土木香乙酸乙酯提取物对人胰腺癌Capan-2细胞增殖的抑制作用及机制研究

摘要

目的:研究土木香乙酸乙酯提取物(IHE)对人胰腺癌Capan-2细胞增殖的抑制作用及机制.方法:采用MTT法测定0、0.5、1、2、4、8μg/mL的IHE作用48 h后细胞的增殖抑制率,克隆形成试验观察0、1、2μg/mL的IHE作用1周后对细胞克隆形成的影响,Hoechst 33342染色法观察0、2、4μg/mL的IHE作用48 h后细胞核形态的变化,流式细胞术检测0、4、8、16μg/mL的IHE作用48 h后细胞的凋亡率,JC-1染色法观察0、4、8、16μg/mL的IHE作用24 h后细胞线粒体膜电位变化,Western blot法检测0、4、8、16μg/mL的IHE作用48 h后细胞线粒体凋亡相关蛋白Bcl-2、Bax、Mcl-1和p53上调凋亡因子(PUMA)、多聚二磷酸腺苷核糖聚合酶(PARP)蛋白的表达.结果:2、4、8μg/mL的IHE对细胞的增殖有明显抑制作用,且呈浓度依赖性,半数抑制浓度为6.6μg/mL;1、2μg/mL的IHE可明显抑制细胞的克隆形成;4μg/mL的IHE可明显造成细胞核固缩;8、16μg/mL的IHE可明显促进细胞凋亡,16μg/mL的IHE作用48 h后细胞凋亡率达到45.53%;16μg/mL的IHE作用24 h后可引起82.47%的细胞线粒体膜电位下降;8μg/mL的IHE可明显下调细胞中Bcl-2、Mcl-1、PUMA、PARP蛋白表达,16μg/mL的IHE可明显下调细胞中Mcl-1、PUMA表达.结论:IHE可能通过引起细胞线粒体膜电位的下降,下调细胞中PUMA、Mcl-1蛋白的表达,引起细胞的凋亡,从而发挥其抑制人胰腺癌Capan-2细胞增殖的作用.%OBJECTIVE:To study the inhibitory effect and mechanism of Inula helenium ethyl acetate extract(IHE)on prolif-eration of human pancreatic cancer Capan-2 cells. METHODS:MTT was used to determine the cell proliferation inhibition rate af-ter treated by 0,0.5,1,2,4,8 μg/mL IHE;clone formation test was used to observe the effects of 0,1,2 μg/mL IHE treating for 1 week on cell clone formation;Hoechest 33342 staining was used to observe the changes of nuclear morphology after treated by 0,2,4 μg/mL IHE for 48 h;flow cytometry was used to detect the cell apoptosis rate after treated by 0,4,8,16 μg/mL IHE for 48 h;JC-1 staining was used to observe the changes of intracellular mitochondrial membrane potential after treated by 0,4,8, 16 μg/mL IHE for 24 h;Western blot was used to detect the expressions of mitochondrial apoptosis-related proteins Bcl-2,Bax, Mcl-1,p53 up-regulated modulator of apoptosis (PUMA),and polymerase (PARP) after treated by 0,4,8,16 μg/mL IHE for 48 h. RESULTS:2,4,8 μg/mL IHE had obvious inhibitory effect on cell proliferation,showing concentration-dependent relation-ship,with IC50 of 6.6 μg/mL;1,2 μg/mL IHE can obviously inhibit the clone formation of cells;4 μg/mL IHE can obviously cause cell nuclear condensation;8,16 μg/mL IHE can obviously promote the cell apoptosis,and the cell apoptosis rate reached 45.53% after treated by 16 μg/mL IHE for 48 h;16 μg/mL IHE treating for 24 h can cause the decrease of 82.47% cells'mito-chondrial membrane potential;8 μg/mL IHE can obviously down-regulate the protein expressions of Bcl-2,Mcl-1,PUMA and PARP,and 16 μg/mL IHE can obviously down-regulate the expressions of Mcl-1 and PUMA. CONCLUSIONS:IHE may show its inhibitory effect on proliferation of human pancreatic cancer Capan-2 cells by causing the decrease of mitochondrial mem-brane potential in cells and down-regulating the protein expres-sions of Mcl-1 and PUMA to cause cell apoptosis.