异土木香内酯诱导慢性粒细胞白血病细胞BCR-ABL融合蛋白降解

Degradation of BCR-ABL fusion protein in chronic myeloid leukemia cells induced by isoalantolactone

目的·探索异土木香内酯(isoalantolactone,Iso)对伊马替尼敏感和耐药的慢性粒细胞白血病(chronic myeloid leukemia,CML)细胞的效应,以及下调BCR-ABL融合蛋白的分子机制。方法·采用不同浓度的Iso分别处理K562和K562R细胞0、24、36、48 h后,用CCK-8法测定Iso对CML细胞的抑制效应。用流式细胞术检测Iso诱导K562和K562R细胞24 h的凋亡效应。蛋白质印迹法(Western blotting)检测不同浓度、不同作用时间下Iso对CML细胞中BCR-ABL融合蛋白水平的影响。采用反转录及实时荧光定量PCR(quantitative real-time PCR,qPCR)检测Iso对CML细胞中BCR-ABL mRNA水平的影响。分别用蛋白酶体抑制剂MG132、自噬抑制剂3-甲基腺嘌呤和溶酶体抑制剂氯喹联合Iso处理K562细胞,用半胱天冬酶抑制剂Z-VAD-FMK联合Iso处理K562与K562R细胞,并采用Western blotting检测BCR-ABL融合蛋白水平的改变。在K562R细胞中敲减半胱天冬酶3(caspase 3,CASP3)、半胱天冬酶7(caspase 7,CASP7),检测其对由Iso诱导BCR-ABL融合蛋白下调的影响。结果·Iso抑制CML细胞的增殖,且呈剂量和时间依赖性。流式细胞术结果显示Iso可增加K562与K562R细胞凋亡的发生(均P<0.05)。Western blotting结果显示Iso能诱导BCR-ABL融合蛋白水平降低,而qPCR结果显示BCR-ABL mRNA水平无显著变化。MG132、3-甲基腺嘌呤以及氯喹不能逆转由Iso诱导的BCR-ABL融合蛋白的下调,而Z-VAD-FMK可部分逆转该下调。敲减CASP3后能部分逆转由Iso诱导的BCR-ABL蛋白的降解,而CASP7敲减后则无影响。结论·Iso可靶向降解BCR-ABL融合蛋白。该结果为克服CML细胞的耐药提供了实验基础。

Objective·To explore the effect of isoalantolactone(Iso)on imatinib-sensitive and drug-resistant chronic myeloid leukemia(CML)cells,and the molecular mechanism of down regulating BCR-ABL fusion protein.Methods·K562 and K562R cells were treated with different concentrations of Iso for 0,24,36 and 48 h,respectively.The inhibitory effect of Iso on CML cells was determined by CCK-8 method.The apoptosis of K562 and K562R cells induced by Iso for 24 h was detected by flow cytometry.CML cells were treated with different concentration of Iso for different time,and the effect of Iso on the level of BCR-ABL fusion protein was detected by Western blotting.The effect of Iso on BCR-ABL mRNA level in CML cells was detected by reverse transcription and quantitative real-time PCR(qPCR).K562 cells were treated with proteasome inhibitor MG132,autophagy inhibitor 3-methyladenine and lysosomal inhibitor chloroquine combined with Iso,respectively,and K562 and K562R cells were treated with caspase inhibitor Z-VAD-FMK combined with Iso.The level of BCR-ABL fusion protein was detected by Western blotting.Caspase 3(CASP3)and caspase 7(CASP7)were knocked down in K562R cells,and their effects on the down-regulation of BCR-ABL fusion protein induced by Iso were detected.Results·The proliferation of CML cells was inhibited by Iso in a dose-and time-dependent manner.Flow cytometry showed that Iso could increase the apoptosis of K562 and K562R cells(both P<0.05).Western blotting showed that Iso could induce the decrease of BCR-ABL fusion protein level,while qPCR showed that BCR-ABL mRNA level had no significant change.MG132,3-methyladenine and chloroquine could not reverse the down-regulation of BCR-ABL fusion protein induced by Iso,but Z-VAD-FMK could partially reverse the down-regulation.Knockdown of CASP3 could partially reverse the degradation of BCR-ABL protein induced by Iso,while CASP7 could not.Conclusion·Iso can target the degradation of BCR-ABL fusion protein,which provides an experimental basis for overcoming drug resistance of CML cells.