CAJ | 학술논문

目的探讨Jaridonin诱导食管癌细胞凋亡的作用机制.方法采用单细胞凝胶电泳法检测DNA损伤.Western blot法检测p53蛋白的表达.谷胱甘肽(GSH)检测试剂盒检测细胞内还原性GSH水平.采用氧化还原敏感探针2’,7'-二氯荧光素二乙酸酯和二氢乙锭染色,荧光显微镜和流式细胞仪分别检测细胞内过氧化氢(H2O2)和超氧阴离子(O2.-)水平.结果 20、40 μmol/L Jaridonin 处理组的Olive尾矩值分别为45.2 ±8.1和89.0±14.2,与对照组(3.2±2.3)比较,差异均有统计学意义(均P＜0.01).Jaridonin处理EC-1细胞2h时,p53表达开始上调,且随作用时间延长,p53表达水平升高.siRNA干扰p53表达后,EC-1细胞对Jaridonin表现出明显的抗性,细胞凋亡率由转染前的38.5％下降为转染后的8.8％.Jaridonin作用EC-1细胞后,H2O2水平显著升高,O 2.-水平无明显变化.20 μmol/L Jaridonin作用EC-1细胞前后,GSH水平分别为(10.3 ±1.6)nmol/mg蛋白和(4.6±2.1)nmol/mg蛋白,差异有统计学意义(P＜0.01),随药物浓度的增加,GSH水平进一步下降.抗氧化剂GSH逆转了Jaridonin诱导的EC-1细胞中H2O2水平的升高、DNA损伤和细胞凋亡.Jaridonin对人正常肝细胞L-02的生长情况无明显影响.结论 Jaridonin通过消耗GSH的含量引起H2O2水平的升高而诱导DNA损伤,最终选择性诱导了食管癌细胞的凋亡.
목적 탐토Jaridonin유도식관암세포조망적작용궤제.방법 채용단세포응효전영법검측DNA손상.Western blot법검측p53단백적표체.곡광감태(GSH)검측시제합검측세포내환원성GSH수평.채용양화환원민감탐침2’,7'-이록형광소이을산지화이경을정염색,형광현미경화류식세포의분별검측세포내과양화경(H2O2)화초양음리자(O2.-)수평.결과 20、40 μmol/L Jaridonin 처리조적Olive미구치분별위45.2 ±8.1화89.0±14.2,여대조조(3.2±2.3)비교,차이균유통계학의의(균P＜0.01).Jaridonin처리EC-1세포2h시,p53표체개시상조,차수작용시간연장,p53표체수평승고.siRNA간우p53표체후,EC-1세포대Jaridonin표현출명현적항성,세포조망솔유전염전적38.5％하강위전염후적8.8％.Jaridonin작용EC-1세포후,H2O2수평현저승고,O 2.-수평무명현변화.20 μmol/L Jaridonin작용EC-1세포전후,GSH수평분별위(10.3 ±1.6)nmol/mg단백화(4.6±2.1)nmol/mg단백,차이유통계학의의(P＜0.01),수약물농도적증가,GSH수평진일보하강.항양화제GSH역전료Jaridonin유도적EC-1세포중H2O2수평적승고、DNA손상화세포조망.Jaridonin대인정상간세포L-02적생장정황무명현영향.결론 Jaridonin통과소모GSH적함량인기H2O2수평적승고이유도DNA손상,최종선택성유도료식관암세포적조망.
Objective The aim of this study was to explore the molecular mechanism of apoptosis in esophageal cancer cells induced by Isodon rubescens.Methods The DNA-damage effect of Jaridonin was detected by single cell gel electrophoresis (SCGE).The p53 protein was determined by Western blot.GSH assay kit was employed to determine the GSH content in human esophageal cancer EC-1 cells.Intracellular levels of hydrogen peroxide (H2O2) or superoxide (Oz-) were determined using the redox-sensitive probes 2',7'-dichlorodihydrofluorescein diacetate (DCF) or dihydroethidium (DHE),and the fluorescence signal was assayed by fluorescence microscopy and by flow cytometry.Results Jaridonin induced DNA damage in EC-1 cells remarkably.The olive tail moments (OTM) of control and 20,40 μmol/L Jaridonin were 3.2,45.2 and 89.0,respectively.Compared with the control,the differences were significant (P ＜ 0.01 for both).Jaridonin resulted in extensive p53 up-regulation in the EC-1 cells.More importantly,the p53 upregulation occurred as early as 2 h after Jaridonin incubation,and in a time-dependent manner (P ＜ 0.05).p53 siRNA transfection inhibited apoptosis in the EC-1 cells,and the Jaridonin-induced apoptosis rate was reduced from 38.5％ to 8.8％.Intracellular level of H2O2 was increased by Jaridonin,whereas the level of O2.-was barely changed.The GSH content in EC-1 cells was reduced from (10.3 ± 1.6) nmol/mg protein to (4.6 ±2.1) nmol/mg protein after 20 μmol/L Jaridonin incubation for 8 h,and it was further reduced with the increase of Jaridonin concentration.Jaridonin induced DNA damage,H2O2 accumulation and apoptosis were significantly attenuated in the presence of GSH,but Jaridonin showed little effect on normal human liver L-02 cells.Conclusions Jaridonin selectively induces apoptosis in esophageal cancer EC-1 cells through H2 O2-mediated DNA damage by depleting GSH.