The Inhibitory Effect Of Keap1 And Bexarotene On The Nrf2 Signalling Pathway In Non-small Cell Lung Cancer Cells

NF-E2-p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that protects cell from oxidative/electrophilic stresses by activating the expression of cytoprotective genes through the antioxidant response element (ARE). Kelch-like ECH-associated protein 1 (KEAP1), a substrate adaptor protein for a cul3-dependent E3 ubiquitin ligase complex, plays a dominant role in negative regulating NRF2 activity. Mutation of KEAP1 in non-small cell lung cancer cells (NSCLC) A549 and H460, leads to the loss of function of KEAP1, and constitutive activation of NRF2 and its downstream cytoprotective genes, which contributes to chemoresistance during therapy. NRF2 is regarded as a pharmacological target to overcome therapeutic resistance in lung and other types of cancers with constititive activation of NRF2.The effect of Keap1 on Nrf2 signal pathway was studied by stably expressing Keapl in A549 and H460. We successfully established Keapl expressing cell lines A549-mKeap1-pEGFP (HI) and H460-mKeapl-pEGFP (N5) via transfecting cells with mKeapl-pEGFP plasmid and screening. Control cell lines were established by transfecting cells with pEGFP plasmids and screening. The expression of Nrf2 and Nrf2-regulated genes discreased in H1 and N5. Compared to control cell line, A549-mKeap1-pEGFP (HI) is more sensitive to Oxaliplatin; the anti-proliferation of Oxaliplatin, Doxorubincin and Etopside increased significantly in H460-N5 cell line. Taken together, over-expression of Keapl in A549 and H460 sensitizes cells to anti-cancer drugs via inhibiting the expression of Nrf2 gene battery.The effect of Bexarotene, a Nrf2 inhibitors on the localization of Nrf2 was investigated. Unitilizing some specific Abs-coilin, SMN, SC35, nuclear pore complex (NPC), RNA polymerase II (Polâ…¡) and Y12-to mark the nuclear subcellular organelles. In A549 cells, Nrf2 colocalized with coilin and SMN. After the cells treated with Bexarotene for 6h, first, the expression of coilin increased together with co-localization of Nrf2 with coilin, which was similar to the effect of DRB treatment; second, the expression of SMN increase together with co-localization of Nrf2 with SMN, which was similar to that of a-amanitin treatment. As a result, Bexarotene regulates Nrf2 through subcellular organelles such as coilin and SMN.The interaction of Nrf2 with colin or SMN was further studied. The co-localization of Nrf2 and coilin was observed in A549 cells transfected with Red Coilin plasmid. The reaction of Nrf2 with SMN was also confirmed by co-immunoprecipitation. Nrf2 colocalized with colin or SMN in other cells. Therefore, the locolization of Nrf2 with coilin or SMN was proved and seems general. In colon cancer cell Caco2, the colocalization was also observed and increased after treatment with Bexarotene, but the result should be further validated.