TAS102选择性抑制ATRX缺失胶质瘤细胞的实验研究

王龙; 蒋航; 陈图南; 李学刚; 黄苏娜; 穆宁; 冯华; 李飞; 张云东

doi:10.16016/j.1000-5404.201907113

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TAS102选择性抑制ATRX缺失胶质瘤细胞的实验研究

基础医学 | 更新时间：2026-01-09

- TAS102选择性抑制ATRX缺失胶质瘤细胞的实验研究
- TAS102 selectively inhibits proliferation of α-thalassemia mental retardation X-linked gene-deficient glioma cells
- 第三军医大学学报 2019年41卷第24期页码：2393-2400
- 作者机构：
  
  1.401120 重庆，重庆医科大学附属第三医院神经疾病中心
  2.400038 重庆，陆军军医大学(第三军医大学)第一附属医院神经外科
- 作者简介：
  
  [ 李飞，E-mail:feifly7887@126.com LI Fei, E-mail: feifly7887@126.com
  [ 张云东，E-mail:zhangyundong6688@163.com ZHANG Yundong, E-mail: zhangyundong6688@163.com
- 基金信息：
  
  国家自然科学基金面上项目(81672783)
- DOI：10.16016/j.1000-5404.201907113
  中图分类号： R730.264;R966;R979.1
- 收稿：2019-07-15，
  
  修回：2019-08-29，
  
  纸质出版：2019-12-30
- 稿件说明：
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王龙, 蒋航, 陈图南, 等. TAS102选择性抑制ATRX缺失胶质瘤细胞的实验研究[J]. 第三军医大学学报, 2019,41(24):2393-2400.

Long WANG, Hang JIANG, Tunan CHEN, et al. TAS102 selectively inhibits proliferation of α-thalassemia mental retardation X-linked gene-deficient glioma cells[J]. Journal of Third Military Medical University, 2019, 41(24): 2393-2400.
王龙, 蒋航, 陈图南, 等. TAS102选择性抑制ATRX缺失胶质瘤细胞的实验研究[J]. 第三军医大学学报, 2019,41(24):2393-2400. DOI： 10.16016/j.1000-5404.201907113.

Long WANG, Hang JIANG, Tunan CHEN, et al. TAS102 selectively inhibits proliferation of α-thalassemia mental retardation X-linked gene-deficient glioma cells[J]. Journal of Third Military Medical University, 2019, 41(24): 2393-2400. DOI： 10.16016/j.1000-5404.201907113.

摘要

目的

探讨TAS102对α地中海贫血伴智力低下综合征X连锁基因(α-thalassemia mental retardation X-linked

ATRX)缺失U87胶质瘤细胞体外增殖的影响及机制。

方法

选择ATRX野生型U87细胞和ATRX敲除的U87

ATRX-/-

胶质瘤细胞为研究对象，设立U87对照组、U87药物组、U87

ATRX-/-

对照组、U87

ATRX-/-

药物组，采用CCK-8法检测细胞存活率，流式细胞术检测细胞周期的变化，β-半乳糖苷酶染色检测细胞老化情况，采用免疫荧光和Western blot检测DNA损伤识别反应通路活化及相关蛋白的表达情况。

结果

CCK-8检测结果显示，经不同浓度的TAS102培养72 h，U87

ATRX-/-

药物组细胞存活率低于U87药物组，U87组细胞IC

值为(10.50±1.52)mg/L，U87

ATRX-/-

细胞IC

为(4.72±0.48)mg/L，与U87组细胞相比，U87

ATRX-/-

组IC

更低(

0.01)。细胞周期检测示:U87对照组G

期细胞百分比为(17.86±2.12)%，药物组G

期细胞百分比为(23.04±3.57)%；U87

ATRX-/-

对照组G

期细胞百分比为(18.94±3.05)%，药物组为(61.67±1.13)%

与U87药物组相比，U87

ATRX-/-

药物组G

期细胞明显增多(

0.01)。β-半乳糖苷酶染色结果示:U87对照组染色阳性细胞百分比为(7.73±0.71)%，药物组为(29.55±4.12)%，U87

ATRX-/-

对照组为(8.70±2.55)%，药物组为(53.64±7.03)%，与U87药物组相比，U87

ATRX-/-

药物组染色阳性细胞增多(

0.01)。免疫荧光检测53BP1的表达显示，U87对照组染色阳性细胞百分比为(14.5±7.39)%，药物组为(21.19±3.68)%，U87

ATRX-/-

对照组为(16.75±2.97)%，药物组为(54.27±8.38)%，U87

ATRX-/-

药物组53BP1表达较U87药物组明显增多(

0.05)。Western blot检测不同时间点ATR、CHK1、ATM、CHK2的磷酸化水平，U87

ATRX-/-

药物组ATM/CHK2的磷酸化水平增高。

结论

TAS102能够选择性抑制U87

ATRX-/-

胶质瘤细胞增殖，通过诱导细胞衰老和活化ATM-CHK2损伤反应通路，引起细胞周期阻滞于G

期，抑制细胞增殖。

Abstract

Objective

To investigate the effect of TAS102 on the proliferation of α-thalassemia mental retardation X-linked (ATRX)-deficient U87 glioma cells

in vitro

and explore the mechanism.

Methods

Wild-type glioma U87 cells and ATRX knockout U87 cells (U87

ATRX-/-

) were both treated with TAS102

and the changes in the cell viability and cell cycle distribution were examined using CCK8 assay and flow cytometry

respectively. Beta-galactosidase staining was used to assess the cell senescence

and immunofluorescence staining and Western blotting were employed to detect the activation of DNA damage recognition reaction pathways and the expression of the related proteins.

Results

CCK-8 assay showed that TAS102 treatment for 72 h resulted in significantly lower cell viability in U87

ATRX-/-

cells than in wild-type U87 cells

and its IC

value was significantly lower in U87

ATRX-/-

cells than in U87 cells (4.72±0.48

10.50 ±1.52 mg/L

0.01). TAS102 treatment resulted in more cells arrested in G

phase in U87 cells [(23.04±3.57)%

(17.86±2.12)%

]

and in U87

ATRX-/-

cells [(61.67±1.13)%

(18.94±3.05)%

]

and the arrest was more obviously in the knockout cells than the wild-type ones(

0.01). Beta-galactosidase staining sho

wed that the positive percentage was (7.73±0.71)% and (29.55±4.12)% in U87 cells and TAS102 treated cells

and (8.70±2.55)% and (53.69±7.03)% in control and treated knockout cells. There were more positive cells in the knockout than the wild-type cells (

0.01). Immunofluorescence assay showed that TAS102 treatment increased the percentage of 53BPI-positive cells in both of the cells [control and treated U87 cells: (14.5±7.39)% and (21.19±3.68)%; control and treated U87

ATRK-/-

A cells: (16.75±2.97)% and (54.27±8.38)%

]

. but the increment was significantly greater in U87

ATRX-/-

cells than in U87 cells (

0.05). In the phosphorylation levels of ATR

CHK1

ATM and CHK2 at different time points following TAS102 treatment

significant increment was seen in ATM/CHK2 levels in U87

ATRX-/-

cells.

Conclusion

TAS102 can selectively inhibit the proliferation of U87

ATRX-/-

cells by inducing cell senescence

activating ATM-CHK2 damage response pathways

and causing cell cycle arrest in G

phase.

关键词

Keywords

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