星形孢菌素诱导TR3由细胞核向线粒体转运

Translocation of Orphan Receptor TR3 from Nuclei to Mitochondria Induced by Staurosporine

背景和目的:星形孢菌素(staurosporine,STS)是一种非特异性蛋白激酶抑制剂,能够诱导多种类型细胞凋亡。孤生受体(orphanreceptor)TR3是一种立早基因(immediate-earlygene)产物,属于类固醇/甲状腺激素受体超家族成员。当细胞受到凋亡因子刺激时,TR3表达并从细胞核转运至线粒体,从而诱导细胞凋亡。本研究旨在探讨STS诱导人胃癌细胞BGC-823凋亡及其与TR3转运的相关性。方法:荧光显微术观察经STS处理3h和24h后BGC-823细胞凋亡的形态变化并测定细胞凋亡率;激光共聚焦显微术和Westernblot对STS处理后BGC-823细胞中TR3蛋白和细胞色素c蛋白进行定位;流式细胞术和激光共聚焦显微术检测细胞线粒体的膜电位。结果:STS处理BGC-823细胞3h和24h后,细胞凋亡率分别从对照组的4.0%和4.7%提高到15.7%和39.4%;STS处理24h后,细胞出现核膜消失、染色体聚集成发亮的小体等典型的细胞凋亡形态变化。BGC-823细胞中TR3蛋白定位于细胞核,STS处理3h后TR3蛋白由细胞核转运至线粒体上,导致线粒体膜电位下降,并伴随着细胞色素c释放至胞浆。结论:孤生受体TR3参与STS诱导的细胞凋亡事件。在STS诱导下,TR3从细胞核移位至线粒体膜,引起线粒体膜电位下降,细胞色素c的释放,进而诱导细胞凋亡。

BACKGROUND &OBJECTIVE: Staurosporine (STS), a non specific protein kinase inhibitor, can extensively induce cell apoptosis. Orphan receptor TR3, an immediate early response gene, belongs to the steroid/thyroid receptor superfamily. After stimulated by apoptosis inducing agents, TR3 is expressed rapidly and translocates from nuclei to mitochondria,which finally induces cell apoptosis. This study was to investigate the mechanism by which STS induces apoptosis of gastric cancer cell line BGC 823,and its correlation with translocation of TR3 in BGC 823 cells. METHODS:Fluorescent microscopy was used to observe apoptotic morphology of BGC 823 cells after treated with STS for 3 and 24 h and stained by DAPI, and determine cell apoptotic rate. Laser scan confocal microscopy and Western blot were performed to investigate translocation of orphan receptor TR3 and the release of cytochrome c. Flow cytometry and confocal microscopy were used to examine mitochondrial membrane potential.RESULTS: After treated for 3 h,and 24 h,apoptosis rates of BGC 823 cells in STS treatment groups were higher than those of cells in control groups (15.7%vs. 4.0%, and 39.4%vs. 4.7%). After BGC 823 cells exposed to STS for 3 h,TR3 translocated from nuclei to mitochondria, mitochondrial membrane potential was decreased, and cytochrome c released from mitochondria to the cytoplasm. CONCLUSION: TR3, in response to STS, translocates from nuclei to cytoplasm, where it targets to mitochondria to induce cytochrome c release, finally results in apoptosis of gastric cancer cells.