安罗替尼对胶质瘤放疗的增敏作用及其机制

Observation and mechanism exploration of the radiotherapy sensitization effect of antinib on glioma

目的观察安罗替尼对胶质瘤的放疗增敏作用并探讨其作用机制。方法(1)培养胶质瘤细胞人脑胶质母细胞瘤系(U87MG),对照组,置于6MV-X射线照射,安罗替尼组,将安罗替尼加入U87MG联合6MV-X射线照射,细胞克隆法绘制生长曲线,比较两组的放疗敏感性,蛋白质印迹法(Western blot)法检测小窝蛋白(Caveolin-1)蛋白及丝裂原活化蛋白激酶(MAPK)表达。(2)建立U87MG动物模型,随机数字表法分为4组,对照组,不处理;单放组,6MV-X射线照射,安罗替尼组,安罗替尼灌胃,联合组,安罗替尼灌胃联合6MV-X射线照射,记录瘤重,计算抑瘤率,检测凋亡表达,Caveolin-1蛋白表达。采用t检验和方差分析。结果细胞实验,对照组的细胞存活分数(sF2)、终斜率的倒数(Do)、准阈剂量(Dq)值分别为0.64、1.83 Gy、1.34 Gy;安罗替尼组的sF2、Do、Dq值为0.47、1.25 Gy、0.92 Gy;增敏比(SER)值为1.47,差异有统计学意义(t=2.066,P<0.05)。Western blot检测安罗替尼组Caveolin-1蛋白水平,丝裂原活化蛋白激酶(MAPK)磷酸化水平低于对照组。动物实验发现单放组、安罗替尼组及联合组小鼠肿瘤体积低于对照组,瘤重分别为(6391.89±114.06)、(3367.19±60.61)、(4763.02±71.65)、(2982.77±54.30)mg,差异有统计学意义(F=331.631,P<0.05)。流式细胞仪检测肿瘤细胞的凋亡,单放组、安罗替尼组及联合组比高于对照组细胞凋亡率,凋亡率分别为(8.92±0.53)%、(26.75±1.19)%、(24.43±0.79)%、(17.64±0.65)%,差异有统计学意义(t=6.873,P<0.05)。免疫组织化学染色法检测单放组、安罗替尼组及联合组小鼠肿瘤Caveolin-1表达率低于对照组。结论体内外实验发现安罗替尼对胶质瘤有放疗增敏作用,Caveolin-1表达下降调控MAPP磷酸化通路改变可能是其作用机制之一。

Objective Observation and mechanism of the radiosensitization of anlotinib on glioma.Methods(1)The glioma cell line U87MG was pretreated with anlotinib and irradiated by 6MV-X ray.The growth curve was drawn and the expression of Caveolin-1 protein and phosphorylation level of mitogen-activated protein kinase 1(MAPK)signaling pathway were detected by Western blotting.(2)anlotinib was fed by gavage on the in vivo glioma U87MG model,and the mice were irradiated under 6MV-X ray.Tumor weight was recorded,tumor inhibition rate was calculated,and apoptosis and Caveolin-1 protein expression were detected.Results Cell experiment:sF2,Do and Dq of the control group were 0.64,1.83 Gy and 1.34 Gy,respectively.The values of sF2,Do and Dq in the anlotinib group were 0.47,1.25 Gy and 0.92 Gy.SER value is 1.47.Caveolin-1 protein and MAPK phosphorylation level was decreased.In vivo experiments showed that the tumor volume the anlotinib group and the combined group was smaller than that in the control group,and the tumor weight was(6391.89±114.06),(3367.19±60.61),(4763.02±71.65)and(2982.77±54.30)mg,respectively.Flow cytometry was used to test the apoptosis of tumor cells.Compared with the control group,the apoptosis rates of the radiation group,the anlotinib group and the combined group were increased,and the apoptosis rates of each group were(8.92±0.53)%,(26.75±1.19)%,(24.43±0.79)%and(17.64±0.65)%,respectively.Caveolin-1 expression by immunohistochemical of tumor in mice of radiation group,anlotinib group and combination group was lower than that of control group.Conclusion In vitro and in vivo experiments showed that anlotinib had radiosensitization effect on glioma cells.The decrease of Caveolin-1 expression that modulates of MAPK pathway may be one of the underlying mechanisms of that radiosensitization.