受体作用蛋白在异硫氰酸苯乙酯诱导人急性白血病细胞凋亡中的作用

Role of receptor-interaction proteins in phenethylisothiocyanate-induced apoptosis in human leukemia cells in vitro

目的探讨受体作用蛋白(receptor-interaction proteins,RIP)在异硫氰酸苯乙酯(phenethy lisothiocyanate,PEITC)诱导人白血病细胞凋亡中的作用。方法将0、2、4、6、8μmol/L异硫氰酸苯乙酯作用于U937细胞3、6h,观察量效关系。用8μmol/L异硫氰酸苯乙酯作用U937细胞1、3、6、9、12、24h,观察时效关系。采用AnnexinV/PI染色和流式细胞仪检测细胞凋亡。用Westernblot法检测细胞凋亡相关蛋白蛋白聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]、Caspasep-3、Caspasep-8及RIP的表达。结果异硫氰酸苯乙酯在2、4、6、8μmol/L作用于U937细胞3h分别引起9%、25%、44%和56%的细胞发生凋亡,6h分别引起16%、28%、51%和82%的细胞发生凋亡,呈明显的量效关系。8μmol/L的异硫氰酸苯乙酯作用U937细胞1、3、6、9、12、24h分别引起12%、57%、79%、86%、90%和91%的细胞发生凋亡,呈明显的时效关系。Westernblot结果表明,异硫氰酸苯乙酯引起凋亡蛋白酶Caspase-3、Caspase-8的降解/活化增加,促进凋亡作用底物PARP的降解增加。异硫氰酸苯乙酯还可引起受体作用蛋白表达降低及RIP降解增加。结论异硫氰酸苯乙酯可引起人白血病细胞发生凋亡,其分子机制可能与死亡受体途径有关,其中受体作用蛋白的降解可能在异硫氰酸苯乙酯诱导细胞凋亡中起着重要作用。

Objective To explore the role of receptor-interaction proteins （RIP） in phenethylisothiocyanate （PEITC）-induced apoptosis in human leukemia cells. Methods U937 cells were exposed to PEITC at various concentrations of 0, 2, 4, 6 and 8 μmol/L for 3 and 6 h, or at 8 μmol/L for different time intervals of 1, 3, 6, 9, 12 and 24 h. Cells were stained with Annexin V/PI, and apoptosis was determined by using flow cytometry. Total protein extracts were prepared and subjected to Western blot assay using antibodies against poly （ADP-ribose） polymerase （PARP） , Caspase-3, Caspase-8, and RIP. β-actin was used to ensure equivalent loading. Results After exposure to PEITC at concentrations of 2, 4, 6 and 8 μmol/L for 3 h, U937 cells showed an increase in cell apoptosis （9% , 25%, 44% and 56% respectively）. While when the time expanded to 6 h, the amount of apoptotic cells was increased to 16%, 28%, 51% and 82% respectively. A dose-dependent manner was seen when U937 cells were exposure to PEITC at different concentrations. When U937 cells were treated by PEITC at concentration of 8 μmol/L for 1, 3, 6, 9, 12, and 24 h, the apoptotic cells covered 12%, 57% , 79%, 86% , 90%, and 91% of all cells respectively, indicating a time-dependent manner. Western blot assay showed that PEITC caused the activation/cleavage of Caspase-3 and -8, and improved the PARP cleavage, and also decreased the expression of RIP. PEITC-induced apoptosis was proceeded by degradation of RIP. Conclusion Our results indicate that PEITC induces the apoptosis in human leukemia cells through a process that involves death receptor pathway, and RIP may play an important role in the apoptosis.