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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (4): 17-23    DOI: 10.13523/j.cb.20180403
研究报告     
白花地胆草单体EM-12诱导2774-C10细胞G1/S期阻滞及细胞凋亡的分子机制研究
黄翔1,杨杰1,何佩彦1,吴志慧1,曾慧兰2,王新宁3(),蒋建伟1()
1 暨南大学医学院 广州 510632
2 暨南大学附属第一医院 广州 510632
3 广东药学院附属第一医院 广州 510080
Molecular Mechanism of Inducing 2774-C10 Cell Apoptosis and G1/S Cell Cycle Arrest by Ethanol Extract from Elephantopus mollis H.B.K.
Xiang HUANG1,Jie YANG1,Pei-yan HE1,Zhi-hui WU1,Hui-lan ZENG2,Xin-Ning WANG3(),Jian-wei JIANG1()
1 Medical College,Jinan University,Guangzhou 510632,China
2 The First Affiliated Hospital of Jinan University, Guangzhou 510632,China
3 First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou 510080, China
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摘要： 目的

研究白花地胆草(Elephantopus mollis H.B.K.)的乙醇提取物EM-12抗肿瘤作用分子机制。

 方法

MTT检测EM-12对卵巢癌细胞活性影响;克隆形成抑制实验检测EM-12对卵巢癌细胞增殖能力的影响; GO功能分析(gene ontology analysis)分析EM-12对卵巢癌2774-C10细胞的影响; PI单染检测细胞周期; Annexin V-FITC/PI双染法检测细胞凋亡; Western blotting检测细胞凋亡、周期相关蛋白。

 结果

MTT实验结果表明,EM-12可以显著抑制卵巢癌细胞的活性;RNA-Seq及GO功能分析表明EM-12诱导2774-C10发生G1/S期阻滞;克隆形成抑制实验结果表明,EM-12可以显著抑制卵巢癌细胞的增殖;流式细胞术结果表明,随着药物浓度的增加,凋亡率逐渐增加,并且G1期细胞比例逐渐增加,S期细胞比例减少,发生G1/S期阻滞。Western blotting结果表明,随着药物浓度的增加cyclin E2、cyclin D1、CDK2、CDK6蛋白水平下降,并伴随caspase-8、caspase-3活化及多聚ADP核糖聚合酶PARP酶切失活。

 结论

EM-12诱导卵巢癌细胞发生G1/S期阻滞,且通过死亡受体通路诱导细胞凋亡。
关键词： 白花地胆草;  卵巢癌;  凋亡;  死亡受体通路;  G1/S期阻滞    
Abstract: Objective:

To investigate the antitumor mechanism of EM-12, an ethanol extracts from Elephantopus mollis H.B.K., in ovarian cancer.

 Methods:

The effects of EM-12 on the cell viability of 2774-C10 and L02 cells were detected by MTT assay, and colony formation assay. Gene ontology analysis plots for EM-12 regulated gene, as determined by RNA-Seq. Cell cycle was evaluated by PI single staining. Cell apoptosis was evaluated by Annexin V FITC/PI staining. Expressions of apoptosis-relative proteins,and cell cycle-relative in 2774-C10 cells were measured by Western blotting.

 Result:

The cell viability of 2774-C10 was inhibited by EM-12 in a dose manner. Colony formation assays showed that EM-12 decreased colony formation compared with control. Flow cytometry analysis showed an increase of the percentage of apoptotic cells and G1/S phase in a dose-dependent manner treated with EM-12. Gene ontology analysis result showed that the gene set related to G1/S cell cycle arrest scores was enriched in EM-12 treated 2774-C10 cell. EM-12 downregulated the expression of cyclin E2, cyclinD1, CDK2, and CDK6 with different concentrations. In addition, active bands of caspase-3 and PARP could be detected using Western blotting.

 Conclusions:

EM-12 induces the G1/S cell cycle arrest. In addition, EM-12 induces apoptosis by death receptor pathway.
Key words: Elephantopus mollis H.B.K.    Ovarian cancer    Apoptosis    Death receptor pathway    G1/S cell cycle arrest
收稿日期: 2017-10-30 出版日期: 2018-05-08
ZTFLH:  Q291  
基金资助: 广州市科技计划(201607010372);广东省自然科学基金(2014A030313356);广东省中医药管理局建设中医药强省科研课题(2015118);华南肿瘤学国家重点实验室开放课题资助项目(HN2017-01)
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黄翔
杨杰
何佩彦
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曾慧兰
王新宁
蒋建伟

引用本文:

黄翔,杨杰,何佩彦,吴志慧,曾慧兰,王新宁,蒋建伟. 白花地胆草单体EM-12诱导2774-C10细胞G1/S期阻滞及细胞凋亡的分子机制研究[J]. 中国生物工程杂志, 2018, 38(4): 17-23.

Xiang HUANG,Jie YANG,Pei-yan HE,Zhi-hui WU,Hui-lan ZENG,Xin-Ning WANG,Jian-wei JIANG. Molecular Mechanism of Inducing 2774-C10 Cell Apoptosis and G1/S Cell Cycle Arrest by Ethanol Extract from Elephantopus mollis H.B.K.. China Biotechnology, 2018, 38(4): 17-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180403    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I4/17

图1  EM-12 单体化学结构
图2  EM-12对卵巢癌2774-C10细胞的活性影响
图3  EM-12对卵巢癌 2774-C10 细胞增殖能力的影响
图4  EM-12对卵巢癌2774-C10 细胞G1/S期阻滞的影响
图5  EM-12对卵巢癌2774-C10 细胞凋亡的影响
图6  G1/S期阻滞相关蛋白表达检测
图7  细胞凋亡相关蛋白表达检测
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