冬凌草甲素上调PLK1对Jurkat细胞细胞周期的影响

doi:10.3969/j.issn.1674-8115.2021.05.007

摘要/Abstract

摘要：

目的·探索冬凌草甲素对Jurkat细胞增殖的抑制作用及其机制。方法·采用CCK-8法、流式细胞术、瑞士吉姆萨染色、蛋白质印迹法，检测冬凌草甲素处理后Jurkat细胞的增殖情况、细胞周期、凋亡情况及有丝分裂相关蛋白激酶表达情况。蛋白质印迹法检测冬凌草甲素处理后，Jurkat细胞中Polo样蛋白激酶1（Polo-like kinase 1，PLK1）下游蛋白的磷酸化水平。通过计算机模拟、免疫沉淀、蛋白质印迹法、细胞热力学迁移实验方法研究冬凌草甲素与PLK1的结合方式及结合位点。组间比较采用t检验，P<0.05为差异具有统计学意义。结果·冬凌草甲素通过上调PLK1蛋白含量并促进其激酶活性，从而诱导Jurkat细胞发生G₂/M期阻滞；冬凌草甲素可与PLK1蛋白直接结合抑制其通过泛素蛋白酶体途径降解；如PLK1蛋白的Cys67或Cys133氨基酸残基发生突变则抑制冬凌草甲素与其直接结合，且抑制冬凌草甲素对PLK1蛋白的稳定作用。结论·冬凌草甲素通过直接结合PLK1蛋白抑制其泛素化修饰及降解，促进其活性，诱导Jurkat细胞发生G₂/M期阻滞；PLK1蛋白的Cys67和Cys133位点是蛋白与冬凌草甲素结合的关键位点。

关键词: 冬凌草甲素, Polo样蛋白激酶1（PLK1）, 细胞周期阻滞, 急性T淋巴细胞白血病

Abstract:

Objective·To explore the inhibitory effect of oridonin on the proliferation of Jurkat cells and its mechanism.

Methods·CCK-8 method, flow cytometry, Swiss Giemsa staining, and Western blotting were used to detect the proliferation, cell cycle and apoptosis of Jurkat cells and the expression of mitosis-related protein kinases after oridonin treatment. Western blotting was used to detect the phosphorylation level of PLK1 downstream proteins in Jurkat cells after oridonin treatment. Computer simulation, immunoprecipitation, Western blotting and Celluar Thermal Shift Assay were used to study the binding mode and binding site of oridonin and PLK1. Student's-t test was performed for comparison between each two groups. Statistical significance was accepted at a value of P<0.05.

Results·Oridonin could induce G₂/M phase arrest in Jurkat cells by up-regulating the content of PLK1 protein and promoting its kinase activity. Oridonin could directly bind to PLK1 protein to inhibit its degradation through the ubiquitin proteasome pathway. The mutation of Cys67 or Cys133 amino acid residues of PLK1 protein inhibited its direct binding to oridonin. And the mutation inhibited the stabilizing effect of oridonin on PLK1 protein.

Conclusion·Oridonin inhibits PLK1 protein ubiquitination modification and degradation by directly binding to its protein. Oridonin can also promote PLK1 protein kinase activity and induce G₂/M phase arrest in Jurkat cells. The Cys67 and Cys133 sites of the PLK1 protein are the key sites for the protein to bind to oridonin.

Key words: oridonin, polo-like kinase 1 (PLK1), cell cycle arrest, acute T-1ymphocytic leukemia (T-ALL)

中图分类号:

R733.7

赫玮, 左勇. 冬凌草甲素上调PLK1对Jurkat细胞细胞周期的影响[J]. 上海交通大学学报(医学版), 2021, 41(5): 603-611.

Wei HE, Yong ZUO. Effect of oridonin up-regulating PLK1 on the cell cycle of Jurkat cells[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(5): 603-611.

图/表 10

表1 PLK1蛋白Cys67位点（M1）及 Cys133位点（M2）突变引物序列(5′→3′)

Tab 1 Cys67 (M1) and Cys133 (M2) mutation primers of PLK1 protein sequence (5′→3′)

Gene locus	Primer	Sequence
M1	Forward	CTTTGCCAAGAGCTTCGAGATCTCGGACGCGGACACCAAG
M1	Reward	TCTCGAAGCTCTTGGCAAAGCCGCCCTTGCCCAAAAAGCG
M2	Forward	TGGTGTTGGAGCTCAGCCGCCGGAGGTCTCTCCTGGAGCT
M2	Reward	GCGGCTGAGCTCCAACACCACGAACACGAAGTCGTTGTCCT

图1 冬凌草甲素对Jurkat细胞增殖的影响Note: A. Jurkat cell proliferation detected by CCK-8. B. Jurkat cell apoptosis detected by flow cytometry. ①P=0.002, ②P=0.000， compared with the control group.

Fig 1 Effect of oridonin on the proliferation in Jurkat cells

图2 低剂量冬凌草甲素处理Jurkat细胞后对细胞周期的影响Note: A?D. Jurkat cell cycle was detected by flow cytometry, and FlowJo software was used to analyze the data to display the results. E. Representative images of Swiss Giemsa stain（×400）. F. Expression of cyclin B protein detected by Western blotting.

Fig 2 Effect of low concentration oridonin on the cell cycle in Jurkat cells

表2 Genecards数据库中检索与有丝分裂相关的基因信息

Tab 2 Searching the Genecards database with "mitosis" as key word

Number	Symbol	Description	Category	GIFtS	GC id	Score
1	BUB1B	BUB1 Mitotic Checkpoint Serine/Threonine Kinase B	Protein Coding	51	GC15P040161	29.43
2	BUB1	BUB1 Mitotic Checkpoint Serine/Threonine Kinase	Protein Coding	51	GC02M110637	26.96
3	PLK1	Polo Like Kinase 1	Protein Coding	50	GC16P023869	25.57
4	AURKA	Aurora Kinase A	Protein Coding	51	GC20M056370	25.15
5	AURKB	Aurora Kinase B	Protein Coding	50	GC17M008770	24.98

图3 低剂量冬凌草甲素对有丝分裂相关蛋白激酶表达的影响Note: Expression of BuBR1、BuB1、PLK1、Aurora A、Aurora B protein detected by Western blotting.

Fig 3 Effect of low concentration oridonin on the expression of mitosis-related kinase

图4 冬凌草甲素对PLK1下游蛋白磷酸化水平的影响Note: A. Expression of BuBR1、CDC25c、CDC27 protein and phosphorylation level detected by Western blotting. B. Expression of WEE1 protein detected by Western blotting.

Fig 4 Effect of oridonin on the phosphorylation level of PLK1 downstream protein

图5 冬凌草甲素对PLK1蛋白泛素化修饰水平的影响Note：Effect of oridonin on the ubiquitination modification level of PLK1 protein detected by Western blotting.

Fig 5 Effect of oridonin on the level of ubiquitination modification for PLK1 protein

图6 不同温度或浓度条件下冬凌草甲素对PLK1稳定性的影响Note: A/B. Expression of PLK1 protein detected by Western blotting (A), and analysis (B) of the data to display the results (temperature=45.0， 46.7， 49.3， 52.5， 56.9 and 60.2 ℃). C/D. Expression of PLK1 protein detected by Western blotting (C), and analysis (D) of the data to display the results (temperature=46.7 ℃).

Fig 6 Effect of oridonin on the stability of PLK1 under different temperatures or concentration

图7 冬凌草甲素与PLK1对接位点预测Note: A. Complex structure of PLK1 protein and BI2536 in the PDB database (PDB number: 2RKU). B. Simulation of docking of oridonin and PLK1 protein (PDB number: 2RKU).

Fig 7 Prediction of the docking site of oridonin and PLK1

图8 PLK1蛋白突变后冬凌草甲素对PLK1稳定性的影响Note: A/B. Expression of Flag-PLK1 protein detected by Western blotting after the mutation of PLK1 protein. C. Ubiquitination modification level of PLK1 protein. ①P=0.000, compared with the control group.

Fig 8 Effect of oridonin on the stability of PLK1 after mutation

参考文献 21

1	Whibley C, Pharoah PDP, Hollstein M. p53 polymorphisms: cancer implications[J]. Nat Rev Cancer, 2009, 9(2): 95-107.
2	Nasmyth K. Segregating sister genomes: the molecular biology of chromosome separation[J]. Science, 2002, 297(5581): 559-565.
3	Cao PF, Yu YL, Wang W, et al. Fluorescence in situ hybridization comparison of the prognostic factors in adult and pediatric acute lymphoblastic leukemia: a retrospective analysis of 282 cases[J]. Exp Ther Med, 2018, 16(6): 4674-4684.
4	Han QG, Xu X, Li J, et al. GATA4 is highly expressed in childhood acute lymphoblastic leukemia, promotes cell proliferation and inhibits apoptosis by activating BCL2 and MDM2[J]. Mol Med Rep, 2017, 16(5): 6290-6298.
5	郭勇, 单卿卿, 龚玉萍, 等. 冬凌草甲素抗Ph染色体阳性急性淋巴细胞白血病效应的实验研究[J]. 中华血液学杂志, 2012, 33(6): 439-443.
6	Zhen T, Wu CF, Liu P, et al. Targeting of AML1-ETO in t(8;21) leukemia by oridonin generates a tumor suppressor-like protein[J]. Sci Transl Med, 2012, 4(127): 127ra38.
7	Moreau P, Attal M, Facon T. Frontline therapy of multiple myeloma[J]. Blood, 2015, 125(20): 3076-3084.
8	Li FF, Yi S, Wen L, et al. Oridonin induces NPM mutant protein translocation and apoptosis in NPM₁c+ acute myeloid leukemia cells in vitro[J]. Acta Pharmacol Sin, 2014, 35(6): 806-813.
9	Gui ZF, Luo F, Yang YY, et al. Oridonin inhibition and miR‑200b‑3p/ZEB1 axis in human pancreatic cancer[J]. Int J Oncol, 2017, 50(1): 111-120.
10	Xu B, Shen W, Liu X, et al. Oridonin inhibits B_xPC-3 cell growth through cell apoptosis[J]. Acta Biochim Biophys Sin (Shanghai), 2015, 47(3): 164-173.
11	齐琦, 张配, 李其响, 等. 冬凌草甲素诱导三阴乳腺癌MDA-MB-231细胞凋亡及对细胞内活性氧水平的影响[J]. 中国中药杂志, 2017, 42(12): 2361-2365.
12	张覃沐. 一种新的抗癌物质冬凌草素[J]. 科学通报, 1978, 23(1): 53-54.
13	叶珍珍, 薛飞龙, 丁文评, 等. 冬凌草甲素通过下调Brg1表达抑制Jurkat细胞生长[J]. 中国当代儿科杂志, 2017, 19(11): 1208-1212.
14	Dal Piaz F, Cotugno R, Lepore L, et al. Chemical proteomics reveals HSP70 1A as a target for the anticancer diterpene oridonin in Jurkat cells[J]. J Proteomics, 2013, 82: 14-26.
15	Lee KS, Yuan YL, Kuriyama R, et al. Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO₁/MKLP-1[J]. Mol Cell Biol, 1995, 15(12): 7143-7151.
16	Park J, Kwon MS, Kim EE, et al. USP35 regulates mitotic progression by modulating the stability of Aurora B[J]. Nat Commun, 2018, 9(1): 688.
17	Wang JS, Maldonado MA. The ubiquitin-proteasome system and its role in inflammatory and autoimmune diseases[J]. Cell Mol Immunol, 2006, 3(4): 255-261.
18	俞卿, 熊秀芳, 孙毅. 靶向Cullin-RING E3泛素连接酶的抗肿瘤策略及相关药物研发进展[J]. 浙江大学学报(医学版), 2020, 49(1): 1-19.
19	Martinez Molina D, Jafari R, Ignatushchenko M, et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay[J]. Science, 2013, 341(6141): 84-87.
20	Almqvist H, Axelsson H, Jafari R, et al. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil[J]. Nat Commun, 2016, 7: 11040.
21	Martinez Molina D, Nordlund P. The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies[J]. Annu Rev Pharmacol Toxicol, 2016, 56: 141-161.

[1]	卿恺, 陈芳源, 沈莉菁, 等. 转Notch1基因T淋巴细胞白血病双荧光示踪斑马鱼模型的建立及鉴定[J]. , 2013, 33(3): 257-.
[2]	颜晶晶, 唐永范, 陆魏杰, 等. 北冬虫夏草水提物对人肺腺癌细胞A549的作用[J]. , 2011, 31(7): 922-.
[3]	王海侠, 郑新勇, 郜尽. 白头翁皂苷B4体外抑制人肝癌细胞HepG2增殖并诱导其凋亡[J]. , 2011, 31(10): 1481-.
[4]	高丰厚, 郭竹英, 徐芒华, 等. 冬凌草甲素诱导结直肠癌细胞SW-1116凋亡与衰老的体内外实验研究[J]. , 2010, 30(6): 683-.