CAJ | 학술논문

目的研究渥曼青霉素(wortmannin,WM)对恶性胶质瘤的辐射增敏作用及机制.方法采用10 μmol/L WM预处理人胶质瘤细胞U251 2 h,并给予10 Gy X射线照射,检测细胞周期及凋亡的变化；通过Western blot和免疫荧光染色方法检测蛋白共剂失调-毛细血管扩张症突变基因(ATM)及其靶基因,以及DNA依赖蛋白激酶的催化亚单位(DNA-PKcs)的表达和功能的变化.结果 WM预处理联合X射线照射(WM+ IR)组,U251细胞的凋亡率由对照组和IR组的(5.3±0.66)％和(11.5±2.0)％增高到(21.8±2.4)％,各组间差异具有统计学意义(F=57.38,P＜0.05).在IR组和WM+ IR组中,伴随着凋亡率的增加,凋亡基因cleaved caspase-3的表达明显高于对照组(q=12.49、19.19,P＜0.05),且WM+ IR组较IR组增高更为明显(q =6.70,P＜0.05).与对照组比较,细胞周期检测IR组G2/M期细胞明显增多(q =9.67,P＜0.05),WM+ IR组进一步增加(q =21.25,P＜0.05),出现明显的G2/M期阻滞.同时,WM有效抑制了X射线诱导的ATM蛋白激酶的磷酸化及其下游基因p53和SMC1的活化,并且抑制射线诱导的DNA-PKcs表达,而增加了DSB标记物phospho-γ-H2A.X(Ser139)的表达.结论 WM通过下调ATM激酶和DNA-PK蛋白的活性来增强恶性胶质瘤细胞的辐射敏感性.
목적 연구악만청매소(wortmannin,WM)대악성효질류적복사증민작용급궤제.방법 채용10 μmol/L WM예처리인효질류세포U251 2 h,병급여10 Gy X사선조사,검측세포주기급조망적변화；통과Western blot화면역형광염색방법검측단백공제실조-모세혈관확장증돌변기인(ATM)급기파기인,이급DNA의뢰단백격매적최화아단위(DNA-PKcs)적표체화공능적변화.결과 WM예처리연합X사선조사(WM+ IR)조,U251세포적조망솔유대조조화IR조적(5.3±0.66)％화(11.5±2.0)％증고도(21.8±2.4)％,각조간차이구유통계학의의(F=57.38,P＜0.05).재IR조화WM+ IR조중,반수착조망솔적증가,조망기인cleaved caspase-3적표체명현고우대조조(q=12.49、19.19,P＜0.05),차WM+ IR조교IR조증고경위명현(q =6.70,P＜0.05).여대조조비교,세포주기검측IR조G2/M기세포명현증다(q =9.67,P＜0.05),WM+ IR조진일보증가(q =21.25,P＜0.05),출현명현적G2/M기조체.동시,WM유효억제료X사선유도적ATM단백격매적린산화급기하유기인p53화SMC1적활화,병차억제사선유도적DNA-PKcs표체,이증가료DSB표기물phospho-γ-H2A.X(Ser139)적표체.결론 WM통과하조ATM격매화DNA-PK단백적활성래증강악성효질류세포적복사민감성.
Objective To study the mechanism of wortmannin (WM) in enhancing radiosensitivity of malignant glioma cells.Methods Human glioma cells (U251) were pretreated with 10 μmol/L WM for 2 h and then irradiated with 10 Gy X-rays.The changes of cell cycle and apoptosis ratio were examined.Western Blot and immunofluorescence staining were used to detect the expression and function of ATM and its target genes as well as DNA-PKcs.Results The ratio of apoptotic cells in the WM combined with X-ray treatment group (WM + IR) was increased from (5.3 ± 0.66) ％ of control group and (11.5 ± 2.0) ％ of IR group to (21.8 ± 2.4) ％ significantly (F =57.38,P ＜ 0.05).Along with the increase of apoptosis,the expression of cleave-caspase-3 was significantly increased in IR or WM + IR group compared to control group (q =12.49,19.19,P ＜ 0.05),and its enhancement in WM + IR group was more significant than that in IR group (q =6.70,P ＜ 0.05).Compared to control group,the cell cycle assay showed that the amount of U251 cells in G2/M phase was increased in IR group (q =9.67,P ＜ 0.05),and further increased in WM + IR group (q =21.25,P ＜ 0.05),indicating an obvious G2/M phase arrest.Moreover,WM could effectively inhibit the phosphorylation of ATM kinase and the activity of ATM downstream target genes of p53 and SMC1.WM also suppressed the IR-induced expressions of DNAPKcs and phospho-γH2AX (Ser139).Conclusions WM effectively increased the radiation sensitivity of malignant glioma cells through down-regulating the activity of ATM kinase and DNA-PK.