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(−)-Epigallocatechin-3-gallate (EGCG) inhibits HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells
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Cancer Letters 271 (2008) 140–152 www.elsevier.com/locate/canlet
()-Epigallocatechin-3-gallate (EGCG) inhibits HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells Young Chang Lim a,b,1, Hun Yi Park c,1, Hye Sook Hwang c, Sung Un Kang c, Jung Hee Pyun c, Mi Hye Lee c, Eun Chang Choi b, Chul-Ho Kim c,* a
Department of Otorhinolaryngology-Head and Neck Surgery, Konkuk University School of Medicine, Republic of Korea b Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Republic of Korea c Department of Otolaryngology, Center for Cell Death Regulating Biodrug, Ajou University College of Medicine, 5 Wonchon-Gu, Suwon 442-749, Republic of Korea Received 8 April 2008; received in revised form 8 April 2008; accepted 28 May 2008
Abstract Hepatocyte growth factor (HGF) has recently attracted a considerable amount of attention as a stromal-derived mediator in tumor-stromal interactions, particularly because of its close involvement in cancer invasion and metastasis, and ()-epigallocatechin-3-gallate (EGCG) can modulate the cell signaling associated with angiogenesis, metastasis, and migration of cancer cells. In the present study, we have investigated the effects of HGF on invasion and metastasis of hypopharyngeal carcinoma cells and the effect of EGCG on blocking HGF-induced invasion and metastasis in these cells. We found that HGF promoted the autophosphorylation of c-Met, HGF receptor, and that HGF-induced proliferation, colony dispersion, migration and invasion of tumors. We also observed that HGF enhanced the activity of matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (uPA) in hypopharyngeal carcinoma cells. In addition, HGFinduced the activation of Akt and Erk pathway as a downstreaming pathway of invasion. On the other hand, EGCG at physiologically relevant concentration (1 lM) suppressed HGF-induced tumor motility and MMP-9 and uPA activities, and the suppression of Akt and Erk pathway by EGCG was one of the downstream mechanisms to facilitate EGCGinduced anti-invasion effects. These results suggest that EGCG may serve as a therapeutic agent to inhibit HGF-induced invasion in hypopharyngeal carcinoma patients. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: HGF; EGCG; MMP; uPA; Hypopharnygeal carcinoma
1. Introduction *
Corresponding author. Tel.: +82 31 219 5269; fax: +82 31 219 5264. E-mail address: [email protected] (C.-H. Kim). 1 The first two authors contributed equally to this work.
Hypopharyngeal squamous cell carcinoma (SCC) is characterized by an aggressive growth pattern and early metastatic spread, making loco-regional control of this cancer difficult [1]. The 5-year overall
0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.05.048
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survival rate for all patients with hypopharyngeal SCC is only 30%, which is one of the cancers with the lowest survival rates among other head and neck subsites [2]. Despite advances in the treatments combined with surgery, radiotherapy, or chemotherapy, only marginal improvement in survival rate has been achieved during the last 20 years. The high mortality rate of patients with hypopharyngeal SCC is in large measure due to uncontrolled loco-regional disease, which includes local tissue invasion by the primary tumor as well as regional lymph node or distant metastasis [3]. The presence of positive cervical lymph node in these patients can reduce the overall survival by 50% [4]. Survival of hypopharyngeal SCC patients with distant metastasis is usually measured in months, and treatment is palliative. Thus, controlling the tumor invasion and metastasis is a crucial goal for the successful treatment of hypopharyngeal SCC. Precise molecular mechanism of tumor invasion and metastasis remains unclear, however these events may be influenced by interactions between cancer cells and neighboring adjacent stromal components, including fibroblasts, blood vessels, inflammatory cells and extracellular matrix (ECM) [5]. Cancer cells themselves alter their adjacent stroma to form a permissive and supportive micro-environment by producing growth factors and cytokines [6]. On the other hand, stromal cells, including fibroblasts, enhance migration and invasion of cancer cells by producing stromal cell-derived factors [6]. Recently, hepatocyte growth factor (HGF) has attracted considerable amount of attention as a stromal-derived mediator in tumor-stromal interactions, particularly because of its close involvement in cancer invasion and metastasis [7]. HGF is a peptide growth factor and mainly produced by adjacent stromal cells, such as fibroblasts or endothelial cells, and its biological signal is transmitted from mesenchymal cells to epithelial cells through HGF receptor, c-Met which is a transmembrane tyrosine kinase receptor [8]. A search of literature shows that green tea, particularly its major polyphenolic constituent ()epigallocatechin-3-gallate (EGCG), possesses remarkable cancer chemopreventive and therapeutic potentials against various cancer sites, due in part to its profound effects in vitro and in vivo on tumor cell signaling pathways regulating growth and apoptosis [9–11]. In addition, another mechanism by which EGCG accomplishes the anti-tumor effect is to inhibit cell signaling associated with tumor invasion and metastasis [12].
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With these considerations, we have hypothesized that EGCG can inhibit HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells which has a highly invasive metastatic behavior. Therefore, we performed the following in vitro experiments: (1) the effects of HGF on migration and metastasis of cancer cells in human hypopharyngeal SCC lines; (2) examination of whether HGF can induce the expression of matrix metalloproteinases (MMP) and urokinase-plasminogen activator (uPA) which have been shown to be associated closely with invasion and metastasis; (3) examination of whether EGCG inhibits HGF-induced invasion and metastatic potential in these cells at a physiologic concentration; and (4) examination of which HGF promoted signaling pathway is blocked by EGCG. The results obtained led us to suggest that EGCG could be a potential therapeutic agent for interference with invasion and metastasis in hypopharyngeal SCC by suppression of HGF. 2. Materials and methods 2.1. Cell lines and reagents Established human HSCC cell line, FaDu cell line, was obtained from American Type Culture Collection (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (100 U/ ml), and streptomycin (100 lg/ml). All experiments were carried out on confluent cells at 37 °C in humidified atmosphere with 5% CO2 and 95% air. For the uPA assay, Madin-Darby canine kidney (MDCK-2) cells were cultured in DMEM with 10% FBS and used in the comparative experiment. The following items were purchased: EGCG from Sigma (St. Louis, MO, USA). Human recombinant HGF, affinity-purified goat anti-HGF polyclonal antibody, and rabbit anti-HGF receptor (c-Met) polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Met, phospho-Erk, and phosphor-Akt from Cell Signaling Technology (Beverly, MA, USA). Plasminogen from (Roche, IN, USA). 2.2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from FaDu cells homogenized in TRIzolÒ reagent (Gibco-BRL, Grand Island, NY, USA). The Omniscript Reverse
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Transcriptase kit (Qiagen, Hilden, Germany) was used for reverse transcription of the RNA. Thus, total RNA (2 lg) was mixed with 20 ll of the mixture; 2.0 ll 10 RT buffer, 2.0 ll dNTPs (5 mM each of dNTP), 2.0 ll of oligo-(dT) primer (10 lM), 1.0 ll RNase inhibitor (10 U/ll), 2 U Omniscript reverse transcriptase, and RNAse-free water. Reverse transcription was carried out, and cDNA was synthesized. The synthesized cDNA was added to a mixture of 1 U of Taq DNA polymerase (Roche Diagnostics, Indianapolis, IN, USA) and the specific primers, and amplified using the MJ Research MinicyclerTM (Bio-Rad Laboratories, Waltham, MA, USA). The following HGFspecific and c-Met-specific primers were used: HGF-F, 50 -ACATCGTCACTTCTGGC-30 and HGF-R, 50 -ATCCATCCTATGTTTGTTCG-30 ; and c-Met-F 50 -AGTAGCCTGATTGTGCATTT30 and c-Met-R, 50 -TCTTTCATGATGCCCTC-30 . PCR was performed under the following conditions: denaturation for 3 min at 96 °C, followed by 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 30 s at 72 °C, with extension for 5 min at 72 °C. Using 50–100 mg of FaDu cells pre-treated with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG, RNA was prepared as described above. The primer pairs used for MMP-2 and MMP-9 were as follows; MMP-2-F, 50 -AC CTGGATGCCGTCGTGGAC-30 and MMP-2-R, 50 -TGTGGCAGCACCAGGGCAGC-30 ; and MMP9-F, 50 -GGGGAAGATGCTGCTGTTCA-30 and MMP-9-R, 50 -GGTCCCAGTGGGGATTTACA-30 . After denaturation for 3 min at 96 °C, the samples were amplified by PCR for 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 30 s in 72 °C, with extension for 5 min at 72 °C. PCR products were separated by electrophoresis in 1.5% agarose gels and were detected under ultraviolet light (Bio-Rad, Hercules, CA, USA).
FaDu cells were plated in culture plates at a density of approximately 1 105/well in the absence of serum and grown to confluency. The cells were deprived of growth factors for 48 h. The monolayer was scratched with a sterile pipette tip, followed by extensive washing to remove cellular debris. Serumfree media with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) were added and cells were incubated. Wound healing was documented by photography at 12, 24, 36 and 48 h.
2.3. Western blot analysis
2.6. Invasion assay
FaDu cells were washed with phosphate-buffered saline (PBS), placed in RIPA (radioimmunoprecipitation) buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.5% deoxycholate, 100 lg of phenylmethylsulfonyl fluoride, and 1 lg/ml leupeptin, and the mixture was homogenized. The reaction mixture was centrifuged at 12,000 rpm and 4 °C for 5 min, and the supernatant was used in Western blot analysis. Protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Sodium
Transwell chambers (Costar) were used to array the invasion level of cells. Initially, type I collagen (6 lg/filter) dissolved in 100 ll of MEM was poured into the upper part of the polyethylene filter (pore size 8 lm), and coating was allowed to proceed overnight in a laminar flow hood. FBS medium (500 ll; 0.5%) was placed in the lower part of the well, and the well was filled with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM). After preprocessing with mitomycin C (8 lg/ml for 30 min),
dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) was used to separate 20 lg of protein per well, and the bands were transferred to a nitrocellulose filter (Amersham, Arlington Heights, IL, USA). The membrane was incubated with specific antibodies overnight at 4 °C. The membrane was then washed with Tris-buffered saline (TBS) that contained 0.1% Tween 20, and reacted with peroxidase-conjugated donkey anti-rabbit antibody (Amersham, Piscataway, NJ) or donkey anti-mouse antibody (Amersham). The membrane was developed using the Enhanced Chemiluminescence Detection System (ECL, Amersham, Piscataway, NJ) and X-ray film. 2.4. Proliferation assay FaDu cells were plated in culture plates (Coster, Cambridge, MA, USA) at a density of approximately 1 105/well in the absence of serum. HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) was added, and cells were cultured for and counted by haemocytometer at day 1, 3, and 5. 2.5. Wound healing assay
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105 cells (in 100 ll of growth medium) were added to the top of the filter in the upper well. The chamber was then cultivated in 5% CO2 at 37 °C for 48 h. The filter in the upper well was removed. Finally, the attached cells in the lower section were stained with hematoxylin and counted by a light microscope.
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2.9. Statistical analysis Student’s t test, one-way ANOVA test and Mann–Whitney test were used for statistical analyses of the data. All statistical analyses were conducted using SPSS 10.0 statistical software (SPSS, Chicago, IL, USA). Cases in which p values of <0.05 were considered statistically significant.
2.7. Zymography 3. Results
Cultures were deprived of growth factors and serum for 24 h prior to treatment with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) for the indicated time periods. one hundred microliters of the supernatant from each sample was mixed with 1 ll of 100 mM APMA, and the samples were activated for 1 h at 37 °C. Then each sample was placed in sample buffer for 10 min, and then electrophoresed in a polyacrylamide gel at 125 V for 120 min at 4 °C using the Novex Xcell II. The gel was incubated in renaturation buffer for 60 min at room temperature, and then incubated in 100 ml of developing buffer at 37 °C for 18 h with light shaking. The gel was then stained with Coomassie blue for 3 h and washed with water. After decolorization in 400 ml of methanol, 100 ml of acetic acid, and 500 ml of distilled water, cell images were taken every 10 min using an image analyzer. 2.8. Urokinase-type plasminogen activator assay FaDu cells (3000 cells/well or 6000 cells/well) were inoculated into 96-well plates in DMEM that contained 10% FBS. MDCK-2 cells were inoculated at 1500 cells/well. Two sets of plates were prepared; uPA activity was measured in one plate and the other plate was used for measuring cell growth. After overnight incubation, HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) were added to the wells, and the plates incubated further for 24 h. The cells were washed with DMEM that lacked phenol red and placed in 200 ll of reaction buffer that contained 50% (v/v) of 0.05 U/ml plasminogen in DMEM (without phenol red) and 40% (v/v) of 50 mM Tris-buffer (pH 8.2), and 10% (v/v) of 2.25 mM chromozyme PL in 100 mM glycine. The mixtures were incubated at 37 °C in 5% CO2 for 3 h, and absorbance at 405 nm was measured in an automated spectrophotometric plate reader.
3.1. Expression of c-Met and HGF in FaDu cells HGF is secreted mainly by surrounding fibroblast, and it’s expression is often limited to cells of mesenchymal origin [13]. To investigate whether HGF and c-Met are expressed in FaDu cells, hypopharyngeal squamous carcinoma cells, we performed RT-PCR and Western blotting were performed to detect m-RNA and protein of HGF and c-Met, respectively. Fig. 1a and b show that the expressions of HGF receptor, c-Met mRNA and protein were detected by RT-PCR and Western blotting respectively, however, the expression of HGF was not detected by these two analytical methods. Next, in order to investigate whether FaDu cells have functional c-Met protein, we tested c-Met phosphorylation after HGF treatment. Fig. 1c shows that HGF(30 ng/ml) increased c-Met tyrosine phosphorylation as early as 5 min, and the high levels of phosphorylated form were sustained for 1 h before beginning to decrease (Fig. 1c). These findings reveal that HGF greatly facilitates autophosphorylation of c-Met in FaDu cells. 3.2. EGCG inhibits HGF-induced proliferation of FaDu cells HGF has been known to stimulate the proliferation of various cancer cells [14–15]. Therefore, we performed the proliferation assay to determine whether HGF also stimulates the proliferation of FaDu cells. As shown in Fig. 2, proliferation assay showed that HGF stimulated the proliferation of FaDu cells in a dose–dependent manner (p* < 0.05). Next, we examined the ability of EGCG to inhibit HGF-induced cell proliferation, and the result showed that HGF-induced proliferative effect was attenuated by EGCG in a dose-dependent manner (p* < 0.05). 3.3. EGCG inhibits HGF-induced motility of FaDu cells It has been shown that HGF is one of the potent tumor motility factors and contributes to metastasis by stimulating of tumor motility [16]. Therefore, to examine the effects of HGF on the motility property of in FaDu cell line and also whether EGCG affects the cell motility induced by HGF, in vitro wound healing assay was
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Fig. 1. Expression of c-Met and HGF in FaDu cell and effects of HGF on c-Met phosphorylation. (a) FaDu cell were cultured in DMEM media with 10% FBS, and following RNA and protein extraction, the expression of HGF and c-Met were evaluated by RT-PCR and (b) Western blotting.(c) FaDu cell was treated with 30 ng/ml HGF for the indicated times and determination of the protein levels of phosphorylated c-Met by Western blot.
Fig. 2. Proliferative assay. FaDu cells were plated at 105 cells/well in a 96-well plate and incubated with HGF (0, 10, 30 ng/ml) ±increasing concentration of EGCG (0, 1, 10 lM) for 5 days. Cell growth was measured using haemocytometer.
performed. Thus FaDu cells were treated with HGF (0, 10, 30 ng/ml) together with increasing concentrations of EGCG (0, 1, 10 lM). As shown in Fig. 3a and b, HGF significantly enhanced the migration and proliferation of
FaDu cells. However this effect of HGF was blocked by EGCG in a dose-dependent manner. EGCG was able to almost completely block HGF-induced motility at concentrations as low as 1 lM.
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Fig. 3. Wound healing assay. FaDu cells were plated in a 6-well plate and grown to confluency in serum containing media. The cells were growth factor starved for 48 h. The monolayer was scratched with a pipette tip and washed with PBS. In order to evaluate the effect of HGF and EGCG on both migratory and proliferative activities, FaDu cells were treated with HGF (0, 10 ng/ml (a), 30 ng/ml (b)) and with increasing concentrations of EGCG (0, 1, 10 lM). Wound healing was documented by photography.
A Transwell chamber invasion assay was performed with FaDu cell line to determine whether HGF contributes to cell invasiveness and whether EGCG is capable of inhibiting HGF-induced invasion. Fig. 4a and b demonstrate that HGF treatment significantly increased dose-dependently the number of cells that invaded, compared to untreated controls, and this invasiveness induced by HGF was inhibited by EGCG in a dose-dependent manner. (Fig 4a and b) (p* < 0.05).
MMP-9 mRNA expression was significantly increased after HGF treatment for 24 h in a dose-dependent manner, however, the expression of MMP-2 mRNA could not be amplified. Next, we examined whether EGCG could influence HGF-induced MMP-9 mRNA expression in FaDu cells, and found that MMP-9 gene expression was decreased by EGCG in a dose-dependant manner(Fig. 5a). In zymogram, MMP-9 activity was also shown to increase after 24 h in the HGF treatment group as compared to the control group, and EGCG inhibited this effect of HGF on MMP-9 activity(Fig. 5b).
3.5. Effect of EGCG on the expression and invasion of HGF-induced MMP-2 and -9 in FaDu cells
3.6. Effect of EGCG on HGF-induced plasmin activity in FaDu cells
To confirm whether HGF can induce MMP gene expression in FaDu cells, we performed RT-PCR after treatment of the cells with HGF (10 or 30 ng/ml).
The ability of HGF to induce the conversion of exogenous plasminogen to plasmin was tested in FaDu cells used as control and MDCK-2. MDCK-2 cells displayed
3.4. EGCG inhibits HGF-induced invasion of FaDu cells
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Fig. 4. Invasion assay. Transwell chambers (Costar) were used to verify the level of cell invasiveness. FaDu cells seeded on upper membrane in HGF (0, 10, 30 ng/ml) ±increasing concentrations of EGCG. After 48-h incubation, plugged cells in 8-lm pore or cells attached to undersurface or membrane were counted and the attached cells in the lower section were stained with hematoxylin and were counted by light microscopy. 10 ng/ml (a) or 30 ng/ml (b) HGF stimulated cell invasion and this effect of HGF was inhibited by EGCG.
a marked increase of plasmin activity following 24 h of stimulation with HGF. HGF enhanced the plasmin activity in FaDu cells and this effect was inhibited by EGCG (Fig. 6). 3.7. EGCG inhibits HGF-activated Akt and Erk downstream pathways in FaDu cells Met/HGF signaling pathways have been known to be capable of activating a number of downstream signaling pathways, including extracellular signal-regulated kinase, src, and phosphatidylinositol 3-kinase pathway [12]. In order to assess the downstream signaling events induced
by HGF in FaDu cells, we treated the cells with 30 ng/ ml HGF, and cell lysates were prepared at regular intervals for up to 24 h and then analyzed for the activity of Erk and Akt. As shown in Fig. 7, Erk and Akt phosphorylation was increased in the HGF (30 ng/ml)-treated cells with strong activation within 5 min and a decrease of phosphorylation by 30 min post-treatment. Next, in order to investigate whether EGCG can block HGF signaling at the level of c-Met receptor activation and also aforementioned HGF-activated downstream Akt and Erk signaling pathway, we examined the phosphorylation activity of Met, Akt, and Erk following treatment with 30 ng/ml HGF for 24 h together with increasing concentrations of
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Fig. 5. Effect of EGCG on the expression (a) and invasion (b) of HGF-induced MMP-2 and -9 in FaDu cells. MMP-9 mRNA expression was significantly increased after HGF treatment for 24 h in a dose-dependent manner, but the expression of MMP-2 mRNA could not be amplified. EGCG inhibits expression and invasion of HGF-induced MMP-9 in FaDu cells in RT-PCR and zymogram, respectively.
Fig. 6. Effect of HGF and EGCG on uPA in FaDu cells. FaDu cells (3000 cells/well or 6000 cells/well) were inoculated into 96-well plates in DMEM that contained 10% FBS. After overnight incubation, HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) were added to the wells, and the plates incubated further for 24 h. The cells were placed in 200 ll of reaction buffer that contained 50% (v/v) of 0.05 U/ml plasminogen. The mixtures were incubated at 37 °C in 5% CO2 for 3 h, and absorbance at 405 nm was measured in an automated spectrophotometric plate reader.
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Fig. 7. Effects of HGF on activation of Met, Akt and Erk. FaDu cells were deprived of serum overnight and then incubated for 24 h. Cells were then stimulated with 30 ng/ml HGF for 15 min. Met, Akt and Erk were analyzed by Western blotting using phosphorylated Met, Akt and Erk, respectively.
Fig. 8. Suppression of EGCG of HGF-activated the Met, Akt and Erk pathways. FaDu cells were deprived of serum overnight and then incubated for 24 h in the absence or presence of HGF (30 ng/ml) with the indicated concentrations of EGCG. Cell lysates were subjected to western analysis with antibodies to phosphorylated form of Met, to phosphorylated Akt and to phosphorylated Erk as well as with antibodies to total Met, Akt, or Erk.
EGCG (0.001–10 lM). The result showed that the phosphorylation of Met, Akt, and Erk was decreased or disappeared at concentrations of 5, 5, and 10 lM EGCG, respectively (Fig. 8).
4. Discussion Hepatocyte growth factor (HGF) was first discovered as a major factor of liver regeneration and is now recognized as a motogen, mitogen and morphogen [17]. Met is the only known functional
receptor for HGF, and HGF is the only natural ligand for Met [8]. Under normal conditions, HGF and Met play roles in embryonic development, epithelial-mesenchymal transition, angiogenesis and tissue regeneration, including the liver [7]. In addition to regulation of normal cell functions, recent studies have shown that it is involved in malignant cell transformation and stimulates the invasive characteristics of various cancer cells, such as hepatocellular carcinoma and pancreatic cancer [11–12]. In several clinical studies, a relationship
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between the concentration of HGF in serum or in cancer tissue and the progression of disease has been noted in patients with gastric, breast and lung cancer [18]. Previously, we reported that the expression of mRNA and protein of both HGF and c-Met was increased in head and neck SCC tissue compared to normal tissue [19]. Immunohistochemical staining of hypopharyngeal cancer tissue showed high expression of HGF and c-Met and the level of HGF was significantly increased in proportion to lymph node metastasis and clinical stage of the tumor [19]. These pre-clinical study implied that HGF may be associated also with tumor progression and invasion of head and neck SCC. Therefore, we have investigated in the present study whether HGF promoted proliferation, migration, and invasion of hypopharyngeal carcinoma cells in vitro situation, and the results showed that FaDu cells, hypopharyngeal carcinoma cells, have functional c-Met protein, and that HGF promotes autophosphorylation of c-Met, and demonstrated that HGF induces proliferation, colony dispersion, migration and invasion of cancer cells. Tumor invasion requires degradation of basement membranes, proteolysis of ECM, pseudopodial extension, and cell migration [20]. Basement membrane, which separates the epithelial and mesenchymal cell compartments and is made up of matrix macromolecules such as type IV collagen, laminin, and heparan sulfate proteoglycans, is the first barrier of the ECM against cancer invasion. A number of proteolytic enzymes, including MMPs and serine proteinases, are involved in this tumorhost interactions, such as degradation of underlying basement membrane. Of these basement membrane degrading enzymes, MMPs, especially activated forms of MMP-2 and MMP-9, are thought to play an important role in its degradation because of their ability to cleave the type IV collagen. Currently, there are 28 different types of MMP identified, which can be classified into eight subgroups, including six secretory and two membrane-bound classes [21]. MMPs are produced by cancer cells or through the induction of surrounding stromal cells. Regarding SCC of the tongue, Yoshizaki et al. reported that activation of MMP-2 predicts poor prognosis of patients [22], Jeon et al. demonstrated that MMP-9 expression is associated with nodal and distant metastasis in nasopharyngeal carcinoma [23], and Pacheco et al. also described a link between MMP-9 expression and head and neck SCC recurrence rate [24]. Therefore, elevated expression of
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MMP appears to be related to the invasion, aggressiveness and overall survival of patients with head and neck cancers. Serine protease, which is also important for tumor invasiveness and metastasis, is usually secreted from endothelial cells and served as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). tPA is usually involved in thrombolysis, whereas uPA is involved in the infiltration of carcinoma [25–26]. The 52-kDa uPA plays a major role in the decomposition of basement membranes, as the expression of uPA is increased in solid tumors. Activation of the uPA/uPAR/plasmin proteolytic network has been shown to play key roles in tumor invasion and dissemination of various malignancies [27–28]. For example, the role of the uPA fibrinolytic network in tumor malignancy has been demonstrated in uPA / mice, in which there was a dramatic reduction in the progression of chemically-induced malignant melanomas [29]. The pro-uPA form (inactive pro-enzyme) is synthesized and secreted from tumor cells and is transformed into active form by plasmin [30]. Activated uPA accelerates the conversion of plasminogen into plasmin. The elevated plasmin degrades the fibrin, fibronectin, proteoglycan, and laminin that compose the matrix around the tumor, activates type IV collagenases, and indirectly destroys type IV collagen, which is an important component of basement membranes [31]. Recently, the presence of uPA in tumor tissues has been proposed as a potential prognostic factor, and an enzyme-linked immunosorbent assay (ELISA) for uPA has been developed as a prognostic test for solid cancers [32]. In addition, the levels of uPA and uPAR expression serve as prognostic markers in various malignancies in which high levels of expression are often associated with a poor prognosis [33]. Thus, we examined the association between HGF and expressions of MMP and uPA in hypopharyngeal carcinoma cells, and observed that RT-PCR revealed a marked increase in the level of MMP-9 mRNA after HGF treatment for 24 h and zymography confirmed the increased MMP-9 activity in the 30 ng/ml HGF treatment group. In addition, HGF increased the activity of uPA in a dose-dependent manner. These results indicated that HGF enhanced the expression of MMP-9 and uPA in hypopharyngeal carcinoma cells. Next, we investigated the downstream signaling pathways in hypopharyngeal carcinoma cells,
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induced by HGF because no information has so far been available regarding Met signaling pathwayassociated with tumor invasion in these cells, although an earlier study found that downstream activation of the PI3-kinase/AKT and the Ras/Erk pathways is necessary for HGF-induced motility in other cancer cells [34]. Our present study also revealed that HGF induced sustained activation of the Akt and Erk pathways in hypopharyngeal carcinoma cells. Epidemiological and pre-clinical studies demonstrated that polyphenols derived from green tea possess profound chemopreventive and anti-tumor effects against various cancers. The major polyphenols in green tea are ()-epigallocatechin-3-gallate (EGCG), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC). Of these, EGCG is a major catechin found in green tea, and has been shown to induce apoptosis and inhibit proliferation in many tumor cell lines [35]. Recent work revealed that EGCG could modulate the cell signaling associated with angiogenesis, metastasis, and migration of cancer cells, such as prostate cancer, liver cancer and breast cancer [36]. Thus, we investigated whether EGCG can block aforementioned HGFinduced invasion in hypopharyngeal carcinoma cell. First, in order to determine if EGCG can inhibit HGF-induced motility and invasion, we performed wound healing and invasion assays, and found that EGCG was able to block HGF-induced motility at physiologically relevant concentration (1 lM). Bigelow et al. also observed that treatment with 5.0 l/l EGCG can inhibit the HGF-induced MMP, and that EGCG blocked the ability of HGF to induce cell motility and invasion in immortalized and tumorigenic breast epithelial cells [37]. We then examined whether EGCG blocks the expression of MMP and uPA which are closely associated with tumor invasion. EGCG has been shown to directly and indirectly repress MMP activity, MMP-2 and MMP-9 are remarkably inhibited by orally administered green tea polyphenols in the prostate in TRAMP mice [38], and EGCG (25–100 lmol/l) has also been shown to inhibit the MMP-2 and MMP-9 in endothelial cells [39]. Thus, it seems quite likely that EGCG could inhibit or delay cancer invasion, metastasis, and angiogenesis via modulation of MMPs. Jankun et al. showed that EGCG inhibits the activity of uPA [40]. With the use of computer-based molecular modeling, they
found that EGCG binds to urokinase, thereby blocking histidine 57 and serine residues of the urokinase catalytic triad and extending arginine 35 from a positively charged loop of urokinase [40]. Based on the above observation, the authors suggested that the anti-tumor effect of EGCG is mediated by inhibition of uPA, one of the most frequently overexpressed enzymes in human cancers. Our present study also demonstrated that EGCG blocked the HGF-induced MMP-9 and uPA activities in hypopharyngeal carcinoma cells at a physiologic relevant concentration. Finally, we examined whether EGCG blocks the downstream pathway of Akt and Erk which are activated by HGF, and demonstrated that EGCG was able to repress the HGF-induced increase of Met phosphorylation, as well as block activation of the downstream kinases, Akt and Erk. The pharmacokinetic studies in humans indicate approximately 1.0 lM as the maximally obtainable plasma concentration after single p.o. dose of EGCG35. Thus, it is important to consider whether blocking effect of EGCG on HGF-induced invasion and metastasis is accomplished at this physiologically relevant concentrations. Our study revealed that 1 lM EGCG is sufficient to block HGFinduced motility and invasion in hypopharyngeal cancer cell. In summary, our study showed that inhibition of HGF/Met by physiologic concentration of EGCG leads to decreased proliferation, migration and invasion of hypopharyngeal carcinoma cells. Thus, these findings indicate that EGCG may serve as a therapeutic agent to inhibit HGF-induced invasion in hypopharyngeal carcinoma patients, and future study on its clinical application seems to be worthwhile. Acknowledgement This research was supported by CCRB through the ‘‘GRRC” Project of Gyeonggi Provincial Government, Republic of Korea. References [1] C.H. Kim, J.H. Kim, H. Kahng, E.C. Choi, Change of Ecadherin by hepatocyte growth factor and effects on the prognosis of hypopharyngeal carcinoma, Ann. Surg. Oncol. 14 (2007) 1565–1574. [2] W.I. Wei, The dilemma of treating hypopharyngeal carcinoma: more or less, Arch. Otolaryngol. Head Neck Surg. 128 (2002) 229–232.
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Cancer Letters 271 (2008) 140–152 www.elsevier.com/locate/canlet
()-Epigallocatechin-3-gallate (EGCG) inhibits HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells Young Chang Lim a,b,1, Hun Yi Park c,1, Hye Sook Hwang c, Sung Un Kang c, Jung Hee Pyun c, Mi Hye Lee c, Eun Chang Choi b, Chul-Ho Kim c,* a
Department of Otorhinolaryngology-Head and Neck Surgery, Konkuk University School of Medicine, Republic of Korea b Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Republic of Korea c Department of Otolaryngology, Center for Cell Death Regulating Biodrug, Ajou University College of Medicine, 5 Wonchon-Gu, Suwon 442-749, Republic of Korea Received 8 April 2008; received in revised form 8 April 2008; accepted 28 May 2008
Abstract Hepatocyte growth factor (HGF) has recently attracted a considerable amount of attention as a stromal-derived mediator in tumor-stromal interactions, particularly because of its close involvement in cancer invasion and metastasis, and ()-epigallocatechin-3-gallate (EGCG) can modulate the cell signaling associated with angiogenesis, metastasis, and migration of cancer cells. In the present study, we have investigated the effects of HGF on invasion and metastasis of hypopharyngeal carcinoma cells and the effect of EGCG on blocking HGF-induced invasion and metastasis in these cells. We found that HGF promoted the autophosphorylation of c-Met, HGF receptor, and that HGF-induced proliferation, colony dispersion, migration and invasion of tumors. We also observed that HGF enhanced the activity of matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (uPA) in hypopharyngeal carcinoma cells. In addition, HGFinduced the activation of Akt and Erk pathway as a downstreaming pathway of invasion. On the other hand, EGCG at physiologically relevant concentration (1 lM) suppressed HGF-induced tumor motility and MMP-9 and uPA activities, and the suppression of Akt and Erk pathway by EGCG was one of the downstream mechanisms to facilitate EGCGinduced anti-invasion effects. These results suggest that EGCG may serve as a therapeutic agent to inhibit HGF-induced invasion in hypopharyngeal carcinoma patients. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: HGF; EGCG; MMP; uPA; Hypopharnygeal carcinoma
1. Introduction *
Corresponding author. Tel.: +82 31 219 5269; fax: +82 31 219 5264. E-mail address: [email protected] (C.-H. Kim). 1 The first two authors contributed equally to this work.
Hypopharyngeal squamous cell carcinoma (SCC) is characterized by an aggressive growth pattern and early metastatic spread, making loco-regional control of this cancer difficult [1]. The 5-year overall
0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.05.048
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survival rate for all patients with hypopharyngeal SCC is only 30%, which is one of the cancers with the lowest survival rates among other head and neck subsites [2]. Despite advances in the treatments combined with surgery, radiotherapy, or chemotherapy, only marginal improvement in survival rate has been achieved during the last 20 years. The high mortality rate of patients with hypopharyngeal SCC is in large measure due to uncontrolled loco-regional disease, which includes local tissue invasion by the primary tumor as well as regional lymph node or distant metastasis [3]. The presence of positive cervical lymph node in these patients can reduce the overall survival by 50% [4]. Survival of hypopharyngeal SCC patients with distant metastasis is usually measured in months, and treatment is palliative. Thus, controlling the tumor invasion and metastasis is a crucial goal for the successful treatment of hypopharyngeal SCC. Precise molecular mechanism of tumor invasion and metastasis remains unclear, however these events may be influenced by interactions between cancer cells and neighboring adjacent stromal components, including fibroblasts, blood vessels, inflammatory cells and extracellular matrix (ECM) [5]. Cancer cells themselves alter their adjacent stroma to form a permissive and supportive micro-environment by producing growth factors and cytokines [6]. On the other hand, stromal cells, including fibroblasts, enhance migration and invasion of cancer cells by producing stromal cell-derived factors [6]. Recently, hepatocyte growth factor (HGF) has attracted considerable amount of attention as a stromal-derived mediator in tumor-stromal interactions, particularly because of its close involvement in cancer invasion and metastasis [7]. HGF is a peptide growth factor and mainly produced by adjacent stromal cells, such as fibroblasts or endothelial cells, and its biological signal is transmitted from mesenchymal cells to epithelial cells through HGF receptor, c-Met which is a transmembrane tyrosine kinase receptor [8]. A search of literature shows that green tea, particularly its major polyphenolic constituent ()epigallocatechin-3-gallate (EGCG), possesses remarkable cancer chemopreventive and therapeutic potentials against various cancer sites, due in part to its profound effects in vitro and in vivo on tumor cell signaling pathways regulating growth and apoptosis [9–11]. In addition, another mechanism by which EGCG accomplishes the anti-tumor effect is to inhibit cell signaling associated with tumor invasion and metastasis [12].
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With these considerations, we have hypothesized that EGCG can inhibit HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells which has a highly invasive metastatic behavior. Therefore, we performed the following in vitro experiments: (1) the effects of HGF on migration and metastasis of cancer cells in human hypopharyngeal SCC lines; (2) examination of whether HGF can induce the expression of matrix metalloproteinases (MMP) and urokinase-plasminogen activator (uPA) which have been shown to be associated closely with invasion and metastasis; (3) examination of whether EGCG inhibits HGF-induced invasion and metastatic potential in these cells at a physiologic concentration; and (4) examination of which HGF promoted signaling pathway is blocked by EGCG. The results obtained led us to suggest that EGCG could be a potential therapeutic agent for interference with invasion and metastasis in hypopharyngeal SCC by suppression of HGF. 2. Materials and methods 2.1. Cell lines and reagents Established human HSCC cell line, FaDu cell line, was obtained from American Type Culture Collection (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (100 U/ ml), and streptomycin (100 lg/ml). All experiments were carried out on confluent cells at 37 °C in humidified atmosphere with 5% CO2 and 95% air. For the uPA assay, Madin-Darby canine kidney (MDCK-2) cells were cultured in DMEM with 10% FBS and used in the comparative experiment. The following items were purchased: EGCG from Sigma (St. Louis, MO, USA). Human recombinant HGF, affinity-purified goat anti-HGF polyclonal antibody, and rabbit anti-HGF receptor (c-Met) polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Met, phospho-Erk, and phosphor-Akt from Cell Signaling Technology (Beverly, MA, USA). Plasminogen from (Roche, IN, USA). 2.2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from FaDu cells homogenized in TRIzolÒ reagent (Gibco-BRL, Grand Island, NY, USA). The Omniscript Reverse
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Transcriptase kit (Qiagen, Hilden, Germany) was used for reverse transcription of the RNA. Thus, total RNA (2 lg) was mixed with 20 ll of the mixture; 2.0 ll 10 RT buffer, 2.0 ll dNTPs (5 mM each of dNTP), 2.0 ll of oligo-(dT) primer (10 lM), 1.0 ll RNase inhibitor (10 U/ll), 2 U Omniscript reverse transcriptase, and RNAse-free water. Reverse transcription was carried out, and cDNA was synthesized. The synthesized cDNA was added to a mixture of 1 U of Taq DNA polymerase (Roche Diagnostics, Indianapolis, IN, USA) and the specific primers, and amplified using the MJ Research MinicyclerTM (Bio-Rad Laboratories, Waltham, MA, USA). The following HGFspecific and c-Met-specific primers were used: HGF-F, 50 -ACATCGTCACTTCTGGC-30 and HGF-R, 50 -ATCCATCCTATGTTTGTTCG-30 ; and c-Met-F 50 -AGTAGCCTGATTGTGCATTT30 and c-Met-R, 50 -TCTTTCATGATGCCCTC-30 . PCR was performed under the following conditions: denaturation for 3 min at 96 °C, followed by 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 30 s at 72 °C, with extension for 5 min at 72 °C. Using 50–100 mg of FaDu cells pre-treated with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG, RNA was prepared as described above. The primer pairs used for MMP-2 and MMP-9 were as follows; MMP-2-F, 50 -AC CTGGATGCCGTCGTGGAC-30 and MMP-2-R, 50 -TGTGGCAGCACCAGGGCAGC-30 ; and MMP9-F, 50 -GGGGAAGATGCTGCTGTTCA-30 and MMP-9-R, 50 -GGTCCCAGTGGGGATTTACA-30 . After denaturation for 3 min at 96 °C, the samples were amplified by PCR for 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 30 s in 72 °C, with extension for 5 min at 72 °C. PCR products were separated by electrophoresis in 1.5% agarose gels and were detected under ultraviolet light (Bio-Rad, Hercules, CA, USA).
FaDu cells were plated in culture plates at a density of approximately 1 105/well in the absence of serum and grown to confluency. The cells were deprived of growth factors for 48 h. The monolayer was scratched with a sterile pipette tip, followed by extensive washing to remove cellular debris. Serumfree media with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) were added and cells were incubated. Wound healing was documented by photography at 12, 24, 36 and 48 h.
2.3. Western blot analysis
2.6. Invasion assay
FaDu cells were washed with phosphate-buffered saline (PBS), placed in RIPA (radioimmunoprecipitation) buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.5% deoxycholate, 100 lg of phenylmethylsulfonyl fluoride, and 1 lg/ml leupeptin, and the mixture was homogenized. The reaction mixture was centrifuged at 12,000 rpm and 4 °C for 5 min, and the supernatant was used in Western blot analysis. Protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Sodium
Transwell chambers (Costar) were used to array the invasion level of cells. Initially, type I collagen (6 lg/filter) dissolved in 100 ll of MEM was poured into the upper part of the polyethylene filter (pore size 8 lm), and coating was allowed to proceed overnight in a laminar flow hood. FBS medium (500 ll; 0.5%) was placed in the lower part of the well, and the well was filled with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM). After preprocessing with mitomycin C (8 lg/ml for 30 min),
dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) was used to separate 20 lg of protein per well, and the bands were transferred to a nitrocellulose filter (Amersham, Arlington Heights, IL, USA). The membrane was incubated with specific antibodies overnight at 4 °C. The membrane was then washed with Tris-buffered saline (TBS) that contained 0.1% Tween 20, and reacted with peroxidase-conjugated donkey anti-rabbit antibody (Amersham, Piscataway, NJ) or donkey anti-mouse antibody (Amersham). The membrane was developed using the Enhanced Chemiluminescence Detection System (ECL, Amersham, Piscataway, NJ) and X-ray film. 2.4. Proliferation assay FaDu cells were plated in culture plates (Coster, Cambridge, MA, USA) at a density of approximately 1 105/well in the absence of serum. HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) was added, and cells were cultured for and counted by haemocytometer at day 1, 3, and 5. 2.5. Wound healing assay
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105 cells (in 100 ll of growth medium) were added to the top of the filter in the upper well. The chamber was then cultivated in 5% CO2 at 37 °C for 48 h. The filter in the upper well was removed. Finally, the attached cells in the lower section were stained with hematoxylin and counted by a light microscope.
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2.9. Statistical analysis Student’s t test, one-way ANOVA test and Mann–Whitney test were used for statistical analyses of the data. All statistical analyses were conducted using SPSS 10.0 statistical software (SPSS, Chicago, IL, USA). Cases in which p values of <0.05 were considered statistically significant.
2.7. Zymography 3. Results
Cultures were deprived of growth factors and serum for 24 h prior to treatment with HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) for the indicated time periods. one hundred microliters of the supernatant from each sample was mixed with 1 ll of 100 mM APMA, and the samples were activated for 1 h at 37 °C. Then each sample was placed in sample buffer for 10 min, and then electrophoresed in a polyacrylamide gel at 125 V for 120 min at 4 °C using the Novex Xcell II. The gel was incubated in renaturation buffer for 60 min at room temperature, and then incubated in 100 ml of developing buffer at 37 °C for 18 h with light shaking. The gel was then stained with Coomassie blue for 3 h and washed with water. After decolorization in 400 ml of methanol, 100 ml of acetic acid, and 500 ml of distilled water, cell images were taken every 10 min using an image analyzer. 2.8. Urokinase-type plasminogen activator assay FaDu cells (3000 cells/well or 6000 cells/well) were inoculated into 96-well plates in DMEM that contained 10% FBS. MDCK-2 cells were inoculated at 1500 cells/well. Two sets of plates were prepared; uPA activity was measured in one plate and the other plate was used for measuring cell growth. After overnight incubation, HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) were added to the wells, and the plates incubated further for 24 h. The cells were washed with DMEM that lacked phenol red and placed in 200 ll of reaction buffer that contained 50% (v/v) of 0.05 U/ml plasminogen in DMEM (without phenol red) and 40% (v/v) of 50 mM Tris-buffer (pH 8.2), and 10% (v/v) of 2.25 mM chromozyme PL in 100 mM glycine. The mixtures were incubated at 37 °C in 5% CO2 for 3 h, and absorbance at 405 nm was measured in an automated spectrophotometric plate reader.
3.1. Expression of c-Met and HGF in FaDu cells HGF is secreted mainly by surrounding fibroblast, and it’s expression is often limited to cells of mesenchymal origin [13]. To investigate whether HGF and c-Met are expressed in FaDu cells, hypopharyngeal squamous carcinoma cells, we performed RT-PCR and Western blotting were performed to detect m-RNA and protein of HGF and c-Met, respectively. Fig. 1a and b show that the expressions of HGF receptor, c-Met mRNA and protein were detected by RT-PCR and Western blotting respectively, however, the expression of HGF was not detected by these two analytical methods. Next, in order to investigate whether FaDu cells have functional c-Met protein, we tested c-Met phosphorylation after HGF treatment. Fig. 1c shows that HGF(30 ng/ml) increased c-Met tyrosine phosphorylation as early as 5 min, and the high levels of phosphorylated form were sustained for 1 h before beginning to decrease (Fig. 1c). These findings reveal that HGF greatly facilitates autophosphorylation of c-Met in FaDu cells. 3.2. EGCG inhibits HGF-induced proliferation of FaDu cells HGF has been known to stimulate the proliferation of various cancer cells [14–15]. Therefore, we performed the proliferation assay to determine whether HGF also stimulates the proliferation of FaDu cells. As shown in Fig. 2, proliferation assay showed that HGF stimulated the proliferation of FaDu cells in a dose–dependent manner (p* < 0.05). Next, we examined the ability of EGCG to inhibit HGF-induced cell proliferation, and the result showed that HGF-induced proliferative effect was attenuated by EGCG in a dose-dependent manner (p* < 0.05). 3.3. EGCG inhibits HGF-induced motility of FaDu cells It has been shown that HGF is one of the potent tumor motility factors and contributes to metastasis by stimulating of tumor motility [16]. Therefore, to examine the effects of HGF on the motility property of in FaDu cell line and also whether EGCG affects the cell motility induced by HGF, in vitro wound healing assay was
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Fig. 1. Expression of c-Met and HGF in FaDu cell and effects of HGF on c-Met phosphorylation. (a) FaDu cell were cultured in DMEM media with 10% FBS, and following RNA and protein extraction, the expression of HGF and c-Met were evaluated by RT-PCR and (b) Western blotting.(c) FaDu cell was treated with 30 ng/ml HGF for the indicated times and determination of the protein levels of phosphorylated c-Met by Western blot.
Fig. 2. Proliferative assay. FaDu cells were plated at 105 cells/well in a 96-well plate and incubated with HGF (0, 10, 30 ng/ml) ±increasing concentration of EGCG (0, 1, 10 lM) for 5 days. Cell growth was measured using haemocytometer.
performed. Thus FaDu cells were treated with HGF (0, 10, 30 ng/ml) together with increasing concentrations of EGCG (0, 1, 10 lM). As shown in Fig. 3a and b, HGF significantly enhanced the migration and proliferation of
FaDu cells. However this effect of HGF was blocked by EGCG in a dose-dependent manner. EGCG was able to almost completely block HGF-induced motility at concentrations as low as 1 lM.
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Fig. 3. Wound healing assay. FaDu cells were plated in a 6-well plate and grown to confluency in serum containing media. The cells were growth factor starved for 48 h. The monolayer was scratched with a pipette tip and washed with PBS. In order to evaluate the effect of HGF and EGCG on both migratory and proliferative activities, FaDu cells were treated with HGF (0, 10 ng/ml (a), 30 ng/ml (b)) and with increasing concentrations of EGCG (0, 1, 10 lM). Wound healing was documented by photography.
A Transwell chamber invasion assay was performed with FaDu cell line to determine whether HGF contributes to cell invasiveness and whether EGCG is capable of inhibiting HGF-induced invasion. Fig. 4a and b demonstrate that HGF treatment significantly increased dose-dependently the number of cells that invaded, compared to untreated controls, and this invasiveness induced by HGF was inhibited by EGCG in a dose-dependent manner. (Fig 4a and b) (p* < 0.05).
MMP-9 mRNA expression was significantly increased after HGF treatment for 24 h in a dose-dependent manner, however, the expression of MMP-2 mRNA could not be amplified. Next, we examined whether EGCG could influence HGF-induced MMP-9 mRNA expression in FaDu cells, and found that MMP-9 gene expression was decreased by EGCG in a dose-dependant manner(Fig. 5a). In zymogram, MMP-9 activity was also shown to increase after 24 h in the HGF treatment group as compared to the control group, and EGCG inhibited this effect of HGF on MMP-9 activity(Fig. 5b).
3.5. Effect of EGCG on the expression and invasion of HGF-induced MMP-2 and -9 in FaDu cells
3.6. Effect of EGCG on HGF-induced plasmin activity in FaDu cells
To confirm whether HGF can induce MMP gene expression in FaDu cells, we performed RT-PCR after treatment of the cells with HGF (10 or 30 ng/ml).
The ability of HGF to induce the conversion of exogenous plasminogen to plasmin was tested in FaDu cells used as control and MDCK-2. MDCK-2 cells displayed
3.4. EGCG inhibits HGF-induced invasion of FaDu cells
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Fig. 4. Invasion assay. Transwell chambers (Costar) were used to verify the level of cell invasiveness. FaDu cells seeded on upper membrane in HGF (0, 10, 30 ng/ml) ±increasing concentrations of EGCG. After 48-h incubation, plugged cells in 8-lm pore or cells attached to undersurface or membrane were counted and the attached cells in the lower section were stained with hematoxylin and were counted by light microscopy. 10 ng/ml (a) or 30 ng/ml (b) HGF stimulated cell invasion and this effect of HGF was inhibited by EGCG.
a marked increase of plasmin activity following 24 h of stimulation with HGF. HGF enhanced the plasmin activity in FaDu cells and this effect was inhibited by EGCG (Fig. 6). 3.7. EGCG inhibits HGF-activated Akt and Erk downstream pathways in FaDu cells Met/HGF signaling pathways have been known to be capable of activating a number of downstream signaling pathways, including extracellular signal-regulated kinase, src, and phosphatidylinositol 3-kinase pathway [12]. In order to assess the downstream signaling events induced
by HGF in FaDu cells, we treated the cells with 30 ng/ ml HGF, and cell lysates were prepared at regular intervals for up to 24 h and then analyzed for the activity of Erk and Akt. As shown in Fig. 7, Erk and Akt phosphorylation was increased in the HGF (30 ng/ml)-treated cells with strong activation within 5 min and a decrease of phosphorylation by 30 min post-treatment. Next, in order to investigate whether EGCG can block HGF signaling at the level of c-Met receptor activation and also aforementioned HGF-activated downstream Akt and Erk signaling pathway, we examined the phosphorylation activity of Met, Akt, and Erk following treatment with 30 ng/ml HGF for 24 h together with increasing concentrations of
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Fig. 5. Effect of EGCG on the expression (a) and invasion (b) of HGF-induced MMP-2 and -9 in FaDu cells. MMP-9 mRNA expression was significantly increased after HGF treatment for 24 h in a dose-dependent manner, but the expression of MMP-2 mRNA could not be amplified. EGCG inhibits expression and invasion of HGF-induced MMP-9 in FaDu cells in RT-PCR and zymogram, respectively.
Fig. 6. Effect of HGF and EGCG on uPA in FaDu cells. FaDu cells (3000 cells/well or 6000 cells/well) were inoculated into 96-well plates in DMEM that contained 10% FBS. After overnight incubation, HGF (0, 10, 30 ng/ml) in the presence and absence of increasing concentrations of EGCG (0, 1, 10 lM) were added to the wells, and the plates incubated further for 24 h. The cells were placed in 200 ll of reaction buffer that contained 50% (v/v) of 0.05 U/ml plasminogen. The mixtures were incubated at 37 °C in 5% CO2 for 3 h, and absorbance at 405 nm was measured in an automated spectrophotometric plate reader.
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Fig. 7. Effects of HGF on activation of Met, Akt and Erk. FaDu cells were deprived of serum overnight and then incubated for 24 h. Cells were then stimulated with 30 ng/ml HGF for 15 min. Met, Akt and Erk were analyzed by Western blotting using phosphorylated Met, Akt and Erk, respectively.
Fig. 8. Suppression of EGCG of HGF-activated the Met, Akt and Erk pathways. FaDu cells were deprived of serum overnight and then incubated for 24 h in the absence or presence of HGF (30 ng/ml) with the indicated concentrations of EGCG. Cell lysates were subjected to western analysis with antibodies to phosphorylated form of Met, to phosphorylated Akt and to phosphorylated Erk as well as with antibodies to total Met, Akt, or Erk.
EGCG (0.001–10 lM). The result showed that the phosphorylation of Met, Akt, and Erk was decreased or disappeared at concentrations of 5, 5, and 10 lM EGCG, respectively (Fig. 8).
4. Discussion Hepatocyte growth factor (HGF) was first discovered as a major factor of liver regeneration and is now recognized as a motogen, mitogen and morphogen [17]. Met is the only known functional
receptor for HGF, and HGF is the only natural ligand for Met [8]. Under normal conditions, HGF and Met play roles in embryonic development, epithelial-mesenchymal transition, angiogenesis and tissue regeneration, including the liver [7]. In addition to regulation of normal cell functions, recent studies have shown that it is involved in malignant cell transformation and stimulates the invasive characteristics of various cancer cells, such as hepatocellular carcinoma and pancreatic cancer [11–12]. In several clinical studies, a relationship
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between the concentration of HGF in serum or in cancer tissue and the progression of disease has been noted in patients with gastric, breast and lung cancer [18]. Previously, we reported that the expression of mRNA and protein of both HGF and c-Met was increased in head and neck SCC tissue compared to normal tissue [19]. Immunohistochemical staining of hypopharyngeal cancer tissue showed high expression of HGF and c-Met and the level of HGF was significantly increased in proportion to lymph node metastasis and clinical stage of the tumor [19]. These pre-clinical study implied that HGF may be associated also with tumor progression and invasion of head and neck SCC. Therefore, we have investigated in the present study whether HGF promoted proliferation, migration, and invasion of hypopharyngeal carcinoma cells in vitro situation, and the results showed that FaDu cells, hypopharyngeal carcinoma cells, have functional c-Met protein, and that HGF promotes autophosphorylation of c-Met, and demonstrated that HGF induces proliferation, colony dispersion, migration and invasion of cancer cells. Tumor invasion requires degradation of basement membranes, proteolysis of ECM, pseudopodial extension, and cell migration [20]. Basement membrane, which separates the epithelial and mesenchymal cell compartments and is made up of matrix macromolecules such as type IV collagen, laminin, and heparan sulfate proteoglycans, is the first barrier of the ECM against cancer invasion. A number of proteolytic enzymes, including MMPs and serine proteinases, are involved in this tumorhost interactions, such as degradation of underlying basement membrane. Of these basement membrane degrading enzymes, MMPs, especially activated forms of MMP-2 and MMP-9, are thought to play an important role in its degradation because of their ability to cleave the type IV collagen. Currently, there are 28 different types of MMP identified, which can be classified into eight subgroups, including six secretory and two membrane-bound classes [21]. MMPs are produced by cancer cells or through the induction of surrounding stromal cells. Regarding SCC of the tongue, Yoshizaki et al. reported that activation of MMP-2 predicts poor prognosis of patients [22], Jeon et al. demonstrated that MMP-9 expression is associated with nodal and distant metastasis in nasopharyngeal carcinoma [23], and Pacheco et al. also described a link between MMP-9 expression and head and neck SCC recurrence rate [24]. Therefore, elevated expression of
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MMP appears to be related to the invasion, aggressiveness and overall survival of patients with head and neck cancers. Serine protease, which is also important for tumor invasiveness and metastasis, is usually secreted from endothelial cells and served as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). tPA is usually involved in thrombolysis, whereas uPA is involved in the infiltration of carcinoma [25–26]. The 52-kDa uPA plays a major role in the decomposition of basement membranes, as the expression of uPA is increased in solid tumors. Activation of the uPA/uPAR/plasmin proteolytic network has been shown to play key roles in tumor invasion and dissemination of various malignancies [27–28]. For example, the role of the uPA fibrinolytic network in tumor malignancy has been demonstrated in uPA / mice, in which there was a dramatic reduction in the progression of chemically-induced malignant melanomas [29]. The pro-uPA form (inactive pro-enzyme) is synthesized and secreted from tumor cells and is transformed into active form by plasmin [30]. Activated uPA accelerates the conversion of plasminogen into plasmin. The elevated plasmin degrades the fibrin, fibronectin, proteoglycan, and laminin that compose the matrix around the tumor, activates type IV collagenases, and indirectly destroys type IV collagen, which is an important component of basement membranes [31]. Recently, the presence of uPA in tumor tissues has been proposed as a potential prognostic factor, and an enzyme-linked immunosorbent assay (ELISA) for uPA has been developed as a prognostic test for solid cancers [32]. In addition, the levels of uPA and uPAR expression serve as prognostic markers in various malignancies in which high levels of expression are often associated with a poor prognosis [33]. Thus, we examined the association between HGF and expressions of MMP and uPA in hypopharyngeal carcinoma cells, and observed that RT-PCR revealed a marked increase in the level of MMP-9 mRNA after HGF treatment for 24 h and zymography confirmed the increased MMP-9 activity in the 30 ng/ml HGF treatment group. In addition, HGF increased the activity of uPA in a dose-dependent manner. These results indicated that HGF enhanced the expression of MMP-9 and uPA in hypopharyngeal carcinoma cells. Next, we investigated the downstream signaling pathways in hypopharyngeal carcinoma cells,
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induced by HGF because no information has so far been available regarding Met signaling pathwayassociated with tumor invasion in these cells, although an earlier study found that downstream activation of the PI3-kinase/AKT and the Ras/Erk pathways is necessary for HGF-induced motility in other cancer cells [34]. Our present study also revealed that HGF induced sustained activation of the Akt and Erk pathways in hypopharyngeal carcinoma cells. Epidemiological and pre-clinical studies demonstrated that polyphenols derived from green tea possess profound chemopreventive and anti-tumor effects against various cancers. The major polyphenols in green tea are ()-epigallocatechin-3-gallate (EGCG), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC). Of these, EGCG is a major catechin found in green tea, and has been shown to induce apoptosis and inhibit proliferation in many tumor cell lines [35]. Recent work revealed that EGCG could modulate the cell signaling associated with angiogenesis, metastasis, and migration of cancer cells, such as prostate cancer, liver cancer and breast cancer [36]. Thus, we investigated whether EGCG can block aforementioned HGFinduced invasion in hypopharyngeal carcinoma cell. First, in order to determine if EGCG can inhibit HGF-induced motility and invasion, we performed wound healing and invasion assays, and found that EGCG was able to block HGF-induced motility at physiologically relevant concentration (1 lM). Bigelow et al. also observed that treatment with 5.0 l/l EGCG can inhibit the HGF-induced MMP, and that EGCG blocked the ability of HGF to induce cell motility and invasion in immortalized and tumorigenic breast epithelial cells [37]. We then examined whether EGCG blocks the expression of MMP and uPA which are closely associated with tumor invasion. EGCG has been shown to directly and indirectly repress MMP activity, MMP-2 and MMP-9 are remarkably inhibited by orally administered green tea polyphenols in the prostate in TRAMP mice [38], and EGCG (25–100 lmol/l) has also been shown to inhibit the MMP-2 and MMP-9 in endothelial cells [39]. Thus, it seems quite likely that EGCG could inhibit or delay cancer invasion, metastasis, and angiogenesis via modulation of MMPs. Jankun et al. showed that EGCG inhibits the activity of uPA [40]. With the use of computer-based molecular modeling, they
found that EGCG binds to urokinase, thereby blocking histidine 57 and serine residues of the urokinase catalytic triad and extending arginine 35 from a positively charged loop of urokinase [40]. Based on the above observation, the authors suggested that the anti-tumor effect of EGCG is mediated by inhibition of uPA, one of the most frequently overexpressed enzymes in human cancers. Our present study also demonstrated that EGCG blocked the HGF-induced MMP-9 and uPA activities in hypopharyngeal carcinoma cells at a physiologic relevant concentration. Finally, we examined whether EGCG blocks the downstream pathway of Akt and Erk which are activated by HGF, and demonstrated that EGCG was able to repress the HGF-induced increase of Met phosphorylation, as well as block activation of the downstream kinases, Akt and Erk. The pharmacokinetic studies in humans indicate approximately 1.0 lM as the maximally obtainable plasma concentration after single p.o. dose of EGCG35. Thus, it is important to consider whether blocking effect of EGCG on HGF-induced invasion and metastasis is accomplished at this physiologically relevant concentrations. Our study revealed that 1 lM EGCG is sufficient to block HGFinduced motility and invasion in hypopharyngeal cancer cell. In summary, our study showed that inhibition of HGF/Met by physiologic concentration of EGCG leads to decreased proliferation, migration and invasion of hypopharyngeal carcinoma cells. Thus, these findings indicate that EGCG may serve as a therapeutic agent to inhibit HGF-induced invasion in hypopharyngeal carcinoma patients, and future study on its clinical application seems to be worthwhile. Acknowledgement This research was supported by CCRB through the ‘‘GRRC” Project of Gyeonggi Provincial Government, Republic of Korea. References [1] C.H. Kim, J.H. Kim, H. Kahng, E.C. Choi, Change of Ecadherin by hepatocyte growth factor and effects on the prognosis of hypopharyngeal carcinoma, Ann. Surg. Oncol. 14 (2007) 1565–1574. [2] W.I. Wei, The dilemma of treating hypopharyngeal carcinoma: more or less, Arch. Otolaryngol. Head Neck Surg. 128 (2002) 229–232.
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