Ye Xiao-Feng, Liu Su, Wu Qiao, Lin Xiao-Feng, Zhang Bing, Wu Jia-Fa, Zhang Ming-Qing, Su Wen-Jin
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China.
World J Gastroenterol. 2003 Sep;9(9):1915-9. doi: 10.3748/wjg.v9.i9.1915.
To investigate and determine the mechanism and signal pathway of tetradecanoylphorbol-1, 3-acetate (TPA) in degradation of RXRalpha.
Gastric cancer cell line, BGC-823 was used in the experiments. The expression level of RXRalpha protein was detected by Western blot. Nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation. Localization and translocation of RXRalpha were observed under laser-scanning confocal microscope through labeling specific anti-RXRalpha antibody and corresponding immunofluorescent antibody as secondary antibody. Different inhibitors were used as required.
In BGC-823 cells, RXRalpha was expressed in the nucleus. When cells were treated with TPA, expression of RXRalpha was repressed in a time-dependent and TPA-concentration-dependent manner. Meanwhile, translocation of RXRalpha from the nucleus to the cytoplasm occurred, also in a time-dependent manner. When cells were pre-incubated with proteasome inhibitor MG132 for 3 hrs, followed by TPA for another 12 hrs, TPA-induced RXRalpha degradation was inhibited. Further observation of RXRalpha translocation in the presence of MG132 showed that MG-132 could block TPA-induced RXRalpha redistribution. Conversely, when RXRalpha translocation was inhibited by LMB, an inhibitor for blocking protein export from the nucleus, TPA could not repress expression of RXRalpha.
TPA could induce the degradation of RXRalpha protein in BGC-823 cells, and this degradation is time- and TPA-concentration-dependent. Furthermore, the degradation of RXRalpha by TPA is via a proteasome pathway and associated with RXRalphatranslocation from the nucleus to the cytoplasm.
研究并确定十四酰佛波醇-1,3-乙酸酯（TPA）降解维甲酸X受体α（RXRα）的机制及信号通路。
实验采用胃癌细胞系BGC-823。通过蛋白质免疫印迹法检测RXRα蛋白的表达水平。经细胞裂解和离心制备细胞核和细胞质蛋白组分。以特异性抗RXRα抗体为一抗，相应的免疫荧光抗体为二抗进行标记，在激光扫描共聚焦显微镜下观察RXRα的定位和转位。根据需要使用不同的抑制剂。
在BGC-823细胞中，RXRα在细胞核中表达。当细胞用TPA处理时，RXRα的表达以时间和TPA浓度依赖性方式受到抑制。同时，RXRα也以时间依赖性方式从细胞核转位至细胞质。当细胞先用蛋白酶体抑制剂MG132预孵育3小时，再用TPA处理12小时时，TPA诱导的RXRα降解受到抑制。在存在MG132的情况下对RXRα转位的进一步观察表明，MG-132可阻断TPA诱导的RXRα重新分布。相反，当用LMB（一种阻断蛋白质从细胞核输出的抑制剂）抑制RXRα转位时，TPA不能抑制RXRα的表达。
TPA可诱导BGC-823细胞中RXRα蛋白的降解，且这种降解具有时间和TPA浓度依赖性。此外，TPA诱导的RXRα降解是通过蛋白酶体途径进行的，并且与RXRα从细胞核向细胞质的转位有关。
