组蛋白去乙酰化酶抑制剂抑制三阴性乳腺癌细胞增殖的实验研究

摘要

背景与目的:三阴性乳腺癌(triple negative breast cancer,TNBC)是乳腺癌中的研究热点和治疗难点,目前尚无十分有效的靶向治疗药物.本研究旨在观察组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor,HDACI)对TNBC细胞增殖的影响,探讨针对组蛋白去乙酰化酶(histone deacetylase,HDAC)的治疗对TNBC治疗的可行性.方法:将HDACI辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)作用于TNBC细胞MDA-MB-468,用MTS比色法观察细胞生长情况；用流式细胞术(the flow cytometry,FCM)检测细胞周期分布、凋亡情况,以及P21、14-3-3 σ、Cyclin D1和Cyclin A2蛋白的表达状况,并进行统计分析；同时用Western blot检测以上4种蛋白的表达情况.结果:MTS比色法显示,MDA-MB-468在加入SAHA 24 h后出现细胞生长抑制并逐渐凋亡,于72 h达高峰；FCM检测显示,在加入SAHA 48 h后MDA-MB-468出现S期细胞比例下降,细胞内Cyclin A2蛋白的表达量下降,Cyclin D1、P21和14-3-3 σ蛋白的表达量上升,细胞凋亡率上升,与MDA-MB-468对照组比较差异有统计学意义(P＜0.05)；Western blot检测结果显示,MDA-MB-468在SAHA加入48 h后Cyclin A2蛋白表达量下降,P21、14-3-3 σ及Cyclin D1蛋白表达量上升.结论:SAHA是TNBC细胞增殖的高效抑制剂并促进其凋亡,其抑制作用有一定的时效关系；抑癌基因表达产物P21、14-3-3 σ蛋白和周期素Cyclin D1、Cyclin A2参与了SAHA对细胞增殖周期的调控.%Background and purpose: TNBC is not only a hot research spot but also difficult type for therapy in breast cancer. Up to date, there is no sufficient effective gene therapy for it. In order to present a new approach to the gene therapy, we treated the TNBC with histone deacetylase inhibitors and observed its effects on the cell growth. Methods: We treated the TNBC cell MDA-MB-468 with histone deacetylase inhibitor SAHA. Chose the MTS to observe the cell growth arrest, selected the flow cytometry(FCM) to see the cell cycle distribution, apoptosis and the expression of Cyclin A2,Cyclin Dl, P21 and 14-3-3o. SPSS 17.0 was used to analyze the data. Meanwhile we used the Western blot to identify the expression of the above four protein. Results: From the MTS we could see that SAHA could not only inhibit the growth of MDA-MB-468 cell after adding it 24 hours but also could cause cell apoptosis and reach their peak at the time of 72 hours. From the FCM we can see the proportion of S-phase cell and the expression of Cyclin A2 were decreased after adding the SAHA 24 hours. Meanwhile the cell apoptosis and the expression of P21, 14-3-3o and Cyclin Dl were increased. The Western blot also showed the above results. Conclusion: SAHA, which could cause the death of TNBC cell MDA-MB-468, is also the most powerful histone deacetylase inhibitor for it. Theeffect of depression of cell growth shows time-effect relation. P21, 14-3-3a, Cyclin A2 and Cyclin Dl are involved in this regulation of cell cycle.