白花蛇舌草总黄酮通过激活半胱氨酸蛋白酶依赖的线粒体凋亡途径抑制胃癌作用机制

WANG Xue-li; CAO Xiao-zheng; WANG Dao-yuan; QIU Ye-bei; DENG Kai-yu; CAO Jian-guo; LIN Shao-qiang; XU Yong; REN Kai-qun

doi:10.1007/s11655-022-3469-z

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白花蛇舌草总黄酮通过激活半胱氨酸蛋白酶依赖的线粒体凋亡途径抑制胃癌作用机制

Original Article | Updated：2023-02-22

- 白花蛇舌草总黄酮通过激活半胱氨酸蛋白酶依赖的线粒体凋亡途径抑制胃癌作用机制
- Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1
- 中国结合医学杂志（英文版） 2023年29卷第3期页码：224-232
- Affiliations：
  
  1.Medical College, Hunan University of Medicine, Huaihua, Hunan Province (418000), China
  2.Clinical Department of Guangdong Metabolic Disease Research Centre of Integrated Chinese and Western Medicine, the First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou (510062), China
  3.Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou (510530), China
  4.The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Medical College, Hunan Normal University, Changsha (410013), China
  5.The Key Laboratory of Study and Discover of Small Targeted Molecules of Hunan Province, Medical College, Hunan Normal University, Changsha (410013), China
- Author bio：
  
  Prof. REN Kai-qun, E-mail: kaiqunren@126.com
- Funds：
  
  the National Natural Science Foundation of China(82074075);Scientific Research Fund of Hunan Provincial Education Department(20C1340);the Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Hunan Normal University(2017TP1020);the Huxiang High-Level Talent Innovation Team(2018RS3072)
- DOI：10.1007/s11655-022-3469-z
  中图分类号：
- 纸质出版日期：2023-03，
  
  网络出版日期：2022-07-09，
  
  录用日期：2021-12-21
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白花蛇舌草总黄酮通过激活半胱氨酸蛋白酶依赖的线粒体凋亡途径抑制胃癌作用机制[J]. 中国结合医学杂志（英文版）, 2023,29(3):224-232.

WANG Xue-li, CAO Xiao-zheng, WANG Dao-yuan, et al. Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1[J]. Chinese Journal of Integrative Medicine, 2023,29(3):224-232.
白花蛇舌草总黄酮通过激活半胱氨酸蛋白酶依赖的线粒体凋亡途径抑制胃癌作用机制[J]. 中国结合医学杂志（英文版）, 2023,29(3):224-232. DOI： 10.1007/s11655-022-3469-z.

WANG Xue-li, CAO Xiao-zheng, WANG Dao-yuan, et al. Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1[J]. Chinese Journal of Integrative Medicine, 2023,29(3):224-232. DOI： 10.1007/s11655-022-3469-z.

摘要

目的:

本研究旨在评估白花蛇舌草总黄酮 (Flavonoids of Oldenlandia diffusa

FOD) 对人胃癌细胞凋亡的诱导作用及其对胃癌细胞周期的影响

探讨FOD与控制活性氧 (Reactive Oxygen Species

ROS) 产生的线粒体凋亡途径相关的作用机制

并探讨FOD对小鼠异种移植模型的影响

以期为临床胃癌防治提供实验依据.

方法:

酶法-超声分离提取及D-101树脂纯化FOD后

利用HPLC对其潜在的生物活性成分进行表征. 不同浓度FOD (64

128

160 μg/mL) 处理MKN-45、AGS及GES-1细胞

采用MTT评价药物对细胞活力的影响

Hoechst 33342染色技术用于观察细胞核形态变化

流式细胞术检测细胞凋亡率及细胞周期变化

激光共聚焦显微镜检测胃癌细胞内ROS的产生

荧光显微镜观察肿瘤细胞线粒体膜电位 (Mitochondrial Membrane Potential

MMP) 变化

免疫印迹检测细胞周期蛋白及线粒体途径相关凋亡蛋白表达. 建立MKN-45细胞小鼠异种移植模型

瘤组织经HE染色后确定造模成功

不同剂量FOD (50

100

200 mg/kg/d) 灌胃干预后

测量瘤体积及瘤重

免疫组化检测增殖标志物PCNA及Ki-67表达水平

ELISA检测肿瘤标志物CA72-4表达水平.

结果:

FOD对MKN-45及AGS增殖有明显抑制作用

使细胞周期阻滞在G1/S期

FOD诱导胃癌细胞凋亡过程中ROS积累

进而引起MMP改变

Cyclin A、Cdk2、Bcl-2、Pro-Caspase-9、Cytochrome C蛋白表达水平以剂量依赖性下降

Apaf-1、Cleaved-Caspase-3、Bax表达水平以剂量依赖性增加 (

0.05) . FOD抑制MKN-45荷瘤小鼠肿瘤生长

FOD各剂量组瘤组织中PCNA、Ki-67的表达水平与模型对照组相比降低

血清肿瘤标志物CA72–4表达水平较模型对照组降低 (

0.05) .

结论:

FOD通过激活线粒体控制的内在途径抑制人胃癌细胞的增殖并诱导其凋亡.

Abstract

Objective:

To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism.

Methods:

Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels

self-renewal capability (Nanog and Sox2)

and cancer stem cell markers (CD133 and CD44) were detected by a colorimetric DNMT activity/inhibition assay kit

quantitative real-time reverse transcription-polymerase chain reaction

sphere and colony formation assays

and immunoblot

respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA

respectively

were performed to explore the mechanism for action of CAS (0

and 100 nmol/L).

Results:

DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (

0.05). In addition

HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities

and lower CD133

CD44

Nanog and Sox2 protein expressions (

0.05). Conversely

overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore

CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (

0.05). Interestingly

DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected

DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells.

Conclusion:

CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation

suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.

关键词

中药白花蛇舌草黄酮癌症凋亡线粒体

Keywords

cervical cancercancer stem cellcasticinDNA methyltransferase 1therapeutic action

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