CAJ | 학술논문

目的探讨雷公藤内酯醇(TPL)对前列腺癌DU145细胞株的作用及其机制.方法分别用0、5、25、50、100 nmol/L浓度的TPL作用于DU145细胞24、48 h.噻唑蓝(MTT)比色法观察TPL对DU145细胞的增殖抑制作用;流式细胞仪采用膜联蛋白V-异硫氰酸荧光素/碘化丙锭(Annexin V-FITC/PI)双染法检测细胞凋亡;PI染色后激光共聚焦显微镜观察凋亡细胞核的形态改变;Western blot检测组蛋白3赖氨酸27三甲基化(H3 K27me3)、果蝇zeste基因增强子同源物2(EZH2)蛋白的表达;实时荧光定量聚合酶链反应(FQ-PCR)检测EZH2基因的表达.结果 TPL对DU145细胞增殖有显著的抑制作用,24 h的半数抑制剂量(IC50)值为(68.71 ±0.15) nmol/L;48 h的IC50值为(36.87 ±5.80) nmol/L,与未加TPL的对照组比较,差异有统计学意义(P＜0.05).与正常对照组比较,随着药物浓度的增加(25、50、100 nmol/L),凋亡率也逐渐增加,早期凋亡率分别为(10.9±1.3)％、(22.4±1.3)％和(31.3±2.4)％,两两比较差异均有统计学意义(P＜0.05).PI染色显示经TPL处理的DU145细胞部分表现出细胞核的高度浓缩,为细胞凋亡的重要特征;用不同浓度的TPL处理细胞24 h后,Western blot检测DU145细胞H3 K27 me3及EZH2的蛋白表达量均降低,差异均有统计学意义(P＜0.05).FQ-PCR检测显示TPL处理组EZH2基因表达量显著降低,并且也表现出浓度-效应关系.结论 TPL可以抑制DU145生长增殖并诱导细胞凋亡,并且可能是通过组蛋白修饰这一表观遗传机制来实现的.
목적 탐토뢰공등내지순(TPL)대전렬선암DU145세포주적작용급기궤제.방법 분별용0、5、25、50、100 nmol/L농도적TPL작용우DU145세포24、48 h.새서람(MTT)비색법관찰TPL대DU145세포적증식억제작용;류식세포의채용막련단백V-이류청산형광소/전화병정(Annexin V-FITC/PI)쌍염법검측세포조망;PI염색후격광공취초현미경관찰조망세포핵적형태개변;Western blot검측조단백3뢰안산27삼갑기화(H3 K27me3)、과승zeste기인증강자동원물2(EZH2)단백적표체;실시형광정량취합매련반응(FQ-PCR)검측EZH2기인적표체.결과 TPL대DU145세포증식유현저적억제작용,24 h적반수억제제량(IC50)치위(68.71 ±0.15) nmol/L;48 h적IC50치위(36.87 ±5.80) nmol/L,여미가TPL적대조조비교,차이유통계학의의(P＜0.05).여정상대조조비교,수착약물농도적증가(25、50、100 nmol/L),조망솔야축점증가,조기조망솔분별위(10.9±1.3)％、(22.4±1.3)％화(31.3±2.4)％,량량비교차이균유통계학의의(P＜0.05).PI염색현시경TPL처리적DU145세포부분표현출세포핵적고도농축,위세포조망적중요특정;용불동농도적TPL처리세포24 h후,Western blot검측DU145세포H3 K27 me3급EZH2적단백표체량균강저,차이균유통계학의의(P＜0.05).FQ-PCR검측현시TPL처리조EZH2기인표체량현저강저,병차야표현출농도-효응관계.결론 TPL가이억제DU145생장증식병유도세포조망,병차가능시통과조단백수식저일표관유전궤제래실현적.
Objective To detect the curative effectiveness and its possible mechanisms of triptolide (TPL) on prostate cancer DU145 cells.Methods After DU145 cells were treated with 0,5,25,50,and 100 mmol/L TPL for 24 and 48 h,methyl thiazol tetrazolium (MTT) assay was used to observe the proliferation inhibition.Apoptosis was evaluated by Annexin V-fluoresceine isothiocyanate (FITC)/propidirum iodide (PI) double staining with flow cytometry and visualized using PI staining of apoptotic nuclei.Protein histone H3 containing the trimethylated lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2) expression levels were detected by Western blotting,and EZH2 gene expression by real-time quantitative polymerase chain reaction (FQ-PCR).Results Treatment with TPL for 24 h or 48 h inhibited proliferation of DU145 cells with 50％ inhibitory dose (IC50) being (68.71 ±0.15) and (36.87 ±5.80)nmol/L respectively,with the difference being statistically significant in comparison to the normal control group (P ＜ 0.05).As compared with the normal control group,with the increases of TPL concentrations (25,50 and 100 nmol/L),the apoptosis rate was gradually increased,and the early apoptosis rate was (10.9 ± 1.3) ％,(22.4 ± 1.3) ％ and (31.3 ± 2.4) ％,respectively.There was statistically significant difference between each two groups (P ＜ 0.05).PI staining results showed typical apoptotic characteristics.After DU145 cells were treated with TPL at different concentrations for 24 h,Western blotting showed the expression levels of trimethylated H3K27me3 and EZH2 were significantly reduced (P ＜ 0.05).Using FQ-PCR,we verified that EZH2 mRNA was decreased by TPL in a concentration-dependent manner (P ＜0.05).Conclusion TPL inhibited the proliferation of DU145 cells in a concentration-and time-dependent manner and induced epigenetic alterations probably by regulating histone methylation.