Curcumin Downregulated Expression Of Keap1-Nrf2Pathway On T24Bladder Carcinoma Cells

Objective: To observe the inhibitory effect of curcumin combined with cisplatin onT24bladder carcinoma cells and verify the down-regulating effect of curcumin on theactivated Keap1-Nrf2pathway.Methods: T24cells were cultured and treated with increasing concentrations ofcisplatin (0.001,0.01,0.1,1,10,30,90μg/ml) for24hours. The inhibitory effect ofcisplatin on T24cells were tested with MTT colorimetric assay. Nrf2and Keap1wereextracted from nuclear or cytoplasm from the druged（0.1,1,10,30,μg/ml） cells orcontrol, respectively. GSTP1and NQO1, Nrf2regulated phase Ⅱ enzymes, wereextreatd from cytoplasm only. The protein level of Nrf2, Keap1, GSTP1and NQO1were checked with Western blotting method. The activation function of cisplatin onKeap1-Nrf2pathway was investigated and the optimum activated dose wasdetermined. The down-regulating effect of curcumin on the activated Keap1-Nrf2pathway was observed after T24cells were treated with different concentrations ofcurcumin (5,10,20,40,80μ mol/l).Results:1. The activation effect of cisplatin on Keap1-Nrf2pathway in T24cell(1) Inhibition effect of cisplatin on T24cells was demonstrated dose-dependly.(2) After the treatment with different concentrations of cisplatin, Nuclear Nrf2increased but keap1decreased. Cytoplasmic Nrf2，GSTP1, NQO1elevated but Keap1increased.(3) The nuclear level of Nrf2and the Cytoplasmic level of GSTP1, NQO1were thehighest at30μg/ml of cisplatin. Thus, the dose of30μg/ml of cisplatin was selectedfor next curcumin-cisplatin combination experiment.2. The down-regulating effect of on T24cell Keap1-Nrf2pathway(1) The proliferation of T24cells was significantly inhibited by differentconcentrations of curcumin combined with cisplatin.(2) After T24cells were treated with different concentrations of curcumin, the level ofNrf2in nuclear was reduced significantly. The level of GSTP1, NQO1in cytoplasmwas inhibited as well. The level of Keap1in nuclear was increased but its level in thecytoplasm was decreased.Conclusion: The activated Keap1-Nrf2pathway in T24cells was down-regulated bycurcumin. The expression typical phase2enzymes mediated by curcumin weredecreased while the sensitivity of tumor cells against chemotherapeutic drugs isenhanced. The mechanism is probably that the levels of Keap1are increased bycurcumin to degrade Nrf2which activated by anticancer drugs.