摘要
目的探讨阿司匹林(ASP)联合肿瘤坏死因子相关凋亡诱导因子(TRAIL)诱导剂TIC10对子宫颈癌Siha、HeLa细胞自噬及凋亡的影响。方法选择HPV16阳性的子宫颈鳞癌细胞系Siha和HPV18阳性的子宫颈腺癌细胞系HeLa作为研究对象。分别单独及联合使用ASP及TIC10作用2种细胞系。用四甲基偶氮唑蓝(MTT)比色法检测ASP及TIC10作用后细胞增殖抑制率,用细胞划痕实验检测细胞迁移率,用平板克隆集落形成实验检测细胞克隆形率,用Western Blot法检测活化型半胱天冬酶3(cleaved Caspase-3)、活化型半胱天冬酶8(cleaved Caspase-8)、自噬相关微管关联蛋白(LC3A/B)和自噬基因(Beclin1)的水平。结果 MTT比色法显示,ASP作用后对Siha、HeLa细胞均有明显的增殖抑制作用,且呈浓度依赖性(均P <0. 01),ASP联合TIC10作用于Siha、HeLa细胞48 h后,其细胞增殖抑制率均明显高于单独使用TIC10 (均P <0. 01)。细胞划痕实验显示,ASP和TIC10作用24 h后,实验组对Siha,HeLa细胞的迁移率[ASP:(19. 61±1. 17)%和(23. 75±0. 78)%,TIC10:(16. 89±1. 47)%和(20. 59±2. 01)%]均明显小于对照组[(41. 18±2. 01)%和40. 83±3. 77)%],差异均有统计学意义(均P <0. 01)。平板克隆形成实验显示,ASP和TIC10作用2周后,实验组对Siha,HeLa细胞的克隆形成率[ASP:(24. 93±2. 12)%和(26. 47±3. 30)%,TIC10:(17. 33±1. 50)%和(19. 13±4. 99)%]均明显小于对照组[(69. 60±3. 54)%和(68. 40±4. 20)%],差异均有统计学意义(均P <0. 01)。Western blot法检测显示,促凋亡蛋白cleaved Caspase-3、cleaved Caspase-8表达上调,细胞自噬相关蛋白LC3A/B、Beclin1表达显著上调(均P <0. 01)。结论 ASP可通过自噬增强TRAIL诱导剂TIC10对宫颈癌细胞的凋亡。
Objective To explore the effect of aspirin( ASP) combined with tumor necrosis factor-related apoptosis-inducing ligand( TRAIL)inducer TIC10 on apoptosis and autophagy of cervical cancer Siha and HeLa cells. Methods HPV16 positive squamous carcinoma Siha cells and HPV18 positive cervical adenocarcinoma HeLa cells were selected as research objects. Two cell lines were treated with ASP,TIC10 or two drugs combination. The effects of ASP and TIC10 on cell proliferation were detected by 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide( MTT) assay,cell migration ability was detected by cell scratch assay,cell clone formation ability was detected by plate clone formation assay,and the levels of cleaved Caspase-3/8,LC3A/B,Beclin1 were detected by Western Blot. Results The MTT showed that the proliferations of Siha and HeLa cells were inhibited by APS in a concentration dependent manner( all P < 0. 01). After treated with TIC10 combined with ASP for 48 h,the cell proliferation inhibition rates of Siha and HeLa cells were higher than thosetreated with TIC10 alone( all P < 0. 01). After treated with TIC10 or ASP for 24 h,the migration rates of Siha and HeLa cells in the experimental groups [ASP:( 19. 61 ± 1. 17) %,( 23. 75 ± 0. 78) %; TIC10:( 16. 89 ± 1. 47) %,( 20. 59 ± 2. 01) %]were lower than those of control group [( 41. 18 ± 2. 01) %,( 40. 83 ± 3. 77) % ],all P < 0. 01.After two weeks of treatmentof ASP and TIC10,the CFE( colony-forming efficiency) rates of Siha and HeLa cells in the experimental groups [ASP:( 24. 93 ± 2. 12) %,( 26. 47 ± 3. 30) %, TIC10:( 17. 33 ± 1. 50) %,( 19. 13 ± 4. 99) %]were lower than those of control group [( 69. 60 ± 3. 54) %,( 68. 40 ± 4. 20) % ],all P < 0. 01.Western blot assays showed that the expression of cleaved Caspase-3/8 and autophagy related protein LC3A/B,Beclin1 were higher than those of control group( all P < 0. 01). Conclusion ASP increases cervical cancer cells apoptosis with TRAIL inducer TIC10 through autophagy enhancement.
引文
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