对香豆酸组合物对白血病细胞的增殖抑制作用及机制

Proliferation Inhibition and Its Mechanism of Total Coumarins on Leukemia Cells

目的研究对香豆酸组合物(total coumarins of Hedyotis diffusa,TCHD)对急性髓系白血病(AML)细胞的增殖抑制作用及其机制。方法采用乙醇回流法提取TCHD,超高效液相色谱串联质谱系统(UPLC-MS/MS)检测其纯度。用不同浓度的TCHD(0.02、0.04、0.06、0.08、0.10 mg·mL^(-1)),作用于对数生长期的Kasumi-1细胞、KG-1细胞、THP-1细胞、U937细胞和K562细胞,四甲基偶氮唑蓝(MTT)法检测各细胞增殖;作用于Kasumi-1细胞24 h后,采用AnnexinⅤ/PI流式细胞术检测细胞凋亡情况;运用流式细胞术检测药物作用后Kasumi-1细胞周期阻滞情况;Western blot法检测PARP、caspase-3、caspase-9、caspase-8蛋白活性的变化;以及CDK4/6、cyclin D_1、CDK2、p-CDK2、cyclin E、p21等细胞周期相关蛋白的变化。结果 TCHD在一定浓度范围内对AML细胞增殖有明显的抑制作用,对Kasumi-1、KG-1、THP-1、U937和K562细胞的IC_(50)(24 h vs 48 h)分别为0.077 vs 0.059(mg·mL^(-1))、0.083 vs 0.067(mg·mL^(-1))、0.096 vs 0.072(mg·mL^(-1))、0.087 vs 0.064(mg·mL^(-1))、0.096 vs 0.068(mg·mL^(-1)),对Kasumi-1的抑制作用表现最明显,并且呈时间-浓度依赖性(r=0.357,P<0.05)。不同浓度(0,0.02,0.04,0.06,0.08,0.10 mg·mL^(-1))的TCHD作用于Kasumi-1细胞株24 h后,凋亡率分别是(5.33±0.41)%、(7.99±0.45)%、(10.22±0.32)%、(20.10±1.99)%、(28.66±0.67)%和(33.24±2.12)%。0.02~0.06 mg·mL^(-1)的TCHD处理Kasumi-1细胞24 h后用流式细胞术检测到该细胞G_0/G_1期的比例依次为(51.43±3.21)%、(62.91±2.35)%和(76.42±4.14)%,与空白对照组[(35.8±5.25)%]比较,差异均有统计学意义(P值均<0.05)。Western blot结果显示,不同浓度的TCHD能浓度依耐性激活caspase-8(P<0.01)、caspase-9、caspase-3和PARP(其余P<0.05),促进细胞色素C的表达;显著下调cyclin E、CDK6、CDK2、p-CDK2、cyclin D_1,和上调p21(P<0.01)。结论 TCHD抑制白血病细胞株Kasumi-1的增殖呈时间浓度依赖性,其凋亡机制一方面与其激活caspase-3、caspase-9、PARP蛋白有关,另一方面则通过影响CDK2、p-CDK2、CDK4/6、cyclin E、cyclin D_1和p21使Kasumi-1细胞被阻滞于G_0/G_1期,抑制细胞增殖。

OBJECTIVE To explore the effect of total coumarins isolated from Hedyotis diffusa （ total coumarins from Hedyotis dif- fusa, TCHD） on proliferation inhibition of leukemia cells, and to explore its related mechanism. METHODS The purity of TCHD prepared by ethanol reflux extraction was tested by ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/ MS） system. The cells （KG-1 Kasumi-1, THP 1 cells, U937 cells and K562 cells） were treated with TCHD（0.02, 0.04, 0.06, 0. 08, 0. 10 mg ·mL-1 ） for 24 or 48 h, the inhibitive effect of TCHD on cells growth were determined by MTI＂ method. After Kasumi-1 cells were incubated with TCHD for 24 h, the apoptosis of ceils were analyzed by flow cytometry stained with Annexin V/PI. The expression levels of caspase-3, caspase-8, caspase-9, PARP and Bcl-2 family protein were assayed by Western blot. RESULTS TCHD in certain concentration range could markedly inhibit the proliferation of AML cells, their ICso on Kasumi-1, THP- 1 KG-1, U937 and K562 cells were 0. 077, 0. 083, 0. 096, 0. 087, 0. 096 mg·mL-1 for 24 h, and 0. 059, 0. 067, 0. 072, 0. 064, 0. 068 mg ·mL-1 for 48 h. TCHD has significant inhibitory effect on Kasumi-1, which was stronger than those on other cell lines, and showed a dose- and time-dependent manner（ r = 0. 357, P 〈 0. 05 ）. The apoptotic proportion of Kasumi-lcells in 0, 0. 02, 0. 04, 0.06, 0.08, 0.10mg·mL-1 TCHD treatment groups for 24 h were （5.33 ±0.41）%, （7.99 ±0.45）%, （10.22 ±0.32）%, （20. 10 ±1.99） %, （28.66 ±0. 67） % and （33.24 ±2. 12） %, respectively. After treated with TCHD（0. 02 -0. 06 mg ·mL-1） for24 h, G0/G1 phase ratio of Kasumi-1 detected by flow cytometry were （51.43 ± 3.21 ）%, （62. 91 ± 2. 35 ）% and （76. 42 ± 4. 14） %, respectively, which were significantly higher than that of the control group （35.8 ± 5.25） % （P 〈 0.05 ）. Western blot re- sults showed that different concentrations of TCHD could activate caspase-8, caspase-9, caspase-3 and PARP, promote the expression of cyto-C, down-regulate the cyclin E and CDK6, CDK2, p-CDK2 and cyclin D1 protein, and up-regulate the expression of p21 proteinin concentration- dependent manner（ P 〈 0.01 ）. CONCLUSION TCHD can obviously inhibit the proliferation of Kasu- mi-1 in a dose- and time-dependent manner, which may relate to the apoptosis of Kasumi 1 induced by activating caspase-3, 9, PARP protein through the mitochondrial pathways and Kasumi-1 cell block in G0/G1 phase through the influence of CDK2, p-CDK2, CDK4/6, cyclin E, cyclin D1 and p21.