新型雌激素受体GPER在乳腺癌MCF-7细胞他莫昔芬耐药中的作用及机制

Role of G protein-coupled estrogen receptor in tamoxifen-resistant breast cancer MCF-7 cells

目的研究乳腺癌他莫昔芬耐药细胞MCF-7R中新型雌激素受体(G protein-coupled estrogen receptor,GPER)对促凋亡蛋白Bim的影响及其机制。方法倒置显微镜下观察MCF-7和MCF-7R经过浓度为1μmol/L的他莫昔芬(tamoxifen,TAM)处理后细胞形态的变化;qRT-PCR检测人乳腺癌细胞MCF-7及其他莫昔芬耐药株MCF-7R中促凋亡蛋白Bim(Bcl-2L11)的mRNA表达水平,Western blot检测两种细胞株Bim蛋白表达水平和两种细胞中总的GPER和膜上GPER的表达水平;两种细胞中分别以(无水乙醇、TAM、GPER特异性抑制剂G15+TAM、GPER特异性激动剂G1)处理后,检测Bim的蛋白表达水平;MCF-7R细胞中分别抑制PI3K-Akt及MAPK/Erk两条信号通路后,再加入TAM,检测Bim的蛋白水平;流式细胞术检测细胞凋亡。结果倒置显微镜下发现MCF-7细胞经TAM处理后细胞大部分变透亮且悬浮,而MCF-7R无明显变化;MCF-7R细胞中促凋亡蛋白Bim的mRNA水平及蛋白水平较MCF-7都降低(P<0.05);耐药细胞中总GPER的蛋白表达量较MCF-7无明显变化而膜上的表达量升高(P<0.05);MCF-7经TAM处理后,Bim蛋白表达量增加(P<0.05),而MCF-7R中却无明显变化,但在MCF-7R中加入GPER特异性抑制剂G15后再用TAM处理,Bim蛋白水平增加(P<0.05);耐药细胞中PI3K/AKT及MAPK/Erk信号通路处于活化状态,抑制MAPK/Erk信号通路后再加入TAM,耐药细胞中促凋亡蛋白Bim的表达量增加(P<0.05);在MCF-7中加入TAM后细胞凋亡率明显升高(P<0.05),MCF-7R经TAM处理后无明显差异,但抑制GPER或Erk信号通路后,再加入TAM处理,细胞凋亡率明显增高(P<0.05)。结论乳腺癌TAM耐药细胞MCF-7R中TAM作为GPER的激动剂,活化信号通路MAPK/Erk抑制Bim表达。抑制GPER或MAPK/Erk信号通路,可使耐药细胞对TAM恢复敏感性。

Objective To investigate the effect of G protein-coupled estrogen receptor（ GPER） on pro-apoptotic protein Bim in tamoxifen（ TAM）-resistant breast cancer cells MCF-7R and related mechanism.Methods Human breast cancer cells MCF-7 and the MCF-7R cells were treated with 1 μmol / L TAM,and the cell morphological change was observed with an inverted microscope. The Bim mRNA expression was detected by quantitative real-time PCR（ qRT-PCR） in the MCF-7 cells and MCF-7R cells. The expression of Bim protein,total GPER,and GPER in the cell membrane was detected by Western blotting. After the two kinds of cells were treated with ethanol,TAM,TAM ＋ G15（ GPER specific inhibitor）,and G1（ GPER specific agonist）, the Bim protein expression was analyzed by Western blotting. After inhibiting the PI3 K / AKT and MAPK / Erk signaling pathways in the MCF-7R cells,TAM was added,and then the Bim protein expression was assayed. Flow cytometry was adopted to detect cell apoptosis. Results After treatment with TAM,most of MCF-7 cells became bright and were suspended,while the MCF-7R cells had no obvious changes. The levels of Bim mRNA and protein in the MCF-7R cells were obviously lower than those in the MCF-7 cells（ P 0. 05）. The total GPER had no significant difference between the 2 kinds of cells,but GPER in the cell membrane was increased in the MCF-7R cells（ P 0. 05）. The expression of Bim protein was increased in the MCF-7 cells treated with TAM（ P 0. 05）,but not in the MCF-7R cells. However,Bim protein was increased in the MCF-7R cells treated with G15 and then TAM（ P 0. 05）. The PI3 K / AKT and MAPK / Erk signaling pathways stayed in the activated stage in the MCF-7R cells. When TAM was added after inhibiting MAPK / Erk pathway rather than PI3 K / AKT pathway,the Bim protein was significantly increased（ P 0. 05）. TAM could promote the apoptosis of the MCF-7 cells,but not the MCF-7R cells. However,the apoptosis of MCF-7R cells was increased after GPER or MAPK / Erk signaling pathway was inhibited and then TAM was added（ P 0. 05）. Conclusion TAM as GPER agonist can activate the MAPK / Erk signaling pathway and inhibit the Bim expression in the MCF-7R cells. The MCF-7R cells are sensitive to TAM again through inhibiting GPER or MAPK / Erk signaling pathway.