上海交通大学学报（医学版）

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2021 , Vol. 41 >Issue 7: 891 - 897

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.07.007

论著 · 基础研究

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

  • 陈晨 ,
  • 高丰厚
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  • 上海交通大学医学院附属第九人民医院肿瘤科，上海 201999
陈晨（1995—），女，硕士生；电子信箱：chenchen951226@163.com。

网络出版日期: 2021-08-03

基金资助

上海市科学技术委员会科研计划项目(14401901500)

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

  • Chen CHEN ,
  • Feng-hou GAO
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  • Department of Oncology, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201999, China

Online published: 2021-08-03

Supported by

Scientific Research Project of Science and Technology Commission of Shanghai Municipality(14401901500)

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摘要

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。

关键词： 异土木香内酯; BCR-ABL融合蛋白; 慢性粒细胞白血病; 蛋白降解; 半胱天冬酶3

本文引用格式

陈晨 , 高丰厚 . 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[J]. 上海交通大学学报（医学版）, 2021 , 41(7) : 891 -897 . DOI: 10.3969/j.issn.1674-8115.2021.07.007

Abstract

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.

Key words： isoalantolactone (Iso); BCR-ABL fusion protein; chronic myeloid leukemia (CML); protein degradation; caspase 3 (CASP3)

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