3MA抑制自噬降低头颈鳞癌细胞放疗抵抗性的实验研究

Study on 3MA reduce radioresistance in squamous cell carcinoma of the head and neck via autophagy inhibition

摘要:

目的探讨6-氨基-3-甲基嘌呤(3MA)对头颈鳞癌细胞放疗抵抗性的影响。方法蛋白印迹法（Western blotting）检测CNE2、610B及其放疗抵抗细胞CNE2Rs、610BRs中LC3B蛋白的表达；3MA作用于CNE2Rs及TU686细胞后LC3B蛋白的表达；克隆集落形成实验检测3MA对头颈鳞癌细胞的放疗抵抗能力及生存分数的改变；细胞免疫荧光法检测3MA对放疗致DNA双链损伤相关指标γH2AX焦点数量的改变。结果LC3B蛋白在放疗抵抗细胞较亲本细胞中表达增加；3MA能有效抑制自噬膜蛋白LC3B的表达；3MA抑制自噬后CNE2Rs及TU686细胞克隆集落形成能力减弱，生存分数降低；3MA抑制自噬后CNE2Rs及TU686细胞放疗组中γH2AX焦点数量较对照组显著增加。结论3MA抑制自噬后可能影响放疗后DNA双链损伤修复，进而降低头颈鳞癌细胞的放疗抵抗性。

关键词:自噬|头颈鳞癌|放疗抵抗|3MA|γ-H2AX

Abstract:

ObjectiveTo study the function of 3MA on the radioresistance in squamous cell carcinoma of the head and neck．MethodsWestern blotting was used to detect the LC3B protein in CNE2,610B and its radioresistance cell lines CNE2Rs,610BRs, in CNE2Rs and TU686 cells after 3MA treatment. Colony formation assay was applied to check the alteration of radioresistant capacity and survival fraction．Immunofluorescence was done to detect the expression of γH2AX foci，which was a surrogate marker of DNA doublestrand breaks．ResultsThe expression of LC3B protein was increased in radioresistant cell lines and inhibited in 3MA treatment groups.3MA weakened the colony formation ability and decreased the survival fraction.Also，3MA significantly increased the γH2AX foci formation compared with the control group after irradiation．ConclusionThe inhibition of autophagy by 3MA may affects the repair of DNA double strand damage after radiotherapy, thereby reducing the radioresistance of squamous cell carcinoma of head and neck.

Key words:Autophagy| Squamous Cell Carcinoma of Head and Neck|Radioresistance|3MA, γ-H2AX