槲皮素对结肠癌细胞抑制作用及对Wnt/β-catenin信号转导通路的影响
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摘要
目的:研究槲皮素对人结肠癌细胞SW480增殖、凋亡、细胞周期的影响,建立Wnt/β-catenin信号转导通路报告基因模型,研究槲皮素对SW480细胞Wnt/β-catenin信号转导的调控作用和对Wnt/β-catenin信号转导通路相关基因的影响。探讨槲皮素抗肿瘤作用机理,为槲皮素作为抗肿瘤药物开发提供理论依据。
方法:1.采用MTT比色法检测槲皮素对人结肠癌细胞SW480细胞增殖反应的作用;2.用流式细胞技术分析槲皮素对人结肠癌细胞SW480细胞周期、凋亡的影响。3.采用Western blot分析槲皮素对SW480细胞cyclin B1蛋白表达的影响。4.建立Wnt/β-catenin信号转导通路报告基因模型,研究槲皮素对SW480细胞Wnt/β-catenin信号转导通路的调控作用。5.用半定量RT-PCR和Western blot方法研究槲皮素对Wnt/β-catenin信号转导通路β-catenin/TCF下游靶基因cyclin D1和survivin mRNA及蛋白表达的影响。6.统计学方法:所有数据采用SPSS 12.0软件进行统计学处理。实验结果均以x±S表示,组内比较采用单因素方差分析,多个实验组均数与一个对照组均数比较用Dunnett-t检验,回归方程间比较用t检验与方差分析,以α=0.05或α=0.01为检验水准。
结果:
1.槲皮素抑制SW480细胞增殖
MTT检测结果显示,40、80、160μmol/L的槲皮素对SW480细胞增殖有明显的抑制作用,与对照组比较有显著性差异(P<0.01),高浓度槲皮素组与低浓度组比较,对SW480细胞抑制作用也明显差异(P<0.05),抑制作用呈剂量依赖性和时间依赖性(r=0.99, P=0.00)。
2.槲皮素可诱导SW480细胞周期阻滞和细胞凋亡
流式细胞术检测结果显示,40、60、80μmol/L的槲皮素作用SW480细胞48 h后,G0/G1期和S期细胞减少,G2/M期细胞显著增多(P<0.01)。槲皮素可以剂量依赖性地诱导SW480细胞凋亡,80μmol/L的槲皮素作用48 h后,细胞凋亡率(13.32%±4.62%)明显升高,与对照组(2.68%±1.04%)相比有显著性差异(P<0.01)。
3.槲皮素可明显下调SW480细胞cyclin B1蛋白的表达
经槲皮素作用48 h后,SW480细胞cyclin B1蛋白表达水平明显下降(P<0.05),抑制作用呈剂量依赖性(r=0.999, P=0.001)。
4.槲皮素可以明显下调SW480细胞TCF-4报告基因TOPFLASH活性
应用双荧光素酶报告基因质粒TOPFLASH/FOPFLASH及内参照质粒pRL-null瞬时转染SW480细胞,建立了Wnt/β-catenin信号转导通路双荧光素酶报告基因模型。经分析结果显示,槲皮素处理转染报告基因的SW480细胞24 h后,TCF-4报告基因TOPFLASH活性显著降低,160μ?mol/L槲皮素作用24 h后,TOPFLASH报告基因RLU值由对照组的195.65±12.25降至11.54±5.60,差异有统计学意义(P<0.05),但FOPFLASH活性没有明显的变化(P>0.05),提示槲皮素可剂量依赖性抑制β-catenin/TCF的转录活性。
5.槲皮素可显著下调SW480细胞cyclin D1和survivin mRNA表达和蛋白表达水平
60、120μmol/L槲皮素作用24 h后,SW480细胞cyclin D1和survivin mRNA表达和蛋白表达水平均显著下降,抑制作用呈剂量依赖性(P<0.05)。
结论:槲皮素可明显抑制SW480细胞增殖,诱导细胞凋亡,将SW480细胞周期阻滞于G2/M期。槲皮素可剂量依赖性抑制β-catenin/TCF的转录活性,下调β-catenin/TCF下游靶基因cyclin D1和survivin的mRNA表达和蛋白表达水平,提示其抗肿瘤机制可能与其抑制Wnt/β-catenin信号转导通路有关。
Objective: Effects of quercetin on proliferation, cell cycle and apoptosis of colon carcinoma cell line SW480 were investigated. To investigate the regulation effect of quercetin on the Wnt/β-catenin signaling transcription, a Wnt/β-catenin signaling pathway reporter gene model was build, and the influence of quercetin on mutuality gene of Wnt/β-catenin signaling pathway was studied. By these analysis, the probable molecular mechanisms of quercetin on SW480 cell was analyzed, and maybe provide some theoretical evidences for developing quercetin as an anti-tumor drug.
Methods:
1. Effect of quercetin on proliferation of SW480 cells was analyzed by MTT assay.
2. Cell cycle and apoptosis rate of SW480 cells induced with quercetin were detected by flowcytometry.
3. After treatment with quercetin, protein expression of cyclin B1 in SW480 cells was analyzed by Western blot analysis.
4. We Build a Wnt/β-catenin signaling pathway reporter gene model. The regulation effect of quercetin on the Wnt/β-catenin signaling transcription was investigated by using the reporter gene model.
5. Effects of quercetin on expression ofβ-catenin/TCF downstream target genes, cyclin D1 and survivin, of Wnt/β-catenin signaling pathway were detected by Semi-quantitative RT-PCR and Western blot analysis.
6. Statistics Methods: Statistic analysis was performed with SPSS 12.0. All experiment data were indicated Mean±STD.
Results:
1. Quercetein can inhibite the proliferation of SW480 cells By MTT analysis, it is shown that proliferation of SW480 cells was significantly inhibited by quercetein at concentrations of 40, 80, 160μmol/L (P < 0.01), and showing a dose- and time-dependent manner.
2. After treatment with 40, 60, 80μmol/L quercetin for 48 h, the percentages of SW480 cells at G0/G1 phase were decreased, and those at G2/M phase were increased markedly (P<0.01). After incubated with 80μmol/L quercetin for 48 h, apoptosis rate of SW480 cells increased from 2.68%±1.04% of control group to 13.32%±4.62% (P<0.01).
3. Expression of cyclin B1 in SW480 cells could be down-regulated by quercetin in a dose-depedent manner (r=0.999, P=0.001).
4. Quercetin can down-regulate TCF-4 reporter gene We built a Wnt/β-catenin signaling pathway reporter gene model. Activities of TCF-4 reporter gene was significantly down-regulated by quercetin analyzed by using the reporter gene model (P<0.05), and showing a dose- and time- dependent manner.
5. Expression of cyclin D1 and survivin in SW480 cells were down-regulated markedly by quercetin in a dose-dependent manner.
Conclusions: Quercetin can inhibit proliferation, induce apoptosis and block the cells at G2/M phase of SW480 cells. Cyclin D1 and survivin expression,β-catenin/TCF downstream target genes, in both mRNA and protein level could be down-regulated markedly by quercetin. The anti-tumor mechanism of quercetin may be related to the inhibition of Wnt/β-catenin signaling pathway.
方法:1.采用MTT比色法检测槲皮素对人结肠癌细胞SW480细胞增殖反应的作用;2.用流式细胞技术分析槲皮素对人结肠癌细胞SW480细胞周期、凋亡的影响。3.采用Western blot分析槲皮素对SW480细胞cyclin B1蛋白表达的影响。4.建立Wnt/β-catenin信号转导通路报告基因模型,研究槲皮素对SW480细胞Wnt/β-catenin信号转导通路的调控作用。5.用半定量RT-PCR和Western blot方法研究槲皮素对Wnt/β-catenin信号转导通路β-catenin/TCF下游靶基因cyclin D1和survivin mRNA及蛋白表达的影响。6.统计学方法:所有数据采用SPSS 12.0软件进行统计学处理。实验结果均以x±S表示,组内比较采用单因素方差分析,多个实验组均数与一个对照组均数比较用Dunnett-t检验,回归方程间比较用t检验与方差分析,以α=0.05或α=0.01为检验水准。
结果:
1.槲皮素抑制SW480细胞增殖
MTT检测结果显示,40、80、160μmol/L的槲皮素对SW480细胞增殖有明显的抑制作用,与对照组比较有显著性差异(P<0.01),高浓度槲皮素组与低浓度组比较,对SW480细胞抑制作用也明显差异(P<0.05),抑制作用呈剂量依赖性和时间依赖性(r=0.99, P=0.00)。
2.槲皮素可诱导SW480细胞周期阻滞和细胞凋亡
流式细胞术检测结果显示,40、60、80μmol/L的槲皮素作用SW480细胞48 h后,G0/G1期和S期细胞减少,G2/M期细胞显著增多(P<0.01)。槲皮素可以剂量依赖性地诱导SW480细胞凋亡,80μmol/L的槲皮素作用48 h后,细胞凋亡率(13.32%±4.62%)明显升高,与对照组(2.68%±1.04%)相比有显著性差异(P<0.01)。
3.槲皮素可明显下调SW480细胞cyclin B1蛋白的表达
经槲皮素作用48 h后,SW480细胞cyclin B1蛋白表达水平明显下降(P<0.05),抑制作用呈剂量依赖性(r=0.999, P=0.001)。
4.槲皮素可以明显下调SW480细胞TCF-4报告基因TOPFLASH活性
应用双荧光素酶报告基因质粒TOPFLASH/FOPFLASH及内参照质粒pRL-null瞬时转染SW480细胞,建立了Wnt/β-catenin信号转导通路双荧光素酶报告基因模型。经分析结果显示,槲皮素处理转染报告基因的SW480细胞24 h后,TCF-4报告基因TOPFLASH活性显著降低,160μ?mol/L槲皮素作用24 h后,TOPFLASH报告基因RLU值由对照组的195.65±12.25降至11.54±5.60,差异有统计学意义(P<0.05),但FOPFLASH活性没有明显的变化(P>0.05),提示槲皮素可剂量依赖性抑制β-catenin/TCF的转录活性。
5.槲皮素可显著下调SW480细胞cyclin D1和survivin mRNA表达和蛋白表达水平
60、120μmol/L槲皮素作用24 h后,SW480细胞cyclin D1和survivin mRNA表达和蛋白表达水平均显著下降,抑制作用呈剂量依赖性(P<0.05)。
结论:槲皮素可明显抑制SW480细胞增殖,诱导细胞凋亡,将SW480细胞周期阻滞于G2/M期。槲皮素可剂量依赖性抑制β-catenin/TCF的转录活性,下调β-catenin/TCF下游靶基因cyclin D1和survivin的mRNA表达和蛋白表达水平,提示其抗肿瘤机制可能与其抑制Wnt/β-catenin信号转导通路有关。
Objective: Effects of quercetin on proliferation, cell cycle and apoptosis of colon carcinoma cell line SW480 were investigated. To investigate the regulation effect of quercetin on the Wnt/β-catenin signaling transcription, a Wnt/β-catenin signaling pathway reporter gene model was build, and the influence of quercetin on mutuality gene of Wnt/β-catenin signaling pathway was studied. By these analysis, the probable molecular mechanisms of quercetin on SW480 cell was analyzed, and maybe provide some theoretical evidences for developing quercetin as an anti-tumor drug.
Methods:
1. Effect of quercetin on proliferation of SW480 cells was analyzed by MTT assay.
2. Cell cycle and apoptosis rate of SW480 cells induced with quercetin were detected by flowcytometry.
3. After treatment with quercetin, protein expression of cyclin B1 in SW480 cells was analyzed by Western blot analysis.
4. We Build a Wnt/β-catenin signaling pathway reporter gene model. The regulation effect of quercetin on the Wnt/β-catenin signaling transcription was investigated by using the reporter gene model.
5. Effects of quercetin on expression ofβ-catenin/TCF downstream target genes, cyclin D1 and survivin, of Wnt/β-catenin signaling pathway were detected by Semi-quantitative RT-PCR and Western blot analysis.
6. Statistics Methods: Statistic analysis was performed with SPSS 12.0. All experiment data were indicated Mean±STD.
Results:
1. Quercetein can inhibite the proliferation of SW480 cells By MTT analysis, it is shown that proliferation of SW480 cells was significantly inhibited by quercetein at concentrations of 40, 80, 160μmol/L (P < 0.01), and showing a dose- and time-dependent manner.
2. After treatment with 40, 60, 80μmol/L quercetin for 48 h, the percentages of SW480 cells at G0/G1 phase were decreased, and those at G2/M phase were increased markedly (P<0.01). After incubated with 80μmol/L quercetin for 48 h, apoptosis rate of SW480 cells increased from 2.68%±1.04% of control group to 13.32%±4.62% (P<0.01).
3. Expression of cyclin B1 in SW480 cells could be down-regulated by quercetin in a dose-depedent manner (r=0.999, P=0.001).
4. Quercetin can down-regulate TCF-4 reporter gene We built a Wnt/β-catenin signaling pathway reporter gene model. Activities of TCF-4 reporter gene was significantly down-regulated by quercetin analyzed by using the reporter gene model (P<0.05), and showing a dose- and time- dependent manner.
5. Expression of cyclin D1 and survivin in SW480 cells were down-regulated markedly by quercetin in a dose-dependent manner.
Conclusions: Quercetin can inhibit proliferation, induce apoptosis and block the cells at G2/M phase of SW480 cells. Cyclin D1 and survivin expression,β-catenin/TCF downstream target genes, in both mRNA and protein level could be down-regulated markedly by quercetin. The anti-tumor mechanism of quercetin may be related to the inhibition of Wnt/β-catenin signaling pathway.
引文
1 Echeverry C, Blasina F, Leighton F, et al. Cytoprotection by neutral fraction of tannat red wine against oxidative stress-induced cell death. Agric Food Chem, 2004, 52(24): 7395～7399
2 Wenzel U, Herzog A, Kuntz S, et al. Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells. Proteomics, 2004, 4(7): 2160～2174
3 Sampson L, Rimm E, Hollman PC, et al. Flavonol and flavone intakes in US health professionals. Am Diet Assoc, 2002, 102(10): 1414～1420
4 Candlish JK. Antioxidants in food and chronic degenerative disease. Biomed Enriron, 1996, (2～3) :117
5 Borska S, Gebarowska E, Wysocka T, et al. The effects of quercetin vs cisplatin on proliferation and the apoptotic process in A549 and SW1271 cell lines in in vitro conditions. Folia Morphol (Warsz), 2004, 63(1): 103～105
6 Akbas SH, Timur M, Ozben T. The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells. Surg Res, 2005, 125(1):49～55
7 Pawlikowska-Pawlega B, Jakubowicz-Gil J, Rzymowska J, et al. The effect of quercetin on apoptosis and necrosis induction in human colon adenocarcinoma cell line LS180. Folia Histochem Cytobiol, 2001, 39(2): 217～218
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1 Akbas SH, Timur M, Ozben T. The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells. Surg Res, 2005, 125(1): 49～55
2 Kawabata K, Murakami A, Ohigashi H. Nobiletin, a citrus flavonoid, down-regulates matrix metalloproteinase-7 (matrilysin) expression in HT-29 human colorectal cancer cells. Biosci Biotechnol Biochem, 2005, 69 (2): 307～314
3 Borska S, Gebarowska E, Wysocka T, et al. The effects of quercetin vs cisplatin on proliferation and the apoptotic process in A549 and SW1271 cell lines in in vitro conditions. Folia Morphol (Warsz), 2004, 63(1): 103～105
4 Pawlikowska-Pawlega B, Jakubowicz-Gil J, Rzymowska J, et al. The effect of quercetin on apoptosis and necrosis induction in human colon adenocarcinoma cell line LS180. Folia Histochem Cytobiol, 2001, 39(2): 217～218
5 Marion R, Robert E, Brigitte M. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutrion and cancer, 1999, 34(1): 88～89
6 Waltzer L, Bienz M. The control of beta-catenin and TCF during embryonic development and cancer. Cancer and Metastasis Reviews, 1999, 18(2): 231～246
7 Behrens J. Control of beta-catenin signaling in tumor development. Ann N Y Acad Sci, 2000, 910: 21～33
8 Marion R, Robert E, Brigitte M. Quercetin-induced apoptosisin colorectal tumor cells: possible role of EGF receptor signaling. Nutrion and cancer, 1999, 34(1): 88～89
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1 Echeverry C, Blasina F, Leighton F, et al. Cytoprotection by neutral fraction of tannat red wine against oxidative stress-induced cell death. Agric Food Chem, 2004, 52(24): 7395～7399
2 Wenzel U, Herzog A, Kuntz S, et al. Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells. Proteomics, 2004, 4(7): 2160～2174
3 Sampson L, Rimm E, Hollman PC, et al. Flavonol and flavone intakes in US health professionals. Am Diet Assoc, 2002, 102(10): 1414～1420
4 Akbas SH, Timur M, Ozben T. The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells. Surg Res, 2005, 125(1): 49～55
5 Kawabata K, Murakami A, Ohigashi H. Nobiletin, a citrus flavonoid, down-regulates matrix metalloproteinase-7 (matrilysin) expression in HT-29 human colorectal cancer cells. Biosci Biotechnol Biochem, 2005, 69 (2): 307～314
6 Borska S, Gebarowska E, Wysocka T, et al. The effects of quercetin vs cisplatin on proliferation and the apoptotic process in A549 and SW1271 cell lines in in vitro conditions. Folia Morphol (Warsz), 2004, 63(1): 103～105
7 Pawlikowska-Pawlega B, Jakubowicz-Gil J, Rzymowska J, et al. The effect of quercetin on apoptosis and necrosisinduction in human colon adenocarcinoma cell line LS180. Folia Histochem Cytobiol, 2001, 39(2): 217～218
8 Marion R, Robert E, Brigitte M. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutrion and cancer, 1999, 34(1): 88～89
9 Kuo SM. Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells. Cancer Lett, 1996, 110: 41～48
10 Agullo G, Gamet Payrastre L, Fernandez Y, et al. Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells). Cancer Lett, 1996, 105(11): 61～70
11 Hosokawa N, Hosokawa Y, Sakai T, et al. Inhibitory effect of quercetin on the synthesis of a possibly cell-cycle-related
17-kDa protein in human colon cancer cells. Int. J. Cancer, 1990, 45(6): 1119～1124
12 Behrens J. Control of beta-catenin signaling in tumor development. Ann N Y Acad Sci, 2000, 910: 21～33
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16 Xing Y, Clements WK, Kimelman D, et al. Crystal structure ofa beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev, 2003, 17:2753～2764
17 Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell, 2000, 103:311～320
18 Mao J, Wang J, Liu B, et al. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell, 2001, 7:801～809
19 Satoh S, Daigo Y, Furukawa Y, et a1. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet, 2000, 24:245～250
20 Kikuchi A. Roles of Axin in the Wnt signalling pathway. Cell Signal, l999, 11:777～788
21 Luo W, Lin SC. Axin:a master scafold for multiple signaling pathways. Neurosignals, 2004, 13:99～l13
22 Salahshor S, Woodget JR. The links between axin and carcinogenesis. J Clin Pathol, 2005, 58:225～236
23 Lee E, Salic A, Kruger R, et al. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol, 2003, 1, El0
24 Tolwinski NS, Wieschaus E. Rethinking WNT signaling. Trends Genet, 2004, 20:l77～18l
25 Cong F, Schweizer L, Varmus H. Wnt signals across the plasmam embrane to activate the beta-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP.Development, 2004, 131:5l03～5115
26 Morin PJ, Sparks AB, Korinek V , et al. Activation of β-catenin-tcf signaling in colon cancer by mutations in β-catenin or APC. Science, 1997, 275:1787～1790
27 Samowitz WS,Powers MD,Spirio LN,et al. Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas. Cancer Res, 1999, 59:1442～1444
28 Polakis P. The oncogenic activation of beta-catenin. Curr Opin Genet Dev, 1999,9:15～21
29 Grady WM. Genomic instability and colon cancer. Cancer Metastasis Rev, 2004, 23:11～27
30 Tighe A, Johnson VL Taylor SS. Truncating APC mutations have dominant effects on proliferation, spindle checkpoint control, survival and chromosome stability. J Cell Sci, 2004, 117:6339～6353
31 Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science, 2000, 287:1606～1609
32 van der Luijt R, Khan PM, Vasen H, van Leeuwen C, et al. Rapid detection of trans lation-terminating mutations at the adenomatous polyposis coli(APC)gene by direct protein truncation test, Genomics, 1994;20:1～4
33 Ballhausen WG. Genetic testing for familial adenomatous polyposis. Ann N Y Acad Sci, 2000, 910:36～47
34 Maruyama K, Ochiai A, Akimoto S, et al. Cytoplasmicbeta-catenin accumulation as a predictor of hematogenous metastasis in human colorectal cancer. Oncology, 2000, 59:302～309
35 Wong SC, Lo ES, Chan AK, et al. Nuclear beta-catenin as a potential prognostic and diagnostic marker in patients with colorectal cancer from Hone Kong. Mol pathol, 2003, 56:347～352
36 Wong SC, Lo ES, Lee KC, et al. Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer. Clin Cancer Res, 2004, 10:1401～1408
37 Dihlmann S, Klein S, Doeberitz Mv MK. Reduction of beta-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated beta-catenin. Mol Cancer Ther, 2003, 2:509～516
38 Nath N, Kashfi K, Chen J, et al. Nitric oxide-donating aspirin inhibits beta-catenin/T cell factor (TCF) signaling in SW480 colon cancer cells by disrupting the nuclear beta-catenin-TCF association. Proc Natl Acad Sci USA, 2003, 100:12584～12589
39 Williams JL, Borgo S, Hasan I, et al. Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) alter the kinetics of human colon cancer cell lines more effectively than traditional NSAIDs: implications for colon cancer chemoprevention. Cancer Res, 2001, 61: 3285～3289
40 Williams IL, Nath N, Chen J, et al. Growth inhibition of humancolon cancer cells by nitric oxide (NO)-donating aspirin is associated with cyclooxygenase-2 induction and beta-catenin/T-cell factor signaling, nuclear factor-kappaB, and NO synthase 2 inhibition: implications for chemoprevention. Cancer Res, 2003, 63:7613～7618
41 Smith ML, Hawcroft G, Hull M A. The effect of non-steroidal anti-inflammatory drugs on human colorectal cancer cells: evidence of different mechanisms of action. Eur J Cancer, 2000, 36:664～674
42 Hawcroft G, D Amico M, A1banese C, et al. Indomethacin induces differential expression of beta-catenin, gamma-catenin and T-cell factor target genes in human colorectal cancer cells. Carcinogenesis, 2002, 23:107～114
43 Lepourcelet M, Chen YN, France DS, et al. Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex. Cancer cell, 2004, 5:91～102
44 Jaiswal AS, Marlow BP, Gupta N, et al. Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells. Oncogene, 2002, 21:8414～8427
45 Joe AK, Liu H, Suzui M, et al. Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines. Clin Cancer Res, 2002, 8:893-903
46 Sun Y, Kolligs FT, Hottiger MO, et a1. Regulation ofbeta-catenin transformation by the p300 transcriptional coactivator. Proc Natl Acad Sci USA, 2000, 97:l26l3～126l8
47 Hecht A, Vleminckx K, Stemmler MP, et a1. The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J, 2000, 19:1839-1850
48 Takemaru KI. Moon RT. The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression. J Cell Biol, 2000, 149:249～254
49 Miyagishi M, Fujii R, Hatta M, et a1. Regulation of Lef-mediated transcription and p53-dependent pathway by associating beta-catenin with CBP/p300. J Bio1 Chem, 2000, 275:35170～35175
50 Ma H. Nguyen C. Lee KS, et al. Differentia1 roles for the coactivators CBP and p300 on TCF/beta-catenin mediated surviving gene expression. Oncogene, 2005, 24:3619～3631
51 Kramps T, Peter O, Brunner E, et a1. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell, 2002, 109:47～60
52 Zhou L, An N, Haydon RC, et al. Tyrosine kinase inhibitor STI-571/Gleevec down-regulates the beta-catenin signaling activity. Cancer Lett, 2003, 193:161～170
53 Brown WA, Skinner SA, Vogiagis D, et al. Inhibition of beta-catenin translocation in rodent colorectal tumors: a novel explanation for the protective effect of nonsteroidal anti-inflammatory drugs in colorectal cancer. Dig Dis Sci, 2001, 46:2314～2321
54 Evans JF. Rofecoxib (Vioxx), a specific cyclooxygenase-2 inhibitor, is chemopreventive in a mouse model of colon cancer. Am J Clin Oncol, 2003, 26:S62～S65
55 Oshima M, Murai N, Kargman S, et al. Chemoprevention of intestinal polyposis in the Apcdelta716 mouse by rofecoxib, a specific cyclooxygenase-2 inhibitor. Cancer Res, 2001, 61:1733～1740
56 Yao M, Kargman S, Lam EC, et al. Inhibition of cyclooxygenase-2 by rofecoxib attenuates the growth and metastatic potential of colorectal carcinoma in mice. Cancer Res, 2003, 63:586～592
57 Griffiths GJ. Exisulind Cell Pathways. Curr Opin Investing Drugs, 2000, 1:386～391
58 Fiorucci S, Santucci L, Gresele P, et al. Gastrointestinal safety of NO-aspirin (NCX-4016)in healthy human volunteers: a proof of concepte endoscopic study. Gastroenterology, 2003, 124:600～607
59 Baron JA, Cole BF, Sandler RS, et al. A randomized trial of aspirin to prevent colorectal adenomas. N Engl J Med, 2003, 348:891～899
60 Sandler RS, Halabi S, Baron JA, et al. A randomized trial of aspirin to prevent colorectal adenomas in patients with previous colorectal cancer. N Engl J Med, 2003, 348:883～890
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1 Akbas SH, Timur M, Ozben T. The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells. Surg Res, 2005, 125(1): 49～55
2 Kawabata K, Murakami A, Ohigashi H. Nobiletin, a citrus flavonoid, down-regulates matrix metalloproteinase-7 (matrilysin) expression in HT-29 human colorectal cancer cells. Biosci Biotechnol Biochem, 2005, 69 (2): 307～314
3 Borska S, Gebarowska E, Wysocka T, et al. The effects of quercetin vs cisplatin on proliferation and the apoptotic process in A549 and SW1271 cell lines in in vitro conditions. Folia Morphol (Warsz), 2004, 63(1): 103～105
4 Pawlikowska-Pawlega B, Jakubowicz-Gil J, Rzymowska J, et al. The effect of quercetin on apoptosis and necrosis induction in human colon adenocarcinoma cell line LS180. Folia Histochem Cytobiol, 2001, 39(2): 217～218
5 Marion R, Robert E, Brigitte M. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutrion and cancer, 1999, 34(1): 88～89
6 Waltzer L, Bienz M. The control of beta-catenin and TCF during embryonic development and cancer. Cancer and Metastasis Reviews, 1999, 18(2): 231～246
7 Behrens J. Control of beta-catenin signaling in tumor development. Ann N Y Acad Sci, 2000, 910: 21～33
8 Marion R, Robert E, Brigitte M. Quercetin-induced apoptosisin colorectal tumor cells: possible role of EGF receptor signaling. Nutrion and cancer, 1999, 34(1): 88～89
9 Kuo SM. Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells. Cancer Lett, 1996, 110: 41～48
10 Agullo G, Gamet Payrastre L, Fernandez Y, et al. Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells). Cancer Lett, 1996, 105(11): 61～70
11 Hosokawa N, Hosokawa Y, Sakai T, et al. Inhibitory effect of quercetin on the synthesis of a possibly cell-cycle-related
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12 Goyette MC, Cho K, Fasching CL, et al. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Biol, 1992, 12(3): 1387～1395
1 Echeverry C, Blasina F, Leighton F, et al. Cytoprotection by neutral fraction of tannat red wine against oxidative stress-induced cell death. Agric Food Chem, 2004, 52(24): 7395～7399
2 Wenzel U, Herzog A, Kuntz S, et al. Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells. Proteomics, 2004, 4(7): 2160～2174
3 Sampson L, Rimm E, Hollman PC, et al. Flavonol and flavone intakes in US health professionals. Am Diet Assoc, 2002, 102(10): 1414～1420
4 Akbas SH, Timur M, Ozben T. The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells. Surg Res, 2005, 125(1): 49～55
5 Kawabata K, Murakami A, Ohigashi H. Nobiletin, a citrus flavonoid, down-regulates matrix metalloproteinase-7 (matrilysin) expression in HT-29 human colorectal cancer cells. Biosci Biotechnol Biochem, 2005, 69 (2): 307～314
6 Borska S, Gebarowska E, Wysocka T, et al. The effects of quercetin vs cisplatin on proliferation and the apoptotic process in A549 and SW1271 cell lines in in vitro conditions. Folia Morphol (Warsz), 2004, 63(1): 103～105
7 Pawlikowska-Pawlega B, Jakubowicz-Gil J, Rzymowska J, et al. The effect of quercetin on apoptosis and necrosisinduction in human colon adenocarcinoma cell line LS180. Folia Histochem Cytobiol, 2001, 39(2): 217～218
8 Marion R, Robert E, Brigitte M. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutrion and cancer, 1999, 34(1): 88～89
9 Kuo SM. Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells. Cancer Lett, 1996, 110: 41～48
10 Agullo G, Gamet Payrastre L, Fernandez Y, et al. Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells). Cancer Lett, 1996, 105(11): 61～70
11 Hosokawa N, Hosokawa Y, Sakai T, et al. Inhibitory effect of quercetin on the synthesis of a possibly cell-cycle-related
17-kDa protein in human colon cancer cells. Int. J. Cancer, 1990, 45(6): 1119～1124
12 Behrens J. Control of beta-catenin signaling in tumor development. Ann N Y Acad Sci, 2000, 910: 21～33
13 Shtutman, M, Zhurinsky, J, Simcha, I, et al. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A, 1999, 96(10):5522～5527
14 Tetsu, O, McCormick, F. Proliferation of cancer cells despite CDK2 inhibition. Cancer Cell, 2003, 3(3): 233～245
15 He TC, Sparks AB, Rago C, et al. Identification of c-MYC as a target of the APC pathway. Science, 1998, 281(5382): 1509～1512
16 Ma H, Nguyen C, Lee KS, et al. Differential roles for the coactivators CBP and p300 on TCF/β-catenin-mediated survivin gene expression. Oncogene, 2005,24(22): 3619～3631
17 Waltzer L, Bienz M. The control of beta-catenin and TCF during embryonic development and cancer. Cancer and Metastasis Reviews, 1999, 18(2): 231～246
18 Goyette MC, Cho K, Fasching CL, et al. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Biol, 1992, 12(3): 1387～1395
1 Thorstensen L, Lind GE, Lovig T, et al. Genetic and epigenetic changes of components affecting the WNT pathway in colorectal carcinomas stratified by microsatellite instability. Neoplasia, 2005, 7:99～108
2 Kang Y, Massague J. Epithe1ia1-mesenchyma1 transitions: twist in deve1opment and metastasis. Ce11, 2004, 118:277～279
3 Arias AM. Epithe1ia1 mesenchyma1 intemctions in cancer and deve1opment. Ce11, 2001, 105:425～431
4 Liu C, Li Y, Semenov M, et al. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell, 2002, 108:837～847
5 Amit S, Hatzubai A, Birman Y, et al. Axin-mediated CK Ⅰ phosphorylation of beta-catenin at Ser45: a molecular switch for the Wnt pathway. Genes Dev, 2002, 16:1066～1076
6 Hart M, Concordet JP, Lassot I, et al. The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cel1. Curr Biol, 1999, 9:207～210
7 Kitagawa M, Hatakeyama S, Shirane M, et al. An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin. EMBO J, 1999, 18:2401～2410
8 Winston JT, Strack P, Beer-Romero P, et al. The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs inIkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro. Genes Dev, 1999, 13:270～283
9 Aberle H, Bauer A, Stappert J, et al. Beta-catenin is a target for the ubiquitin-proteasome pathway. EMB0 J, 1997, 16:3797～3804
10 Lee JS, Ishimoto A, Yanagawa S. Characterization of mouse dishevelled (Dv1) proteins in Wnt/Wingless signaling pathway. J Biol Chem, 1999, 274:21464～21470
11 Smalley MJ, Sara E, Paterson H, et al. Interaction of axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription. EMB0 J, 1999, 18:2823～2835
12 Boutros M, Mihaly I, Bouwmeester T, et al. Signaling specificity by Frizzled receptors in Drosophila. Science, 2000, 288:1825～1828
13 Kolligs FT, Bommer G, Goke B. Wnt/beta-catenin/tcf signaling: a critical pathway in gastrointestinal tumorigenesis. Digestion, 2002, 66, 131～144
14 Ha NC, Tonozuka T, Stamos JL, et al. Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. Mol Cell, 2004, 15:511～52l
15 Xing Y, Clements WK, Le Trong I, et a1. Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function. Mol Cell, 2004, 15:523～533
16 Xing Y, Clements WK, Kimelman D, et al. Crystal structure ofa beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev, 2003, 17:2753～2764
17 Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell, 2000, 103:311～320
18 Mao J, Wang J, Liu B, et al. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell, 2001, 7:801～809
19 Satoh S, Daigo Y, Furukawa Y, et a1. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet, 2000, 24:245～250
20 Kikuchi A. Roles of Axin in the Wnt signalling pathway. Cell Signal, l999, 11:777～788
21 Luo W, Lin SC. Axin:a master scafold for multiple signaling pathways. Neurosignals, 2004, 13:99～l13
22 Salahshor S, Woodget JR. The links between axin and carcinogenesis. J Clin Pathol, 2005, 58:225～236
23 Lee E, Salic A, Kruger R, et al. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol, 2003, 1, El0
24 Tolwinski NS, Wieschaus E. Rethinking WNT signaling. Trends Genet, 2004, 20:l77～18l
25 Cong F, Schweizer L, Varmus H. Wnt signals across the plasmam embrane to activate the beta-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP.Development, 2004, 131:5l03～5115
26 Morin PJ, Sparks AB, Korinek V , et al. Activation of β-catenin-tcf signaling in colon cancer by mutations in β-catenin or APC. Science, 1997, 275:1787～1790
27 Samowitz WS,Powers MD,Spirio LN,et al. Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas. Cancer Res, 1999, 59:1442～1444
28 Polakis P. The oncogenic activation of beta-catenin. Curr Opin Genet Dev, 1999,9:15～21
29 Grady WM. Genomic instability and colon cancer. Cancer Metastasis Rev, 2004, 23:11～27
30 Tighe A, Johnson VL Taylor SS. Truncating APC mutations have dominant effects on proliferation, spindle checkpoint control, survival and chromosome stability. J Cell Sci, 2004, 117:6339～6353
31 Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science, 2000, 287:1606～1609
32 van der Luijt R, Khan PM, Vasen H, van Leeuwen C, et al. Rapid detection of trans lation-terminating mutations at the adenomatous polyposis coli(APC)gene by direct protein truncation test, Genomics, 1994;20:1～4
33 Ballhausen WG. Genetic testing for familial adenomatous polyposis. Ann N Y Acad Sci, 2000, 910:36～47
34 Maruyama K, Ochiai A, Akimoto S, et al. Cytoplasmicbeta-catenin accumulation as a predictor of hematogenous metastasis in human colorectal cancer. Oncology, 2000, 59:302～309
35 Wong SC, Lo ES, Chan AK, et al. Nuclear beta-catenin as a potential prognostic and diagnostic marker in patients with colorectal cancer from Hone Kong. Mol pathol, 2003, 56:347～352
36 Wong SC, Lo ES, Lee KC, et al. Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer. Clin Cancer Res, 2004, 10:1401～1408
37 Dihlmann S, Klein S, Doeberitz Mv MK. Reduction of beta-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated beta-catenin. Mol Cancer Ther, 2003, 2:509～516
38 Nath N, Kashfi K, Chen J, et al. Nitric oxide-donating aspirin inhibits beta-catenin/T cell factor (TCF) signaling in SW480 colon cancer cells by disrupting the nuclear beta-catenin-TCF association. Proc Natl Acad Sci USA, 2003, 100:12584～12589
39 Williams JL, Borgo S, Hasan I, et al. Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) alter the kinetics of human colon cancer cell lines more effectively than traditional NSAIDs: implications for colon cancer chemoprevention. Cancer Res, 2001, 61: 3285～3289
40 Williams IL, Nath N, Chen J, et al. Growth inhibition of humancolon cancer cells by nitric oxide (NO)-donating aspirin is associated with cyclooxygenase-2 induction and beta-catenin/T-cell factor signaling, nuclear factor-kappaB, and NO synthase 2 inhibition: implications for chemoprevention. Cancer Res, 2003, 63:7613～7618
41 Smith ML, Hawcroft G, Hull M A. The effect of non-steroidal anti-inflammatory drugs on human colorectal cancer cells: evidence of different mechanisms of action. Eur J Cancer, 2000, 36:664～674
42 Hawcroft G, D Amico M, A1banese C, et al. Indomethacin induces differential expression of beta-catenin, gamma-catenin and T-cell factor target genes in human colorectal cancer cells. Carcinogenesis, 2002, 23:107～114
43 Lepourcelet M, Chen YN, France DS, et al. Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex. Cancer cell, 2004, 5:91～102
44 Jaiswal AS, Marlow BP, Gupta N, et al. Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells. Oncogene, 2002, 21:8414～8427
45 Joe AK, Liu H, Suzui M, et al. Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines. Clin Cancer Res, 2002, 8:893-903
46 Sun Y, Kolligs FT, Hottiger MO, et a1. Regulation ofbeta-catenin transformation by the p300 transcriptional coactivator. Proc Natl Acad Sci USA, 2000, 97:l26l3～126l8
47 Hecht A, Vleminckx K, Stemmler MP, et a1. The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J, 2000, 19:1839-1850
48 Takemaru KI. Moon RT. The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression. J Cell Biol, 2000, 149:249～254
49 Miyagishi M, Fujii R, Hatta M, et a1. Regulation of Lef-mediated transcription and p53-dependent pathway by associating beta-catenin with CBP/p300. J Bio1 Chem, 2000, 275:35170～35175
50 Ma H. Nguyen C. Lee KS, et al. Differentia1 roles for the coactivators CBP and p300 on TCF/beta-catenin mediated surviving gene expression. Oncogene, 2005, 24:3619～3631
51 Kramps T, Peter O, Brunner E, et a1. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell, 2002, 109:47～60
52 Zhou L, An N, Haydon RC, et al. Tyrosine kinase inhibitor STI-571/Gleevec down-regulates the beta-catenin signaling activity. Cancer Lett, 2003, 193:161～170
53 Brown WA, Skinner SA, Vogiagis D, et al. Inhibition of beta-catenin translocation in rodent colorectal tumors: a novel explanation for the protective effect of nonsteroidal anti-inflammatory drugs in colorectal cancer. Dig Dis Sci, 2001, 46:2314～2321
54 Evans JF. Rofecoxib (Vioxx), a specific cyclooxygenase-2 inhibitor, is chemopreventive in a mouse model of colon cancer. Am J Clin Oncol, 2003, 26:S62～S65
55 Oshima M, Murai N, Kargman S, et al. Chemoprevention of intestinal polyposis in the Apcdelta716 mouse by rofecoxib, a specific cyclooxygenase-2 inhibitor. Cancer Res, 2001, 61:1733～1740
56 Yao M, Kargman S, Lam EC, et al. Inhibition of cyclooxygenase-2 by rofecoxib attenuates the growth and metastatic potential of colorectal carcinoma in mice. Cancer Res, 2003, 63:586～592
57 Griffiths GJ. Exisulind Cell Pathways. Curr Opin Investing Drugs, 2000, 1:386～391
58 Fiorucci S, Santucci L, Gresele P, et al. Gastrointestinal safety of NO-aspirin (NCX-4016)in healthy human volunteers: a proof of concepte endoscopic study. Gastroenterology, 2003, 124:600～607
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