The Role Of Nrf2-ARE Signal Pathway In Drug Resistance

First part:We have established stable cell lines with Nrf2 down-regulated for researching the inhibitors of Nrf2-ARE signal pathway.Nrf2 down-regulated cell lines (A549-C27,H460-C9 and H460-C13)were obtained by screening cells transfected with shRNA-Nrf2 for Nrf2 expression through Western blotting and GSH detection.After exposed to Luteolin which is the inhibitor of Nrf2-ARE signal pathway,in A549-C27 cells,the decrease of GSH was not obvious,and Luteolin did not sensitized the Oxaliplatin,Doxorubicin and Bleomycin.But in the control cells,GSH reduced obviously and Luteolin enhanced the cytotoxicity of the anti-cancer drugs significantly. We found Nrf2 an important path for the Luteolin to sensitize tumor cells. Down-regulation of Nrf2 in H460-C9 and H460-C13 also sensitized tumor cells from the results of MTS assay.The stable cell lines with Nrf2 down-regulated can be used to study the role of Nrf2 in anti-cancer drugs resistance,and to research the inhibitors of Nrf2-ARE signal pathway.Second part:We have researched the role of Nrf2-ARE signal pathway in drug resistance in Caco2 cells.After the Caco2 cells were treated with Oxaliplatin,the levels of mRNA and proteins of AKR1C1,NQO1 and HO-1 increased according to the results of Luciferase assay,Real-time PCR and Western blotting,together with the significantly increased cellular GSH level and reduced ROS level.These results showed that, Oxaliplatin activated the Nrf2-ARE signal pathway.Then we carried out further studies on the mechanism of Oxaliplatin to the Nrf2-ARE signal pathway:Oxaliplatin promoted the transcription of Nrf2 through the results of ChIP(Chromatin immunoprecipitation,CHIP);in A549-C1 cells with over expression of Keapl,the mRNA level of HO-1 increased via Real-time PCR.Keapl was involved in the activation of Nrf2 by Oxaliplatin;Oxaliplatin induced the expression of the p-ERK in MAPK signal pathway via Western blotting.The inhibitor UO126 reduced the induction of Oxaliplatin via Dual-luciferase assay.This study builts the foundation of futher research of the relation about Nrf2-ARE signal pathway and cancer drug resistance,also provides a new direction of Oxaliplatin clinical treatment.