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中国癌症防治杂志

中国癌症防治杂志 ›› 2013, Vol. 5 ›› Issue (4): 296-299.doi: 10.3969/j.issn.1674-5671.2013.04.02

• 基础研究 • 上一篇    下一篇

DNA-PK/AKT/GSK3β通路与结肠癌细胞的放射抗拒性

  

  1. 广西医科大学附属肿瘤医院物理诊断中心；广西医科大学研究生学院
  • 出版日期:2013-12-25 发布日期:2013-12-31
  • 通讯作者: 葛连英 E-mail:gelianying@hotmail.com
  • 基金资助:

    广西自然科学基金资助项目（2011GXSFA018205）；广西医疗卫生重点科研课题（重2011086）

The DNA-PK/AKT/GSK3β pathway and acquired radioresistance in human colon cancer cells

  • Online:2013-12-25 Published:2013-12-31

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摘要： 目的 探索结肠癌细胞株SW480在长期分割放疗下获得放射抗拒性的可能机制。方法 经不同剂量0 Gy、2 Gy、5 Gy、10 Gy单次X射线照射后，以RT-PCR法检测结肠癌亲本细胞SW480和放射抗拒性细胞SW480-R中CCND1 mRNA的表达；Western blot法检测DNA-PK/AKT/GSK3β通路中两种细胞株cyclin D1、CDK4、Rb、p-Rb-Ser795、AKT、p-AKT-Ser473、GSK3β、p-GSK3β-Ser9、DNA-PKcs、p-DNA-PKcs蛋白的表达。结果 经单次X射线照射0 Gy、2 Gy、5 Gy、10 Gy后，RT-PCR法检测显示SW480-R细胞中CCND1 mRNA的表达明显低于SW480的表达（P＜0.05），其相对表达水平分别为0.31±0.02、0.32±0.03、0.34±0.05、0.44±0.04；Western blot法检测显示SW480和SW480-R中的cyclin D1、CDK4、Rb、p-Rb-Ser795、AKT、p-AKT-Ser473、p-GSK3β-Ser9、DNA-PKcs及p-DNA-PKcs蛋白的表达含量在两种细胞中显著不同，SW480-R的蛋白表达明显高于SW480（P＜0.05），而GSK3β蛋白的表达含量SW480-R却低于SW480（P＜0.05）。结论 经过长期分割放疗的结肠癌细胞获得的放射抗拒性可能与激活DNA-PK/AKT/GSK3β通路中cyclin D1蛋白过表达介导的DNA损伤反应（DDR）的改变有关。

关键词:  , 结肠肿瘤, cyclin D1, 分割放疗, 放射抗拒性, 实时荧光定量聚合酶链式反应vWestern blot法

Abstract: Objective  To investigate the molecular mechanism of radioresistance acquisition after long-term fractionated radiation （FR） in human colon cancer cell line SW480. Methods  Parental SW480 cells and radioresistant subline SW480-R cells were irradiated with single doses of 0，2，5 or 10 Gy.CCND1 mRNA levels were measured using real-time fluorescent quantitative polymerase chain reaction（PCR）.Western blotting was used to measure levels of proteins involved in the DNA-PK/AKT/GSK3β pathway of SW480 cells and SW480-R cells，including cyclin D1，CDK4，Rb，p-Rb-Sebr795，AKT，p-AKT-Ser473，GSK3β， p-GSK3β-Ser9，DNA-PKcs and p-DNA-PKcs. Results  CCND1 mRNA levels were significantly lower in the radioresistant subline SW480-R than in parental SW480 cells after single irradiation doses （P<0.05），with relative expression being 0.31±0.02 in the resistant subline compared to the parental line after 0 Gy，0.32±0.03 after 2 Gy，0.34±0.05 after 5 Gy and 0.44±0.04 after 10 Gy.Conversely levels of all proteins examined were higher in SW480-R cells than in SW480 cells（P<0.05） except the level of GSK3β， which was lower in SW480-R cells（P<0.05）. Conclusions  Acquired radioresistance in the human colon cancer cell line was correlated with alterations in the DNA damage response；these alterations were activated by long-term FR and mediated by cyclin D1 overexpression in the DNA-PK/AKT/GSK3β pathway.

Key words: Colon neoplasms, Cyclin D1, Fractionated-radiation；Radioresistance, Real-time fluorescent quantitative polymerase chain reaction, Western blot