莪术醇逆转胶质瘤细胞对替莫唑胺的耐药:基于调节UTX/MGMT轴

Curcumol reverses temozolomide resistance in glioma cells by regulating the UTX/ MGMT axis

目的探讨莪术醇在逆转胶质瘤原发性耐药方面的作用及其潜在机制。方法将胶质瘤细胞分为以下主要分组:以0、10、20、40μg/mL浓度莪术醇处理胶质瘤细胞,选择40μg/mL莪术醇处理;通过慢病毒转染构建UTX过表达的胶质瘤细胞,空白对照组、莪术醇组(40μg/mL)、UTX过表达组(UTX过表达细胞)、UTX过表达和莪术醇联合处理组(40μg/mL莪术醇处理UTX过表达细胞);空白对照组、莪术醇组(40μg/mL)、莫唑胺(TMZ)组(10μg/mL)、莪术醇(40μg/mL)和TMZ(10μg/mL)联合处理组。将16只裸鼠随机分为空白对照组、莪术醇组(20 mg/kg)、TMZ组(20 mg/kg)、莪术醇(20 mg/kg)和TMZ(20 mg/kg)联合处理组,4只/组,构建BALB/c裸鼠移植瘤模型。MTT法、细胞克隆形成实验检测细胞的活力和克隆形成能力;流式细胞术检测细胞凋亡情况(TMZ预处理10μg/mL);UTX活性试剂盒检测细胞内的UTX活性;ChiP-qPCR检测MGMT启动子区域的UTX、H3K27me3的富集程度;Westernblot和免疫组化检测胶质瘤细胞内的蛋白表达情况。结果莪术醇能显著抑制胶质瘤细胞的增殖能力(P<0.05,P<0.01),并促进胶质瘤细胞的凋亡(P<0.01)。莪术醇能抑制体内胶质瘤瘤体的生长增殖(P<0.01),而TMZ则无显著效果(P>0.05)。莪术醇能显著抑制胶质瘤细胞内的UTX活性(P<0.01),增加H3K27me3的蛋白表达水平。UTX过表达细胞系的UTX活性显著升高(P<0.01),UTX蛋白表达增加且H3K27me3蛋白表达减少,UTX过表达能显著逆转莪术醇抑制胶质瘤细胞增殖并促进其凋亡的作用(P<0.01)。莪术醇能降低UTX和H3K27me3在MGMT启动子区域的富集程度(P<0.05,P<0.01)。莪术醇能降低MGMT蛋白表达水平,UTX过表达则能逆转该作用。在体内和体外实验中,莪术醇与TMZ联用均能提高胶质瘤细胞内的H3K27me3蛋白表达水平,降低下游靶基因MGMT的表达,进而增强胶质瘤细胞对TMZ的敏感性。结论莪术醇能够通过调控UTX/MGMT轴增强胶质瘤细胞对TMZ的敏感性。

Objective To explore the mechanism through which curcumol reverses primary drug resistance in glioma cells.Methods The inhibitory effect of 10,20,and 40μg/mL curcumol were observed in human glioma cell lines A172 and U251.UTX-overexpressing glioma cells constructed by lentiviral transfection were treated with curcumol(40μg/mL),temozolomide(TMZ;10μg/mL),or both,and the changes in cell viability,clone formation capacity and apoptosis were assessed using MTT assay,cell clone formation experiment,and flow cytometry;UTX activity in the cells was determined using a UTX detection kit,and the enrichment of UTX and H3K27me3 in the MGMT promoter region was detected with ChiP-qPCR.The protein expressions in glioma cells were detected using Western blotting and immunohistochemistry.In a nude mouse model bearing glioma xenografts,the effects of curcumol(20 mg/kg),TMZ(20 mg/kg)and their combination on tumor growth and expressions of UTX,H3K27me3 and MGMT were evaluated.Results Curcumol significantly inhibited the proliferation(P<0.05)and promoted apoptosis of cultured glioma cells(P<0.01).Curcumol,but not TMZ,produced significant inhibitory effect on tumor growth in the tumor-bearing mice(P<0.01).Curcumol significantly inhibited UTX activity and increased the expression level of H3K27me3 protein in the glioma cells.UTX overexpression obviously decreased H3K27me3 protein expression and reversed the effects of curcumol on glioma cell proliferation and apoptosis(P<0.01).Curcumol reduced the enrichment of UTX and H3K27me3 in the MGMT promoter region(P<0.05)and decreased MGMT protein expression,which was reversed by UTX overexpression.In both the in vivo and in vitro experiments,curcumol combined with TMZ significantly increased H3K27me3 protein expression in the glioma cells,reduced the expression of its downstream target gene MGMT,and enhanced TMZ sensitivity of the glioma cells.Conclusion Curcumol can enhance glioma cell sensitivity to TMZ by regulating the UTX/MGMT axis.