ABSTRACT
<p><b>OBJECTIVE</b>To maked a Nrf2 down-regulated cell line by over-expressing Keap1 in H460 cells to study the role of Nrf2 in drug resistance.</p><p><b>METHODS</b>Transfecting H460 cells with mKeap1-pEGFP and screenig for Keap1 expressing clones by Western blotting with antibodies against Nrf2, HO-1, NQO1 and AKR1C. The cell line with Keap1 over-expression was further confirmed by real-time PCR. The cytotoxicity of H460-N5 to anti-cancer drugs was evaluated by MTS assay.</p><p><b>RESULT</b>MTS assay results showed the enhanced cytotoxicity of anticancer drugs (Oxaliplatin, Doxorubicin and Etopside) to the H460 cell line with keap1 overexpression compared to the control cell line. In H460-N0 cells, the IC(50) values of Oxaliplation and Etopside were 93 micromol/L and 100 micromol/L respectively whereas the IC(50) values of the two drugs were 42 micromol/L and 30 micromol/L correspondingly in H460-N5 cells. A Nrf2 down-regulated cell line H460-N5 and a control cell line with GFP over-expression have been identified.Down-regulation of Nrf2 enhanced the cytotoxicity of Oxaliplatin, Doxorubicin and Etopside. The IC(50) value of Doxorubicin to H460-N0 cell was above 3 mg/L, but that to H460-N5 cell was about 2 mg/L.</p><p><b>CONCLUSION</b>A Nrf2 down-regulated cell line H460-N5 and a control cell line with GFP over-expression have been identified. Down-regulation of Nrf2 enhanced the cytotoxicity of Oxaliplatin, Doxorubicin and Etopside.</p>
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Antioxidants , Metabolism , Pharmacology , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Cell Line, Tumor , Down-Regulation , Drug Resistance, Neoplasm , Genetics , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins , Genetics , Metabolism , Kelch-Like ECH-Associated Protein 1 , Lung Neoplasms , Metabolism , Pathology , NF-E2-Related Factor 2 , Genetics , Metabolism , Physiology , Response Elements , Physiology , Signal Transduction , Physiology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of tBHQ and sulforaphane on the protein expression in Nrf2-ARE signaling pathway of Caco2 cells.</p><p><b>METHODS</b>Human colorectal carcinoma Caco2 cells were treated with 20 micromol/L tBHQ and 5 micromol/L sulforaphane (SFN) respectively. Real time PCR, Western blotting and immunoflourescence staining (IF) were performed to measure the target gene expression.</p><p><b>RESULTS</b>Nrf2, AKR1C1 and NQO1 protein expressions were increased time-dependently in Caco2 cells after treatment with tBHQ and SFN. Time-course experiments showed that tBHQ and SFN increased the accumulation of Nrf2, and concomitantly increased the protein levels of AKR1C1 and NQO1. Real-time PCR and Western blotting showed that tBHQ and SFN significantly increased the expression of Nrf2 at 8h after the treatment, and AKR1C1 and NQO1 at 16 h. Confocal microscopy technique showed that Nrf2 accumulated in the nucleus at 6-8 h after treatment with tBHQ. After 1 h treatment with tBHQ the nuclear Nrf2 maintained at elevated level for at least 4 h with tBHQ withdrawn.</p><p><b>CONCLUSION</b>tBHQ and SFN induced nuclear accumulation of Nrf2 and activated Nrf2-dependent regulation of ARE-mediated gene expression in Caco2 cells. In addition, the results provide experimental evidence for choosing the dose and frequency of the inducer in cancer chemoprevention study and in developing inhibitors of Nrf2-ARE signaling pathway.</p>
Subject(s)
Humans , Anticarcinogenic Agents , Pharmacology , Antioxidants , Metabolism , Pharmacology , Caco-2 Cells , Calcium-Transporting ATPases , Hydroquinones , Pharmacology , Isothiocyanates , NF-E2-Related Factor 2 , Genetics , Metabolism , Physiology , Oxidative Stress , Genetics , Physiology , Response Elements , Physiology , Signal Transduction , Thiocyanates , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.</p><p><b>METHODS</b>A549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.</p><p><b>RESULTS</b>The expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.</p><p><b>CONCLUSION</b>DRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.</p>