上调Keap-1抑制Nrf-2核转运和降低卵巢癌细胞Skov3/DPP对顺铂耐药  

Up-regulation of Keap-1 inhibits the nuclear import of Nrf-2and decreases cisplatin resistance in ovarian cancer Skov3/DDP cells

作　　者：邓晶荣[1] 邵林[1] 

机构地区：[1]重庆三峡医药高等专科学校医学技术系,重庆404120

出　　处：《基础医学与临床》2017年第7期994-999,共6页Basic and Clinical Medicine

基　　金：重庆三峡医药高等专科学校重大项目(2016zdxm023)

摘　　要：目的分析Keap-1/Nrf-2通路在耐顺铂(cis-pt)的卵巢癌细胞Skov3/DDP中的活化状态,及其对细胞耐药的影响。方法构建p Flag-CMV-Keap-1过表达载体。细胞分4组:空白、cis-pt、Keap-1过表达及cis-pt联合Keap-1过表达组;Western blot检测Keap-1和Nrf-2蛋白表达及亚细胞定位;流式细胞计量术分析细胞的凋亡;DHE探针检测胞内活性氧簇(ROS)积累效应。结果相比Skov3,Skov3/DDP细胞中Keap-1水平明显下调(P<0.05),Nrf-2在细胞核中含量增多(P<0.05);过表达Keap-1后,Keap-1水平明显增加(P<0.05);细胞核中Nrf-2含量明显减少(P<0.05);20 mg/L顺铂能够明显诱导凋亡,24及48 h的凋亡率分别为24.36%和53.81%,显著高于未过表达Keap-1组(P<0.05),并激活caspase-3及PARP;DHE过表达Keap-1联合顺铂能够明显诱导ROS在细胞内累积。结论 Nrf-2能够在药物刺激时降低ROS保护细胞,Skov3/DDP细胞中Keap-1表达下调导致Nrf-2活化入核,可能与药物作用时ROS的毒性作用降低及细胞耐药有关。Objective To test the status of Keap- 1/Nrf- 2 pathway in cisplatin-resistant Skov3/DDP cells, and the contribution of these molecular in cisplatin （cis-pt） resistance. Methods Skov3/DDP was divided into four groups：blank, cis-pt, Keap-1 up-regulated and Keap-1 plus cis-pt. Keap-1 was up-regulated in Skov3/DDP cells by trans-faction with pFlag-CMV-Keap- 1 plasmid which was validated by expression of flag and increase of Keap- 1 level using Western blot. The effect of Keap-1 up-regulation on Nrf-2 re-distribution was analyzed by Western blot. FCM was employed to detect apoptosis. ROS level was measured by DHE staining. Results The expression of Keap-1 was decreased obviously in Skov3/DDP cells compared with Skov3 cells （P〈0. 05）, Nrf-2 in Skov3/DDP cells detected mainly in nucleus. Then, the Keap-1 was successfully up-regulated （3.5 fold） by exogenous expres- sion of pFlag-CMV-Keap- 1 plasmid, as a result, nucleus portion of Nrf- 2 was significantly reduced （ 0. 2 fold）. Next, the FCM results showed significant increase of apoptosis in Keap- 1 up-regulated Skov3/DDP cells after cis-pt administration （P〈0.05）. Finally, ROS level was significantly raised in Skov3/DDP cells treated with cis-pt plus Keap- 1 up-regulation. Conclusions Up-regulation of Keap- 1 reduced the nucleus portion of Nrf-2 and reverses cisplatin resistance in Skov3/DDP cells.

关 键 词：卵巢癌 顺铂耐药 Keap-1 Nrf-2 ROS 

分 类 号：R737.31[医药卫生—肿瘤]