鞘氨醇激酶2抑制5-氟尿嘧啶诱导的人结肠癌HCT116细胞凋亡

Sphingosine kinase 2 negatively regulates 5-fluorouracil-induced apoptosis in human colon cancer cells

目的探讨鞘氨醇激酶2(SphK2)在5-氟尿嘧啶(5-FU)诱导的人结肠癌HCT116细胞凋亡中的作用。方法采用Hoechst33342染色检测5-FU作用48h后HCT116细胞凋亡形态学的变化;流式细胞术检测5-FU作用48h后HCT116细胞的凋亡率,并分析SphK2的干扰或过表达对5-FU诱导的HCT116细胞凋亡率的影响;用蛋白质印迹法观察5-FU诱导HCT116细胞SphK2活性的改变及干扰或过表达SphK2对5-FU诱导凋亡中凋亡标记蛋白的影响。结果 5-FU能诱导HCT116细胞凋亡,细胞出现了明显的核固缩、染色质凝集等凋亡形态变化。细胞SphK2被干扰后,与未被干扰的细胞相比,5-FU诱导后的细胞凋亡率增高(P<0.01),凋亡标记蛋白表达水平升高;而过表达SphK2的细胞在5-FU作用下其凋亡率较对照组(空质粒处理)下降(P<0.01),凋亡标记蛋白表达水平亦下降;5-FU可诱导磷酸化SphK2水平增高。结论 SphK2对5-FU诱导的HCT116细胞凋亡具有负调控作用。

Objective To explore the role of sphingosine kinase 2（SphK2）in 5-fluorouracil（5-FU）-induced apoptosis in human colon cancer HCT116 cells.Methods Hoechst33342 staining was used to examine the apoptosis morphological changes of HCT116 cells 48hafter treatment with 5-FU;flow cytometry was used to detect the apoptotic ratio of HCT116 cells and the effects of SphK2（down-regulation and over-expression）on 5-FU-induced apoptosis were analyzed.Western blotting analysis was used to examine the changes in the activated forms of SphK2 and apoptotic marker protein in HCT116 cells after exposure to 5-FU.Results 5-FU induced significant apoptosis in HCT116 cells as reflected by apoptosis characteristics-condensation and fragmentation of chromatin.Interference of SphK2 significantly increased 5-FU-induced apoptosis in HCT116 cells compared with non-interfered group（P〈0.01）,accompanied by increased apoptotic marker protein.On the contrary,the apoptosis ratio of the cells with SphK2over-expression was significantly decreased compared with vehicle plasmid-transfected group（P〈0.01）,accompanied by decreased apoptotic marker protein. Moreover,5-FU greatly increased activated SphK2.Conclusion SphK2 negatively regulates 5-FU-induced apoptosis in HCT116 cells.