AICAR对三阴性乳腺癌体外抑制作用及其机制的探讨

Antitumor effect of AMPK activator AICAR on triple negative breast cancer in vitro

目的:探讨AMPK激活剂AICAR对三阴性乳腺癌(triple negative breast cancer,TNBC)细胞的增殖作用及可能机制。方法:不同浓度的AMPK激活剂AICAR(0、0.25、0.5、1.0、2.0mmol/L)作用于TNBC细胞系MDA-MB-231后,采用MTS法检测细胞增殖,流式细胞仪检测AICAR处理后细胞周期改变,蛋白质印迹法检测pAMPK、EGFR、pERK1/2和Cyclin D1等增殖相关蛋白及细胞周期相关蛋白的表达水平。结果:不同浓度的AMPK激活剂AICAR处理乳腺癌MDA-MB-231细胞系24h后,随着AICAR浓度的逐渐增加,对MDA-MB-231细胞的增殖抑制率逐渐增加,呈浓度依赖性。流式细胞结果显示,AICAR阻滞细胞进入有丝分裂期,饥饿(含0.5%血清的培养基)培养24h后的TNBC MDA-MB-231细胞经含有AICAR(0.5与1.0mmol/L)的FBS(含10%血清培养基)处理24h后,细胞周期阻滞于G0/G1期,与单纯FBS处理的对照组比较,差异均有统计学意义,t值分别为5.416和5.578,P值分别为0.000 99和0.000 84;与单纯FBS处理的对照组比较,S期细胞减少,差异有统计学意义,t值分别为4.404和5.941,P值分别为0.003 14和0.000 58。同时,蛋白质印迹法检测发现,AICAR下调细胞周期蛋白Cyclin D1表达,1.0、2.0mmol/L AICAR处理组与对照组相比Cyclin D1蛋白均明显降低。蛋白质印迹法结果还显示,经AICAR处理的MDA-MB-231细胞,AMPK通路被激活,其EGFR/AKT/ERK1/2磷酸化表达水平均明显降低,和对照组相比差异有统计学意义,P值均<0.001。结论:AMPK激活剂AICAR对TNBC细胞有增殖抑制作用,其机制一方面可能是通过激活AMPK信号通路,抑制EGFR/pAKT/ERK通路的活化,另一方面是抑制细胞周期蛋白Cyclin D1表达,使细胞周期阻滞于G0/G1时相,阻止细胞进入S期,从而减少细胞进入有丝分裂。

OBJECTIVE：To investigate the effects of AMPK activator AICAR on triple negative breast cancer （TN- BC） in vitro and explore the possible signaling mechanisms by which AICAR exerts its effects. METItODS：TNBC cell line MDA-MB-231 were treated with different concentration of AMPK activator AICAR. Growth inhibition ratio of the cells were measured by MTS assay;Cell cycle distribution was analyzed by Flow Cytometry and the expressive level of cell pro- liferation protein AMPK/pAMPK, EGFR/pEGFR, AKT/pAKT, ERK/pERK and cell cycle protein Cyclin D1 were detec- ted by Western blot assay. RESULTS： AICAR significantly inhibited the proliferation of MDA-MB-231 cell lines in a dosedependent manner. After being starved for 24 h, MDA-MB-231 cells treated with AICAR（0. 5 mmol/L and 1 retool/L） were arrested at G0/G1 phase, ceils were significantly higher than that in control group （t= 5. 416, P= 0. 000 99; t= 5. 578 ,P〈0. 000 84） ;Compared with control group, the S-phase cells in AICAR treated group were decreased （t= 4. 404, P= 0. 003 14;t= 5. 941, P= 0. 000 58）. Also the results of Western blot demonstrated that cell cycle protein Cyclin D1 in AICAR treated group （0.5 mmol/L and 1.0 mmol/L） were significantly decreased compared with control group. Western blot showed that AICAR activated AMPK in MDA-MB-231 cells after 24 h incubation. Phosphorylation of EGFR/AKT/ ERK in AICAR treated group were lower than that in the control group （all P〈0. 001）. CONCLUSIONS： AICAR can activate AMPK and inhibit the proliferation of TNBC cell line MDA-MB-231. The inhibition of EGFR/pAKT/ERK signaling pathway is the possible underlying molecular mechanism. That AICAR prevents the mitotic effects of cells is another possible way. F