熊果酸诱导人急性白血病细胞凋亡及其作用机制

Mechanism of ursolic acid-induced apoptosis in human leukemia cells

目的探讨熊果酸诱导人急性白血病细胞凋亡的作用机制。方法将0、5、10、15、20μmol/L的熊果酸作用于U937细胞6、12 h,观察量效关系。用20μmol/L熊果酸作用U937细胞1、3、6、9、12、24 h,观察时效关系。采用Annexin V/PI染色和流式细胞仪检测细胞凋亡。用Western blot法检测细胞凋亡相关蛋白PARP、Caspase-3、Caspase-9及Bcl-2家族基因Mcl-1、Bcl-2、Bcl-xL、Bax、Bad的表达,用β-actin做内参照。20μmol/L熊果酸作用于不同类型的急性白血病细胞(U937、Jurkat、HL60)12 h,用流式细胞仪检测细胞凋亡,并用Western blot方法检测Caspase-3、Caspase-9、PARP的表达。结果熊果酸作用于U937细胞引起细胞发生凋亡,并呈明显的量效和时效关系。Western blot结果表明,熊果酸引起凋亡蛋白酶Caspase-3、Caspase-9的降解/活化增加,促进凋亡作用底物PARP的降解增加。熊果酸还可引起抗凋亡蛋白Mcl-1的表达降低,但对Bcl-2、Bcl-xL、Bax、Bad的表达均无明显影响。结论熊果酸可诱导多种急性白血病U937、Jurkat、HL60细胞发生凋亡。Mcl-1的下调可能在熊果酸诱导急性白血病细胞凋亡中起着重要的作用,其作用机制可能为熊果酸引起Mcl-1的下调,随后引起凋亡相关蛋白Caspase-3、Caspase-9的降解/活化及PARP的降解,最终促进细胞凋亡发生。

Objective To investigate the mechanism of ursolic acid-induced apoptosis in human leukemia cells. Methods U937 cells were exposed to ursolic acid （UA） at various concentrations for 6 h and 12 h, or at 20 μmol/L for different time intervals. Cells were stained with Annexin V/PI, and their apoptosis was determined through flow cytometry. Total protein extracts were prepared and subjected to Western blot assay using antibodies against PARP, C-Caspase-3, C-Caspase-9, Mcl-1, Bcl-2, Bel-xL, Bax, and Bad. Parallel studies were performed in human leukemia cell lines Jurkat and HL-60. Results Exposure of U937 cells to UA resulted in increase in apoptosis in dose and time-dependent manners. We found that UA-induced lethality was asso- ciated with Caspase activation and PARP cleavage, and that UA-induced apoptosis was proceeded by the downregulation of Mcl-1. Jurkat and HL-60 leukemia cells in parallel studies exhibited apoptosis after UA exposure similar to those observed in U937 cells, although HL-60 cells were less sensitive than U937 cells in UA-induced apoptosis, PARP degradation as well as Caspases activation. Conclusion UA induces apoptosis in human leukemia cells through a process that involves Mcl-1 downregulation, followed by Caspase activation and PARP degradation.