力达霉素抑制K562细胞增殖和诱导凋亡作用及其机制

Inhibitory effect of lidamycin on cell proliferation and induction of apoptosis in human leukemia K562 cells and their mechanisms

目的:研究力达霉素(LDM)对人慢性粒细胞白血病(CML)K562细胞的增殖抑制和凋亡诱导作用,并探讨其机制。方法:选取处于对数生长期的人CML K562细胞,采用不同浓度(0.01、0.10和1.00nmol·L-1)LDM处理48h,作为0.01、0.10和1.00nmol·L-1 LDM组,不加药物的正常细胞为对照组。台盼蓝染色观察各组K562细胞生长抑制率,MTT法检测细胞存活率,吖啶橙/溴化乙锭(AO/EB)双荧光染色法观察凋亡细胞的形态表现,AnnexinⅤ-FITC/PI双染结合流式细胞术检测细胞凋亡率,Western blotting法检测细胞中caspase-3和caspase-8蛋白相对表达量。结果:台盼蓝染色,LDM对K562细胞增殖有一定的抑制作用,各浓度LDM组细胞生长抑制率均高于对照组(P<0.05);MTT法检测,随LDM作用浓度的升高,细胞存活率下降,LDM对K562细胞作用的半数抑制浓度(IC50)为(0.1±3.2)nmol·L-1。AO/EB染色,荧光显微镜下观察到LDM能够引起细胞发生凋亡(胞质呈芽状突起,胞核碎裂成点状)。流式细胞术检测,各浓度LDM组K562细胞凋亡率均高于对照组(P<0.05)。Western blotting法检测,0.10和1.00nmol·L-1 LDM组K562细胞中caspase-8和caspase-3蛋白相对表达量明显高于对照组(P<0.01)。0.01nmol·L-1 LDM组K562细胞中caspase-8和caspase-3蛋白表达量与对照组比较差异无统计学意义(P>0.05)。结论:LDM能够有效抑制K562细胞增殖,低浓度LDM可能通过上调细胞中caspase-8和caspase-3表达诱导K562细胞发生凋亡。

Objective：To explore the inhibitory effect of lidamycin（LDM）on the cell proliferation and apoptosis induction effect in the human chronic myeloid leukemia（CML）K562cells,and to discuss the mechanisms.Methods：The K562 cells at logarithmic growth phase were selected and treated with different concentrations（0.01,0.10 and 1.00nmol·L-1）of LDM for 48 hand they were used as 0.01,0.10,and 1.00nmol·L-1 LDM groups,and the normal cells without treatment were regarded as control group.Trypan blue staining was used to detect the inhibitory rates of growth of the K562 cells in various groups;the cell viability was determined by MTT assay;the apoptotic morphology of cells was investigated by acridine orange/ethidium bromide（AO/EB）staining;the apoptotic rate of cells was detected by Annexin Ⅴ-FITC/PI double staining and flow cytometry;the relative expression levels of caspase-3 and caspase-8 in K562 cells were measured by Western blotting analysis.Results：The Trypan blue staining results showed that LDM could inhibit the proliferation of K562 cells and the inhibitory rates of growth of the K562 cells in different concentrations of LDM groups were higher than that in control group（P〈0.05）.The MTT results showed that the cell viability was decreased with the increasing of LDM concentration and the 50%inhibitory concentration（IC50）value was（0.1 ± 3.2）nmol·L-1.The AO/EB double staining results showed that LDM could induce the apoptosis of K562 cells under fluorescence microscope（the cytoplasm was in buds and the nucleus were broken into points）.The flow cytometry results showed that the apoptotic rates of cells in different concentrations of LDM groups were higher than that in control group（P〈0.05）.The Western blotting analysis results showed that the relative expression levels of caspase-8and caspase-3in the K562 cells in 0.10nmol·L-1 and 1.00nmol·L-1 LDM groups were higher than those in control group（P〈0.01）;the relative expression levels of caspase-8and caspase-3in the K562 cells in 0.01nmol·L-1 LDM group had no significant differences compared with control group（P〉0.05）.Conclusion：LDM could inhibit the proliferation of K562 cells.Moreover,low concentration of LDM may induce the apoptosis by up-regulating the expressions of caspase-8and caspase-3.