鱼藤素对急性髓系白血病KG-1a细胞增殖和凋亡的影响及机制

Effect of deguelin on the proliferation and apoptosis of acute myeloid leukemia KG-1a cells and its mechanism

目的探讨鱼藤素对急性髓系白血病(AML)KG-1a细胞增殖、凋亡的影响及机制。方法将对数生长期KG-1a细胞随机分为0.00 nmol·L^(-1)鱼藤素组、10.00 nmol·L^(-1)鱼藤素组、20.00 nmol·L^(-1)鱼藤素组、40.00 nmol·L^(-1)鱼藤素组、80.00 nmol·L^(-1)鱼藤素组,分别用含有0.00、10.00、20.00、40.00、80.00 nmol·L^(-1)鱼藤素的培养基培养。鱼藤素干预48 h后,采用倒置显微镜观察5组细胞的形态学变化。采用二甲基噻唑(MTT)实验检测5组细胞增殖能力。另取对数生长期KG-1a细胞随机分为空白组、鱼藤素组、氯化锂(LiCl)组、鱼藤素+LiCl组,空白组细胞用正常培养基培养,鱼藤素组细胞用含80.00 nmol·L^(-1)鱼藤素的培养基干预72 h, LiCl组细胞用含30 mmol·L^(-1)LiCl的培养基干预72 h,鱼藤素+LiCl组细胞用含80.00 nmol·L^(-1)鱼藤素和30 mmol·L^(-1)LiCl的培养基干预72 h。采用流式细胞术检测4组细胞凋亡情况及细胞周期分布。采用实时荧光定量聚合酶链反应(qRT-PCR)法检测4组细胞S期激酶相关蛋白2(SKP2)、β-链接素(β-catenin)、聚腺苷酸二磷酸核糖转移酶(PARP) mRNA相对表达量。采用Western blot法检测4组细胞中SKP2、β-catenin、PARP蛋白相对表达量。结果鱼藤素干预48 h时,与0.00 nmol·L^(-1)鱼藤素组比较,10.00 nmol·L^(-1)鱼藤素组、20.00 nmol·L^(-1)鱼藤素组、40.00 nmol·L^(-1)鱼藤素组、80.00 nmol·L^(-1)鱼藤素组细胞间隙增大,细胞数量显著减少,形态不规则,细胞核固缩、碎片增加,且随着鱼藤素药物干预浓度的增加,细胞数量逐渐减少,形态变化逐渐明显。鱼藤素干预24、48、72 h时,10.00 nmol·L^(-1)鱼藤素组、20.00 nmol·L^(-1)鱼藤素组、40.00 nmol·L^(-1)鱼藤素组、80.00 nmol·L^(-1)鱼藤素组细胞增殖能力显著低于0.00 nmol·L^(-1)鱼藤素组(P<0.05),20.00 nmol·L^(-1)鱼藤素组、40.00 nmol·L^(-1)鱼藤素组、80.00 nmol·L^(-1)鱼藤素组细胞增殖能力显著低于10.00 nmol·L^(-1)鱼藤素组(P<0.05),40.00 nmol·L^(-1)鱼藤素组、80.00 nmol·L^(-1)鱼藤素组细胞增殖能力显著低于20.00 nmol·L^(-1)鱼藤素组(P<0.05),80.00 nmol·L^(-1)鱼藤素组细胞增殖能力显著低于40.00 nmol·L^(-1)鱼藤素组(P<0.05);鱼藤素干预24、48、72 h时,5组细胞的增殖能力均随培养时间延长显著降低,组内各时间点之间两两比较差异有统计学意义(P<0.05)。空白组、鱼藤素组、LiCl组、鱼藤素+LiCl组细胞凋亡率分别为(5.37±0.61)%、(34.89±4.57)%、(2.54±0.36)%、(21.10±4.69)%;空白组、LiCl组、鱼藤素+LiCl组细胞凋亡率显著低于鱼藤素组(P<0.05);空白组、LiCl组细胞凋亡率均显著低于鱼藤素+LiCl组;LiCl组细胞凋亡率均显著低于空白组(P<0.05)。空白组、鱼藤素+LiCl组、鱼藤素组的G_(0)/G_(1)细胞占比显著低于LiCl组,G_(2)/M细胞占比显著高于LiCl组(P<0.05);鱼藤素+LiCl组、鱼藤素组的G_(0)/G_(1)细胞占比显著低于空白组,G_(2)/M细胞占比显著高于空白组(P<0.05);鱼藤素组的G_(0)/G_(1)细胞占比显著低于鱼藤素+LiCl组,G_(2)/M细胞占比显著高于鱼藤素+LiCl组(P<0.05);4组S期细胞百分比比较差异无统计学意义(F=0.696,P>0.05)。空白组、鱼藤素+LiCl组、鱼藤素组细胞SKP2、β-catenin、PARP mRNA及蛋白相对表达量显著低于LiCl组(P<0.05),鱼藤素+LiCl组、鱼藤素组细胞SKP2、β-catenin、PARP mRNA及蛋白相对表达量显著低于空白组(P<0.05),鱼藤素组细胞SKP2、β-catenin、PARP mRNA及蛋白相对表达量显著低于鱼藤素+LiCl组(P<0.05)。结论鱼藤素对AML KG-1a细胞具有增殖抑制、细胞周期阻滞及凋亡促进作用,其作用机制可能与抑制SKP2、β-catenin、PARP表达有关。

Objective To explore the effect and mechanism of deguelin on the proliferation and apoptosis of acute myeloid leukemia(AML) KG-1 a cells.Methods The logarithmic growth phase KG-1 a cells were randomly divided into 0.00 nmol·L^(-1) deguelin group, 10.00 nmol·L^(-1) deguelin group, 20.00 nmol·L^(-1) deguelin group, 40.00 nmol·L^(-1)deguelin group and 80.00 nmol·L^(-1) deguelin group, and they were cultured with 0.00,10.00,20.00,40.00 and 80.00 nmol·L^(-1) deguelin respectively.After intervention with deguelin for 48 h, the morphological changes of cells in the five groups were observed with the inverted microscope.The proliferation ability of cells in the five groups were detected by the dimethylthiazole(MTT) experiment.In addition, KG-1 a cells in the logarithmic growth phase were randomly divided into blank group, deguelin group, lithium chloride(LiCl) group, deguelin + LiCl group, the cells in the blank group were cultured in normal medium, the cells in the deguelin group were intervened with medium containing 80.00 nmol·L^(-1) deguelin for 72 h, the cells in the LiCl group were intervened with medium containing 30 mmol·L^(-1) LiCl for 72 h, the cells in the deguelin + LiCl group were intervened with medium containing 80.00 nmol·L^(-1) deguelin and 30 mmol·L^(-1) LiCl for 72 h.The apoptosis and cell cycle distribution of cells in the four groups were detected by flow cytometry.The relative expression levels of S-phase kinase-related protein 2(SKP2),β-catenin and polyadenylic acid diphosphate ribose transferase(PARP) mRNA of cells in the four groups were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) method.The relative expression levels of SKP2,β-catenin and PARP protein of cells in the four groups were detected by Western blot.Results After deguelin intervened for 48 h, compared with the 0.00 nmol·L^(-1) deguelin group, the cells in the 10.00 nmol·L^(-1) deguelin group, 20.00 nmol·L^(-1) deguelin group, 40.00 nmol·L^(-1) deguelin group and 80.00 nmol·L^(-1) deguelin group had larger intercellular spaces, significantly reduced number, irregular morphology, pyknosis of the nucleus and increased fragments.With the increase in the concentration of deguelin intervention, the number of cells gradually decreased, the morphological change was gradually obvious.After deguelin intervened for 24 h, 48 h and 72 h, the cell proliferation capacity of cells in the 10.00 nmol·L^(-1) deguelin group, 20.00 nmol·L^(-1) deguelin group, 40.00 nmol·L^(-1) deguelin group and 80.00 nmol·L^(-1) deguelin group was significantly lower than that in the 0.00 nmol·L^(-1) deguelin group(P<0.05);the cell proliferation capacity of cells in the 20.00 nmol·L^(-1) deguelin group, 40.00 nmol·L^(-1) deguelin group and 80.00 nmol·L^(-1) deguelin group was significantly lower than that in the 10.00 nmol·L^(-1) deguelin group(P<0.05);the cell proliferation capacity of cells in the 40.00 nmol·L^(-1) deguelin group and 80.00 nmol·L^(-1) deguelin group was significantly lower than that in the 20.00 nmol·L^(-1) deguelin group(P<0.05);the cell proliferation capacity of cells in the 80.00 nmol·L^(-1) deguelin group was significantly lower than that in the 40.00 nmol·L^(-1) deguelin group(P<0.05).After deguelin intervened for 24 h, 48 h and 72 h, the proliferation ability of the cells in the five groups decreased with time, and the difference between each time point in the five groups was statistically significant(P<0.05).The apoptotic rate of cells in the blank group, deguelin group, LiCl group and deguelin+LiCl group was(5.37±0.61)%,(34.89±4.57)%,(2.54±0.36)%,(21.10±4.69)%,respectively;the apoptotic rate of cells in the blank group, LiCl group and deguelin+LiCl group was significantly lower than that in the deguelin group(P<0.05);the apoptotic rate of cells in the blank group and LiCl group was significantly lower than that in the deguelin+LiCl group(P<0.05);the apoptotic rate of cells in the LiCl group was significantly lower than that in the blank group(P<0.05).The proportion of cells in G_(0)/G_(1) in the blank group, the deguelin + LiCl group and the deguelin group were significantly lower than that in the LiCl group, and the proportion of cells in G_(2)/M was significantly higher than that in the LiCl group(P<0.05);the proportion of cells in G_(0)/G_(1) in the deguelin + LiCl group and the deguelin group was significantly lower than that in the blank group, and proportion of cells in G_(2)/M was significantly higher than that in the blank group(P<0.05);the proportion of cells in G_(0)/G_(1) in the deguelin group was significantly lower than that in the deguelin + LiCl group, and proportion of cells in G_(2)/M was significantly higher than that in the deguelin + LiCl group(P<0.05);there was no significant difference in the percentage of S-phase cells among the four groups(F=0.696,P>0.05).The relative expression levels of SKP2,β-catenin, PARP mRNA and protein in the blank group, deguelin + LiCl group and deguelin group were significantly lower than those in the LiCl group(P<0.05);the relative expression levels of SKP2,β-catenin, PARP mRNA and protein in the deguelin + LiCl group and deguelin group were significantly lower than those in the blank group(P<0.05);the relative expression levels of SKP2,β-catenin, PARP mRNA and protein in the deguelin group were significantly lower than those in the deguelin + LiCl group(P<0.05).Conclusion Deguelin has proliferation inhibition, cell cycle arrest and apoptosis promotion effects on AML KG-1 a cells, and its mechanism of action may be related to the inhibition of SKP2,β-catenin and PARP expression.