(-)-Epigallocatechin 3-gallate inhibits invasion by inducing the expression of Raf kinase inhibitor protein in AsPC‑1 human pancreatic adenocarcinoma cells through the modulation of histone deacetylase activity

  • Authors:
    • Sung Ok Kim
    • Mi Ryeo Kim
  • View Affiliations

  • Published online on: November 6, 2012     https://doi.org/10.3892/ijo.2012.1686
  • Pages: 349-358
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The aim of the present study was to assess whether (-)-epigallocatechin 3-gallate (EGCG) via epigenetic modifications, regulates Raf kinase inhibitor protein (RKIP) expression and invasive metastatic activity in AsPC-1 pancreatic adenocarcinoma cells. Basal levels of RKIP were examined in various human pancreatic cancer cell lines and MTT assay was used to assess cell viability. AsPC-1 cells were treated with EGCG with/without trichostatin A (TSA), as the positive control, for 24 h. The levels of RKIP and histone H3 induction were analyzed by immunoblot analysis. In order to determine the role of RKIP induction in NF-κB translocation and invasive metastatic activity in AsPC-1 cells, we examined NF-κB translocation, invasive metastatic parameters by RT-PCR, metastasis-related proteins by western blot analysis and matrix metalloproteinase (MMP)-2 and -9 activity by gelatin zymography. To validate RKIP induction through the extracellular signal regulated kinase (ERK) pathway, the cells were treated with U0126, an ERK inhibitor. Our results showed that EGCG induced RKIP upregulation via the inhibition of histone deacetylase (HDAC) activity which increased histone H3 expression and inhibited Snail expression, NF-κB nuclear translocation, MMP-2 and -9 activity and Matrigel invasion in AsPC-1 cells. The expression of E-cadherin in the cells was upregulated. The phosphorylation of ERK was decreased by RKIP induction following EGCG treatment. Furthermore, our results confirmed that U0126 treatment repressed ERK phosphorylation and induced RKIP expression. Taken together, our results strongly suggest that EGCG regulates RKIP/ERK/NF-κB and/or RKIP/NF-κB/Snail and inhibits invasive metastasis in the AsPC-1 human pancreatic adenocarcinoma cell line.

Introduction

Cancer is a serious public health issue worldwide. Pancreatic cancer (PC) is the fourth to fifth most common type of cancer in Western countries and has the highest case-fatality rate of any of the major cancer types (1). In Korea, the prevalence of PC is generally increasing by 1.7% per year and in 2008, the mortality rate from PC was ranked fifth (2). The only effective therapy is surgical excision, yet only 10 to 15% of patients have disease localized to the pancreas at the time of diagnosis (3). The majority of patients with PC succumb to the disease due to recurrence and metastatis. However, the etiology of PC is not yet fully understood. Previous studies have demonstrated that inflammation, which occurs as a response to most chronic illnesses, may play a significant role in this form of carcinogenesis (35). For example, pro-inflammatory cytokines induce the activation of NF-κB via the phosphorylation of the extracellular signal regulated kinase (ERK) pathway, increase Snail expression and the secretion of invasion-promorting matrix metalloproteinases (MMPs) (58). Therefore, controlling inflammation is a significant factor in decreasing the risk of developing human PC.

Protein acetylation influences cellular processes including transcriptional regulation via the recruitment of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) (9). Acetylation of specific lysine residues within the amino-terminal tails of nucleosomal histones is generally linked to chromatin disruption and the transcriptional activation of genes (10). HDAC inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells (10,11). Thus, dysregulation of the balance between protein acetylation and deacetylation is often associated with the initiation of tumorigenesis, cancer metastasis and other diseases.

The antioxidant and/or anti-inflammatory effects of dietary polyphenols (curcumin and resveratrol) have been shown to play a role in either controlling chromatin remodelling or NF-κB activation through the modulation of HDAC activity and subsequently, the expression of various genes (11,12). (-)-Epigallocatechin-3-gallate (EGCG), an antioxidant found in green tea, has been reported to inhibit HAT and/or HDAC activity by a number of studies (1318). EGCG decreases the hyperacetylation of p65 by directly inhibiting the activity of HAT enzymes. This hypoacetylation of p65 leads to the downregulation of NF-κB activity by diverse inflammatory signals (13). Although the regulation of NF-κB activity by histone deacetylation is a potential anti-metastatic mechanism in various types of cancer, the precise role of EGCG in human PC cells remains unclear. Thus, we hypothesized that EGCG inhibits invasive metastaic activity through the suppression of the NF-κB signaling pathway by inducing RKIP expression and inhibiting HDAC activity in human PC cells.

RKIP, a 23-kDa protein, was identified as a physiological inhibitor of the Raf-MEK-ERK pathway (6), it is a member of the phosphatidylethanolamine-binding protein (PEBP) family (19) and is well known for its metastasis suppressor function in various types of cancer, including prostate, breast, colorectal, cervical cancer and malanoma. The loss or diminution of RKIP expression has been associated with the increasing number of aggressive cancers (2024). Studies have suggested that NF-κB activation by RKIP diminution negatively interferes with NF-κB signaling (2527). Several genes with tumor suppressive functions are thought to be silenced by aberrant histone modifications (28). Inhibitors of HDAC, such as trichostatin A (TSA), sodium butyrate (NaBT) and suberoylanilide hydroxamic acid (SAHA), have displayed anti-cancer activities by inducing the expression of downregulated genes related to metastasis, motility and invasiveness (2831). First of all, we tested that RKIP expression in PC cell lines was induced by TSA through the modulation of HDAC activity. And then we examined whether EGCG can prevent PC metastasis by inhibiting HDAC activity and inducing RKIP expression, thus inhibiting cell signaling pathways involved in proliferation, survival and metastasis.

MMPs, a family of Zinc-dependent endopeptidases, are collectively capable of cleaving virtually all extracellular matrix (ECM) substrates, and play an important role in some physiological and pathological processes (32,33). MMPs have also been implicated as possible mediators of invasion and metastasis in breast cancer, colon cancer and melanoma cell lines (34,35). Since MMPs have many physiological functions in metastasis, the inhibition of the activity of MMPs holds great promise for the prevention or inhibition of metastasis. MMP-2 (gelatinases) is preferentially secreted from fibroblasts and various epithelial cells, while MMP-9 (gelatinases) is preferentially expressed by inflammatory cells (36), and both have been frequently associated with the invasive metastatic potential of tumor cells. However, the biochemical mechanisms underlying the EGCG-induced inhibition of metastasis and invasion are not clear in AsPC-1 PC cells.

Snail was identified as a transcription factor implicated in the epithelial-mesenchymal transition (EMT) during embryonic development (37). The increased expression of Snail in invasive cancer cells accompanied with the loss of E-cadherin expression has been reported in many types of human cancer, including melanoma, hepatocarcinoma and squamous cell carcinoma (3840). E-cadherin is a cell-cell adhesion molecule that is specifically expressed on the membranes of epithelial cells, and its decreased expression has been reported to play a role in the invasion and metastasis of cancers (41,42). The downregulation of E-cadherin expression has been explained by the DNA hypermethylation of its promoter region in breast and prostate cancer cells (43).

Therefore, in this study, we investigated the induction of RKIP expression by EGCG through the modification of histone deacetlyation. We also examined the effects of RKIP induction by EGCG on metastasis and invasive parameters, including MMP activity by gelatin zymography, Matrigel membrane invasion and metastasis-related proteins by western blot analysis using the AsPC-1 human adenocarcinoma metastatic cell line.

Materials and methods

Cell culture and cell viability

The human PC cell lines (Table I), MIA PaCa-2, AsPC-1, PANC-1 and BxPC-3, were obtained from the American Type Culture Collection (ATCC; (Rockville, MD) and cultured in DMBM and IMDM supplemented with 10% fetal bovine serum (FBS) and fetal calf serum (FCS) (Gibco BRL, Grand Island, NY) at 37°C in a humidified atmosphere containing 5% CO2. For the cell viability assay, the cells were plated at 1×104 cells/well in 200 μl medium containing 10 μM EGCG with/without 1 μM TSA (Sigma-Aldrich, St. Louis, MO) in a 96-well plate (Nunc™, Roskilde, Denmark). After incubation with concentrations of 0, 5, 10, 15 μM EGCG for 24 h, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the conversion of MTT to MTT-formazan by mitochondria. Cells were incubated with 1 mg/ml MTT (Chemicon, Temecula, CA) in PBS for 4 h at 37°C in 5% CO2. Isopropanol and hydrochloric acid were then added at final concentrations of 50% and 20 mM, respectively. The optical density at 570 nm (OD570) was determined using an ELISA plate reader (MN 3663 Molecular Devices Co., Sunnyvale, CA) with a reference wavelength of 630 nm.

Table I.

Comparisons of pancreatic cancer cell lines.a

Table I.

Comparisons of pancreatic cancer cell lines.a

OrganismGrowth propertiesDisease
MIA PaCa-2Homo sapiens (human)Adherent, single cells, loosely attached clustersCarcinoma
BxPC-3Homo sapiens (human)AdherentAdenocarcinoma
PANC-1Homo sapiens (human)AdherentEpithelioid carcinoma
AsPC-1Homo sapiens (human)AdherentAdenocarcinoma (metastatic cells)

a www.atcc.org (American Type Culture Collection).

MMP activity using gelatin zymography

Cell-free supernatant was collected and mixed with 2X sample buffer and then subjected to Novex Zymogram gels (Invitrogen, Camarillo, CA). After electrophoresis, the gels were washed twice at room temperature or 30 min in 2.5% Triton X-100, subsequently washed in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 1 μM ZnCl2, 0.02% NaN3 at pH 7.5 and incubated in this buffer at 37°C for 24 h. Thereafter, the gels were stained with 0.25% (w/v) Coomassie brilliant blue G-250 (Bio-Rad Laboratoties, Hercules, CA) and then lightly destained in water solution containing methanol and acetic acid for 1 h, respectively. The gelatinolytic activity was evidenced as clear bands (area of gelatin degradation) against the blue background of stained gelatin.

Matrigel invasion assay

In order to determine the effects of EGCG on AsPC-1 cell invasivness, the cells were exposed to 10 μM of EGCG for 6 h and the pre-treated cells were plated onto the apical side of the Matrigel-coated filters (Sigma-Aldrich) in serum-free medium containing either EGCG or DMSO. Medium containing 20% FBS was placed in the basolateral chamber to act as a chemoattractant. After 72 h, cells on the apical side were wiped off using a Q-tip. The cells on the bottom of the filter were stained with hematoxylin and eosin Y and then counted (three fields of each triplicate filter) using an inverted microscope (Nikon, Tokyo, Japan).

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using RNeasy (Qiagen, Valencia, CA). RNA (1 μg) was reverse-transcribed in a 20 μl reaction mixture using MMLV reverse transcriptase (Invitrogen). The cDNA was amplified in a 20 μl reaction mixture. The PCR conditions were as follows: 0.4 μM of each primer (Table II), 0.2 mM deoxynucleoside triphosphate mixture (Perkin-Elmer, Norwalk, CT, USA), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 1.0 U of Taq DNA polymerase (Perkin-Elmer). The reaction mixtures were incubated in a thermal controller (Model PTC-100; MJ Research, Ramsey, MN) for 35 cycles (denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 90 sec). The PCR products were resolved on 1% agarose gels containing ethidum bromide (EtBr). The intensities of the bands were measured using an image documentation system (ImageMaster VDS; Pharmacia, Uppsala, Sweden) with image analysis software (ImageMaste TotalLab; Pharmacia). The DNA size marker was run in parallel to validate the predicted sizes of the amplified bands (GeneRuler 1 kb DNA Ladder; MBI, Amherst, NY). GADPH, MMP-2 and -9, RKIP, Snail, E-cadherin and ERK primer sequences were designed using Beacon Designer software (Premier Biosoft, Palo Alto, CA) and synthesized by IDT (Skokie, IL).

Table II.

Oligonucleotides used in RT-PCR.

Table II.

Oligonucleotides used in RT-PCR.

GenePrimer sequence
GAPDH
  Sense5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′
  Antisense5′-AGC CTT CTC CAT GGT GGT GAA GAC-3′
RKIP
  Sense5′- CAC AAT GTG ATT TTA TGG T -3′
  Antisense5′- TCT TCA TTC AGG TTT CTA T -3′
E-cadherin
  Sense5′- GAA CAG CAC GTA CAC AGC CCT-3′
  Antisense5′- GCA GAA GTG TCC CTG TTC CAG-3′
Snail
  Sense5′- TAT GCT GCC TTC CCA GGC TTG-3′
  Antisense5′- ATG TGC ATC TTG AGG GCA CCC-3′
MMP-2
  Sense5′-CAG GCT CTT CTC CTT TCG CAA C-3′
  Antisense5′-AAG CCA CGG CTT GGT TTT CCT C-3′
MMP-9
  Sense5′-TGG GCT ACG TGA CCT ATG ACC AT-3′
  Antisense5′-GCC CAG CCC ACC TCC ACT CCT C-3′
Immunoblot analysis

Western immunoblots were prepared to analyze the protein levels. After pre-treatment with the ERK inhibitor, U0126, for 1 h and treatment with/without EGCG for 24 h, the cells were harvested and then proteins were extracted with NP-40 protein lysis buffer compositions; 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 10 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 0.2 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, protease inhibitor and 1 mM PMSF. Cytoplasmic and nuclear extracts were prepared using NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL). Quantification of protein concentration was carried out using the Bradford method (Bio-Rad protein assay reagent) and total protein was resuspended in Laemmli sample buffer containing 5% β-mercaptoethanol and heated at 65°C for 10 min. Aliquots containing ∼20–50 μg of total cell proteins were resolved on 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Amersham, Arlington Heights, IL). Membranes were blocked in 5% non-fat milk (w/v) in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h at room temperature and the membranes were subjected to immunoblot analysis with the desired antibodies (Table III). After an overnight incubation at 4°C, the membranes were washed in TBST and incubated with the appropriate peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were developed using chemiluminescence according to the enhanced chemiluminescence western blotting detection reagent (Pierce).

Table III.

List of antibodies used in this study.

Table III.

List of antibodies used in this study.

AntibodyDilutionSpecies originCompany
MMP-21:500Rabbit polyclonalSanta Cruz Biotechnology
MMP-91:500Rabbit polyclonalSanta Cruz Biotechnology
ERK1:1000Rabbit polyclonalCell signaling
pERK1:1000Rabbit polyclonalCell signaling
NF-κB1:1000Rabbit polyclonalSanta Cruz Biotechnology
IκB1:1000Rabbit polyclonalSanta Cruz Biotechnology
p651:500Mouse polyclonalSanta Cruz Biotechnology
p501:500Rabbit polyclonalSanta Cruz Biotechnology
Snail1:500Rabbit polyclonalAbcam
E-cadherin1:500Mouse monoclonalSanta Cruz Biotechnology
Lamin B1:1000Rabbit polyclonalSanta Cruz Biotechnology
Actin1:1000Mouse monoclonalSigma
Statistical analysis

All data are presented as the means ± SD. Statistical analysis (Student’s t-test) was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). Densitometry was performed using L process V2.01 and Muti Gauge V2.02 (Fuji Film, Stenford, USA). A value of *p<0.05 was considered to indicate a statistically significant difference. All the results presented in the figures shown in this study were obtained from at least three independent experiments.

Results

RKIP expression levels differ in human PC cell lines

The levels of baseline RKIP were investigated in the PC cell lines, MIA PaCa-2, PANC-1, AsPC-1 and BxPC-3. As shown in Fig. 1, the order of the basal levels of RKIP between the cell lines was: MIA PaCa-2 > PANC-1 > BxPC-3 > AsPC-1. To investigate whether RKIP expression was induced by EGCG treatment, we selected the AsPC-1 cell line, which had the lowest RKIP level of all the human PC cell lines. For further investigation, we examined the effect of EGCG on the viability of AsPC-1 cells by MTT assay. There was no toxicity up to 10 μM EGCG (usual EGCG treatment, 15 μM). The dose of 10 μM EGCG with/without TSA was used for the following experiment.

RKIP expression in AsPC-1 cells is upregulated by EGCG treatment

As shown in Fig. 2, treatment with 10 μM EGCG, a major polyphenol in green tea, for 24 h induced RKIP expression in the AsPC-1 cells compared to the control cells. In order to confirm whether RKIP regulation in PC is due, in part, to histone modifications we performed the same experiments while causing RKIP induction with TSA, a known HDAC inhibitor. TSA induced RKIP expression and the effects were synergistic to those of EGCG. Histone H3 expression was significantly increased in the cells in which RKIP expression was induced compared to the control cells. Therefore, RKIP induction in AsPC-1 cells treated with EGCG is due, in part, to HDAC modifications.

EGCG inhibits translocation of NF-κB into the nucleus in AsPC-1 cells

The NF-κB signaling pathway is considerably involved in cancer cell metastasis and invasion. To investigate whether EGCG inhibits NF-κB translocation in AsPC-1 cells, cytosolic and nuclear fractions were isolated in AsPC-1 cells. As shown in Fig. 3A and B, treatment with EGCG decreased the levels of p65 and p50 proteins in the nuclear fractions and elevated the levels of NF-κB and IκB in the cytosolic fractions. These results indicte that the metastasis and invasion of cancer cells are inhibited by the regulation of NF-κB nuclear translocation. Therefore, we suggest that EGCG exerts anti-metastatic and anti-invasion activities through the inhibition of NF-κB nuclear translocation by the induction of RKIP and the inhibition of HDAC.

EGCG inhibits MMP activity and invasion in AsPC-1 cells

To investigate whether EGCG suppresses the activity of MMP-2 and -9, gelatin zymography was performed on the AsPC-1 cells treated with EGCG. The activities of MMP-2 and -9 were decreased in the EGCG-treated cells compared to the control cells (Fig. 4A). EGCG had a greater inhibitory effect on MMP-9 activity than on MMP-2 activity. MMP activity is considered to be critically involved in cancer cell invasion. We then assessed the effects of EGCG on the invasiveness of AsPC-1 cells using Matrigel chamber invasion assay. The results of the Matrigel invasion assay (Fig. 4B) showed that the invasive capability of the AsPC-1 cells treated with EGCG was decreased compared to that of the control cells. These results indicate that EGCG inhibits the metastasis and invasion of human PC cells through HDAC modulation and the repression of MMP activity.

EGCG regulates expression levels of metastasis-related genes in AsPC-1 cells

To elucidate the mechanism by which EGCG inhibits metastasis and invasion, we examined the levels of metastasis-related genes, such as MMP-2 and -9, Snail and E-cadherin, using RT-PCR and western blot analysis. As shown in Fig. 5A and B, mRNA and protein levels of MMP-2 and -9, pERK and Snail were significantly downregulated in the EGCG-treated AsPC-1 cells compared to those of the control cells. The level of E-cadherin, a Snail nuclear translocation inhibitor, was markedly increased by EGCG treatment. As RKIP regulates the activation of NF-κB via the MEK/ERK signaling pathway (44), we investigated whether treatment with EGCG inhibits the phosphorylation of ERK. As shown in Fig. 5C, the treatment of cells with EGCG suppressed the phosphorylation of ERK compared to the control cells and increased RKIP expression. These results prove that the induction of RKIP expression by EGCG inhibits the phosphorylation of ERK and NF-κB activation and decreases Snail expression. Therefore, EGCG as a HDAC inhibitor, can prevent the invasive metastasis of human PC cells.

Discussion

PC is the most severe gastrointestinal malignancy due to its propensity for local invasion and early metastasis (45,46). Modulation of histone deacetylation by a HDAC inhibitor has been associated with transcriptional repression, an important epigenetic event that plays a critical role in inflammation (47). Inhibitors of HDACs, such as TSA, have been shown to display anti-cancer activities by inducing the expression of otherwise silenced genes (28). In this study, our objective was to treat PC cells with EGCG and investigate its effects on RKIP levels within the cells and determine whether an association exists between RKIP levels and cell metastasis.

RKIP was initially identified as a PEBP in the bovine brain. It was later identified as a protein that inhibits the Raf kinase activation of MEK (47). RKIP has a multi-functional role, exerting an inhibitory effect on pathways involved in cell proliferation, survival and metastasis (4749). Beach et al, as well as others have reported that the expression levels of RKIP proteins progressively decrease in breast and prostate cancer cell lines of increasing metastatic potential (48,50). A decrease in RKIP expression can promote invasion in other types of cancer and the induction of RKIP expression with HDAC inhibitors exerts anti-cancer effects in the majority of cancer cells by suppressing the activity of metastasis-related genes (26,49). In this study, treatment with EGCG caused the induction of RKIP expression in PC cells. Our results confirmed that the regulation of RKIP expression in PC cells was due, in part, to histone modifications following treatment with HDAC inhibitors, such as TSA. EGCG suppressed the invasive and metastatic abilities of the PC cells. These effects correlated with RKIP expression. In accordance with our results, Fujimori et al also reported that the loss of RKIP expression was associated with tumor progression and poor survival. Negative RKIP expression combined with positive p-ERK expression is an independent predictor of poor outcomes in patients with gastric cancer (51). Evidence has emerged that RKIP can function as a suppressor of cancer metastasis.

EGCG, a green tea-derived polyphenol, has been reported to exert an inhibitory effect on NF-κB activity in various human malignancies (1316). However, the mechanisms behind the EGCG regulation of NF-κB-dependent activation in PC cells remain unclear (whether EGCG acts by regulating the stability or inhibiting the activity of invasive metastatic proteins). In this study, to determine whether RKIP is repressed by HDAC in PC cells, we investigated the effects of EGCG on RKIP expression levels in AsPC-1 cells. EGCG treatment led to the induction of RKIP expression within 24 h and inhibited NF-κB nuclear translocation by suppressing ERK phosphorylation in the cells. The same experiments performed using TSA in the cells showed similar results at 1 μM TSA concentration. These results confirm that EGCG acts as a HDAC inhibitor to suppress pro-inflammatory events, such as NF-κB activation. Tang et al and Yeung et al reported that RKIP negatively regulates the Raf/MEK/ERK and NF-κB pathways (5,6). Li et al reported that EGCG, in combination with the HDAC inhibitor, TSA, led to the synergistic epigenetic reactivation of estrogen receptor-α (ERα) in ERα-negative breast cancer cells (16).

MMPs are considered important proteolytic enzymes during organ development and tissue regeneration. However, they also play important roles in cancer invasion and metastasis. In particular, MMP-2 and -9 play important roles in tumor invasion and angiogenesis; therefore, tumor metastasis may be inhibited by blocking MMP synthesis and activity (32,33). Hazgui et al(52) reported that the anti-metastatic activity of a polyphenolic constituent of green tea, EGCG, was associated with a reduction in MMP-2 activity. In a study by Shankar et al(53), it was reported that EGCG-treated mice with PC had a significantly reduced ERK activity, invasion, metastasis and angiogenesis. Takada et al(54) demonstrated that indole-3-carbinol, a phytochemical agent, inhibited the cell proliferation and invasion of leukemic cells by inhibiting MMP-9 activity. Additionally, Jin et al(55) reported that Snail plays a critical role in tumor growth and metastasis of ovarian carcinoma through the regulation of MMP activity. These results may explain why the expression of MMP-2 and -9 is elevated in parametrial invasion. Our results indicated a marked inhibition of MMP-2 and -9 mRNA, protein levels and activities following treatment with EGCG with/without TSA. Snail and E-cadherin expression has also been related to cancer metastasis and MMP activity. In accordance with our results, Miyoshi et al suggested that Snail, a zinc-finger transcription factor, represses E-cadherin expression and induces MMP expression, which causes vascular invasion and intrahepatic metastasis in primary hepatocellular carcinoma tumors (56). Batlle et al reported that Snail regulates other genes required for the scattering process, in conjunction with E-cadherin downregulation (38).

Collectively, these data demonstrate that the green tea-derived polyphenol, EGCG, regulates RKIP expression by modulating histone deacetylation. The results from the present study demonstrate that EGCG treatment leads to the inactivation of NF-κB and downstream target genes, such as Snail, as well as to the inactivation of MMPs, resulting in the inhibition of the invasive metastatic ability of PC cells. Therefore, we suggest that EGCG can potentially be a used as a therapeutic agent for PC. Further studies are required in order to gain a deeper understanding of the efficacy of EGCG in PC prevention. Future studies should focus on elucidating the molecular mechanisms involved by using in vivo methods with proper animal models of PC.

References

1. 

Jemal A, Siegel R, Xu J and Ward E: Cancer statistics 2010. CA Cancer J Clin. 60:277–300. 2010. View Article : Google Scholar

2. 

Korea Ministry of Health and Welfare: Annual report of cancer statistics in Korea in 2008. http://mw.go.krurisimplehttp://mw.go.kr. 2011

3. 

Warshaw AL, Gu ZY, Wittenberg J and Waltman AC: Preoperative staging and assessment of resectability of pancreatic cancer. Arch Surg. 125:230–233. 1990. View Article : Google Scholar : PubMed/NCBI

4. 

Whitcomb DC: Inflammation and Cancer V. Chronic pancreatitis and pancreatic cancer. Am J Physiol Gastrointest Liver Physiol. 287:G315–G319. 2004. View Article : Google Scholar : PubMed/NCBI

5. 

Tang H, Park S, Sun SC, Trumbly R, Ren G, Tsung E and Yeung KC: RKIP inhibits NF-kappaB in cancer cells by regulating upstream signaling components of the IkappaB kinase complex. FEBS Lett. 4:662–668. 2009.PubMed/NCBI

6. 

Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, Sedivy JM and Kolch W: Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. Mol Cell Biol. 20:3079–3085. 2000. View Article : Google Scholar : PubMed/NCBI

7. 

Barbera MJ, Puig I, Dominguez D, Julien-Grille S, Guaita-Esteruelas S, Peiró S, Baulida J, Francí C, Dedhar S, Larue L and García de Herreros A: Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells. Oncogene. 23:7345–7354. 2004. View Article : Google Scholar : PubMed/NCBI

8. 

Munshi HG and Stack MS: Reciprocal interactions between adhesion receptor signaling and MMP regulation. Cancer Metastasis Rev. 25:45–56. 2006. View Article : Google Scholar : PubMed/NCBI

9. 

Gregory PD, Wagner K and Horz W: Histone acetylation and chromatin remodeling. Exp Cell Res. 265:195–202. 2001. View Article : Google Scholar : PubMed/NCBI

10. 

Struhl K: Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12:599–606. 1998. View Article : Google Scholar : PubMed/NCBI

11. 

Gray SG and Teh BT: Histone acetylation/deacetylation and cancer: an ‘open’ and ‘shut’ case? Curr Mol Med. 1:401–429. 2001.

12. 

Hayden MS and Ghosh S: Signaling to NF-κB. Genes Dev. 18:2195–2224. 2004.

13. 

Choi KC, Jung MG, Lee YH, Yoon JC, Kwon SH, Kang HB, Kim MJ, Cha JH, Kim YJ, Jun WJ, Lee JM and Yoon HG: Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibits EBV-induced B lymphocyte transformation via suppression of RelA acetylation. Cancer Res. 69:583–592. 2009. View Article : Google Scholar : PubMed/NCBI

14. 

Thakur VS, Gupta K and Gupta S: Green tea polyphenols increase p53 transcriptional activity and acetylation by suppressing class I histone deacetylases. Int J Oncol. 41:353–361. 2012.PubMed/NCBI

15. 

Choudhury SR, Balasubramanian S, Chew YC, Han B, Marquez VE and Eckert RL: (-)-Epigallocatechin-3-gallate and DZNep reduce polycomb protein level via a proteasome-dependent mechanism in skin cancer cells. Carcinogenesis. 32:1525–1532. 2011. View Article : Google Scholar : PubMed/NCBI

16. 

Li Y, Yuan YY, Meeran SM and Tollefsbol TO: Synergistic epigenetic reactivation of estrogen receptor-α (ERα) by combined green tea polyphenol and histone deacetylase inhibitor in ERα-negative breast cancer cells. Mol Cancer. 9:274–286. 2010.PubMed/NCBI

17. 

Yun JM, Jialal I and Devaraj S: Effects of epigallocatechin gallate on regulatory T cell number and function in obese v. lean volunteers. Br J Nutr. 103:1771–1777. 2010. View Article : Google Scholar : PubMed/NCBI

18. 

Fang M, Chen D and Yang CS: Dietary polyphenols may affect DNA methylation. J Nutr. 137:223S–228S. 2007.PubMed/NCBI

19. 

Yeung K, Seitz T, Li S, Janosch P, McFerran B, Kaiser C, Fee F, Katsanakis KD, Rose DW, Mischak H, Sedivy JM and Kolch W: Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature. 401:173–177. 1999. View Article : Google Scholar : PubMed/NCBI

20. 

Ren G, Baritaki S, Marathe H, Feng J, Park S, Beach S, Bazeley PS, Beshir AB, Fenteany G, Mehra R, Daignault S, Al-Mulla F, Keller E, Bonavida B, de la Serna I and Yeung KC: Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer. Cancer Res. 72:3091–3104. 2012. View Article : Google Scholar : PubMed/NCBI

21. 

Minn AJ, Bevilacqua E, Yun J and Rosner MR: Identification of novel metastasis suppressor signaling pathways for breast cancer. Cell Cycle. 11:2452–2457. 2012. View Article : Google Scholar : PubMed/NCBI

22. 

Karamitopoulou E, Zlobec I, Panayiotides I, Patsouris ES, Peros G, Rallis G, Lapas C, Karakitsos P, Terracciano LM and Lugli A: Systematic analysis of proteins from different signaling pathways in the tumor center and the invasive front of colorectal cancer. Hum Pathol. 42:1888–1896. 2011. View Article : Google Scholar : PubMed/NCBI

23. 

Hu CJ, Zhou L, Zhang J, Huang C and Zhang GM: Immunohistochemical detection of Raf kinase inhibitor protein in normal cervical tissue and cervical cancer tissue. J Int Med Res. 39:229–237. 2011. View Article : Google Scholar : PubMed/NCBI

24. 

Zebisch A, Wölfler A, Fried I, Wolf O, Lind K, Bodner C, Haller M, Drasche A, Pirkebner D, Matallanas D, Rath O, Blyth K, Delwel R, Taskesen E, Quehenberger F, Kolch W, Troppmair J and Sill H: Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia. Leukemia. 26:1842–1849. 2012. View Article : Google Scholar : PubMed/NCBI

25. 

Wu K and Bonavida B: The activated NF-kappaB-Snail-RKIP circuitry in cancer regulates both the metastatic cascade and resistance to apoptosis by cytotoxic drugs. Crit Rev Immunol. 29:241–254. 2009. View Article : Google Scholar : PubMed/NCBI

26. 

Yeung KC, Rose DW, Dhillon AS, Yaros D, Gustafsson M, Chatterjee D, McFerran B, Wyche J, Kolch W and Sedivy JM: Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation. Mol Cell Biol. 21:7207–7217. 2001. View Article : Google Scholar : PubMed/NCBI

27. 

Karin M and Ben-Neriah Y: Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol. 18:621–663. 2000.PubMed/NCBI

28. 

Emonds E, Fitzner B and Jaster R: Molecular determinants of the antitumor effects of trichostatin A in pancreatic cancer cells. World J Gastroenterol. 16:1970–1978. 2010. View Article : Google Scholar : PubMed/NCBI

29. 

Farrow B, Rychahou P, O’Connor KL and Evers BM: Butyrate inhibits pancreatic cancer invasion. J Gastrointest Surg. 7:864–870. 2003. View Article : Google Scholar : PubMed/NCBI

30. 

Hardtner C, Multhoff G, Falk W and Radons J: (-)-Epigallocatechin-3-gallate, a green tea-derived catechin, synergizes with celecoxib to inhibit IL-1-induced tumorigenic mediators by human pancreatic adenocarcinoma cells Colo357. Eur J Pharmacol. 684:36–43. 2012. View Article : Google Scholar

31. 

Choi KC, Lee YH, Jung MG, Kwon SH, Kim MJ, Jun WJ, Lee J, Lee JM and Yoon HG: Gallic acid suppresses lipopolysaccharide-induced nuclear factor-kappaB signaling by preventing RelA acetylation in A549 lung cancer cells. Mol Cancer Res. 7:2011–2021. 2009. View Article : Google Scholar

32. 

Zucker S, Cao J and Chen WT: Critical appraisal of the use of matrix metalloproteinase inhibitors in cancer treatment. Oncogene. 19:6642–6650. 2000. View Article : Google Scholar : PubMed/NCBI

33. 

Mook OR, Frederiks WM and Van Noorden CJ: The role of gelatinases in colorectal cancer progression and metastasis. Biochim Biophys Acta. 1705:69–89. 2004.PubMed/NCBI

34. 

Duffy MJ, Maguire TM, Hill A, McDermott E and O’Higgins N: Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res. 2:252–257. 2000. View Article : Google Scholar : PubMed/NCBI

35. 

Vihinen P, Ala-aho R and Kähäri VM: Matrix metalloproteinases as therapeutic targets in cancer. Curr Cancer Drug Targets. 5:203–220. 2005. View Article : Google Scholar : PubMed/NCBI

36. 

Gibbs DF, Warner RL, Weiss SJ, Johnson KJ and Varani J: Characterization of matrix metalloproteinases produced by rat alveolar macrophages. Am J Respir Cell Mol Biol. 20:1136–1144. 1999. View Article : Google Scholar : PubMed/NCBI

37. 

Yokoyama K, Kamata N, Hayashi E, Hoteiya T, Ueda N, Fujimoto R and Nagayama M: Reverse correlation of E-cadherin and snail expression in oral squamous cell carcinoma cells in vitro. Oral Oncol. 37:65–71. 2001. View Article : Google Scholar : PubMed/NCBI

38. 

Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J and García De Herreros A: The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumor cells. Nat Cell Biol. 2:84–89. 2000. View Article : Google Scholar : PubMed/NCBI

39. 

Jiao W, Miyazaki K and Kitajima Y: Inverse correlation of E-cadherin and Snail expression in hepatocellular cell lines in vitro and in vivo. Br J Cancer. 86:98–101. 2002. View Article : Google Scholar : PubMed/NCBI

40. 

Poser I, Domínguez D, de Herreros AG, Varnai A, Buettner R and Bosserhoff AK: Loss of E-cadherin expression in melanoma cells involves up-regulation of the transcriptional repression Snail. J Biol Chem. 276:24661–24666. 2001. View Article : Google Scholar : PubMed/NCBI

41. 

Luo J, Lubaroff DM and Hendrix MJ: Suppression of prostate cancer invasive potential and matrix metalloproteinase activity by E-cadherin transfection. Cancer Res. 59:3552–3556. 1999.PubMed/NCBI

42. 

Oka H, Shiozaki H, Kobayashi K, Inoue M, Tahara H, Kobayashi T, Takatsuka Y, Matsuyoshi N, Hirano S, Takeichi M and Mori T: Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis. Cancer Res. 53:1696–1701. 1993.PubMed/NCBI

43. 

Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, Isaacs WB, Pitha PM, Davidson NE and Baylin SB: E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res. 55:5195–5199. 1995.PubMed/NCBI

44. 

Odabaei G, Chatterjee D, Jazirehi AR, Goodglick L, Yeung K and Bonavida B: Raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis. Adv Cancer Res. 91:169–200. 2004. View Article : Google Scholar : PubMed/NCBI

45. 

Eckel F, Schneider G and Schmid RM: Pancreatic cancer: a review of recent advances. Expert Opin Investig Drugs. 15:1395–1410. 2006. View Article : Google Scholar : PubMed/NCBI

46. 

Schneider G, Siveke JT, Eckel F and Schmid RM: Pancreatic cancer: basic and clinical aspects. Gastroenterology. 128:1606–1625. 2005. View Article : Google Scholar : PubMed/NCBI

47. 

Escara-Wilke J, Yeung K and Keller ET: Raf kinase inhibitor protein (RKIP) in cancer. Cancer Metastasis Rev. Jun 9–2012.(Epub ahead of print).

48. 

Klysik J, Theroux SJ, Sedivy JM, Moffit JS and Boekelheide K: Signaling crossroads: the function of Raf kinase inhibitory protein in cancer, the central nervous system and reproduction. Cell Signal. 20:1–9. 2008. View Article : Google Scholar : PubMed/NCBI

49. 

Zeng L, Imamoto A and Rosner MR: Raf kinase inhibitory protein (RKIP): a physiological regulator and future therapeutic target. Expert Opin Ther Targets. 12:1275–1287. 2008. View Article : Google Scholar : PubMed/NCBI

50. 

Beach S, Tang H, Park S, Dhillon AS, Keller ET, Kolch W and Yeung KC: Snail is a repressor of RKIP transcription in metastatic prostate cancer cells. Oncogene. 27:2243–2248. 2008. View Article : Google Scholar : PubMed/NCBI

51. 

Fujimori Y, Inokuchi M, Takagi Y, Kato K, Kojima K and Sugihara K: Prognostic value of RKIP and p-ERK in gastric cancer. J Exp Clin Cancer Res. 31:30–38. 2012. View Article : Google Scholar : PubMed/NCBI

52. 

Hazgui S, Bonnomet A, Nawrocki-Raby B, Milliot M, Terryn C, Cutrona J, Polette M, Birembaut P and Zahm JM: Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells. Respir Res. 9:33–46. 2008. View Article : Google Scholar : PubMed/NCBI

53. 

Shankar S, Ganapathy S, Hingorani SR and Srivastava RK: EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer. Front Biosci. 13:440–452. 2008. View Article : Google Scholar : PubMed/NCBI

54. 

Takada Y, Andreeff M and Aggarwal BB: Indole-3-carbinol suppresses NF-κB and IκBα kinase activation, causing inhibition of expression of NF-κB-regulated antiapoptotic and metastatic gene products and enhancement of apoptosis in myeloid and leukemia cells. Blood. 106:641–649. 2005.

55. 

Jin H, Yu Y, Zhang T, Zhou X, Zhou J, Jia L, Wu Y, Zhou BP and Feng Y: Snail is critical for tumor growth and metastasis of ovarian carcinoma. Int J Cancer. 126:2102–2111. 2010.PubMed/NCBI

56. 

Miyoshi A, Kitajima Y, Kido S, Shimonishi T, Matsuyama S, Kitahara K and Miyazaki K: Snail accelerates cancer invasion by upregulating MMP expression and is associated with poor prognosis of hepatocellular carcinoma. Br J Cancer. 92:252–258. 2005.PubMed/NCBI

Related Articles

Journal Cover

January 2013
Volume 42 Issue 1

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Kim, S.O., & Kim, S.O. (2013). (-)-Epigallocatechin 3-gallate inhibits invasion by inducing the expression of Raf kinase inhibitor protein in AsPC‑1 human pancreatic adenocarcinoma cells through the modulation of histone deacetylase activity. International Journal of Oncology, 42, 349-358. https://doi.org/10.3892/ijo.2012.1686
MLA
Kim, S. O., Kim, M. R."(-)-Epigallocatechin 3-gallate inhibits invasion by inducing the expression of Raf kinase inhibitor protein in AsPC‑1 human pancreatic adenocarcinoma cells through the modulation of histone deacetylase activity". International Journal of Oncology 42.1 (2013): 349-358.
Chicago
Kim, S. O., Kim, M. R."(-)-Epigallocatechin 3-gallate inhibits invasion by inducing the expression of Raf kinase inhibitor protein in AsPC‑1 human pancreatic adenocarcinoma cells through the modulation of histone deacetylase activity". International Journal of Oncology 42, no. 1 (2013): 349-358. https://doi.org/10.3892/ijo.2012.1686