替莫唑胺通过自噬诱导焦亡抑制人脑胶质瘤细胞增殖作用的研究  

Inhibition effect of temozolomide on proliferation of human glioma cells by autophagy-induced pyroptosis

作　　者：吕世文 黄准 朱亚兰 洪春霞 Lyu Shiwen;Huang Zhun;Zhu Yalan;Hong Chunxia(Department of Pharmacy,Jinhua Hospital,Zhejiang University School of Medicine,Jinhua 321000,China;Department of Pharmacy,Jinhua Second Hospital,Jinhua 321016,China)

机构地区：[1]浙江大学医学院附属金华医院药学部,金华321000 [2]金华市第二医院药剂科,金华321016

出　　处：《中华内分泌外科杂志》2023年第5期624-629,共6页Chinese Journal of Endocrine Surgery

基　　金：金华市科技计划项目(2022-4-191)。

摘　　要：目的探讨替莫唑胺(Temozolomide,TMZ)对人脑胶质瘤细胞自噬的影响,及自噬诱导焦亡对人脑胶质瘤细胞的增殖抑制作用。方法用2~64μmol/L的TMZ处理体外培养的脑胶质瘤U251细胞,分为对照组、不同浓度TMZ处理组(16、32、64μmol/L)、TMZ+巴伐络霉素(Bafilomycin A1,BafA1)组。MTT实验检测细胞活力,MDC染色检测细胞中自噬泡,克隆形成实验检测细胞增殖能力,RT-qPCR检测细胞中焦亡相关mRNA表达水平、Western Blot法检测细胞中自噬相关蛋白,焦亡相关蛋白表达情况,并通过加入自噬抑制剂检测自噬与焦亡的关系。结果TMZ能诱导人脑胶质瘤细胞发生自噬,且对肿瘤细胞有明显的焦亡诱导作用,从而抑制肿瘤细胞的增殖;RT-qPCR结果表明,与正常组相比,替莫唑胺给药后caspase-1(2.109±0.183 vs 1.057±0.043)、GSDMD(2.495±0.091 vs 0.957±0.098)、IL-1β(2.709±0.193 vs 0.973±0.029)、IL-18(2.553±0.113 vs 1.095±0.108)和NLRP3(2.755±0.123 vs 1.072±0.045)的mRNA表达水平显著上升,并且呈现浓度相关性;Western blot结果表明Cleaved-caspase-1(2.595±0.123 vs 1.057±0.043)、Cleaved-N-terminal GSDMD(3.145±0.141 vs 0.957±0.098)、IL-1β(2.776±0.093 vs 1.095±0.108)、IL-18(2.659±0.108 vs 0.973±0.029)和NLRP3(2.715±0.113 vs 1.072±0.045)蛋白上调;加入自噬抑制剂后细胞焦亡发生率减少。结论TMZ能诱导人脑胶质瘤细胞产生自噬,进而产生细胞焦亡,从而发挥增殖抑制作用。Objective To investigate the effect of temozolomide on autophagy of human glioma cells,and the inhibition of autophagy induced pyrocytosis on the proliferation of human glioma cells.Methods 2-64μmol/L of temozolomide was used to treat glioma U251 cells cultured in vitro.MTT assay was used to detect cell viability,MDC staining was used to detect autophagic vesicles in cells,cloning assay was used to detect cell proliferation,RT qPCR was used to detect the expression level of pyroptosis related mRNA in cells,Western blot was used to detect the expression of autophagy related proteins and pyroptosis related proteins in cells,and the relationship between autophagy and pyroptosis was detected by adding autophagy inhibitors.Results Temozolamide could induce autophagy of human glioma cells,and significantly induce tumor cells to pyroptosis,thereby inhibiting the proliferation of tumor cells;RT qPCR results showed that caspase-1,GSDMD,IL-1 after temozolomide administration compared with the normal groupβ,the mRNA expression levels of IL-18 and NLRP3 increased significantly;Western blot results showed that Cleaved-caspase-1,Cleaved-N-terminalGSDMD,IL-1β、IL-18 and NLRP3 protein were up-regulated;The incidence of pyroptosis decreased after the addition of autophagy inhibitors.Conclusion Temozolamide can induce autophagy of human glioma cells,and then lead to pyroptosis,which plays an inhibitory role in proliferation.

关 键 词：替莫唑胺 脑胶质瘤 自噬 焦亡 

分 类 号：R739.4[医药卫生—肿瘤]