EGCG对急性早幼粒细胞白血病细胞株NB4增殖、细胞周期及DAPK1基因甲基化影响的研究
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摘要
目的:探讨表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)对急性早幼粒细胞白血病细胞株NB4增殖、细胞周期的影响及其作用机制。方法:应用EGCG 0、50、75、100及125μmol/L分别处理NB4细胞24、48、72、96 h后,使用CCK-8法检测各时间点和浓度的细胞增殖水平,流式细胞术检测不同浓度EGCG处理NB4细胞48 h后的细胞周期情况,RT-PCR检测DNMT1、DNMT3a、DAPK1 mRNA的表达水平,甲基化特异性PCR检测DAPK1基因的甲基化状态,Western blot检测DAPK1蛋白的表达情况。结果:EGCG明显抑制NB4细胞增殖并诱导细胞阻滞于G_0/G_1期,且呈时间-浓度依赖性(r=0. 916)。EGCG呈浓度依赖性下调NB4细胞DNMT1、DNMT3a的表达,且对DNM T3a的作用更为明显。随EGCG浓度增加,DAPK1基因的甲基化程度减弱(r=-0. 813),DAPK1 mRNA和蛋白表达上调(r=0. 96,r=0. 991)。结论:EGCG通过抑制DNMT1和DNMT3a基因的表达,下调DAPK1S基因的甲基化程度,从而抑制NB4细胞增殖和诱导其细胞周期阻滞。
Objective: To investigate the effects of epigallocatechin-3-gallate(EGCG) on proliferation and cell cycle of acute promyelocytic leukemia NB4 cell line and to clarify the molecular mechanism. Methods: NB4 cells were treated with 0,50,75,100 and 125 μmol/L of EGCG for 24, 48, 72 and 96 h, respectively. The proliferation level of NB4 cells was measured by CCK-8 assay. The cell cycle progression of NB4 cells was assayed by flow cytometry. The mRNA expression levels of DNMT1, DNMT3 a and DAPKl were detected by RT-PCR. The methylation status of DAPKl gene was tested by methylation specific PCR, and the expression level of DAPK1 protein was detected by Western blot.Results: The proliferation and cell cycle progression of NB4 cells treated with EGCG were inhibited and showed the characteristic of time-dependent and dose-dependent manner. The expression level of DAPK1 and DNMT3 a decreased in NB4 cells treated with EGCG. The expression level of DAPK increased in NB4 cells treated with EGCG, while the methylation of DAPK1 gene decreased. Conclusion: EGCG inhibits the proliferation and cell cycle progression of NB4 cells by inhibiting the expression of DNMT1 and DNMT3 a and down-regulating the methylation status of DAPK1 gene.
Objective: To investigate the effects of epigallocatechin-3-gallate(EGCG) on proliferation and cell cycle of acute promyelocytic leukemia NB4 cell line and to clarify the molecular mechanism. Methods: NB4 cells were treated with 0,50,75,100 and 125 μmol/L of EGCG for 24, 48, 72 and 96 h, respectively. The proliferation level of NB4 cells was measured by CCK-8 assay. The cell cycle progression of NB4 cells was assayed by flow cytometry. The mRNA expression levels of DNMT1, DNMT3 a and DAPKl were detected by RT-PCR. The methylation status of DAPKl gene was tested by methylation specific PCR, and the expression level of DAPK1 protein was detected by Western blot.Results: The proliferation and cell cycle progression of NB4 cells treated with EGCG were inhibited and showed the characteristic of time-dependent and dose-dependent manner. The expression level of DAPK1 and DNMT3 a decreased in NB4 cells treated with EGCG. The expression level of DAPK increased in NB4 cells treated with EGCG, while the methylation of DAPK1 gene decreased. Conclusion: EGCG inhibits the proliferation and cell cycle progression of NB4 cells by inhibiting the expression of DNMT1 and DNMT3 a and down-regulating the methylation status of DAPK1 gene.
引文
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6 Amara K,Tfimeehe M,Ziadi S,et al.Prognostic significance of aberrant promoter hypermethylation of cpg islands in patients with diffuse large B-cell lymphomas.Ann Oncol.2008;19(10):1774-1786.
7 Voso MT,Gumiero D,D'Alo F,et al.DAP-kinase hypermethylation in the bonce marrow of patients with follicular lymphoma.Haematologica,2006;91(9):1252-1256.
8 Braggio E,Maiolino A,Gouveia ME,et al.Methylation status of nine tumor suppresor genes in multiple myeloma.Int J Hematol,2010;91(1):87-96.
9 Bialik S,Kimchi A.DAP-kinase as a target for drug design in cancer and diseases associated with accelerated cell death.Semin Cancer Biol,2004;14(4):283-294.
10 Valcic S,Timmermann BN,Alberts DS,et al.Inhibitory effect of six green tea atechins and caffeine on the growth of four selectedhuman tumor cell lines.Anticancer Drugs,1996;7(4):461-468.
11 Islam S,Islam N,Kemode T,et al.Involvement of caspase-3 in epigallocatechin-3-gallate mediated apoptosis of human chondrosarcoma cells.Biochem Biophys Res Commun,2000;270(3):793-797.
12 Wu KM,Ghantous H,Birnkrant DB.Current regulatory toxicology perspectives on the development of herbal medicines to prescription drug products in the United States.Food Chem Toxicol,2008;46(8):2606-2610.
13 Khan N,Mukhtar H.Multitargeted therapy of cancer by green teapolyphenols.Cancer Lett,2008;269(2):269-280.
14 Zhu YM,Huang Q,Lin J,et al.Expression of human DNA methyl transferase 1 in colorectal cancer tissues and their corresponding distant normal tissue.Int J Colorectal Dis,2007;22(6):661-666.
15 Navada SC,Steinmann J,Lubbert M,et al.Clinical development of demethylation agents in hematology.J Clin Invest,2014;124(1):40-46.
16 Kanai Y.Genome-wide DNA methylation profiles in precancerous conditions and cancers.Cancer Sci,2010;101(1):36-45.
2 Evens AM,Tallman MS,Gartenhaus RB.2004.The potential of arenic trioxide in the treatment of malignant disease:past,present and future.Leuk Res,2004;28(9):891-900.
3 Momparler RL,Bovenzi V.DNA methylation and cancer.J Cell Physiol,2000;183(2):145-154.
4 Chim CS,Fung TK,Wong KF,et al.Frequent DAP kinase but not p14 or Apaf-1 hypermethylation in B-cell chronic lymphocytic leukemia.J Hum Genet,2006;51(9):832-838.
5钱军,姚冬明,林江,等.慢性粒细胞白血病患者死亡相关蛋白激酶基因甲基化.中华血液学杂志.2008;29(12):850-851.
6 Amara K,Tfimeehe M,Ziadi S,et al.Prognostic significance of aberrant promoter hypermethylation of cpg islands in patients with diffuse large B-cell lymphomas.Ann Oncol.2008;19(10):1774-1786.
7 Voso MT,Gumiero D,D'Alo F,et al.DAP-kinase hypermethylation in the bonce marrow of patients with follicular lymphoma.Haematologica,2006;91(9):1252-1256.
8 Braggio E,Maiolino A,Gouveia ME,et al.Methylation status of nine tumor suppresor genes in multiple myeloma.Int J Hematol,2010;91(1):87-96.
9 Bialik S,Kimchi A.DAP-kinase as a target for drug design in cancer and diseases associated with accelerated cell death.Semin Cancer Biol,2004;14(4):283-294.
10 Valcic S,Timmermann BN,Alberts DS,et al.Inhibitory effect of six green tea atechins and caffeine on the growth of four selectedhuman tumor cell lines.Anticancer Drugs,1996;7(4):461-468.
11 Islam S,Islam N,Kemode T,et al.Involvement of caspase-3 in epigallocatechin-3-gallate mediated apoptosis of human chondrosarcoma cells.Biochem Biophys Res Commun,2000;270(3):793-797.
12 Wu KM,Ghantous H,Birnkrant DB.Current regulatory toxicology perspectives on the development of herbal medicines to prescription drug products in the United States.Food Chem Toxicol,2008;46(8):2606-2610.
13 Khan N,Mukhtar H.Multitargeted therapy of cancer by green teapolyphenols.Cancer Lett,2008;269(2):269-280.
14 Zhu YM,Huang Q,Lin J,et al.Expression of human DNA methyl transferase 1 in colorectal cancer tissues and their corresponding distant normal tissue.Int J Colorectal Dis,2007;22(6):661-666.
15 Navada SC,Steinmann J,Lubbert M,et al.Clinical development of demethylation agents in hematology.J Clin Invest,2014;124(1):40-46.
16 Kanai Y.Genome-wide DNA methylation profiles in precancerous conditions and cancers.Cancer Sci,2010;101(1):36-45.