Celecoxib对SHG-44细胞株的放射增敏作用

Radiosensitizing Effects of Celecoxib on SHG-44 Cell Lines

目的评价Celecoxib联合放射作用对SHG-44细胞周期时相分布的影响,探讨Celecoxib调控细胞周期可能的信号通路机制。方法体外培养胶质瘤SHG-44细胞,以不同浓度Celecoxib(0μmol/L、30μmol/L、50μmol/L、100μmol/L)设立药物组(D组),不同剂量(0Gy、2Gy、4Gy、6Gy、8Gy)6MV-X线照射设立放射组(R组),二者交互设立药物+放射组(D+R组),流式细胞术(FCM)检测细胞周期时相分布,逆转录PCR及实时PCR检测Cy-clinB1mRNA表达及水平。结果 FCM周期分析显示,细胞周期各时相分布在D组、R组及D+R组中均有差异。其中D+R组50μmol/LCelecoxib时,各照射剂量下与R组比较,G2/M期阻滞均进一步增强(P<0.05),但30μmol/L时各照射剂量下的G2/M期阻滞较R组均无明显增加(P>0.05)。逆转录PCR显示各实验组Cyclin B1均表达;实时定量PCR进一步检测发现,D组30、50、100μmol/L时Cyclin B1表达均较空白对照(0μmol/L)明显降低(P<0.05),其中50μmol/L较0和30μmol/L下降明显(P<0.05),但50μmol/L和100μmol/L之间差异无统计学意义(P>0.05);D+R组仅在100μmol/L时Cyclin B1表达较D组明显下降,其余浓度(0、30、50μmol/L)D+R组较D组无明显下降(P>0.05)。结论 Celecoxib在一定浓度下使SHG-44细胞发生G2/M期阻滞,并可进一步增强放射作用后的G2/M期阻滞,其放射增敏效应可能与下调细胞周期信号传导通路中的下游靶基因Cyclin B1有关。

Objective To evaluate the COX-2 inhibitor Celecoxib ’s radiosensitizing effects on SHG-44 cell lines in vitro,to characterize the effects on cell cycle redistribution when radiation combined with Celecoxib,and to probe the underlying signal pathway mechanism of Celecoxib ’s regulation of cell cycle . Methods Human glioma cell SHG-44 was managed in vitro. A group of drug（ Group D） was divided according to the different concentration of Celecoxib（ 0 μmol/L,30 μmol/L,50 μmol/L,100 μmol/ L） ,and the groups of radiation（ Group R） was divided according to the different dose of 6MV X-ray（ 0 Gy,2 Gy,4 Gy,6 Gy,8Gy） . The other group was formed by cells treated with Celecoxib combined with radiation（ Group D ＋ R） . The cell cycle redistribution was analyzed in different groups by flow-cytometric analysis; the expression of Cyclin B1 mRNA in SHG-44 cells was detected by RT-PCR,and real time-PCR was used to determine the relative level of Cyclin B1 mRNA. Results Flow-cytometric analysis showed cell cycle redistribution varied in Group D,Group R and Group D ＋ R. There was more G2 /M phase arrest with 50 μmol/L Celecoxib than those with 0μmol/L and 30 μmol/L in both Group D and Group D ＋ R （ P 0. 05） ,while there was no significant difference between 30 μmol/L and 0 μmol/L（ P 0. 05） ; the expression of Cyclin B1 gene was proved by RT-PCR in all groups,and real-time PCR was performed to determine the level of cyclin B1 mRNA： compared with 0 μmol/L Celecoxib,the expression level of Cyclin B1 was downregulated with 30 μmol/L,50 μmol/L and 100 μmol/L Celecoxib in Group D（ P 0. 05） ,but there were no significant differences within the three different concentration （ P 0. 05） . Compared with Group D,the expression level of Cyclin B1 was downregulated significantly with the dose of 100 μmol/L Celecoxib in Group D ＋R,while with the other concentration of Celecoxib（ 0,30,50 μmol/L ） in Group D ＋ R,there were no significant difference（ P 0. 05） . Conclusion Celecoxib enhanced G2 /M arrest at a certain concentration,and could strengthen the radiation-induced G2 /M arrest in SHG-44 cells. One of the underlying mechanisms of radiosensitizing effects of Celecoxib maybe the downregulation of Cyclin B1 acted as a key downstream gene in the signal pathway for the G2 /M phase transition.