Article pubs.acs.org/JAFC
Inhibition of the β‑Catenin/Tcf Signaling by Caffeoylquinic Acids in Sweet Potato Leaf through down Regulation of the Tcf‑4 Transcription Junsei Taira,* Masatsugu Uehara, Eito Tsuchida, and Wakana Ohmine Department of Bioresource Technology, Okinawa National College of Technology, 905 Henoko, Nago City, Okinawa, 905-2192 Japan ABSTRACT: Sweet potato leaves contain the highest levels of functional polyphenols. In this study the effects of the sweet potato leaf extract and its contents, such as mono (3, 4, and 5)-caffeoylquinic acid (CQA), di-CQA (4,5-diCQA, 3,5-diCQA, and 3,4-diCQA) and caffeic acid (CA), were evaluated on the β-catenin/Tcf-4 signaling in human colorectal cancer HCT116 cells. The extract and the CQA derivatives inhibited the β-catenin/Tcf-4 signaling, and the inhibition of the di-CQA (with two caffeoyl groups) was higher than that of the mono-CQA (one-caffeoyl group) and CA, suggesting that the caffeoyl structure in the presence of a catechol group plays a significant role in interfering with the β-catenin/Tcf-4 signaling. In addition, the CQA derivatives had no effect on the β-catenin protein expression, but all test compounds inhibited the expression of the Tcf-4 transcription, and the inhibition of the di-CQA derivatives was stronger than those of the mono-CQA derivatives as well as the βcatenin/Tcf-4 transcriptional activity. These compounds can modulate the downstream Wnt signaling pathway, suggesting that sweet potato leaves can be a protective food for colorectal cancer. KEYWORDS: caffeoylquinic acid, HCT 116 cells, colorectal cancer, β-catenin/Tcf signaling, sweet potato
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β-catenin signaling pathway.13,14 These active compounds have shown antioxidant activity; thus, caffeoylquinic acids will be expected to have a similar activity. This study was designed to confirm whether caffeoylquinic acid derivatives can modulate βcatenin/Tcf-4 signaling.
INTRODUCTION Recent studies have demonstrated that sweet potato (Ipomoea batatas L.) leaves contain the highest levels of polyphenols compared to other commercial vegetables.1,2 The leaves were found to be an inhibitor of the growth of colon and stomach cancer cells, leukemia, and viruses3,4 and to reduce the effects of diabetes in humans.5 The sweet potato leaves contain phenolic compounds, such as caffeoylquinic acid (CQA) derivatives, a family of esters formed from certain cinnamic acids and quinic acid, including the mono-CQA (chlorogenic acid derivatives) and di-CQA, such as 3,5-diCQA, 3,4-diCQA, and 4,5diCQA.6−8 The CQA derivatives are the main sweet potato polyphenols and they have an anti-LDL oxidation activity, DPPH-radical scavenging action, and antimutagen and antitumor activities.8−11 Colorectal cancer is a human malignance as well as stomach and lung cancer which are causes of human death. The incidence tends to increase due to changing Japanese dietary habits. Recent studies have indicated that in most of the colorectal cancers associated with the aberrant Wnt signaling pathway, the expression of β-catenin plays critical roles in cell proliferation and differentiation. The glycogen synthase kinase (GSK)-3β/Axin complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. When the β-catenin destruction complex is inhibited, a pool of cytoplasmic β-catenin stabilizes and is able to enter the nucleus and interact with the T-cell factor/lymphoid enhancer-binding factor (Tcf/Lef) family transcription factors to promote a specific gene expression and the signaling is an early progression event in colorectal cancer.12 In previous studies over the past decade, several naturally occurring dietary compounds, such as flavonoids, curcumin, and the tea polyphenol epigallocatechin-3-gallate (EGCG), have been shown to target intermediates in the Wnt/ © 2013 American Chemical Society
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MATERIALS AND METHODS
Materials. Caffeic acid (CA) and 3-caffeoylquinic acid were obtained from the Wako Pure Chemical Co. (Osaka, Japan). FuGENE 6 as a transfection reagent and the Lucyferase Assay System containing the luciferase substrate and lysis buffer to assess the reporter assay were obtained from the Promega Corporation (CA). Polyclonal antiβ-catenin (mouse monoclonal antibody, BD Biosciences, CA) and Tcf4 monoclonal antibody (mouse monoclonal antibody, Exalpha Biologicals, Inc., MA) were used to detect the protein expression. Isolation of CQA Derivatives. The CQA derivatives were isolated from the methanol (MeOH) extract of the sweet potato leaf as previously reported.8,9 Briefly, the MeOH extract was partitioned between dichloromethane and H2O (50/50 v/v) and the H2O fraction was continuously partitioned with n-BuOH. The H2O fraction was freeze-dried, then separated by HPLC (Agilent 1200, Agilent Technologies, CA) with an ODS-column (COAMOSIL Packed column 5C18-MS-II, 10 I.D. × 250 mm, Nacalai Tesque, Tokyo, Japan), and monitored at 326 nm at the flow rate of 2.0 mL/min at 40 °C. The mobile phase consisting of a formic acid aqueous solution (0.5%) and acetonitrile was carried out by a linear gradient to 2, 45, and 100% for 15, 50, and 60 min, respectively. Each peak of the mono (4 and 5)-CQA and di-CQA, such as 4,5- diCQA, 3,5-diCQA, and 3,4diCQA, was collected by a fraction collector (Analyte FC, Agilent Technologies). These compounds were used in the β-catenin/Tcf-4 Received: Revised: Accepted: Published: 167
October 1, 2013 December 2, 2013 December 5, 2013 December 5, 2013 dx.doi.org/10.1021/jf404411r | J. Agric. Food Chem. 2014, 62, 167−172
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reporter assay and as the standard substances for determining the quantity of the content in the sweet potato leaf extract. Sample Preparation. The sweet potato leaf powder (1 g) was extracted with distilled water (50 mL) at 85 °C for 45 min. The extract was filtered and then freeze-dried (Tokyo Rikakiki Co. Ltd., Tokyo, Japan). The sample (430.1 mg) dissolved in MQ-water (1 mL) was filtered using a disk filter (0.45 μm, Millipore, MA) and used for the assay and analysis of the contents in the sweet potato leaf. LC/MS Analysis. The content of each component in the extract of the sweet potato leaves was determined by an LC/MS analysis. The LC/MS (Agilent1200, Agilent Technologies) was carried out using a photodiode array detector and monitored in the operating wavelength range from 210 to 700 nm at the flow rate of 0.80 mL/min on a reversed-phase chromatographic column, YMC-Pack Pro C18 (100 × 4.6 mm I.D., 5 μm particle size, YMC Co., Ltd., Kyoto, Japan) at 40 °C. The mass spectra were measured under the following conditions: ESI negative ion mode; desolvation temperature, 350 °C; desolvation pressure, 35 psi; and desolvation gas flow, 12.01 mL/min (6120 Quadrupole LC/MS spectrometer, Agilent Technologies). Cell Culture. HCT116 human colon cancer cells (American Type Culture Collection) were cultured in DMEM (Gibco-BRL, Life Technologies, CA) medium (including 10% FBS, 100 U/ml penicillin and 100 μg/mL streptomycin) at 37 °C in a 5% CO2 atmosphere. Cell Viability. The cell viability due to treatment with a test sample was examined by an MTT assay as previously reported.15 Briefly, the cells were seeded at a density of 5.0 × 105 cells/mL and cultured for 24 h with or without the test sample. After the culture, MTT (0.05%) was added to each well and incubated for 3 h, and then the suspension was carefully removed. Extraction with DMSO (50 μL) was measured at 540 nm with the reference at 655 nm using a microplate reader (BIORAD model 550, BIO-RAD, CA). Reporter Assay. The HCT116 cells with or without the test sample were cultured on a 12-well microplate (5 × 105 cells/well) for 24 h the day before being transiently cotransfected in triplicate with Tcf-4 and the β-catenin plasmid using the FuGENE 6 transfection reagent. After 24 h, the cells were washed twice in phosphate-buffered saline (PBS) and lysed in 100 μL of lysis buffer. The lucyferase activity of the lysate cells (50 μL) was assayed with a luciferase substrate, then the chemiluminescence (CL) was measured by a microplate reader (GLOMAX MULTI Detection system, Promega). The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc., IL). Western Blot. After stimulation, the cells were washed with PBS and then treated with the lysis buffer. The cellular lysates were centrifuged at 13800g for 5 min. The total cellular extracts were separated on SDS-polyacrylamide gels (4−12% SDS-polyacrylamide, Invitrogen, CA) and transferred to a nitrocellulose membrane (iBlot Gel Transfer Mini, Invitrogen) using an iBlot Gel Transfer Device (Invitrogen). The membrane treatment and detection of the protein expression were carried out by manual procedures using an immunodetection system (Invitrogen) while incubated with the polyclonal anti-β-catenin or Tcf-4 overnight at 4 °C. Statistical Analysis. Data were expressed as mean ± SD. Analysis of data was carried out using one-way analyses of variance (ANOVA) and Tukeỳs method for multiple comparison. Significance P < 0.05 was considered statistically significant.
Figure 1. LC/MS chromatogram obtained from the hot-water extract of the sweet potato leaves. The LC/MS was carried out by the analytical condition as shown in the text. Each peak indicated the mono (3, 4, and 5)-caffeoylquinic acid (CQA, m/z = 353.0), di-CQA (4,5-diCQA, 3,5-diCQA, and 3,4-diCQA, m/z = 515.1), and caffeic acid (CA, m/z = 179.1).
the other mono-CQA derivatives and CA, particularly, the 4,5diCQA had the highest content. Similar results were obtained from the ethanol extract of the sweet potato leaves.8,9 The CQA derivatives are the antitumor main sweet potato polyphenols and they have DPPH-radical scavenging action, antimutagen, and activities.10,11 A recent study revealed that the CQA derivatives have an anti-LDL oxidation activity suggesting that they may prevent the development of atherosclerosis caused by the oxidative modification of LDL.8,9 In previous studies over the past decade, several naturally occurring dietary polyphenols, such as quercetin, EGCG, and curcumin, have been shown to modulate genes in the Wnt/β-catenin signaling pathway.16−18 Based on the sweet potato leaf extract and the main polyphenol contents, the CQA derivatives were expected to modulate the Wnt/β-catenin signaling pathway; thus, the reporter assay of the test sample was carried out on the β-catenin/Tcf-4 signaling pathway in the human colorectal cancer HCT116 cells. Figure 4 shows the result of the reporter assay by the combination of βcatenin and Tcf-4 plasmid DNAs with or without the test sample. As shown in Figure 4, the chemiluminescence (CL) reporter activity of β-catenin and Tcf-4 transfected in cells increased when compared to Tcf-4 alone. When cells transfected with the β-catenin and Tcf-4 genes were treated with the sweet potato leaf extract, the induction of the CL reporter activity, suppressed in a dose dependent manner (0.22, 1.10, and 2.20 mg/mL). Each compound from the extract also inhibited the CL reporter activity, and the activity of the diCQA derivatives was higher than those of mono-CQA. The concentration range of these compounds used in the reporter assay did not show any cytotoxicity, indicating that the reduction of the CL reporter activity is due to the interference of the β-catenin/Tcf-4 signaling (Table 1). The antioxidant activity of the phenolic compound is due to the function of the OH group in the molecule, specifically, the presence of a catechol group contributes to the effective radical scavenging activity.15,19 The CQA derivatives having a catechol group in the caffeoyl structure have an effective antioxidant activity, such as the DPPH-radical scavenging action and the anti LDLoxidation activity.7−9 The antioxidant activity of the CQA derivatives was related to the number of caffeoyl groups in the structure, for example, the antioxidant activity is decreasing as follows: tri-CQA, which has three caffeoyl groups, di-CQA
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RESULTS AND DISCUSSION Figure 1 shows the LC−MS chromatogram of the CQA derivatives obtained from the hot-water extract of the sweet potato leaves. It indicated mono-CQA, 5-CQA (24.70 min, 353.0 m/z), 3-CQA (27.19 min, 353.0 m/z), 4-CQA (27.55, 353.0 m/z), CA (27.71 min, 179.1 m/z), and di-CQA, such as 4,5-diCQA (33.62 min, 515.1 m/z), 3,5-diCQA (33.94 min, 515.1 m/z), and 3,4-diCQA (34.89 min, 515.1 m/z). These chemical structures of the CQA derivatives in the sweet potato leaf extract were depicted in Figure 2. Figure 3 shows that the content in the extract and the di-CQA was higher than those of 168
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Figure 2. Chemical structures of caffeoylquinic acid (CQA) derivatives including caffeic acid (CA) from sweet potato leaves. The sweet potato leaves were extracted with methanol. The CQA derivatives, except for CA and 3-CQA, were separated from the extract by the procedures shown in the text.
that of the antioxidant activity. Quercetin, the major representative of the flavonol subclass of flavonoids, has been shown to be a potent inhibitor of the β-catenin/Tcf signaling in SW480 human colon adenocarcinoma cells. Quercetin also has a catechol function in the A-ring of the molecule.16 Caffeic acid phenethyl esters, a phenolic component of propolis, having a catechol structure in the molecule inhibited the colorectal cancer cell growth in a dose and time-dependent manner by decreasing the β-catenin.20,21 Therefore, the caffeoyl structure in the presence of a catechol group of the CQA derivatives may play a functional role in interfering the Wnt/β-catenin signaling pathway. The cytoplasmic β-catenin levels are normally low through continuous proteasome-mediated degradation by an APC/ GSK-3β/Axin complex. When cells receive the Wnt signal, the degradation of β-catenin is inhibited and the levels of β-catenin increase in the cytoplasm and nucleus. Nuclear β-catenin interacts with the transcription factors, such as the Tcf/Lef, and lead to tumor formation and progression.12 The inhibition of the GSK-3β kinase activity might lead to the onset of the βcatenin/Tcf/Lef-mediated gene transcription, representing a potentially mitogenic stimulus. The molecules, such as GSK-3β, β-catenin, and the transcription factor, and Tcf are the key factors in the Wnt pathway involved in the regulation of the βcatenin homeostasis. In previous studies, it was reported that the naturally occurring dietary compounds inhibited the βcatenin/Tcf-4 transcriptional signaling in the cancer cells. A
Figure 3. Composition of the CQA derivatives in the sweet potato leaf. The content of each component in the hot-water extract of the sweet potato leaf was determined by an LC/MS analysis using the procedures shown in the text. The content of each compound is expressed as mean ± SD of three experiments.
(with 2 caffeoyl groups) derivatives, and mono-CQA (with one caffeoyl group). The CL reporter activity of the CQA derivatives including CA having a catechol structure similar to 169
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Figure 4. Inhibition of the chemiluminescence reporter activity on βcatenin/Tcf-4 signaling in the HCT116 colorectal cancer cells. The chemiluminescence (CL) of the β-catenin/Tcf-4 signaling in the HCT116 colorectal cancer cells with or without the test sample was measured by the CL reporter assay and indicated as the CL reporter activity rate (%) for the control and β-catenin/Tcf-4 plasmid transfected cells. Data were expressed as mean ± SD of three experiments and groups with different letters are significantly different (p < 0.05).
polyphenol-rich apple juice extract was found to effectively inhibit the kinase activity of GSK-3β.22 Curcumin derivative treatment of a variety of colon cancer cell lines reduced in a dose dependent manner the β-catenin transcriptional activity by increasing the degradation of the cytoplasmic β-catenin.17 EGCG treatment in HEK293 cells inhibited the β-catenin transcriptional activity,18 and resveratrol also significantly decreased the level of β-catenin in the nucleus of colon cancer cells.23 In this study, the target molecule by the CQA derivatives in the β-catenin/Tcf-4 signaling was examined in cells transfected with the β-catenin/Tcf-4 plasmid DNAs with or without test compounds (Figure 5). The expression of βcatenin in cells transfected with the β-catenin/Tcf-4 plasmid DNAs was at a level similar to that of the vector alone. As shown in Figure 5a, the CQA derivatives treatment in cells had no effect on the reduction of the β-catenin protein suggesting
Figure 5. Suppression of the CQA derivatives for the β-catenin and Tcf-4 protein expression in HCT116 colorectal cancer cells. Western blot analysis of protein expression for the β-catenin/Tcf-4 plasmid transfected cells with or without the test sample (12.5, 25, and 50 μM). (a) Effect of the CQA derivatives on β-catenin protein expression and (b) inhibition of the Tcf-4 protein expression. A representative example of two experiments is shown.
that the inhibition of the β-catenin/Tcf-4 transcriptional activity is due to the downregulation of the signaling pathway. In the same experiment, the Tcf-4 protein expression in cells transfected with the β-catenin/Tcf-4 plasmid DNAs was greater than that of the vector alone (control). When all of the test compounds were treated at 25 μM in the cells, the production of the Tcf-4 protein was completely suppressed (Figure 5b).
Table 1. Cytotoxicity of Sweet Potato Leaf Extract and the CQA Derivatives in HCT 116 Colorectal Cancer Cells cells survival rate (%)b extract (mg/mL) group
control
a
CQA derivatives (μM)
0.22
1.10
2.20
97.64 ± 3.38
98.16 ± 5.16
95.15 ± 4.81
12.5
25
50
100 ± 9.28 extract CA 3-CQA 4-CQA 5-CQA 3,4-diCQA 3,5-diCQA 4,5-diCQA
96.06 101.28 99.29 97.54 91.78 96.13 94.48
± ± ± ± ± ± ±
4.24 10.63 7.33 7.23 6.80 2.98 8.75
87.54 86.85 97.07 105.27 93.55 92.72 95.70
± ± ± ± ± ± ±
1.97 3.37 6.59 5.88 5.12 2.47 7.77
85.75 91.82 94.53 97.17 91.31 90.21 93.34
± ± ± ± ± ± ±
6.47 3.37 7.22 2.51 1.18 1.45 3.20
β-catenin/Tcf-4 plasmid transfected cells without test sample. bCells survival rate (%) for control was determined by MTT assay. Data are expressed as mean ± SD of four experiments. a
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caffeoylquinic acid derivatives. Antivial Chem. Chemother. 1993, 4, 235−240. (5) Ludivik, B.; Hanefeld, M.; Pacini, M. Improved metabolic control by Ipomoea batatas (Caiapo) is associated with increased adiponectin and decreased fibrinogen levels in type 2 diabetic subjects. Diab. Obes. Metabol. 2008, 10, 586−592. (6) Cliffold, M. N.; Johnson, K. L.; Knight, S.; Kuhnert, N. Hierarchical scheme for LC-MS identification of chlorogenic acids. J. Agric. Food Chem. 2003, 51, 2900−2911. (7) Ishiguro, K.; Yahara, S.; Yoshimoto, M. Changes in polyphenols contents and radical-scavenging activity of sweetpotato (Ipomoea batatas L.) during storage at optimal and low temperature. J. Agric. Food Chem. 2007, 55, 10773−10778. (8) Taira, J.; Ohmine, W. Characteristics of Caffeic acid derivatives in Okinawan sweet potato (Ipomoea batatas L.) leaves and the anti-LDL oxidation activity. Nippon Shokuhin Kagaku Kogaku Kaishi 2011, 58, 16−20. (9) Taira, J.; Taira, K.; Ohmine, W.; Nagata, J. Mineral determination and anti-LDL oxidation activity of sweet potato (Ipomoea batatas L.) leaves. J. Food Comp. Anal. 2013, 29, 117−125. (10) Oki, T.; Masuda, M.; Furuya, S.; Nishiba, Y.; Terahara, N.; Suda, I. Involvement of anthocyanins and other phenolic compounds in radical-scavenging activity of purple-fleshed sweet potato cultivars. J. Food Sci. 2002, 67, 1752−1756. (11) Yoshimoto, M.; Yahara, S.; Okuno, S.; Islam, M. S.; Ishiguro, K.; Yamakawa, O. Antimutagenecity of mono-, di-, and tricaffeoylquinic acid derivatives isolated from sweetpotato (Impomoae batatas L.) leaf. Biosci. Biotechnol. Biochem. 2002, 66, 2336−2341. (12) Bienz, M.; Clevers, H. Linking Colorectal Cancer to Wnt Signaling Review. Cell 2000, 103, 311−320. (13) Tarapore, R. S.; Siddique, I. A.; Mukhter, H. A.; Mukhtar, H. Modulating of Wnt/β-catenin signaling pathway by bioactive food components. Carcinogenesis 2011, 0, 1−9. (14) Teiten, M.-H.; Gaascht, F.; Dicato, M.; Diederich, M. Targeting the Wingless signaling pathway with natural compounds as chemopreventive or chemotherapeutic agents. Curr. Pharmaceut. Biotechnol. 2012, 13, 245−254. (15) Taira, J.; Nanbu, H.; Ueda, K. Nitric oxide-scavenging compounds in Agrimonia pilosa Ledeb on LPS-induced RAW264.7 macrophages. Food Chem. 2009, 115, 1221. (16) Park, C. H.; Chang, J.-Y.; Hahm, E. R.; Park, S.; Kim, H.-K.; Yang, C. H. Quercetin, a potent inhibitor against β-catenin/Tcf signaling in SW480 colon cancer cells. Biochem. Biophys. Res. Commun. 2005, 328, 227−254. (17) Ryu, M.-J.; Cho, M.; Song, J.-Y.; Yun, Y.-S.; Choi, II-W.; Kim, D.-E.; Park, B.-S.; Oh, S. Natural derivatives of curcumin attenuate the Wnt/β-catenin pathway through down regulation of the transcriptional coactivator p300. Biochem. Biophys. Res. Commun. 2008, 377, 1304− 1308. (18) Dashwood, W.-M.; Orner, G. A.; Dashwood, R. H. Inhibition of β-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3gallate (EGCG): minor contribution of H2O2 at physiologically relevant EGCG concentrations. Biochem. Biophys. Res. Commun. 2002, 296, 584−588. (19) Haenen, G. R. M. M.; Paqary, J. B. G.; Korthouwer, R. E. M.; Bast, A. Peroxynitrite scavenging by flavonoids. Biochem. Biophys. Res. Commun. 1997, 236, 591−593. (20) Wang, D.; Xiang, D.-B.; He, Y.-J.; Li, Z.-P.; Wu, X.-H.; Mou, J.H.; Xiao, H.-L.; Zhang, Q.-H. Effect of caffeic acid phenythyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J. Gastroenterol. 2005, 11, 4008−4012. (21) He, Y.-J.; Liu, B.-H.; Xiang, D.-B.; Qiao, Z-Yi.; Fu, T.; He, Yu-H. Inhibitory effect of caffeic acid phenythyl ester on the growth of SW480 colorectal tumor cells involves β-catenin associated signaling pathway down-regulation. World J. Gastroenterol. 2006, 31, 4981− 4985. (22) Kern, M.; Pahlkb, G.; Ngiewjh, Y.; Marko, D. Modulation of key elements of the Wnt pathway by apple polyphenols. J. Agric. Food Chem. 2006, 54, 7011−7046.
The result clearly indicated that the CQA derivatives targeted the transcription factor Tcf-4 on the Wnt/β-catenin signaling pathway. In previous papers, it was reported that the naturally occurring dietary compounds inhibited the β-catenin/Tcf-4 transcriptional signaling due to modulating the Wnt signaling gene expression. Quercetin inhibits the β-catenin/Tcf-4 signaling in SW480 human colon adenocarcinoma cells by decreasing the levels of β-catenin and Tcf-4 in the nucleus, resulting in a decreased downstream Wnt signaling gene expression of c-myc.16 Curcumin treatment inhibits the mRNA expression of the Wnt target genes c-myc, c-fos, and c-jun in a variety of cancer cells.24 EGCG reduced the expression of the β-catenin/Tcf-4 signaling target genes c-jun and cyclin D1.25,26 Resveratrol decreased the localization of β-catenin in the nucleus due to the expression of lgs and pygol, the activator of β-catenin in all cell lines.23 The caffeic acid phenethyl ester significantly suppressed the β-catenin, c-myc, and cyclin D1 protein expression in SW480 cells.21 The CQA derivatives may have also affected the Wnt target genes, such as the cell cycle prompting genes c-myc and cyclin D1. In addition, the CQA derivatives may have a potential modulation of the Wnt/βcatenin signaling pathway in various cancer cells. These subjects will be the focus of future research. In conclusion, this study demonstrated that the edible sweet potato leaf extract and its contents, such as mono (3, 4, and 5)caffeoylquinic acid (CQA), di-CQA (4,5-diCQA, 3,5-diCQA, and 3,4-diCQA), and caffeic acid (CA), inhibit the β-catenin/ Tcf-4 transcriptional activity in HCT116 cells. Particularly, the di-CQA derivatives (with 2 caffeoyl groups) were greater than that of the mono-CQA derivatives (one caffeoyl group) and CA, suggesting that the caffeoyl structure in the presence of a catechol group plays a significant role in the reduction of the βcatenin transcriptional activity. In addition, the compounds targeted the molecular transcription factor Tcf-4 for the inhibition of the β-catenin/Tcf-4 transcriptional activity. Sweet potato leaf containing the β-catenin signaling inhibitor, CQA derivatives, may be a protective diet for colorectal cancer.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. A. Nagafuchi (Kumamoto University, Japan) for providing a constitutively active form of mouse β-catenin (caβcatenin) cDNA. We thank Dr. T. Shimotono (University of Kyoto, Japan) for providing TCF-BP-Luc plasmid.
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REFERENCES
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(23) Hope, C.; Kestutis, P.; Marina, P.; Moyer, M. P.; Johal, K. S.; Woo, J.; Santoso, C.; Hanson, J. A.; Holcombe, R. F. Low concentration of resveratrol inhibit Wnt signal throughout in colonderived cells: Implications for colon cancer prevention. Mol. Nutr. Food Res. 2008, 52 (Suppl 1), 852−861. (24) Lin, J.-K. Molecular targets of curcumin. Adv. Exp. Med. Biol. 2007, 595, 227−243. (25) Issa, A. Y.; Volate, S. R.; Muga1, S. J.; Nitcheva, D.; Smith, T.; Wargovich1, M. J. Green tea selectively targets initial stages of intestinal carcinogenesis in the AOM-ApcMin mouse model. Carcinogenesis 2007, 28, 1978−1984. (26) Ju, J.; Hong, J.; Zhou, J.; Pan, Z.; Bose, M.; Liao, J.; Yang, G-yu.; Liu, Y. Y.; Hou, Z.; Lin, Y.; Ma, J.; Shih, W. J.; Carothers, M.; Yang, C. S. Inhibition of intestinal tumorigenesis in Apcmin/+Mice by (-)-epigallocatechin-3-gallate, the major catechin in green tea. Cancer Res. 2005, 65, 10623−10631.
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Inhibition of the β‑Catenin/Tcf Signaling by Caffeoylquinic Acids in Sweet Potato Leaf through down Regulation of the Tcf‑4 Transcription Junsei Taira,* Masatsugu Uehara, Eito Tsuchida, and Wakana Ohmine Department of Bioresource Technology, Okinawa National College of Technology, 905 Henoko, Nago City, Okinawa, 905-2192 Japan ABSTRACT: Sweet potato leaves contain the highest levels of functional polyphenols. In this study the effects of the sweet potato leaf extract and its contents, such as mono (3, 4, and 5)-caffeoylquinic acid (CQA), di-CQA (4,5-diCQA, 3,5-diCQA, and 3,4-diCQA) and caffeic acid (CA), were evaluated on the β-catenin/Tcf-4 signaling in human colorectal cancer HCT116 cells. The extract and the CQA derivatives inhibited the β-catenin/Tcf-4 signaling, and the inhibition of the di-CQA (with two caffeoyl groups) was higher than that of the mono-CQA (one-caffeoyl group) and CA, suggesting that the caffeoyl structure in the presence of a catechol group plays a significant role in interfering with the β-catenin/Tcf-4 signaling. In addition, the CQA derivatives had no effect on the β-catenin protein expression, but all test compounds inhibited the expression of the Tcf-4 transcription, and the inhibition of the di-CQA derivatives was stronger than those of the mono-CQA derivatives as well as the βcatenin/Tcf-4 transcriptional activity. These compounds can modulate the downstream Wnt signaling pathway, suggesting that sweet potato leaves can be a protective food for colorectal cancer. KEYWORDS: caffeoylquinic acid, HCT 116 cells, colorectal cancer, β-catenin/Tcf signaling, sweet potato
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β-catenin signaling pathway.13,14 These active compounds have shown antioxidant activity; thus, caffeoylquinic acids will be expected to have a similar activity. This study was designed to confirm whether caffeoylquinic acid derivatives can modulate βcatenin/Tcf-4 signaling.
INTRODUCTION Recent studies have demonstrated that sweet potato (Ipomoea batatas L.) leaves contain the highest levels of polyphenols compared to other commercial vegetables.1,2 The leaves were found to be an inhibitor of the growth of colon and stomach cancer cells, leukemia, and viruses3,4 and to reduce the effects of diabetes in humans.5 The sweet potato leaves contain phenolic compounds, such as caffeoylquinic acid (CQA) derivatives, a family of esters formed from certain cinnamic acids and quinic acid, including the mono-CQA (chlorogenic acid derivatives) and di-CQA, such as 3,5-diCQA, 3,4-diCQA, and 4,5diCQA.6−8 The CQA derivatives are the main sweet potato polyphenols and they have an anti-LDL oxidation activity, DPPH-radical scavenging action, and antimutagen and antitumor activities.8−11 Colorectal cancer is a human malignance as well as stomach and lung cancer which are causes of human death. The incidence tends to increase due to changing Japanese dietary habits. Recent studies have indicated that in most of the colorectal cancers associated with the aberrant Wnt signaling pathway, the expression of β-catenin plays critical roles in cell proliferation and differentiation. The glycogen synthase kinase (GSK)-3β/Axin complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. When the β-catenin destruction complex is inhibited, a pool of cytoplasmic β-catenin stabilizes and is able to enter the nucleus and interact with the T-cell factor/lymphoid enhancer-binding factor (Tcf/Lef) family transcription factors to promote a specific gene expression and the signaling is an early progression event in colorectal cancer.12 In previous studies over the past decade, several naturally occurring dietary compounds, such as flavonoids, curcumin, and the tea polyphenol epigallocatechin-3-gallate (EGCG), have been shown to target intermediates in the Wnt/ © 2013 American Chemical Society
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MATERIALS AND METHODS
Materials. Caffeic acid (CA) and 3-caffeoylquinic acid were obtained from the Wako Pure Chemical Co. (Osaka, Japan). FuGENE 6 as a transfection reagent and the Lucyferase Assay System containing the luciferase substrate and lysis buffer to assess the reporter assay were obtained from the Promega Corporation (CA). Polyclonal antiβ-catenin (mouse monoclonal antibody, BD Biosciences, CA) and Tcf4 monoclonal antibody (mouse monoclonal antibody, Exalpha Biologicals, Inc., MA) were used to detect the protein expression. Isolation of CQA Derivatives. The CQA derivatives were isolated from the methanol (MeOH) extract of the sweet potato leaf as previously reported.8,9 Briefly, the MeOH extract was partitioned between dichloromethane and H2O (50/50 v/v) and the H2O fraction was continuously partitioned with n-BuOH. The H2O fraction was freeze-dried, then separated by HPLC (Agilent 1200, Agilent Technologies, CA) with an ODS-column (COAMOSIL Packed column 5C18-MS-II, 10 I.D. × 250 mm, Nacalai Tesque, Tokyo, Japan), and monitored at 326 nm at the flow rate of 2.0 mL/min at 40 °C. The mobile phase consisting of a formic acid aqueous solution (0.5%) and acetonitrile was carried out by a linear gradient to 2, 45, and 100% for 15, 50, and 60 min, respectively. Each peak of the mono (4 and 5)-CQA and di-CQA, such as 4,5- diCQA, 3,5-diCQA, and 3,4diCQA, was collected by a fraction collector (Analyte FC, Agilent Technologies). These compounds were used in the β-catenin/Tcf-4 Received: Revised: Accepted: Published: 167
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reporter assay and as the standard substances for determining the quantity of the content in the sweet potato leaf extract. Sample Preparation. The sweet potato leaf powder (1 g) was extracted with distilled water (50 mL) at 85 °C for 45 min. The extract was filtered and then freeze-dried (Tokyo Rikakiki Co. Ltd., Tokyo, Japan). The sample (430.1 mg) dissolved in MQ-water (1 mL) was filtered using a disk filter (0.45 μm, Millipore, MA) and used for the assay and analysis of the contents in the sweet potato leaf. LC/MS Analysis. The content of each component in the extract of the sweet potato leaves was determined by an LC/MS analysis. The LC/MS (Agilent1200, Agilent Technologies) was carried out using a photodiode array detector and monitored in the operating wavelength range from 210 to 700 nm at the flow rate of 0.80 mL/min on a reversed-phase chromatographic column, YMC-Pack Pro C18 (100 × 4.6 mm I.D., 5 μm particle size, YMC Co., Ltd., Kyoto, Japan) at 40 °C. The mass spectra were measured under the following conditions: ESI negative ion mode; desolvation temperature, 350 °C; desolvation pressure, 35 psi; and desolvation gas flow, 12.01 mL/min (6120 Quadrupole LC/MS spectrometer, Agilent Technologies). Cell Culture. HCT116 human colon cancer cells (American Type Culture Collection) were cultured in DMEM (Gibco-BRL, Life Technologies, CA) medium (including 10% FBS, 100 U/ml penicillin and 100 μg/mL streptomycin) at 37 °C in a 5% CO2 atmosphere. Cell Viability. The cell viability due to treatment with a test sample was examined by an MTT assay as previously reported.15 Briefly, the cells were seeded at a density of 5.0 × 105 cells/mL and cultured for 24 h with or without the test sample. After the culture, MTT (0.05%) was added to each well and incubated for 3 h, and then the suspension was carefully removed. Extraction with DMSO (50 μL) was measured at 540 nm with the reference at 655 nm using a microplate reader (BIORAD model 550, BIO-RAD, CA). Reporter Assay. The HCT116 cells with or without the test sample were cultured on a 12-well microplate (5 × 105 cells/well) for 24 h the day before being transiently cotransfected in triplicate with Tcf-4 and the β-catenin plasmid using the FuGENE 6 transfection reagent. After 24 h, the cells were washed twice in phosphate-buffered saline (PBS) and lysed in 100 μL of lysis buffer. The lucyferase activity of the lysate cells (50 μL) was assayed with a luciferase substrate, then the chemiluminescence (CL) was measured by a microplate reader (GLOMAX MULTI Detection system, Promega). The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc., IL). Western Blot. After stimulation, the cells were washed with PBS and then treated with the lysis buffer. The cellular lysates were centrifuged at 13800g for 5 min. The total cellular extracts were separated on SDS-polyacrylamide gels (4−12% SDS-polyacrylamide, Invitrogen, CA) and transferred to a nitrocellulose membrane (iBlot Gel Transfer Mini, Invitrogen) using an iBlot Gel Transfer Device (Invitrogen). The membrane treatment and detection of the protein expression were carried out by manual procedures using an immunodetection system (Invitrogen) while incubated with the polyclonal anti-β-catenin or Tcf-4 overnight at 4 °C. Statistical Analysis. Data were expressed as mean ± SD. Analysis of data was carried out using one-way analyses of variance (ANOVA) and Tukeỳs method for multiple comparison. Significance P < 0.05 was considered statistically significant.
Figure 1. LC/MS chromatogram obtained from the hot-water extract of the sweet potato leaves. The LC/MS was carried out by the analytical condition as shown in the text. Each peak indicated the mono (3, 4, and 5)-caffeoylquinic acid (CQA, m/z = 353.0), di-CQA (4,5-diCQA, 3,5-diCQA, and 3,4-diCQA, m/z = 515.1), and caffeic acid (CA, m/z = 179.1).
the other mono-CQA derivatives and CA, particularly, the 4,5diCQA had the highest content. Similar results were obtained from the ethanol extract of the sweet potato leaves.8,9 The CQA derivatives are the antitumor main sweet potato polyphenols and they have DPPH-radical scavenging action, antimutagen, and activities.10,11 A recent study revealed that the CQA derivatives have an anti-LDL oxidation activity suggesting that they may prevent the development of atherosclerosis caused by the oxidative modification of LDL.8,9 In previous studies over the past decade, several naturally occurring dietary polyphenols, such as quercetin, EGCG, and curcumin, have been shown to modulate genes in the Wnt/β-catenin signaling pathway.16−18 Based on the sweet potato leaf extract and the main polyphenol contents, the CQA derivatives were expected to modulate the Wnt/β-catenin signaling pathway; thus, the reporter assay of the test sample was carried out on the β-catenin/Tcf-4 signaling pathway in the human colorectal cancer HCT116 cells. Figure 4 shows the result of the reporter assay by the combination of βcatenin and Tcf-4 plasmid DNAs with or without the test sample. As shown in Figure 4, the chemiluminescence (CL) reporter activity of β-catenin and Tcf-4 transfected in cells increased when compared to Tcf-4 alone. When cells transfected with the β-catenin and Tcf-4 genes were treated with the sweet potato leaf extract, the induction of the CL reporter activity, suppressed in a dose dependent manner (0.22, 1.10, and 2.20 mg/mL). Each compound from the extract also inhibited the CL reporter activity, and the activity of the diCQA derivatives was higher than those of mono-CQA. The concentration range of these compounds used in the reporter assay did not show any cytotoxicity, indicating that the reduction of the CL reporter activity is due to the interference of the β-catenin/Tcf-4 signaling (Table 1). The antioxidant activity of the phenolic compound is due to the function of the OH group in the molecule, specifically, the presence of a catechol group contributes to the effective radical scavenging activity.15,19 The CQA derivatives having a catechol group in the caffeoyl structure have an effective antioxidant activity, such as the DPPH-radical scavenging action and the anti LDLoxidation activity.7−9 The antioxidant activity of the CQA derivatives was related to the number of caffeoyl groups in the structure, for example, the antioxidant activity is decreasing as follows: tri-CQA, which has three caffeoyl groups, di-CQA
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RESULTS AND DISCUSSION Figure 1 shows the LC−MS chromatogram of the CQA derivatives obtained from the hot-water extract of the sweet potato leaves. It indicated mono-CQA, 5-CQA (24.70 min, 353.0 m/z), 3-CQA (27.19 min, 353.0 m/z), 4-CQA (27.55, 353.0 m/z), CA (27.71 min, 179.1 m/z), and di-CQA, such as 4,5-diCQA (33.62 min, 515.1 m/z), 3,5-diCQA (33.94 min, 515.1 m/z), and 3,4-diCQA (34.89 min, 515.1 m/z). These chemical structures of the CQA derivatives in the sweet potato leaf extract were depicted in Figure 2. Figure 3 shows that the content in the extract and the di-CQA was higher than those of 168
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Figure 2. Chemical structures of caffeoylquinic acid (CQA) derivatives including caffeic acid (CA) from sweet potato leaves. The sweet potato leaves were extracted with methanol. The CQA derivatives, except for CA and 3-CQA, were separated from the extract by the procedures shown in the text.
that of the antioxidant activity. Quercetin, the major representative of the flavonol subclass of flavonoids, has been shown to be a potent inhibitor of the β-catenin/Tcf signaling in SW480 human colon adenocarcinoma cells. Quercetin also has a catechol function in the A-ring of the molecule.16 Caffeic acid phenethyl esters, a phenolic component of propolis, having a catechol structure in the molecule inhibited the colorectal cancer cell growth in a dose and time-dependent manner by decreasing the β-catenin.20,21 Therefore, the caffeoyl structure in the presence of a catechol group of the CQA derivatives may play a functional role in interfering the Wnt/β-catenin signaling pathway. The cytoplasmic β-catenin levels are normally low through continuous proteasome-mediated degradation by an APC/ GSK-3β/Axin complex. When cells receive the Wnt signal, the degradation of β-catenin is inhibited and the levels of β-catenin increase in the cytoplasm and nucleus. Nuclear β-catenin interacts with the transcription factors, such as the Tcf/Lef, and lead to tumor formation and progression.12 The inhibition of the GSK-3β kinase activity might lead to the onset of the βcatenin/Tcf/Lef-mediated gene transcription, representing a potentially mitogenic stimulus. The molecules, such as GSK-3β, β-catenin, and the transcription factor, and Tcf are the key factors in the Wnt pathway involved in the regulation of the βcatenin homeostasis. In previous studies, it was reported that the naturally occurring dietary compounds inhibited the βcatenin/Tcf-4 transcriptional signaling in the cancer cells. A
Figure 3. Composition of the CQA derivatives in the sweet potato leaf. The content of each component in the hot-water extract of the sweet potato leaf was determined by an LC/MS analysis using the procedures shown in the text. The content of each compound is expressed as mean ± SD of three experiments.
(with 2 caffeoyl groups) derivatives, and mono-CQA (with one caffeoyl group). The CL reporter activity of the CQA derivatives including CA having a catechol structure similar to 169
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Figure 4. Inhibition of the chemiluminescence reporter activity on βcatenin/Tcf-4 signaling in the HCT116 colorectal cancer cells. The chemiluminescence (CL) of the β-catenin/Tcf-4 signaling in the HCT116 colorectal cancer cells with or without the test sample was measured by the CL reporter assay and indicated as the CL reporter activity rate (%) for the control and β-catenin/Tcf-4 plasmid transfected cells. Data were expressed as mean ± SD of three experiments and groups with different letters are significantly different (p < 0.05).
polyphenol-rich apple juice extract was found to effectively inhibit the kinase activity of GSK-3β.22 Curcumin derivative treatment of a variety of colon cancer cell lines reduced in a dose dependent manner the β-catenin transcriptional activity by increasing the degradation of the cytoplasmic β-catenin.17 EGCG treatment in HEK293 cells inhibited the β-catenin transcriptional activity,18 and resveratrol also significantly decreased the level of β-catenin in the nucleus of colon cancer cells.23 In this study, the target molecule by the CQA derivatives in the β-catenin/Tcf-4 signaling was examined in cells transfected with the β-catenin/Tcf-4 plasmid DNAs with or without test compounds (Figure 5). The expression of βcatenin in cells transfected with the β-catenin/Tcf-4 plasmid DNAs was at a level similar to that of the vector alone. As shown in Figure 5a, the CQA derivatives treatment in cells had no effect on the reduction of the β-catenin protein suggesting
Figure 5. Suppression of the CQA derivatives for the β-catenin and Tcf-4 protein expression in HCT116 colorectal cancer cells. Western blot analysis of protein expression for the β-catenin/Tcf-4 plasmid transfected cells with or without the test sample (12.5, 25, and 50 μM). (a) Effect of the CQA derivatives on β-catenin protein expression and (b) inhibition of the Tcf-4 protein expression. A representative example of two experiments is shown.
that the inhibition of the β-catenin/Tcf-4 transcriptional activity is due to the downregulation of the signaling pathway. In the same experiment, the Tcf-4 protein expression in cells transfected with the β-catenin/Tcf-4 plasmid DNAs was greater than that of the vector alone (control). When all of the test compounds were treated at 25 μM in the cells, the production of the Tcf-4 protein was completely suppressed (Figure 5b).
Table 1. Cytotoxicity of Sweet Potato Leaf Extract and the CQA Derivatives in HCT 116 Colorectal Cancer Cells cells survival rate (%)b extract (mg/mL) group
control
a
CQA derivatives (μM)
0.22
1.10
2.20
97.64 ± 3.38
98.16 ± 5.16
95.15 ± 4.81
12.5
25
50
100 ± 9.28 extract CA 3-CQA 4-CQA 5-CQA 3,4-diCQA 3,5-diCQA 4,5-diCQA
96.06 101.28 99.29 97.54 91.78 96.13 94.48
± ± ± ± ± ± ±
4.24 10.63 7.33 7.23 6.80 2.98 8.75
87.54 86.85 97.07 105.27 93.55 92.72 95.70
± ± ± ± ± ± ±
1.97 3.37 6.59 5.88 5.12 2.47 7.77
85.75 91.82 94.53 97.17 91.31 90.21 93.34
± ± ± ± ± ± ±
6.47 3.37 7.22 2.51 1.18 1.45 3.20
β-catenin/Tcf-4 plasmid transfected cells without test sample. bCells survival rate (%) for control was determined by MTT assay. Data are expressed as mean ± SD of four experiments. a
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caffeoylquinic acid derivatives. Antivial Chem. Chemother. 1993, 4, 235−240. (5) Ludivik, B.; Hanefeld, M.; Pacini, M. Improved metabolic control by Ipomoea batatas (Caiapo) is associated with increased adiponectin and decreased fibrinogen levels in type 2 diabetic subjects. Diab. Obes. Metabol. 2008, 10, 586−592. (6) Cliffold, M. N.; Johnson, K. L.; Knight, S.; Kuhnert, N. Hierarchical scheme for LC-MS identification of chlorogenic acids. J. Agric. Food Chem. 2003, 51, 2900−2911. (7) Ishiguro, K.; Yahara, S.; Yoshimoto, M. Changes in polyphenols contents and radical-scavenging activity of sweetpotato (Ipomoea batatas L.) during storage at optimal and low temperature. J. Agric. Food Chem. 2007, 55, 10773−10778. (8) Taira, J.; Ohmine, W. Characteristics of Caffeic acid derivatives in Okinawan sweet potato (Ipomoea batatas L.) leaves and the anti-LDL oxidation activity. Nippon Shokuhin Kagaku Kogaku Kaishi 2011, 58, 16−20. (9) Taira, J.; Taira, K.; Ohmine, W.; Nagata, J. Mineral determination and anti-LDL oxidation activity of sweet potato (Ipomoea batatas L.) leaves. J. Food Comp. Anal. 2013, 29, 117−125. (10) Oki, T.; Masuda, M.; Furuya, S.; Nishiba, Y.; Terahara, N.; Suda, I. Involvement of anthocyanins and other phenolic compounds in radical-scavenging activity of purple-fleshed sweet potato cultivars. J. Food Sci. 2002, 67, 1752−1756. (11) Yoshimoto, M.; Yahara, S.; Okuno, S.; Islam, M. S.; Ishiguro, K.; Yamakawa, O. Antimutagenecity of mono-, di-, and tricaffeoylquinic acid derivatives isolated from sweetpotato (Impomoae batatas L.) leaf. Biosci. Biotechnol. Biochem. 2002, 66, 2336−2341. (12) Bienz, M.; Clevers, H. Linking Colorectal Cancer to Wnt Signaling Review. Cell 2000, 103, 311−320. (13) Tarapore, R. S.; Siddique, I. A.; Mukhter, H. A.; Mukhtar, H. Modulating of Wnt/β-catenin signaling pathway by bioactive food components. Carcinogenesis 2011, 0, 1−9. (14) Teiten, M.-H.; Gaascht, F.; Dicato, M.; Diederich, M. Targeting the Wingless signaling pathway with natural compounds as chemopreventive or chemotherapeutic agents. Curr. Pharmaceut. Biotechnol. 2012, 13, 245−254. (15) Taira, J.; Nanbu, H.; Ueda, K. Nitric oxide-scavenging compounds in Agrimonia pilosa Ledeb on LPS-induced RAW264.7 macrophages. Food Chem. 2009, 115, 1221. (16) Park, C. H.; Chang, J.-Y.; Hahm, E. R.; Park, S.; Kim, H.-K.; Yang, C. H. Quercetin, a potent inhibitor against β-catenin/Tcf signaling in SW480 colon cancer cells. Biochem. Biophys. Res. Commun. 2005, 328, 227−254. (17) Ryu, M.-J.; Cho, M.; Song, J.-Y.; Yun, Y.-S.; Choi, II-W.; Kim, D.-E.; Park, B.-S.; Oh, S. Natural derivatives of curcumin attenuate the Wnt/β-catenin pathway through down regulation of the transcriptional coactivator p300. Biochem. Biophys. Res. Commun. 2008, 377, 1304− 1308. (18) Dashwood, W.-M.; Orner, G. A.; Dashwood, R. H. Inhibition of β-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3gallate (EGCG): minor contribution of H2O2 at physiologically relevant EGCG concentrations. Biochem. Biophys. Res. Commun. 2002, 296, 584−588. (19) Haenen, G. R. M. M.; Paqary, J. B. G.; Korthouwer, R. E. M.; Bast, A. Peroxynitrite scavenging by flavonoids. Biochem. Biophys. Res. Commun. 1997, 236, 591−593. (20) Wang, D.; Xiang, D.-B.; He, Y.-J.; Li, Z.-P.; Wu, X.-H.; Mou, J.H.; Xiao, H.-L.; Zhang, Q.-H. Effect of caffeic acid phenythyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J. Gastroenterol. 2005, 11, 4008−4012. (21) He, Y.-J.; Liu, B.-H.; Xiang, D.-B.; Qiao, Z-Yi.; Fu, T.; He, Yu-H. Inhibitory effect of caffeic acid phenythyl ester on the growth of SW480 colorectal tumor cells involves β-catenin associated signaling pathway down-regulation. World J. Gastroenterol. 2006, 31, 4981− 4985. (22) Kern, M.; Pahlkb, G.; Ngiewjh, Y.; Marko, D. Modulation of key elements of the Wnt pathway by apple polyphenols. J. Agric. Food Chem. 2006, 54, 7011−7046.
The result clearly indicated that the CQA derivatives targeted the transcription factor Tcf-4 on the Wnt/β-catenin signaling pathway. In previous papers, it was reported that the naturally occurring dietary compounds inhibited the β-catenin/Tcf-4 transcriptional signaling due to modulating the Wnt signaling gene expression. Quercetin inhibits the β-catenin/Tcf-4 signaling in SW480 human colon adenocarcinoma cells by decreasing the levels of β-catenin and Tcf-4 in the nucleus, resulting in a decreased downstream Wnt signaling gene expression of c-myc.16 Curcumin treatment inhibits the mRNA expression of the Wnt target genes c-myc, c-fos, and c-jun in a variety of cancer cells.24 EGCG reduced the expression of the β-catenin/Tcf-4 signaling target genes c-jun and cyclin D1.25,26 Resveratrol decreased the localization of β-catenin in the nucleus due to the expression of lgs and pygol, the activator of β-catenin in all cell lines.23 The caffeic acid phenethyl ester significantly suppressed the β-catenin, c-myc, and cyclin D1 protein expression in SW480 cells.21 The CQA derivatives may have also affected the Wnt target genes, such as the cell cycle prompting genes c-myc and cyclin D1. In addition, the CQA derivatives may have a potential modulation of the Wnt/βcatenin signaling pathway in various cancer cells. These subjects will be the focus of future research. In conclusion, this study demonstrated that the edible sweet potato leaf extract and its contents, such as mono (3, 4, and 5)caffeoylquinic acid (CQA), di-CQA (4,5-diCQA, 3,5-diCQA, and 3,4-diCQA), and caffeic acid (CA), inhibit the β-catenin/ Tcf-4 transcriptional activity in HCT116 cells. Particularly, the di-CQA derivatives (with 2 caffeoyl groups) were greater than that of the mono-CQA derivatives (one caffeoyl group) and CA, suggesting that the caffeoyl structure in the presence of a catechol group plays a significant role in the reduction of the βcatenin transcriptional activity. In addition, the compounds targeted the molecular transcription factor Tcf-4 for the inhibition of the β-catenin/Tcf-4 transcriptional activity. Sweet potato leaf containing the β-catenin signaling inhibitor, CQA derivatives, may be a protective diet for colorectal cancer.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. A. Nagafuchi (Kumamoto University, Japan) for providing a constitutively active form of mouse β-catenin (caβcatenin) cDNA. We thank Dr. T. Shimotono (University of Kyoto, Japan) for providing TCF-BP-Luc plasmid.
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REFERENCES
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