摘要
目的研究沙蟾毒精(arenobufagin,ARE)对急性白血病细胞的凋亡诱导作用及可能的分子机制。方法急性白血病细胞Jurkat和MV-4-11经不同浓度(0、10、20、40、80 nmol/L)ARE作用24 h,80 nmol/L ARE作用不同时间(0、6、12、18、24 h)或ROCK1抑制剂Y-27632(20μmon/L)预处理1 h后80 nmol/L ARE作用24 h,采用MTT检测ARE抑制急性白血病细胞增殖的能力;采用流式细胞术检测ARE诱导急性白血病细胞凋亡的能力;采用Western blot和CoIP实验检测急性白血病细胞经ARE作用后细胞中凋亡相关蛋白和Rho A/ROCK1信号通路相关蛋白的变化情况。结果ARE对Jurkat和MV-4-11细胞的抑制增殖作用呈剂量依赖性和时间依赖性(P<0.01),对Jurkat和MV-4-11细胞凋亡诱导作用亦呈剂量和时间依赖性(P<0.01);Western blot检测结果显示:ARE可激活ROCK1信号通路,促进BAX移位至线粒体,Cytochrome c(Cyto c)和AIF由线粒体释放入细胞质,增加Caspase 3和Caspase 7剪切激活,增加PARP1剪切,并减少XIAP表达,其上述作用具有剂量依赖性和时间依赖性(P<0.01);使用Y-27632抑制ROCK1信号通路可明显阻断ARE诱导的BAX移位至线粒体、Cyto c和AIF向细胞质的释放、Caspase 3和Caspase 7的剪切激活、PARP1的剪切、XIAP表达的下调以及细胞凋亡(P<0.01)。结论ARE通过激活Rho A/ROCK1信号通路有效诱导急性白血病细胞(Jurkat、MV-4-11)发生凋亡。
Objective To investigate the apoptosis-inducing effect of arenobufagin(ARE)on acute leukemia cells and explore its possible molecular mechanism.Methods Acute leukemia cell lines Jurkat and MV-4-11 were treated with different concentrations of ARE(0,10,20,40 or 80 nmol/L)for 24 h,or with 80 nmol/L ARE for different durations(0,6,12,18 or 24 h),or with 80 nmol/L ARE for 24 h after being pretreated with 20μmol/L Y-27632(inhibitor of ROCK1)for 1 h,respectively.Then,MTT assay and flow cytometry were used to respectively detect the effect of ARE on cell proliferation and apoptosis in acute leukemia cells;Western blotting and co-immunoprecipitation(CoIP)assay were performed to determine the expression of apoptosis-related and Rho A/ROCK1 pathway-related proteins in the acute leukemia cells after ARE treatment.Results ARE inhibited the proliferation and induced apoptosis of acute leukemia cells in dose-and time-dependent manners(P<0.01).Western blotting results showed that ARE activated ROCK1 signaling pathway,facilitated the translocation of BAX from cytoplasm to mitochondria,promoted the release of mitochondrial proteins(Cytochrome c,AIF),increased the cleavage/activation of Caspase 3 and Caspase 7 as well as the degradation of Poly(ADP-ribose)polymerase 1(PARP1),and then reduced the expression of X-linked inhibitor of apoptosis(XIAP).The above effects presented dose-and time-dependence(P<0.01).Interruption of ROCK1 pathway by the pharmacological inhibitor Y-27632 significantly attenuated ARE-mediated mitochondrial translocation of BAX,reduced cytosolic release of Cytochrome c and AIF from mitochondria,suppressed the activation of Caspase 3 and Caspase 7,the degradation of cleavage of PARP1,and down-regulated XIAP expression as well as blocked apoptosis(P<0.01).Conclusion ARE effectively induces apoptosis of acute leukemia cells(Jurkat,MV-4-11)through the activation of Rho A/ROCK1 signaling pathway.
作者
李志强
姜秀星
胡金娇
丁鑫
雷令
高宁
LI Zhiqiang;JIANG Xiuxing;HU Jinjiao;DING Xin;LEI Ling;GAO Ning(Department of Pharmacognosy and Traditional Chinese Medicine,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2022年第7期700-710,共11页
Journal of Army Medical University
基金
国家自然科学基金面上项目(31571425)
