羟基脲对人脑胶质瘤U251细胞放化疗增敏作用的研究

Study of sensitization effect of hydroxyurea on chemoradiotherapy of human glioma U251 cells

目的探讨羟基脲(HU)联合替莫唑胺(TMZ)加放疗(RT)对人脑胶质瘤U251细胞放化疗(CRT)敏感性的影响。方法体外培养U251细胞,采用CCK8实验检测不同浓度HU、TMZ及不同条件处理后细胞的增殖能力;流式细胞术检测细胞凋亡及细胞周期分布情况;Transwell小室、划痕实验评估细胞侵袭、迁移能力变化;Western blot实验检测凋亡蛋白表达情况;克隆形成实验检测克隆源细胞存活分数。结果HU浓度≤50μmol/L时不会显著影响U251细胞增殖(P>0.05);低剂量HU联合CRT组较CRT组可抑制细胞增殖(P<0.05)、侵袭(P<0.01)、迁移(12h时P<0.001,24h时P<0.01)能力,并促进细胞凋亡(P<0.01)。50μmol/L HU联合RT后可增加细胞放射敏感性;细胞周期S及G2期显著延长(均P<0.05);凋亡蛋白Caspase-3及Bax表达水平上升,抗凋亡蛋白Bcl-2水平下降(均P<0.001)。结论HU联合CRT较单纯CRT进一步抑制U251细胞增殖、侵袭及迁移能力,促进细胞凋亡及增加放射敏感性,且出现S期及G2期阻滞,从而增加了U251细胞的CRT敏感性。

Objective To investigate the effect of hydroxyurea(HU)combined with temozolomide(TMZ)and radiotherapy(RT)on the sensitivity of human glioma U251 cells to chemoradiotherapy(CRT).Methods Human glioma U251 cell line was cultured in vitro.CCK8 cell assay was used to detect the proliferation activity of U251 cells treated with different concentrations of HU/TMZ under different conditions.Flow cytometry was utilized to detect apoptosis rate and cell cycle distribution of U251 cells.Transwell chamber assay and scratch test were performed to evaluate the changes of cell invasion and migration.The expression levels of apoptosis proteins were determined by Western blot.Colony formation assay was adopted to detect the cell survival fraction.Results HU concentration at 50μmol/L and below did not affect the proliferation of human glioma U251 cells(P>0.05).Low-dose HU combined with CRT significantly inhibited cell proliferation(P<0.05),invasion(P<0.01)and migration(12h P<0.001,24h P<0.01),and promoted cell apoptosis(P<0.01)compared with the use of CRT alone.Application of 50μmol/L HU combined with RT increased the radiosensitivity of cells(SER=1.49),significantly prolonged the cell cycle of S phase and G2 phase(both P<0.05),considerably up-regulated the expression levels of the apoptosis-associated proteins of Caspase-3 and Bax and significantly down-regulated the expression level of anti-apoptosis protein of Bcl-2(all P<0.001).Conclusions Compared with CRT,HU combined with CRT can further inhibit the proliferation,invasion and migration of human glioma U251 cells,promote cell apoptosis,increase the radiosensitivity and prolong the cell cycle of S and G2 phases,thereby enhancing the sensitivity of human glioma U251 cells to CRT.