expression via a protein kinase C, Ras, p38 mitogen-activated protein kinase signaling pathway. J Biol Chem 2003;278:45419 – 34.
30. Hah N, Lee ST. An absolute role of the PKC-dependent NF-nB activation for induction of MMP-9 in hepatocellular carcinoma cells. Biochem Biophys Res Commun 2003;305:428 – 33.
31. Garbisa S, Sartor L, Biggin S, Salvato B, Benelli R, Albini A. Tumor gelatinases and invasion inhibited by the green tea flavanol epigalloca-techin-3-gallate. Cancer 2001;91:822 – 32.
32. Woo JH, Lim JH, Kim YH, et al. Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC y signal transduction. Oncogene 2004;23:1845 – 53.
33. Shishodia S, Potdar P, Gairola CG, Aggarwal BB. Curcumin (diferuloyl-methane) down-regulates cigarette smoke-induced NF-nB activation through inhibition of InBa kinase in human lung epithelial cells: correlation with suppression of COX-2, MMP-9 and cyclin D1. Carcinogenesis 2003; 24:1269 – 79.
34. Mira L, Fernandez MT, Santos M, Rocha R, Florencio MH, Jennings KR. Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity. Free Radic Res 2002;36:1199 – 208.
35. Fernandez MT, Mira ML, Florencio MH, Jennings KR. Iron and copper chelation by flavonoids: an electrospray mass spectrometry study. J Inorg Biochem 2002;92:105 – 11.
