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

doi:10.3969/j.issn.1674-8115.2021.07.007

摘要/Abstract

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

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)

中图分类号:

R733.72

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.

Chen CHEN, Feng-hou GAO. Degradation of BCR-ABL fusion protein in chronic myeloid leukemia cells induced by isoalantolactone[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 891-897.

图/表 9

表1 qPCR引物序列

Tab 1 Primer sequence for qPCR

Gene	Forward primer （5′→3′）	Reverse primer （5′→3′）
β-actin	TCGTGCGTGACATTAAGGAG	GTCAGGCAGCTCGTAGCTCT
BCR-ABL	CGGGAGCAGCAGAAGAAGTGT	CGAAAAGGTTGGGGTCATTTTC

表2 靶向敲减序列

Tab 2 Target sequence of knockdown

Name	Target gene	Target sequence （5′→3′）
shCASP7-1	CASP7	GGCAAATGCATCATAATAA
shCASP7-2	CASP7	GCCCATCAATGACACAGAT
shCASP3-1	CASP3	CCCTGGACAACAGTTATAA
shCASP3-2	CASP3	GCGTGATGTTTCTAAAGAA
NC	Limulus polyphemus $δ$ -like protein B	GTTCTCCGAACGTGCACGT

表2 靶向敲减序列

Tab 2 Target sequence of knockdown

Name	Target gene	Target sequence （5′→3′）
shCASP7-1	CASP7	GGCAAATGCATCATAATAA
shCASP7-2	CASP7	GCCCATCAATGACACAGAT
shCASP3-1	CASP3	CCCTGGACAACAGTTATAA
shCASP3-2	CASP3	GCGTGATGTTTCTAAAGAA
NC	Limulus polyphemus $δ$ -like protein B	GTTCTCCGAACGTGCACGT

表3 Iso对K562/K562R细胞的增殖抑制率分析（%）

Tab 3 Analysis of proliferation inhibition rate of K562/K562R cells by Iso （%）

Group	Phase of treatment with Iso in K562 cells			Phase of treatment with Iso in K562R cells
Group	24 h	36 h	48 h	24 h	36 h	48 h
Control group （0 μmol·L^-1 Iso）	0	0	0	0	0	0
Iso treatment group
2.5 μmol·L^-1	2.9±0.8	5.1±2.7	1.9±1.0	0.1±1.0	0.7±1.4	6.2±3.1
5 μmol·L^-1	11.7±1.8	33.5±0.9^①	44.7±3.5^①	1.9±4.1	4.6±1.2	15.2±0.7^①
10 μmol·L^-1	34.1±1.7^①	54.4±0.7^①	68.9±0.5^①	15.5±1.0^①	23.9±2.0^①	33.6±0.9^①
20 μmol·L^-1	41.7±0.7^①	60.6±1.0^①	73.4±0.5^①	23.7±0.7^①	31.6±1.7^①	55.5±0.6^①
40 μmol·L^-1	52.4±1.0^①	66.6±1.1^①	77.9±0.4^①	30.9±0.7^①	45.2±1.4^①	63.5±1.3^①
80 μmol·L^-1	53.3±0.8^①	70.8±0.6^①	82.7±0.4^①	38.2±0.8^①	48.7±0.2^①	67.9±1.5^①

图1 流式细胞术检测Iso对K562和K562R细胞的凋亡诱导效应Note： A. Result of K562 cell apoptosis. B. Result of K562R cell apoptosis. ①P=0.034, ②P=0.000, compared with the control group.

Fig 1 Apoptosis inducing effects of Iso on K562 and K562R cells by flow cytometry

图2 不同浓度及作用时间下Iso对K562和K562R细胞中凋亡相关蛋白和BCR-ABL融合蛋白水平的影响Note： A/B. Apoptosis-related protein and BCR-ABL fusion protein expression in K562 cells (A) and K562R cells (B) treated with different concentrations of Iso. C?D. Apoptosis-related protein and BCR-ABL fusion protein expression in K562 cells (C) and K562R cells (D) treated with Iso for different time.

Fig 2 Effects of Iso on the levels of apoptosis-related protein and BCR-ABL fusion protein in K562 and K562R cells at different concentrations and time

图3 Iso对K562细胞（A）和K562R细胞（B）中BCR-ABL mRNA水平的影响Note： NS—no significance.

Fig 3 Effects of Iso on the level of BCR-ABL mRNA in K562 cells (A) and K562R cells (B)

图4 蛋白酶体抑制剂MG132（A）、自噬抑制剂3-MA（B）和溶酶体抑制剂CQ（C）对Iso诱导BCR-ABL融合蛋白下调的影响

Fig 4 Effects of proteasome inhibitor MG132 (A), autophagy inhibitor 3-MA (B) and lysosomal inhibitor CQ (C) on the down-regulation of BCR-ABL fusion protein induced by Iso

图5 半胱天冬酶抑制剂Z-VAD-FMK对Iso诱导BCR-ABL融合蛋白下调的影响Note： A. K562 cells. B. K562R cells.

Fig 5 Effects of caspase inhibition Z-VAD-FMK on the down-regulation of BCR-ABL fusion protein induced by Iso

图6 CASP7（A）与CASP3（B）敲减对Iso诱导BCR-ABL融合蛋白下调的影响

Fig 6 Effects of CASP7 (A) and CASP3 (B) knockdown on the down-regulation of BCR-ABL fusion protein induced by Iso

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