乙酰化酶抑制剂Garcinol对雌激素促乳腺癌细胞MCF-7增殖的作用与机制

Effect of acetyltransferase inhibitor Garcinol on estrogen-promoted proliferation of breast cancer cell line MCF-7 and the related mechanism

目的研究乙酰化酶抑制剂Garcinol对雌激素促乳腺癌细胞MCF-7增殖、细胞周期转化及凋亡抑制的影响及机制。方法应用CCK-8法检测Garcinol对17β-雌二醇(17β-E2)促MCF-7细胞增殖的影响;流式细胞术检测细胞周期和细胞凋亡情况;细胞免疫荧光检测NF-κB/p65核转运情况;RT-PCR检测cyclin D1、Bcl-2、Bcl-xLmRNA水平;蛋白质印迹分析检测乙酰化(ac)-H3、ac-H4、NF-κB/ac-p65、cyclin D1、Bcl-2、Bcl-xL蛋白的表达水平。结果 Garcinol能抑制17β-E2促细胞增殖作用,使细胞周期阻滞于G0/G1期,细胞凋亡率增加(P<0.01);17β-E2促进MCF-7细胞ac-H3、ac-H4、NF-κB/ac-p65表达水平(P<0.01,P<0.05,P<0.01),Garcinol能抑制17β-E2对ac-H3、NF-κB/acp65的表达促进作用(P<0.01),对ac-H4影响无统计学意义;17β-E2促进NF-κB/p65核转运相应受到Garcinol抑制(P<0.01);17β-E2促进cyclin D1、Bcl-2、Bcl-xL的mRNA转录和蛋白表达水平的作用受到Garcinol抑制(P<0.05)。结论17β-E2促MCF-7细胞增殖和凋亡抑制作用与组蛋白和非组蛋白NF-κB/p65乙酰化水平增高有关,乙酰化酶抑制剂Garcinol通过降低乙酰化水平对雌激素促乳腺癌细胞增殖具有明显抑制作用,降低NF-κB通路的ac-p65蛋白水平,从而下调cyclin D1、Bcl-2、Bcl-xL可能是其重要机制。

Objective To investigate the effect of acetyltransferase inhibitor Garcinol on estrogen-induced proliferation,cell cycle promotion and apoptosis inhibition of human breast cancer MCF-7 cells and the related mechanism.Methods The effect of Garcinol on 17β-estradiol（17β-E2）-induced proliferation of MCF-7 cells was investigated using CCK-8 assay,and the optimal concentration of Garcinol was determine according to the inhibition rate.The cell cycle and apoptosis were analyzed by flow cytometry;the nuclear translocation of NF-κB/p65was examined by immune cell fluorescence.RT-PCR was used to determine the expressions of cyclin D1,Bcl-2 and Bcl-xLmRNA in MCF-7cells;and the expressions of acH3,ac-H4,NF-κB/ac-p65,cyclin D1,Bcl-2,and Bcl-xLprotein were determined by Western blotting analysis.Results Acetyltransferase inhibitor Garcinol inhibited 17β-E2-induced proliferation of MCF-7cells,arrested MCF-7cell cycle at G0/G1 phase,and increased the cell apoptosis（P0.01）.17β-E2 increased the expressions of ac-H3,ac-H4 and NF-κB/ac-p65 protein in MCF-7cells（P0.01,P0.05,P0.01）,while Garcinol significantly inhibited the increase of ac-H3and NF-κB/ac-p65（P0.01）but not ac-H4（P0.05）.17β-E2-induced nuclear translocation of NF-κB/p65in MCF-7cells was also significantly inhibited by Garcinol（P0.01）.Garcinol also significantly inhibited 17β-E2-induced transcription and protein expression of cyclin D1,Bcl-2and Bcl-xL mRNA in MCF-7 cells（P0.05）.Conclusion 17β-E2-induced proliferation and apoptosisinhibition of MCF-7 cells are associated with elevated acetylation level of histone and nonhistone NF-κB/p65,and acetyltransferase inhibitor Garcinol may inhibit the effect of 17β-E2 by decreasing acetylation,probably through decreasing acp65 expression of NF-κB pathway,and subsequently down-regulating cyclin D1,Bcl-2,and Bcl-xL.