雷帕霉素对莱菔硫烷诱导人结肠癌细胞UGT1A同工酶及CYP3A4表达的调控

Regulations of rapamycin on gene expressions of UDP-glucuronosyltransferase 1A isforms and cytochrome P450 3A4 induced by sulforaphane in human colon cancer cells

目的以人结肠癌Caco-2细胞为模型,观察雷帕霉素(Rapa)对莱菔硫烷(SFN)诱导葡萄糖醛酸转移酶(UGT)1A1、UGT1A8、UGT1A10及细胞色素P450(CYP)3A4的调控,探讨Rapa对SFN化学预防效应的影响。方法实验分为对照组、10 nmol/L Rapa组、25μmol/L SFN组和25μmol/L SFN+10 nmol/L Rapa组。透射电镜观察细胞超微结构,Western blotting法测定微管相关蛋白1轻链3(LC3)和核因子E2 P45相关因子2(Nrf2)的表达,实时荧光定量PCR法测定UGT1A1、UGT1A8、UGT1A10及CYP3A4 mRNA的表达,免疫荧光法观察Nrf2的核内转位。结果透射电镜观察到10 nmol/L Rapa组、25μmol/L SFN组和25μmol/L SFN+10 nmol/L Rapa组细胞中自噬体及自噬溶酶体的形成;与对照组相比,25μmol/L SFN组、25μmol/L SFN+10 nmol/L Rapa组可诱导LC3-II蛋白及UGT1A1、UGT1A8、UGT1A10 mRNA的表达增加,诱导Nrf2蛋白的表达及核内转位增强,且25μmol/L SFN+10 nmol/L Rapa组的作用更显著;而10 nmol/L Rapa组、25μmol/L SFN组和25μmol/L SFN+10 nmol/L Rapa组中,CYP3A4 mRNA的表达均受到抑制。结论 Rapa可协同增强SFN对Caco-2细胞自噬效应的诱导;Rapa可能通过上调Nrf2信号通路,使SFN诱导UGT1A1、UGT1A8和UGT1A10表达增加,进而增强SFN的化学预防效应,同时未影响SFN对CYP3A4转录抑制效应。

Objective To observe the regulations of rapamycin （Rapa） on UDP-glucuronosyltransferase （UGT） 1A1, UGT1A8, UGT1A10 and cytochrome P450 （CYP） 3A4 induced by sulforaphane （SFN） in human colon cancer Caco-2 cells and to explore the influence of Rapa on chemopreventive effect induced by SFN. Methods Experiments were divided into control group, 10nmol/L Rapa group, 25μmol/L SFN group, and 25μmol/L SFN＋10nmol/L Rapa group. The ultrastructures of Caco-2 cells were observed by transmission electron microscope. Western blotting was used to detect the expression of microtubule-associated protein 1 light chain 3 （LC3） and NF-E2-related factor 2 （Nrf2） proteins. Quantitative real-time RT-PCR was employed to examine the mRNA expression of UGT1A1, UGT1A8, UGT1A10 and CYP3A4. Immunocytochemistry was used to observe the nuclear localization of Nrf2. Results Autophagysomes and autolysosomes could be found in the 10nmol/L Rapa, 25μmol/L SFN and 25μmol/L SFN＋10nmol/L Rapa groups by transmission electron microscope. In comparison to the control group, the proteins of LC3-II and Nrf2 and the mRNA of UGT1A1, UGT1A8 and UGT1A10 were increased significantly in the 25μmol/L SFN group and 25μmol/L SFN＋10nmol/L Rapa group, and 25μmol/L SFN＋10nmol/L Rapa group possessed the highest level. An intense nuclear labeling of Nrf2 could also be observed in SFN-treated cells, especially in 25μmol/L SFN＋10nmol/L Rapa group. CYP3A4 mRNA expression could be inhibited in the 10nmol/L Rapa, 25μmol/L SFN and 25μmol/L SFN＋10nmol/L Rapa groups. Conclusion Rapa can enhance SFN-induced autophagy, and improve SFN-induced mRNA expression of UGT1A1, UGT1A8 and UGT1A10 through the Nrf2 signaling pathway up-regulated by Rapa. There is no effect of Rapa on CYP3A4 mRNA down-regulated by SFN.