内质网应激介导的凋亡耐受在甲氨蝶呤耐药绒癌发生机制中的作用

The effect mechanism of the tolerance of ER stress-induced apoptosis in methotrexate resistant human choriocarcinoma cells

目的:初步探讨内质网应激介导的凋亡耐受在甲氨蝶呤耐药绒癌发生机制中的作用。方法:以人绒毛膜上皮癌JEG-3细胞及本课题组前期构建的甲氨蝶呤耐药绒癌细胞(JEG-3/MTXR)为研究对象。采用WST法评估甲氨蝶呤处理后JEG-3/MTXR和JEG-3亲本细胞的细胞活力,Western blot法测定两种细胞中caspase-9激活、GRP78和GADD153蛋白的表达。RNA干扰法沉默JEG/MTXR细胞中GADD153基因,Western blot法测定caspase-9蛋白激活的情况。结果:不同浓度梯度的甲氨蝶呤诱导后,JEG-3亲本细胞系的细胞生存率显著降低。相同浓度甲氨蝶呤作用后,JEG-3/MTXR细胞内切割caspase-9无增加,JEG-3亲本细胞内caspase-9激活并切割。JEG-3/MTXR和JEG-3细胞中GRP78表达水平呈时间依赖性上调;JEG-3/MTXR细胞中GADD153蛋白水平无显著上调,而JEG-3亲本细胞内出现显著上调。沉默GADD153基因可抑制MTX引起的切割caspase-9蛋白水平上调。结论:内质网应激介导的凋亡耐受可能参与甲氨蝶呤耐药绒癌发生机制。

Objective ：To explore the effect mechanism of the tolerance of ER stress-in- duced apoptosis in methotrexate resistant human choriocarcinoma JEG-3 cell lines. Methods： Human choriocarcinoma JEG-3 cell line, and methotrexate resistant choriocarcinoma JEG-3 （JEG/MTXR） cell line were used in our present study. Cell viability was evaluated with WST in both cells. Cleavage caspase 9, GRP78 and GADD153 proteins were evaluated with Western blot in both cells after exposure to methotrexate （0.02ng/ml） for Oh, 12h,24h,36h,48h and 60h. GADD153 was knockdown by siRNA in MTXR/JEG-3 cell lines, and cleaved caspase 9 was evaluated by Western blot. Results：The cell viability was significantly decreased in JEG-3 parental cells after the exposure of different concentration methotrexate. The cleavage of caspase 9 was increased in JEG-3 parental cells but not in JEG-3/MTXR cells. ER stress related protein GRP78 was also upregulated in JEG-3 parental cells. ER stress-induced apoptotic factor GADD153 was increased in parental cells. Further investigation demonstrated that GADD153- siRNA could inhibit the upregulation of cleaved caspase 9 after 0.02ng/ml methotrexate expo- sure for 48h. Conclusion：The tolerance of ER stress-induced apoptosis was involved in the de- velopment of methotrexate resistance in choriocarcinoma JEG-3 cells.