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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 investigate the pro-apoptotic effect of Ligustrazine (TMP) on PC-3 in hormone-refractory prostate cancer (HRPC) cells and to explore the molecular mechanism of TMP on the expression of RWPE-1 in normal prostate epithelial cells influences. Methods The PC-3 cells and RWPE-1 cells were treated with different concentrations of TMP for 48 and 72 h, respectively. MTT assay and Annexin V / PI staining were used to evaluate the effect of TMP on the apoptosis and apoptosis of these two cell lines. Western blot The effects of TMP on the activation of mTOR signaling pathway and the expression of apoptosis-related proteins in PC-3 cells were detected by pull-down and luciferase reporter assay. The effects of TMP on translation initiation complex formation and cap-dependent The impact of translation. Results TMP had no significant effect on the activity and survival of RWPE-1 cells, but inhibited the activity of PC-3 cells in a dose-dependent and time-dependent manner. When TMP concentration was 25μmol / L, the difference was statistically significant (P <0.05 ). Compared with 0μmol / L treatment group, Bcl-2 and Bax / Bcl-2 ratio and Caspase-3 expression in PC-3 cells were significantly increased after treatment with 50 and 100μmol / L TMP for 24 h, 3 cells significantly (P <0.01). Compared with 0μmol / L treatment group, 50,100μmol / L TMP significantly inhibited mammalian target of rapamycin (mTOR) in PC-3 cells ) And the phosphorylation of 4EBP1 and p70S6K, the downstream key proteins of mTOR, affect the formation of translation initiation complex eIF4F and further inhibit the cap-dependent translation and synthesis of related proteins. Conclusion TMP can reduce the cap-dependent translation of proliferation and anti-apoptosis related proteins in HRPC cells by inhibiting the activation of mTOR signaling pathway, thereby promoting its apoptosis.