米哚妥林衍生物5'''-methoxyfradcarbazole A对小鼠白血病CB3细胞的作用及机制研究

Effect and Mechanism of Midostaurin Derivatives 5'''-methoxyfradcarbazole A on Mouse Leukemia CB3 Cells

目的研究米哚妥林衍生物5'''-methoxyfradcarbazole A对小鼠白血病细胞CB3生长抑制的作用机制。方法四甲基偶氮唑蓝(MTT)法检测化合物5'''-methoxyfradcarbazole A对小鼠白血病细胞CB3细胞增殖的影响,并测定其生长抑制曲线;利用流式细胞术测定细胞凋亡、周期和分化的结果;Western blot分析5'''-methoxyfradcarbazole A对细胞周期、凋亡相关蛋白的影响。结果化合物5'''-methoxyfradcarbazole A对小鼠白血病细胞具有显著的生长抑制作用,其IC50值为(0.587±0.135)μmol·L-1;5'''-methoxyfradcarbazole A能够诱导CB3细胞产生早期凋亡和晚期凋亡,并表现出时间和浓度的依赖性;其还能影响CB3的细胞周期,明显增高G2期比例;5'''-methoxyfradcarbazole A促进血小板分化标志物CD41和红细胞分化标志物Ter119表达的增加;此外,5'''-methoxyfradcarbazole A作用CB3细胞后显著增加凋亡相关蛋白Bim、PARP1和细胞周期蛋白P21的表达,降低磷酸化ERK蛋白的表达。结论5'''-methoxyfradcarbazole A通过促进凋亡蛋白Bim的表达增加小鼠白血病细胞CB3的凋亡,通过增加细胞周期蛋白P21的表达使CB3细胞发生G2期阻滞;此外,PARP1及磷酸化的ERK蛋白表达水平变化也在一定程度上解释了5'''-methoxyfradcarbazole A对CB3细胞的生长抑制作用。

OBJECTIVE To investigate the mechanism of midostaurin derivative 5'''-methoxyfradcarbazole A of action in inhibiting mouse leukemia cells(CB3)growth.METHODS MTT assay was employed to evaluate the effect of compound 5'''-methoxyfradcarbazole A on the proliferation of CB3 cells,and generate the growth inhibition curves.Flow cytometry and Annexin V-FITC/PI double staining were used to determine the changes of the cell cycle,cell differentiation and apoptosis.Western blot analysis was applied to test the effects of 5'''-methoxyfradcarbazole A on cyclin and apoptosis-related proteins.RESULTS The compound 5'''-methoxyfradcarbazole A could significantly inhibit the growth of CB3 cells,and the half maximal inhibitory concentration(IC50)of the compound was(0.587±0.135)μmol·L-1.5'''-Methoxyfradcarbazole A was able to induce early apoptosis and late apoptosis of CB3 cells in a time-and dose-dependent manner.At the same time,it also affected the cell cycle of CB3 and significantly increased the proportion of G2 phase.The expression of CD41,a platelet differentiation marker,and Ter119,an erythrocyte differentiation marker,were also increased in CB3 cells treated with 5'''-methoxyfradcarbazole A.Besides,the expressions of apoptosis-related proteins Bim,PARP1 and cyclin-related protein P21 were significantly increased,and the phosphorylated ERK protein was decreased.CONCLUSION Midostaurin derivative 5'''-methoxyfradcarbazole A could increase the apoptosis of CB3 cells by promoting the expression of apoptotic protein Bim,and cause G2 phase arrest by increasing the expression of cyclin protein P21.Also,the changes of the expression levels of PARP1 and phosphorylated ERK protein also partly explain the inhibition of 5'''-methoxyfradcarbazole A on CB3 cells growth.