三氧化二砷诱导耐药和敏感白血病细胞凋亡机制的比较

Comparative study on the mechanism of arsenic trioxide-induced apoptosis in drug-resistant and sensitive leukemia cells

目的探讨三氧化二砷(As2O3)诱导耐药白血病细胞(K562/ADM细胞)凋亡的是否有异于对药物敏感的白血病细胞(K562细胞)。方法采用流式细胞术(FCM)AnnexinV/PI双标检测细胞凋亡,通过电镜观察凋亡细胞内质网和线粒体形态结构变化;FCM测定细胞内Ca2+和Caspase-3活性;Western blot检测葡萄糖调节蛋白78(GRP78/Bip)的表达。结果 As2O3诱导K562和K562/ADM细胞发生凋亡过程中,内质网明显扩张和脱颗粒,线粒体内、外膜融合,内膜扩张呈空泡样变,嵴紊乱、肿胀,细胞质Ca2+浓度升高和Caspase-3活性明显增强,K562/ADM细胞的变化更为明显。K562/ADM细胞GRP78表达增高,而K562细胞并无明显变化。结论 As2O3可诱导K562/ADM细胞发生内质网应激反应,使得该细胞通过内质网-线粒体途径凋亡。其诱导K562细胞凋亡的机制与之不同,是通过经典的线粒体途径导致细胞凋亡。

Objective To investigate whether the mechanism of arsenic trioxide（As2O3）-induced apopotosis in drug-resistant leukemia cells（K562/ADM） is different from that in sensitive leukemia cells（K562）.Methods The apoptosis of K562 cells and K562/ADM cells were detected by flow cytometry（FCM）,in which cells were double labled with FITC-Annexin V and PI.The morphological ultrastructures of the cells,including endoplasmic reticulum and mitochondria were observed through transmission electron microscopy.FCM was employed to detect intracellular calcium concentration and caspase-3 activity.The expression of GRP78 was detected by Western blot.Results During the apoptotic process of the two cell types,the endoplasmic reticulum exhibited obvious expansion and degranulation,the mitochondria showed fusing inner and outer membranes,disorder and swelling cristae.In addition to that,the inner membrane of mitochondria expanded and exhibited vacuolar degeneration.Meanwhile,The intracellular calcium concentration increased and caspase-3 was activated,especially obviously in K562/ADM cells.Upregulation of GRP78 at endoplasmic reticulum in apopototic K562/ADM cells were detected by Western blot,but not in K562 cells.Conclusion As2O3 can initiate the endoplasmic reticulum stress in K562/ADM cells,and causes the apoptosis of drug-resistant cell via endoplasmic reticulum-mitochondrial pathway,while induces K562 cells to apoptosis via the classic mitochondrial pathway.