冬凌草乙素对白血病K562细胞的诱导凋亡作用及机制研究  

Apoptosis inducing effect of ponicidin in leukemia K562 cells and its mechanisms of action

作　　者：刘晓丹[1] 刘文达[2] 徐妍[1] 刘培庆[3] 王春芝[1] 林东军[1] 黄河清[3] 吴传斌[3] 肖若芝[1] 黄仁魏[1] 刘加军[1] 

机构地区：[1]中山大学附属第三医院血液科/血液病研究所,广东广州510630 [2]中山大学附属第三医院输血科,广东广州510630 [3]中山大学药学院药理学与毒理学实验室,广东广州510080

出　　处：《中国中药杂志》2010年第16期2161-2165,共5页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(30772782);教育部新世纪优秀人才支持计划项目(NCET-0721)

摘　　要：目的：探讨冬凌草乙素（ponicidin,PON）对白血病K562细胞的诱导凋亡作用及其作用机制。方法：以不同浓度的PON（10~50μmol.L-1）作用于体外培养的K562细胞24,48,72 h,应用MTT法检测细胞生长抑制率,收集药物作用72h后的细胞Annexin V染色并通过流式细胞术（FCM）检测细胞凋亡率,瑞氏-姬姆萨染色观察细胞凋亡时的形态学变化。应用免疫印迹法（Western blot）检测caspase-3及其裂解底物多聚（ADP-核糖）聚合酶PARP的表达水平,并对MAPKs（mitogen-acti-vated protein kinase）信号转导通路相关蛋白（p-P38,p-ERK和p-JNK）以及PI3K（磷脂酰肌醇-3-激酶）/AKT信号通路中的p-AKT,p-P85蛋白的表达水平进行检测。结果：30μmol.L-1以上的PON可显著抑制细胞的生长,并呈现出明显的量-效与时-效关系,FCM检测结果表明,不同浓度的药物作用72 h后,随药物浓度的增加细胞凋亡率逐渐升高,50μmol.L-1的PON作用不同时间后在瑞氏-姬姆萨染色下可见典型的核浓缩及核碎裂等细胞凋亡现象。Western blot检测结果表明,药物作用48 h后caspase-3被活化出现17 kD的亚单位,同时PARP被裂解出现89 kD的亚单位片段。Western blot检测结果进一步显示药物作用48 h后MAPKs信号途径中p-P38,p-ERK和p-JNK蛋白的表达水平无明显变化,而PI3K/AKT信号通路中的p-AKT,p-P85蛋白的表达水平则随着药物浓度增加而逐渐下降。结论：PON可以通过诱导白血病K562细胞调亡而发挥体外抗白血病作用。PON对K562细胞的诱导凋亡作用机制与caspse-3的活化以及降低PI3K/AKT信号通路中p-AKT及p-P85蛋白的表达水平有关,而与MAPKs信号通路中的相关调节蛋白无关;这些研究结果为进一步研究冬凌草素的抗白血病作用提供了有力的实验依据和技术平台。Objective： To investigate the apoptosis inducing effects of ponicidin（PON） on leukemic K562 cells and its mechanisms of action.Method： K562 cells in culture medium in vitro were given different concentrations of PON（10-50 μmol·L-1） for 24,48 and 72 h.The inhibitory rate of the cells was measured by MTT assay,cell apoptotic rates were detected by flow cytometry（FCM） by using Annexin V staining after K562 cells were treated with different concentrations of PON for 72 hours,and cell morphology was observed by Wright-Giemsa staining.Western blot was used to detect caspase-3 and poly（ADP-ribose） polymerase（PARP） expression,and the protein levels in mitogen-activated protein kinase signaling pathways（MAPKs,p-P38,p-ERK and p-JNK）as well as p-AKT and p-P85 in PI3K/AKT signaling pathways were also detected.Result： PON（over 30 μmol·L-1） could inhibit the growth of K562 cells in both time-and dose-dependent manner.FCM analysis revealed that apoptotic cells were gradually increased in a dose-dependent manner after treatment for 72 hours,and that marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Wright-Giemsa staining after treatment by 50 μmol·L-1 PON.Western blot showed cleavage of the caspase-3 zymogen protein（32 kD）,with the appearance of its 17 kD subunit,and a cleaved 89 kD fragment of 116 kD PARP was also found.Furthermore,Western blotting also showed that expression of p-AKT and p-P85 in PI3K/AKT signaling pathways was downregulated dramatically whereas the expression of p-P38 as well as p-ERK and p-JNK remained unchanged after the cells were treated by PON for 48 h.Conclusion： The results demonstrate that PON exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells,and that PON induced apoptosis in K562 cells mainly related to activation of caspase-3 as well as inactivation of PI3K/AKT signaling pathway via down regulation of the expression of p-AKT and p-P85 protein levels.These results provide

关 键 词：冬凌草乙素 白血病 K562细胞 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]