新型组蛋白去乙酰化酶抑制剂对结肠癌细胞的增殖抑制作用

A novel histone deacetylase inhibitor induces proliferation inhibition of human colon cancer cells

目的观察组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor,HDACi)JZ004对结肠癌细胞系HCT-8和HT-29细胞增殖的影响,并初步探讨其抗癌机制。方法用不同浓度JZ004分别处理HCT-8和HT-29细胞,MTT法检测细胞的增殖情况;流式细胞仪检测细胞周期停滞、细胞凋亡;若丹明(rhodamine)123和二氯二氢荧光素二乙酸酯(DCFH-DA)测定细胞线粒体跨膜电位及活性氧(reactive oxygen species,ROS)产生的变化;免疫印迹法检测细胞内乙酰化组蛋白H3、p21、细胞周期蛋白依赖激酶(CDK)4、Bcl-2、Mcl-1、Bax等相关蛋白的表达水平。结果MTT结果显示,JZ004对结肠癌细胞系的增殖有明显抑制作用,其抑制效果与时间、剂量呈正相关;流式分析结果显示,细胞凋亡率随JZ004浓度提高而增加;JZ004处理72 h后,p21表达水平上调,CDK4蛋白表达下调,细胞周期停滞于G0/G1期而抑制细胞的生长,细胞内ROS产生增多,且线粒体跨膜电位显著下降,促凋亡蛋白Bax的表达呈上调趋势,而抑凋亡蛋白Bcl-2、Mcl-1表达则受到抑制。结论 JZ004作为一种新型的HDACi对结肠癌细胞具有明显的抑制生长和诱导凋亡作用,凋亡诱导机制可能与Bcl-2家族蛋白表达水平变化有关。提示JZ004具有成为结肠癌临床化疗药物的潜能。

Objective To examine the anticancer effect of a novel histone deacetylase inhibitor（ HDACi）,JZ004,on colon cancer cells HCT-8 and HT-29,and to investigate the molecular mechanisms of proliferation inhibition and apoptosis induction of cancer cells treated by JZ004. Methods Colon cancer cells were treated with a series of concentrations of JZ004. MTT assay was used to detect the proliferation of cancer cells. The cell cycle distribution and apoptosis were determined by flow cytometry. Rhodamine 123 and DCFH-DA were applied to detect the mitochondrial membrane potential（ ΔΨm） and reactive oxygen species（ ROS） production. The protein expressions of acetyl-histone H3,p21,cyclindependent kinase（ CDK） 4,Bcl-2,Mcl-1 and Bax were assayed by Western blotting. Results JZ004 was found to inhibit proliferation and induce apoptosis of colon cancer cells in a time-and dose-dependent manner,accompanied by a dosedependent hyperacetylation of histone H3. JZ004 induced the cancer cell arrest in G0/G1 phase by increasing the expression level of p21 while CDK4 was downregulated. JZ004 also increased cellular ROS production and reduced ΔΨm by regulating the expressions of Bcl-2 family proteins. Conclusion As a novel HDACi,JZ004 effectively inhibits proliferation and increases ROS production to induce apoptosis of colon cancer cells. The results indicate that JZ004 is a potential compound to be developed as an anti-colon cancer agent for clinic application.