NRF2 mediated redox stress in arsenic induced human keratinocytes malignant transformation
OBJECTIVE To explore the role of nuclear transcription factor E2-related factor 2(NRF2)-mediated reductive stress in arsenite induced malignant transformation in human keratinocytes.METHODS HaCaT cells and fluorescent labeled mitochondrial glutathione HaCaT cells(Mito-Grx1-roGFP2 HaCaT)were cultured to 35 passages in medium containing 0.0 and 1.0 μmol/L NaAsO2 to establish a model of malignant transformation of cells.Cellular and mitochondrial reduced glutathione/oxidized glutathione(GSH/GSSG)and reduced coenzyme Ⅱ/oxidized coenzyme Ⅱ(NADPH/NADP+)ratios were measured in HaCaT cells.Cell doubling time,cell migration ability,soft agar clone formation ability and GSH/GSSG at different times in the 0 passage,the early stage(1st,7th and 14th passages)and later stage(21st,28th and 35th passages)were measured in Mito-Grx1-roGFP2 HaCaT cells.NaAsO2 induced malignant transformation cells were transfected with NRF2 siRNA,and detected the expression level of NRF2 and the redox-related indexes and malignant transformation indexes.RESULTS Compared with the control group,the GSH/GSSG ratio in 1.0 μmol/L NaAsO2 treated HaCaT cells significantly decreased in the 1st and 7th generations,but significantly increased after the 21st generation,and the NADPH/NADP+ratio significantly increased in the 1st,14th,21st,28th and 35th generations;The levels of GSH/GSSG in mitochondria significantly increased from 1st to 35th generation,and the levels of NADPH/NADP+in mitochondria significantly increased at 1st,7th,21st,28th and 35th generation.After continuous treatment of Mito-Grx1-roGFP2 HaCaT cells with 0.0 or 1.0μmol/L NaAsO2 to 35 passages,the doubling time of cells treated with 1.0 pmol/L NaAsO2 was significantly shortened,the cell migration rate was increased greatly,and more clones with larger volumes than the control cells formed.The GSH/GSSG ratio in mitochondria of Mito-Grx1-roGFP2 HaCaT cells showed a significant decrease in the 1st generation and increased from the 7th generation onwards(all P＜0.05).After transfection of NaAsO2 treated cells with NRF2 siRNA,the levels of hydrogen peroxide and superoxide increased compared with the siRNA controls.The levels of cell and mitochondrial NADPH/NADP+and GSH/GSSG decreased and the level of mitochondrial GSH/GSSG in Mito-Grx1-roGFP2 HaCaT cells decreased.Cell doubling time increased,cell migration rate and soft agar clone formation ability decreased(all P＜0.05).The malignant phenotype was reversed.CONCLUSION In the early stage(1st,7th and 14th passages)of NaAsO2 treated HaCaT cells,oxidative stress occurred with continuous high NRF2 expression.Later(21st,28th and 35th passages),NRF2 induced reductive stress,leading to malignant transformation.
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