4-氨基-2-三氟甲基苯基维甲酸酯对K562细胞分化和细胞周期的影响

目的本研究探讨新型维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯(4-amino-2-trifluoromethyl-phenyl retinate,ATPR)对K562细胞株的抑制增殖和诱导分化活性并对其机制进行研究。方法ATPR作用于K562细胞3d后,通过MTT法检测细胞的增殖,NBT还原实验法分析细胞的分化指标,瑞氏染色法在油镜下观察加药前后细胞形态学变化,FCM检测分析细胞周期,RT-PCR法检测cyclinE、cyclinD1、CDK2、CDK4、CDK6、p21cip1、p27kip1、p57kip2和PCNA mRNA的变化情况。Western blot法检测cyclin D1和CDK4蛋白表达的改变。结果ATPR呈浓度依赖性抑制K562细胞增殖的作用。ATPR诱导分化活性表现为NBT阳性细胞率增加,油镜下观察K562细胞有分化成熟的改变,G0/G1期细胞表达量增加,S期细胞表达量减少,呈G1期阻滞。RT-PCR检测发现cyclin E、cyclin D1、CDK2、CDK4、CDK6表达减少,PC-NA、P21cip1、P27kip1改变不明显,P57kip2表达增加。Western blot检测cyclin D1和CDK4蛋白表达减少。结论ATPR有较强的抑制K562细胞增殖并诱导其分化的活性,并通过上调P57kip2的表达,抑制Cyclin-CDK激酶复合物,发挥细胞周期阻滞的作用。

4-amino-2-trifluoromethyl-phenyl retinate induced K562 cell differentiation and cell cycle influence

Aim To explore the effect of 4-amino-2-trifluoromethyl-phenyl retinate(ATPR)on proliferation,differentiation activity in K562 cell line,and to research the mechanisms.Methods Cell proliferation was assessed by MTT assay.Cell differentiation index was analyzed by NBT reduction test.Morphologic changes were observed by Wright's staining in the light microscope. Cell cycle was determined by FCM.The mRNA expression of Cyclin D1,Cyclin E,CDK2,CDK4,CDK6,P21cip1,P27kip1,P57kip2,PCNA mRNA were detected by RT-PCR.While the protein expression of cyclin D1 and CDK4 was detected by Western blot.Results The growth of K562 cells was inhibited in a dose-dependent manner.NBT reduction test indicated that the ATPR could induce differentiation of K562 cells and increase the positive cell ratio.Morphologic changes were observed after Wright's staining using inverted phase contrast microscope.The proportion of cells in G0/G1 phase increased while S phase cells decreased.Cell cycle progression was blocked in the G1 phase.The expression of Cyclin E,cyclin D1,CDK2,CDK4,CDK6 mRNA decreased,while PCNA,P21 cip1,P27 kip1 change was not obvious,but P57 (kip2) mRNA expression was increased.Cyclin D1 and CDK4 protein expressions were reduced as well.Conclusions ATPR inhibits the growth of K562 cells and induces differentiation.P57 kip2 plays a key role in differentiation.Moreover,high level of P57kip2 is regulated via inhibiting its degradation through reducing proteasome-dependent proteolysis,and ATPR plays a role in cell cycle arrest.