摘要
目的:探讨细胞焦亡在紫杉醇诱导的鼻咽癌(nasopharyngeal carcinoma,NPC)细胞死亡中的作用,及可能的分子作用机制。方法:采用紫杉醇处理NPC细胞HNE-2和5-8F,光学显微镜下观察紫杉醇对细胞形态学的影响,乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验检测对HNE-2和5-8F细胞的毒性作用,酶联免疫吸附实验(enzymelinked immunosorbent assay,ELISA)检测对HNE-2和5-8F细胞中白细胞介素-1β(interleukin-1β,IL-1β)和IL-18释放水平的影响,FCM法检测对HNE-2和5-8F细胞中焦亡效应分子Caspase-1活性和细胞膜膜孔形成水平的影响,同时采用蛋白质印迹法检测细胞焦亡关键效应分子Caspase-1、人GasderminD蛋白(GSDMD)-全长(fulllength,FL)和GSDMD-N端片段表达水平的变化,以观察紫杉醇是否可以诱导NPC细胞发生细胞焦亡。随后,予以Caspase-1特异性抑制剂Z-YVAD-FMK和细胞焦亡抑制剂甘氨酸预处理HNE-2和5-8F细胞,再用LDH释放实验、ELISA法、FCM法以及蛋白质印迹法检测对紫杉醇诱导的HNE-2和5-8F细胞发生焦亡的影响。结果:紫杉醇可以诱导HNE-2和5-8F细胞发生细胞焦亡形态学改变,紫杉醇处理HNE-2和5-8F细胞后LDH释放量明显增加(P均<0.01),IL-1β和IL-18的释放量均明显增加(P均<0.05);FLICA660-YVADFMK和SYTOX1Green双阳性细胞所占的比例较对照组明显增加(P均<0.001),提示紫杉醇诱导了细胞焦亡;蛋白质印迹法结果进一步提示,紫杉醇处理的HNE-2和5-8F细胞中,经典焦亡中的关键效应分子的Caspase-1、GSDMD-FL蛋白和GSDMD-N端片段和IL-1β蛋白表达明显上调(P均<0.001)。Caspase-1特异性抑制剂Z-YVAD-FMK和细胞焦亡抑制剂甘氨酸都可以阻断上述紫杉醇诱导的经典细胞焦亡途径(P均<0.05)。结论:紫杉醇可以诱导NPC细胞通过经典途径发生细胞焦亡。
Objective:To investigate the role and the underlying molecular mechanisms of pyroptosis in taxol-induced cell death in nasopharyngeal carcinoma(NPC).Methods:The effect of taxol on cell morphology was observed under light microscope,the killing effect of taxol on HNE-2 and 5-8F cells was assessed by lactate dehydrogenase(LDH)release assay,the enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of secreted interleukin-1β(IL-1β)and IL-18 in HNE-2 and 5-8F cells,and the Caspase-1activity and cell membrane pore formation in HNE-2 and 5-8F cells were analyzed by FCM method.The expression levels of Caspase-1,human Gasdermin D protein(GSDMD)-full length(FL)and GSDMD-N-terminal fragment were detected by Western blotting to examine whether taxol could induce pyroptosis in NPC cells.Subsequently,HNE-2 and 5-8F cells were pretreated with the Caspase-1-specific inhibitor Z-YVAD-FMK and the pyroptosis inhibitor glycine,and then the effect on taxol-induced pyroptosis in HNE-2 and 5-8F cells was examined by LDH release assay,ELISA,FCM method and Western blotting.Results:Taxol could induce pyroptotic morphological changes in HNE-2 and 5-8F cells.The level of released of LDH was significantly increased in taxol-treated HNE-2 and 5-8F cells(both P<0.01),and the level of released of IL-1βand IL-18 were significantly increased(both P<0.05).the proportion of FLICA 660-YVAD-FMK and SYTOX1 Green double-positive cells was significantly increased compared with the control group(both P<0.001),suggesting that taxol induced pyroptotic.The expression levels of Caspase-1,GSDMD-FL protein,GSDMD-N-terminal fragment and IL-1βprotein,which are key effector molecules in classical pyroptosis,were significantly up-regulated in taxol-treated HNE-2 and 5-8F cells(both P<0.01).Both the Caspase-1 specific inhibitor Z-YVAD-FMK and the pyroptosis inhibitor glycine blocked the classical pyroptosis pathway induced by taxol as described above(all P<0.05).Conclusion:Taxol could induce Caspase-1/GSDMD-mediated canonical pyroptosis in NPC cells.
作者
刘霞静
张永全
宋业勋
刘陶文
LIU Xiajing;ZHANG Yongquan;SONG Yexun;LIU Taowen(Graduate School of Guilin Medical University,Guangxi Zhuang Autonomous Region,Guilin 541004,China;Department of Oncology,Nanxishan Hospital of Guangxi Zhuang Autonomous Region/Affiliated Nanxishan Hospital of Guilin Medical University,Guilin 541002,China;Department of Otorhinolaryngology Head and Neck Surgery,Third Xiangya Hospital,Central South University,Hunan Province,Changsha 410013,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2021年第7期475-486,共12页
Tumor
基金
国家自然科学基金(81702706)
湖南省自然科学基金(2020JJ5866)。