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目的探讨组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂曲古抑霉素A(trichostatin A,TSA)通过HIF-2α途径抗肿瘤作用机制。方法体外模拟低氧环境培养骨肉瘤MG-63细胞,RT-PCR、westernblot检测不同浓度的TSA对骨肉瘤MG-63细胞HIF-2α、VEGF及Glut-1 mRNA、蛋白表达的影响。使用蛋白酶体抑制剂MG132处理MG-63细胞和VHL基因缺陷的786-0细胞,western blot检测TSA对HIF-2α表达的影响。骨肉瘤MG-63细胞注入裸鼠皮下,待肿瘤体积长至20 mm~3时,分为TSA组(N=6)和生理盐水组(N=6),每3天给药1次,17天后镜下观察肿瘤组织形态;裸鼠处死后肿瘤组织提取蛋白,western blot检测HIF-2α表达情况。结果体外低氧状态下,在培养的MG-63细胞系中,TSA呈浓度依赖性抑制HIF-2α蛋白及靶基因VEGF和Glut-1mRNA的表达;在蛋白酶体抑制剂MG132存在的情况下,可以逆转TSA对HIF-2α的抑制作用。在VHL基因缺陷的786-0细胞系中,TSA呈浓度依赖性抑制HIF-2α蛋白表达,MG132可以逆转TSA的作用。在动物移植瘤中,TSA抑制HIF-2α的表达。结论实验表明,TSA通过一个VHL非依赖性的蛋白酶体依赖的途径抑制HIF-2α表达,TSA的抗肿瘤作用有一部分是通过抑制HIF-2α介导的,这为研究组蛋白去乙酰化酶抑制剂抗肿瘤作用的分子机制提供了一个新的方向。
Objective To investigate the antitumor mechanism of histone deacetylase (HDAC) inhibitor trichostatin A (TSA) via the HIF-2α pathway. Methods The osteosarcoma MG-63 cells were cultured in hypoxia environment in vitro. The effects of different concentrations of TSA on the expression of HIF-2α, VEGF and Glut-1 mRNA and protein in osteosarcoma MG-63 cells were detected by RT-PCR and western blot. 786-0 cells deficient in MG-63 cells and VHL gene were treated with proteasome inhibitor MG132, and western blot was used to detect the effect of TSA on HIF-2α expression. Osteosarcoma MG-63 cells were subcutaneously injected into nude mice. Tumor volume was increased to 20 mm ~ 3 and divided into TSA group (N = 6) and saline group (N = 6) The morphology of the tumor was observed under the microscope after daylight; the tumor tissue was extracted after the death of the nude mouse, and the expression of HIF-2α was detected by western blot. Results In vitro hypoxia, TSA inhibited the expression of HIF-2αprotein and VEGF and Glut-1mRNA in a concentration-dependent manner in cultured MG-63cells. In the presence of the proteasome inhibitor MG132, Reverse TSA inhibition of HIF-2α. In the VHL gene-deficient 786-0 cell line, TSA inhibited HIF-2α protein expression in a concentration-dependent manner, and MG132 reversed the effect of TSA. In animal xenografts, TSA inhibits the expression of HIF-2α. Conclusions Experiments have shown that TSA inhibits HIF-2α expression through a VHL-independent proteasome-dependent pathway. The anti-tumor effect of TSA is partly mediated by inhibition of HIF-2α, which is an experimental study of histone deacetylase inhibition The molecular mechanism of antitumor agents provides a new direction.