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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 effect of ponicidin (PON) on leukemia K562 cells and its mechanism. Methods: K562 cells cultured in vitro with different concentrations of PON (10 ~ 50μmol.L-1) for 24, 48, 72 h, MTT assay was used to detect the cell growth inhibition rate. After 72 hours, the cells were stained with Annexin V The apoptosis rate was detected by flow cytometry (FCM). The Wright-Giemsa staining was used to observe the morphological changes of apoptosis. Western blot was used to detect the expression of caspase-3 and PARP, an inhibitor of MAPKs (ADP-ribose) polymerase (PARP) p-P38, p-ERK and p-JNK) and p-AKT and p-P85 protein in PI3K (phosphatidylinositol 3-kinase) / AKT signaling pathway. Results: PON at 30μmol.L-1 significantly inhibited the cell growth and showed a dose-effect-time-effect relationship. FCM results showed that with different concentrations of PON for 72 h, with the increase of drug concentration Apoptosis rate gradually increased. After PON treatment at 50μmol.L-1 for different time, typical apoptotic phenomena such as nuclear condensation and nuclear fragmentation were observed under Wright-Giemsa staining. The results of Western blot showed that caspase-3 was activated to 17 kD subunit after 48 h treatment, and 89 kD subunit fragment was cleaved in PARP. The result of Western blot further showed that the expression of p-P38, p-ERK and p-JNK protein in MAPKs signal pathway did not change significantly after 48 h treatment, while p-AKT and p-P85 protein in PI3K / AKT signaling pathway The level of expression decreased with the increase of drug concentration. CONCLUSION: PON can exert anti-leukemia effect in vitro by inducing apoptosis of leukemia K562 cells. The mechanism of apoptosis induced by PON in K562 cells is related to the activation of caspase-3 and the expression of p-AKT and p-P85 in the PI3K / AKT signaling pathway, but not to the related regulatory proteins in MAPKs signaling pathway. The results provide a powerful experimental basis and technology platform for further study of the anti-leukemia effect of Rubescensin.