Up-regulation of Keap-1 inhibits the nuclear import of Nrf-2 and decreases cisplatin resistance in ovarian cancer Skov3/DDP cells / 基础医学与临床

ABSTRACT

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 expression 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.