川芎嗪抑制mTOR信号通路介导的帽依赖性翻译而诱导HRPC细胞凋亡

Ligustrazine suppresses apoptosis in HRPC cells through inhibition of cap-dependent translation via mTOR pathway

目的明确川芎嗪(Ligustrazine,TMP)对激素抵抗性前列腺癌(hormone-refractory prostate cancer,HRPC)细胞PC-3的促凋亡效应并探讨其分子机制,评价TMP对正常前列腺上皮细胞RWPE-1的影响。方法分别用不同终浓度的TMP处理PC-3细胞和RWPE-1细胞48、72 h后,行MTT实验及Annexin V/PI染色法评价TMP对这两种细胞活性及凋亡的影响;Western blot检测TMP对PC-3细胞中mTOR信号通路活化情况及凋亡相关蛋白表达的影响;pull-down及荧光素酶报告基因实验研究TMP对PC-3细胞中翻译起始复合物形成及帽依赖性翻译的影响。结果 TMP对RWPE-1细胞的活性及存活无显著影响,但以剂量依赖及时间依赖的方式抑制PC-3细胞的活性,当TMP浓度≥25μmol/L时,差异具有统计学意义(P<0.05);与0μmol/L处理组相比,50、100μmol/L TMP处理24 h即可显著上调PC-3细胞中Bax/Bcl-2比值及Caspase-3的表达,处理后48 h检测发现PC-3细胞发生明显的凋亡(P<0.01);与0μmol/L处理组相比,50、100μmol/L TMP显著抑制PC-3细胞中哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及mTOR下游关键蛋白4EBP1和p70S6K的磷酸化,影响翻译起始复合物eIF4F的形成,进而抑制相关蛋白质的帽依赖性翻译及合成。结论 TMP可通过抑制mTOR信号通路的活化来降低HRPC细胞中增殖及抗凋亡相关蛋白的帽依赖性翻译,进而促进其凋亡。

Objective To determine the pro-apoptotic effect of ligustrazine（ TMP） in human hormone-refractory prostate cancer（ HRPC） cell line PC-3,investigate its underlying mechanism,and evaluate its effect on normal prostate epithelial cell line RWPE-1. Methods Different concentrations of TMP were used to treat RWPE-1 or PC-3 cells for 48 and 72 h,and then the proliferation and apoptosis in the treated cells were measured by MTT assay and Annexin V / PI assay respectively. The expression levels and phosphorylation of several key proteins in the mammalian target of rapamycin（ mTOR） pathway were detected by Western blotting. The formation of eukaryotic translation initiation complex（ eIF4F） was analyzed by pulldown assay with 7-methyl-GTP Sepharose 4B. The activity of cap-dependent translation was analyzed by capdependent reporter gene assay. Results TMP barely affected the proliferation and survival of RWPE-1 cells,but affected the growth and survival of PC-3 cells in dose- and time-dependent manners,and there was statistical difference when the final concentration of TMP was higher than 25 μmol / L（ P〈0. 05）. Compared with the control cells（ 0 μmol / L）,TMP（ 50 or 100 μmol / L） treatment for 24 h resulted in a increase of ratio of Bax to Bcl-2 and obvious up-regulation of Caspase-3,and the treatment for 48 h induced obvious apoptosis in PC-3 cells（ P〈0. 01）. Meanwhile,50 or 100 μmol / L TMP also significantly inhibited the expression of mTOR and the phosphorylation of its related downstream targets,4EBP1 and p70S6 K. Furthermore,TMP reduced the formation of translation initiation complex eIF4E for cap-dependent translation through increasing the binding of 4EBP-1 with eIF4E,and thus resulted in the inhibition of cap-dependent translation and synthesis.Conclusion TMP reduces the cap-dependent translation of proliferative and anti-apoptotic proteins by inhibiting the activation of mTOR pathway,and thus affects the growth and survival of HRPC cells.