茶叶提取物EGCG对人乳腺癌细胞侵袭转移的影响及其可能机制的探讨
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摘要
目的: 研究茶叶提取物EGCG对人乳腺癌细胞株MDA-MB-231侵袭转移能力的影响及其可能机制。
方法：以高转移人乳腺癌细胞株MDA-MB-231为研究对象，应用糖基化抑制剂苯甲基-α-N-乙酰半乳糖胺为去糖基化阳性对照，以RPMI-1640为阴性对照。MTT法检测EGCG对肿瘤细胞增殖的抑制作用，求出IC50。免疫细胞化学染色法检测MUC1的表达，MTT法检测EGCG对乳腺癌细胞黏附Matrigel能力的影响，Transwell小室进行人工重组基底膜侵袭实验和运动实验,明胶酶谱法(SDS-PAGE)检测肿瘤细胞用药前后分泌Ⅳ型胶原酶MMP-2、MMP-9的差异。
结果：EGCG对人乳腺癌细胞株MDA-MB-231的细胞增殖抑制作用随药物浓度增大和时间延长而增强，其IC50为72.45μg/ml ，用其半量（36μg/ml）作为实验用药浓度。EGCG实验组与未处理组相比，MUC1表达降低，阳性率分别为64%和100%，具有显著性差异（P<0.01），与苯甲基-α-N-乙酰半乳糖胺组的MUC1抑制效应一致。EGCG实验组肿瘤细胞对基底膜的黏附能力下降（P<0.01），未处理组、
苯甲基处理组和EGCG处理组侵袭迁移能力分别为86.5±14.9、64.7±1.2、40.9±10.8和109.0±8.30、76.2±10.69、50.8±5.30。其分泌Ⅳ型胶原酶MMP-2、MMP-9的能力亦明显降低。
结论： 体外实验中，EGCG对MDA-MB-231细胞株的增殖有抑制作用，与时间和剂量呈依赖性；EGCG具有抑制人乳腺癌细胞黏附、侵袭、迁移能力，抑制IV型胶原酶MMP-2、MMP-9的分泌，其机理可能是通过去糖基化干预MUC1的生成。
Objective: To study the effects of tea extract EGCG on invasion and metastasis of breast cancer line MDA-MB-231 and the possible mechanisms.
Methods: Our study was conducted on a highly metastatic human breast cancer cell line MDA-MB-231. Taking benzyl-α-GalNAc(a glycosilation inhinbitor) and RPMI-1640 group as positive and negative control respectively. MTT is performed to determine the growth inhibiting effect of EGCG for getting its IC50. Expression of MUCI was observed by immunocytochemistry. Adhesion of MDA-MB-231 to Matrigel was measured with MTT assay. Invasion and metastatic potential were evaluated with transwell chamber. Collagenase-Ⅳ secretion was dectected
with SDS-PAGE before and after the administration of EGCG .
Results: Our study demonstrated that EGCG had a time/dose- dependent growth inhibiting effect on MDA-MB-231 and the IC50 was 72.45μg/ml. Compared with untreated group, MDA-MB-231 treated with 36μg/ml EGCG displayed decreased MUCI expression (64% vs 100%), while MUC1 expression was not significantly different between benzyl-α-GalNAc group and EGCG group. Compared with untreated group，Adhesion is remarkably decreased. Invasive and metastatic abilities of three groups are 85.5±14.9、64.7±1.2、40.9±10.8 and 109.0±8.30、76.2±10.69、50.8±5.30 respectively,the Collagenase-Ⅳ secretion of MDA-MB-231 cells was also decreased.
Conclusion: In vitro, EGCG has a time/dose-dependent growth inhibiting effect on MDA-MB-231 cells. EGCG may suppress adhesion , metastasis, Invasion, and Collagenase-Ⅳsecretion of MDA-MB-231 cells by inhibiting the production of MUC1 .
方法：以高转移人乳腺癌细胞株MDA-MB-231为研究对象，应用糖基化抑制剂苯甲基-α-N-乙酰半乳糖胺为去糖基化阳性对照，以RPMI-1640为阴性对照。MTT法检测EGCG对肿瘤细胞增殖的抑制作用，求出IC50。免疫细胞化学染色法检测MUC1的表达，MTT法检测EGCG对乳腺癌细胞黏附Matrigel能力的影响，Transwell小室进行人工重组基底膜侵袭实验和运动实验,明胶酶谱法(SDS-PAGE)检测肿瘤细胞用药前后分泌Ⅳ型胶原酶MMP-2、MMP-9的差异。
结果：EGCG对人乳腺癌细胞株MDA-MB-231的细胞增殖抑制作用随药物浓度增大和时间延长而增强，其IC50为72.45μg/ml ，用其半量（36μg/ml）作为实验用药浓度。EGCG实验组与未处理组相比，MUC1表达降低，阳性率分别为64%和100%，具有显著性差异（P<0.01），与苯甲基-α-N-乙酰半乳糖胺组的MUC1抑制效应一致。EGCG实验组肿瘤细胞对基底膜的黏附能力下降（P<0.01），未处理组、
苯甲基处理组和EGCG处理组侵袭迁移能力分别为86.5±14.9、64.7±1.2、40.9±10.8和109.0±8.30、76.2±10.69、50.8±5.30。其分泌Ⅳ型胶原酶MMP-2、MMP-9的能力亦明显降低。
结论： 体外实验中，EGCG对MDA-MB-231细胞株的增殖有抑制作用，与时间和剂量呈依赖性；EGCG具有抑制人乳腺癌细胞黏附、侵袭、迁移能力，抑制IV型胶原酶MMP-2、MMP-9的分泌，其机理可能是通过去糖基化干预MUC1的生成。
Objective: To study the effects of tea extract EGCG on invasion and metastasis of breast cancer line MDA-MB-231 and the possible mechanisms.
Methods: Our study was conducted on a highly metastatic human breast cancer cell line MDA-MB-231. Taking benzyl-α-GalNAc(a glycosilation inhinbitor) and RPMI-1640 group as positive and negative control respectively. MTT is performed to determine the growth inhibiting effect of EGCG for getting its IC50. Expression of MUCI was observed by immunocytochemistry. Adhesion of MDA-MB-231 to Matrigel was measured with MTT assay. Invasion and metastatic potential were evaluated with transwell chamber. Collagenase-Ⅳ secretion was dectected
with SDS-PAGE before and after the administration of EGCG .
Results: Our study demonstrated that EGCG had a time/dose- dependent growth inhibiting effect on MDA-MB-231 and the IC50 was 72.45μg/ml. Compared with untreated group, MDA-MB-231 treated with 36μg/ml EGCG displayed decreased MUCI expression (64% vs 100%), while MUC1 expression was not significantly different between benzyl-α-GalNAc group and EGCG group. Compared with untreated group，Adhesion is remarkably decreased. Invasive and metastatic abilities of three groups are 85.5±14.9、64.7±1.2、40.9±10.8 and 109.0±8.30、76.2±10.69、50.8±5.30 respectively,the Collagenase-Ⅳ secretion of MDA-MB-231 cells was also decreased.
Conclusion: In vitro, EGCG has a time/dose-dependent growth inhibiting effect on MDA-MB-231 cells. EGCG may suppress adhesion , metastasis, Invasion, and Collagenase-Ⅳsecretion of MDA-MB-231 cells by inhibiting the production of MUC1 .
引文
Sazuka, M.;Murakami, S.;Isemura, M.; Satoh, K.; Nukiwa, T. Inhibitory effects of green tea infusion on in vitro invasion and in vivo metastasis of mouse lung carcinoma cells. Cancer Lett.1995;98,27-31.
Aznavoorian S, Murphy AN, Stetler, Stevenson WG, et al. Molecular aspects of tumor cell invasion and metastasis. Cancer,1993;71(4):1368-1383.
Deborah JH, Bruce AL, Vilram R, et al . Tenascin is differentially expressed in endometriun and endometriosis[J]. Pathol.1999;187:242-248.
Rafael F. The role of cell adhesion and migration in the in vitro invasiveness of mouse adrenal carcinoma cells[J]. Invasion Metastasis,1990;10:208-24.
Pereda MP, Hopfner U, Pagotto U, et al. Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion[J]. Br J Cancer,1999;81(3):382-386.
Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells[J]. Cancer Res, 1987;47:3239-3245.
刘红岩,韩锐. 金荞麦提取物抑制肿瘤细胞侵袭转移和HT-1080细胞产生Ⅳ型胶原酶的研究[J]. 中国药理学通报.1998,14(1):36-39.
Junger WG, Cardoza TA, Liu FC et al. Improved rapid photometric assay for quantitative measurement of PMN migration[J]. Immunol Methods,1993;160:73-9.
Zucker S, Mancuso P, DiMassimo B, et al. Comparison of techniques for measurement of gelatinases/type Ⅳ collagenase :enzyme-linked immunoassays versus substrate degradation assays[J]. Clin Exp Metastasis,1994,12:13-23.
朱峰,刘新光,梁念慈.大黄素、芹菜素抑制人卵巢癌细胞侵袭的体外实验研究[J].癌症,2003,22(4):358-362.
张天泽，主编.肿瘤学[M].天津科学技术出版社,1993;1410.
Wojtowicz Praga S, Low J,Marshall J,et al. Phase I trial of novel matrix metal oproteinase inhibitor batimastat(BB-94) in patients with advanced cancer[J]. Invest
New Drugs,1996,14(2):193-202.
Zhang Y, Griffith EC, Sage J,et al. Cell cycle inhibition by the antiangiogenic agent TNP-470 is mediated by P53 and P21 WAF1/CIP1[J]. Proc Natl Acad Sci USA,2000;97(12):6427-6432.
陈宗懋.中国茶经[M].上海:上海文化出版社,1999:1.
Graham HN.Green tea composition,consumption,and polyphenolchemistry[J]. Prev Med,1992,21(3):334-350.
Nakachi-K: Influence of drinking green tea on breast cancer malignancy among Japanese patients. Jpn-J-Cancer-Res. 1998 ; 89(3):254-61
Gao-YT: Reduced risk of esophageal cancer associated with green tea consumption. J-Natl-Cancer-Inst. 1994 Jun 1; 86(11): 855-8
Yu-Gp: Green-tea consumption and risk of stomach cancer: a population-based case-control study in Shanghai, China. Cancer-Causes-Control. 1995 Nov;6(6); 532-8
Ji-BTF: Green tea consumption and the risk of pancreatic and colorectal cancers. Int-J-Cancer. 1997 ; 70(3):255-8
Yamane-T: Inhibitory effects and toxicity of green tea polyphenols for gastrointestinal carcinogenesis. Cancer.1996; 77(8 suppl):1662-7
陈宗懋.中国茶经[M].上海：上海文化出版社,1999:102-109.
Ahmad N, Feyes DK, Nieminen AL, et al. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells.[J].J Natl Cancer Inst 1997;89(24):1881-1886.
Liang YC, Lin Shiau SY, Chen CF, et al. Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-)-epigallocatechin-3-gallate in human A431 epidermoid carcinoma cells[J]. J Cell Biochem 1997;67(1):55-56.
Lin YL, Lin JK. (-)-epigallacatechin-3-gallate blocks the induction of nitric oxidesynthase by down-regulating lipopolysaccharide-induced activity of transcription factor NF-κB[J]. Mol Pharmacol 1997;52(3):465-472.
Hendrix MJC, Wood WR, Seftor EA et al. Retinoic acid inhibition of human melanoma cell invasion throuth a reconstituted basement membrane and its relation to decreases in the expression of proteolytic enzymes and motility factor receptor. Cancer Res 1990;50;4121-30
Timar J,Chen YQ, Liu B, et al. The lipoxygenase metabolite 12(s)-HETE promotes α11bβ3 integrin-mediated tumor-cell spreading on fibronection. Int J cancer 1992;52:594-603
Yan C, Han R. Suppression of adhesion-induced protein tyrosine phosphorylation decreases the invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor. Invasion Metastasis 1997;17:189-198
Aznavoorian S, Syetler-Stevenson WG, Liotta LA. Molecular aspects of tumor cell invasion and metastasis[J]. Cancer 1993;71:1368.
Stetler-Stevenson WG. Type Ⅳ collagenases in tumor invasion and metastasis. Cancer Metast Rev 1990;31:19.
Joyce A.Schroeder,Melissa C.Thompson,Melissa Mockensturm et al. Tansgenic MUC1 interacts with Epidermal Growth Factor Receptor and correlates with Mitogen-activated Protein Kinase activation in the mouse mammary gland[J]. J Biol Chem 2001;276(16):13057-13064.
张立新,李春海.MUC1粘蛋白的免疫生物学作用及其在肿瘤生物治疗中的应用[J].中国肿瘤生物治疗杂志 2000;7(3):165-170.
UlloaF,Real FX.Benzyl-GalNAc induces a storage disease-like phenotype by perturbing the endocytic pathway[J]. J Biol Chem 2003;278(14):12374-83.
Schwartz B,Robert S,Bresalier,et al. The role of mucin in colon-cancer metastasis[J]. Cancer 1992;52(1):60-65
郑宏强,刘德育.蛇葡萄素抗黑色素瘤侵袭和转移的作用[J].癌症2003;22(4):363-367.
Zuker S, Wieman J, Lysik RM, et al. Gelatin-degrading type Ⅳcollagenases isolated from human small cell lung cancer. Invasion Metastasis 1989;9:167
Aznavoorian S, Murphy AN, Stetler, Stevenson WG, et al. Molecular aspects of tumor cell invasion and metastasis. Cancer,1993;71(4):1368-1383.
Deborah JH, Bruce AL, Vilram R, et al . Tenascin is differentially expressed in endometriun and endometriosis[J]. Pathol.1999;187:242-248.
Rafael F. The role of cell adhesion and migration in the in vitro invasiveness of mouse adrenal carcinoma cells[J]. Invasion Metastasis,1990;10:208-24.
Pereda MP, Hopfner U, Pagotto U, et al. Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion[J]. Br J Cancer,1999;81(3):382-386.
Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells[J]. Cancer Res, 1987;47:3239-3245.
刘红岩,韩锐. 金荞麦提取物抑制肿瘤细胞侵袭转移和HT-1080细胞产生Ⅳ型胶原酶的研究[J]. 中国药理学通报.1998,14(1):36-39.
Junger WG, Cardoza TA, Liu FC et al. Improved rapid photometric assay for quantitative measurement of PMN migration[J]. Immunol Methods,1993;160:73-9.
Zucker S, Mancuso P, DiMassimo B, et al. Comparison of techniques for measurement of gelatinases/type Ⅳ collagenase :enzyme-linked immunoassays versus substrate degradation assays[J]. Clin Exp Metastasis,1994,12:13-23.
朱峰,刘新光,梁念慈.大黄素、芹菜素抑制人卵巢癌细胞侵袭的体外实验研究[J].癌症,2003,22(4):358-362.
张天泽，主编.肿瘤学[M].天津科学技术出版社,1993;1410.
Wojtowicz Praga S, Low J,Marshall J,et al. Phase I trial of novel matrix metal oproteinase inhibitor batimastat(BB-94) in patients with advanced cancer[J]. Invest
New Drugs,1996,14(2):193-202.
Zhang Y, Griffith EC, Sage J,et al. Cell cycle inhibition by the antiangiogenic agent TNP-470 is mediated by P53 and P21 WAF1/CIP1[J]. Proc Natl Acad Sci USA,2000;97(12):6427-6432.
陈宗懋.中国茶经[M].上海:上海文化出版社,1999:1.
Graham HN.Green tea composition,consumption,and polyphenolchemistry[J]. Prev Med,1992,21(3):334-350.
Nakachi-K: Influence of drinking green tea on breast cancer malignancy among Japanese patients. Jpn-J-Cancer-Res. 1998 ; 89(3):254-61
Gao-YT: Reduced risk of esophageal cancer associated with green tea consumption. J-Natl-Cancer-Inst. 1994 Jun 1; 86(11): 855-8
Yu-Gp: Green-tea consumption and risk of stomach cancer: a population-based case-control study in Shanghai, China. Cancer-Causes-Control. 1995 Nov;6(6); 532-8
Ji-BTF: Green tea consumption and the risk of pancreatic and colorectal cancers. Int-J-Cancer. 1997 ; 70(3):255-8
Yamane-T: Inhibitory effects and toxicity of green tea polyphenols for gastrointestinal carcinogenesis. Cancer.1996; 77(8 suppl):1662-7
陈宗懋.中国茶经[M].上海：上海文化出版社,1999:102-109.
Ahmad N, Feyes DK, Nieminen AL, et al. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells.[J].J Natl Cancer Inst 1997;89(24):1881-1886.
Liang YC, Lin Shiau SY, Chen CF, et al. Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-)-epigallocatechin-3-gallate in human A431 epidermoid carcinoma cells[J]. J Cell Biochem 1997;67(1):55-56.
Lin YL, Lin JK. (-)-epigallacatechin-3-gallate blocks the induction of nitric oxidesynthase by down-regulating lipopolysaccharide-induced activity of transcription factor NF-κB[J]. Mol Pharmacol 1997;52(3):465-472.
Hendrix MJC, Wood WR, Seftor EA et al. Retinoic acid inhibition of human melanoma cell invasion throuth a reconstituted basement membrane and its relation to decreases in the expression of proteolytic enzymes and motility factor receptor. Cancer Res 1990;50;4121-30
Timar J,Chen YQ, Liu B, et al. The lipoxygenase metabolite 12(s)-HETE promotes α11bβ3 integrin-mediated tumor-cell spreading on fibronection. Int J cancer 1992;52:594-603
Yan C, Han R. Suppression of adhesion-induced protein tyrosine phosphorylation decreases the invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor. Invasion Metastasis 1997;17:189-198
Aznavoorian S, Syetler-Stevenson WG, Liotta LA. Molecular aspects of tumor cell invasion and metastasis[J]. Cancer 1993;71:1368.
Stetler-Stevenson WG. Type Ⅳ collagenases in tumor invasion and metastasis. Cancer Metast Rev 1990;31:19.
Joyce A.Schroeder,Melissa C.Thompson,Melissa Mockensturm et al. Tansgenic MUC1 interacts with Epidermal Growth Factor Receptor and correlates with Mitogen-activated Protein Kinase activation in the mouse mammary gland[J]. J Biol Chem 2001;276(16):13057-13064.
张立新,李春海.MUC1粘蛋白的免疫生物学作用及其在肿瘤生物治疗中的应用[J].中国肿瘤生物治疗杂志 2000;7(3):165-170.
UlloaF,Real FX.Benzyl-GalNAc induces a storage disease-like phenotype by perturbing the endocytic pathway[J]. J Biol Chem 2003;278(14):12374-83.
Schwartz B,Robert S,Bresalier,et al. The role of mucin in colon-cancer metastasis[J]. Cancer 1992;52(1):60-65
郑宏强,刘德育.蛇葡萄素抗黑色素瘤侵袭和转移的作用[J].癌症2003;22(4):363-367.
Zuker S, Wieman J, Lysik RM, et al. Gelatin-degrading type Ⅳcollagenases isolated from human small cell lung cancer. Invasion Metastasis 1989;9:167