三乙酰穿心莲内酯对人乳腺癌细胞MCF-7凋亡的影响及其机制

Effect of Andrographolide Derivative ADA on Apoptosis and Its Mechanism of Breast Cancer MCF-7 Cells

目的研究三乙酰穿心莲内酯(ADA)对人乳腺癌MCF-7细胞的增殖抑制作用和凋亡的影响,并对其作用机制进行研究和探讨。方法采用四甲基偶氮唑蓝(MTT)法检测细胞增殖。4',6-二脒基-2-苯基吲哚(DAPI)、吖啶橙/溴乙啶(AO/EB)双染法、以及Annexin V-FITC/PI双染法流式细胞仪测定细胞凋亡。Western bolt法检测NF-κB信号通路相关蛋白NF-κB p65核位移、磷酸化IκBɑ、磷酸化IKKβ的表达以及凋亡通路中相关蛋白Bcl-2、Bax和caspase 3的表达。结果 MTT结果显示,ADA对MCF-7细胞增殖有非常显著的生长抑制作用(P<0.01),并呈剂量依赖性;ADA可以诱导MCF-7细胞凋亡,具体可能机制为MCF-7细胞caspase 3被激活并表达增加(P<0.01);促凋亡蛋白Bax的表达增加(P<0.01),抗凋亡Bcl-2的表达降低(P<0.05),提示ADA参与Bax、Bcl-2、caspase 3的调控,同时磷酸化IκBɑ、磷酸化IKKβ、细胞核内NF-κB p65表达均降低(P<0.01),细胞质内p65表达增加(P<0.01),表明ADA也参与NF-κB信号通路的调控。结论 ADA可诱导MCF-7细胞凋亡,可能参与Bax、Bcl-2、caspase 3与NF-κB信号通路的调控。

OBJECTIVE To investigate the anti-proliferation effects of andrographolide Derivative （ADA） and its potential anti- tumor mechanisms in MCF-7 cells. METHODS The anti-proliferation effects of ADA were assessed by MTT assay in MCF-7 cells. Apoptotic morphological changes of MCF-7 cells after ADA treatment were observed by high content screening （ HCS ） after AO/EB double staining and DAPI, Apoptotie number was investigated by using flow cytometry. The expression of Bcl-2, Bax, caspase 3, NF-κB p65 ,p-IκBα and p-IKKβ were measured using Western blot. RESULTS MTT assay demonstrated that ADA exerted potent anti-tumor effect in a significant concentration-dependent manner;ADA could promote the apoptosis of MCF-7 cells. Western blot results showed that Bcl-2 families and NF-κB p65 and caspase 3 involved in the process of apoptosis, Bcl-2 expression lowered（ P 〈 0. 05 ） , p-IκBα and p-IKKβ expression decreased（P 〈0. 01 ）, Bax, NF-κB p65 and caspase 3 expression increased（P 〈 0. 01 ）. CONCLUSION ADA could inhibit the proliferation of MCF-7 cells and mechanism may be that it might induce apoptosis by regulated the expression of Bax, Bcl-2 and caspase 3 protein and NF-κB passway.