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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 investigate the effect of HDACi JZ004 on the proliferation of human colon cancer cell lines HCT-8 and HT-29 and to investigate its anticancer mechanism. Methods HCT-8 and HT-29 cells were treated with different concentrations of JZ004 respectively. Cell proliferation was detected by MTT assay. Cell cycle arrest and apoptosis were detected by flow cytometry. Rhodamine 123 and dichlorodihydrofluorescein (DCFH-DA) was used to measure the changes of cell mitochondrial transmembrane potential and reactive oxygen species (ROS). The levels of acetylated histone H3, p21 and cyclin dependent kinase CDK) 4, Bcl-2, Mcl-1, Bax and other related protein expression levels. Results The results of MTT showed that JZ004 could inhibit the proliferation of colon cancer cell lines, and the inhibitory effect was positively correlated with time and dose. Flow cytometry analysis showed that the apoptosis rate of JZ004 increased with the increase of JZ004 concentration. After 72 h of JZ004 treatment, , the expression of p21 was upregulated, the expression of CDK4 was down-regulated, the cell cycle arrested in G0 / G1 phase, the growth of cells was inhibited, the intracellular ROS production was increased, the mitochondrial transmembrane potential was significantly decreased, the expression of pro-apoptotic protein Bax was up- The inhibition of apoptosis protein Bcl-2, Mcl-1 expression was inhibited. Conclusion As a novel HDACi, JZ004 can significantly inhibit the growth and induce apoptosis of colon cancer cells. The mechanism of apoptosis induction may be related to the changes of Bcl-2 family protein expression. Suggesting that JZ004 has the potential to become a clinical chemotherapeutic agent for colon cancer.