PEITC诱导人AML-M2白血病细胞凋亡机制的初步探讨

Explore the mechanism of PEITC on human Acute Myeloid leukemia-M2 cells' apoptosis

目的证实苯乙基异硫氰酸酯(phenethyl isothiocyanate,PEITC)诱导人急性髓系白血病(AML)M2白血病细胞系Kasumi-1细胞凋亡的效应,初步探讨PEITC诱导Kasumi-1细胞凋亡的机制。方法培养人M2白血病细胞株Kasumi-1细胞,CCK-8法检测不同浓度PEITC处理后Kasumi-1细胞的增殖抑制率;流式细胞术检测药物诱导细胞凋亡和活性氧(reactive oxygen species,ROS)生成的情况;Real-time PCR及Western blot检测HO-1以及凋亡相关基因caspase3、caspase8、caspase9mRNA及蛋白水平的变化。结果 CCK-8结果显示PEITC作用Kasumi-1细胞24h、48h、72h后,细胞增殖抑制率呈现浓度-时间依赖性。流式细胞术显示PEITC能够诱导Kasumi-1细胞的凋亡。Real-time PCR及Western blot结果显示PEITC处理细胞后,HO-1表达下降,而凋亡相关基因Caspase3、Caspase8、Caspase9的表达上调。DCFH-DA探针法显示PEITC作用Kasumi-1细胞后,ROS生成增多。结论 PEITC能够促进Kasumi-1细胞凋亡,呈时间剂量梯度依赖关系;PEITC诱导Kasumi-1细胞凋亡可能通过的机制包括下调HO-1表达促进ROS生成及凋亡相关基因Caspase3、Caspase8、Caspase9的激活。

Objective To investigate the effect of Phenethyl isothiocyanate in inducing acute mye‐locytic leukemia （AML） Kasumi‐1 cell line apoptosis ,explore the possible mechanisms ,thus provi‐ding new idea for the treatment of AML .Methods Kasumi‐1 cells were cultured .The influences of PEITC on the growth inhibition rate of cells were detected by CCK‐8 .Annexin‐V‐PI method was used to detect cell apoptosis by flow cytometry .The mRNA and proteins expressions of HO‐1 and apopto‐sis‐related genes ,caspase3 ,caspase8 and caspase9 were detected by Real‐time PCR and western blot （WB） respectively .The ROS generation was detected by flow cytometry .Results CCK‐8 methods showed that PEITC inhibited the proliferation of Kasumi‐1 cells in a dose and time‐dependent manner . Flow cytometry showed that PEITC could induced Kasumi‐1 cell apoptosis .Real‐time PCR and WB methods showed that the mRNA and protein expressions of apoptosis related genes caspase3 ,caspase8 and caspase9 increased after PEITC treatment .DCFH‐DA probe methods showed that PEITC could increased the ROS generation .Conclusion PEITC is found to suppress cell grow th and cause apoptosis by down regulating HO‐1 expression to increase ROS generation and by the activation of caspase3 , caspase8 and caspase9 .The results of the present study suggest that PEITC may be a potential anti‐tumor compound for AML‐M2 therapy .