CAJ | 학술논문

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、western blot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 mm3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-1 mRNA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.
목적탐토조단백거을선화매(histone deacetylase,HDAC)억제제곡고억매소A (trichostatin A, TSA)통과HIF-2α도경항종류작용궤제.방법체외모의저양배경배양골육류MG-63세포,RT-PCR、western blot검측불동농도적TSA대골육류MG-63세포HIF-2α、VEGF급Glut-1 mRNA、단백표체적영향.사용단백매체억제제MG132처리MG-63세포화VHL기인결함적786-0세포,western blot검측TSA대HIF-2α표체적영향.골육류MG-63세포주입라서피하,대종류체적장지20 mm3시,분위TSA조(N=6)화생리염수조(N=6),매3천급약1차,17천후경하관찰종류조직형태;라서처사후종류조직제취단백,western blot검측HIF-2α표체정황.결과체외저양상태하,재배양적MG-63세포계중,TSA정농도의뢰성억제HIF-2α단백급파기인VEGF화Glut-1 mRNA적표체;재단백매체억제제MG132존재적정황하,가이역전TSA대HIF-2α적억제작용.재VHL기인결함적786-0세포계중,TSA정농도의뢰성억제HIF-2α단백표체,MG132가이역전TSA적작용.재동물이식류중, TSA억제HIF-2α적표체.결론실험표명,TSA통과일개VHL비의뢰성적단백매체의뢰적도경억제HIF-2α표체,TSA적항종류작용유일부분시통과억제HIF-2α개도적,저위연구조단백거을선화매억제제항종류작용적분자궤제제공료일개신적방향.
Objective To investigate the antitumor mechanism of histone deacetylase (HDAC) inhibitor trichostatin A (TSA) via hypoxia-inducible factor-2α (HIF-2α). Methods Osteosarcoma MG-63 cells were cultured under the hypoxia condition in vitro, and then treated with TSA. The effects of TSA at different concentrations on the expression of HIF-2α, vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and glucose transporter-1 (GLUT-1) mRNA were tested by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. MG-63 cells and von Hippel-Lindau (VHL) deficient 786-0 cells were treated by the proteasome inhibitor MG132, and the effects of TSA on the expression of HIF-2α were tested by western blotting. Osteosarcoma MG-63 cells were injected subcutaneously into nude mice. When tumors were about 20 mm3 in size, the mice were randomly divided into 2 groups: TSA group (N=6) and normal saline group (N=6). The mice were dosed each 3 days. The tumor tissue morphology was observed under the microscope 17 days later. After the mice were sacrificed, the protein was extracted from the tumor tissues, and the expression of HIF-2α was tested by western blotting. Results In cultured MG-63 cell lines, TSA inhibited the expression of HIF-2α, VEGF mRNA and Glut-1 mRNA in a concentration-dependent manner under the hypoxia condition in vitro; The proteasome inhibitor MG132 could reverse the inhibitory effects of TSA on HIF-2α. In VHL deficient 786-0 cell lines, TSA could also inhibit the HIF-2α expression in a concentration-dependent manner, and MG132 could reverse the effects. In animal xenografts, TSA inhibited the expression of HIF-2α. Conclusions The experiments show that TSA inhibits the HIF-2α expression by a VHL-independent but proteasome-dependent pathway, which suggests that the anticancer effects of TSA are partially mediated by its inhibition of HIF-2α. It provides new insights into the molecular mechanism of the antitumor effects of HDAC inhibitors.