维生素E对前列腺癌微小染色体维持蛋白4的抑制作用及信号通路

The suppressive effect and signal pathway of vitamin E on the expression of minichromosome maintenance protein 4 in prostate cancer cells

目的探讨维生素E调节前列腺癌细胞DNA复制的新分子机制及信传导通路。方法利用基因转染、免疫印迹、实时定量聚合酶链反应(PCR)、免疫沉淀等方法研究不同浓度、不同时间琥珀酸维生素E(VES)对前列腺癌细胞内的微小染色体维持蛋白4(MCM4)的mRNA及蛋白表达水平的影响。结果VES可抑制前列腺癌MCM4 mRNA的表达,并存在时效与量效关系,20μmoL/L VES处理LNCaP细胞36 h,MCM4的mRNA表达水平较对照组下降24.5%(P<0.05),作用96 h下降达60.3%;随着VES浓度的增加,MCM4 mRNA的表达水平受抑制的程度逐步加深。Western blot表明VES可抑制MCM4蛋白的表达。过表达E2F1可拮抗VES对MCM4的抑制作用。VES抑制Rb磷酸化,增加Rb与E2F1的结合程度。BrdU摄入试验表明20μmol/L与40μmol/L VES处理LNCaP细胞24h后,癌细胞内DNA合成较对照组下降63.4%与71.6%(P<0.05);48 h后分别达74.8%与81.5%(P<0.05)。结论我们识别了维生素E抑制前列腺癌细胞DNA复制的一种新机制是下调MCM4的表达,E2F1-Rb蛋白参与这一信号传导通路。

Objective To investigate the mechanisms and signal pathways by which vitamin E succinate （VES） regulates the DNA replication in prostate cancer cells. Methods Gene transfection,immunoblotting, real-time quantitative polymerase chain reaction （ PCR）, immunoprecipitation were applied to investigate the effects of different concentrations and treating periods of VES on the mRNA and protein levels of minichromosome maintenance protein 4 （MCM4） in prostate cancer cells. Results VES inhibited the expression of MCM4 mRNA level in time- and dose-dependent manners. When LNCaP cells were treated with 20 μmol/L of VES for 36 h,the mRNA expression of MCM4 was inhibited by 24.5% as compared with that in the control group （P 〈 0.05 ）. VES inhibited 60.3% of the MCM4 mRNA expression at 96 h （P 〈 0.05）. Western blot analysis revealed that VES could inhibit the expression of MCM4 protein. The suppressive effect of VES on MCM4 could be reduced by ectopic-expression of E2F1. VES inhibited Rb-phosphorylation as well as increased the binding of Rb with E2F1. BrdU incorporation studies showed that 20 and 40 μmol/L VES inhibited DNA synthesis in the prostate cancer cells by 63.4% and 71.6% （ P 〈 0.05 ） at the time point of 24 h ,74.8% and 81.5 % at 48 h （ P 〈 0.05 ）, respectively. Conclusion We have identified a novel mechanism of Vitamin E inhibiting the DNA replication in prostate cancer cells as suppressing the expression of MCM4 ,and the proteins of E2F1 and Rb are involved in this signal pathway.