Roles Of Nrf2-NQO1 Signaling Pathway In Promoting The Proliferation In The Process Of Arsenite-induced Malignant Transformation

Objective:Inorganic arsenic is confirmed human carcinogen;chronic arsenic exposure can cause skin cancer and lung cancer.However due to the arsenite-carcinogenic animal models have not been established successfully,the mechanism of arsenic toxicity is still unrevealed.Oxidative stress hypothesis and abnormal cell proliferation,apoptosis are hot hypotheses of arsenite-carcinogenic mechanism.A series of studies have shown that chronic exposure to inorganic arsenic can promote cell proliferation and cause malignant transformation.However,the exact mechanism that arsenic induced cell malignant transformation remains to be elucidated.The aim of present study was to elucidate dynamic changes of cell proliferation,cell cycle,cell cycle proteins and cyclin dependent kinase in different passages of arsenite-induced malignant transformation models about human bronchial epithelial cells?HBE?and human keratinocyte cells?HaCaT?,further to explore specific molecular mechanism that leads to cell proliferation and malignant transformation.This study has a certain theoretical significance and practical application value for exploring arsenite-carcinogenic mechanisms and providing new ideas for prevention and control of arsenic poisoning.Methods:1.Detection of cell proliferation related indicators in different passages of malignant transformed HBE and HaCaT cell models induced by NaAsO₂?HBE including 0,1,8,15,22,29,36,43 passages,HaCaT cells including 0,1,7,14,21,28,35 passages?:The cell proliferation was determined by MTT experiment;Using flow cytometry to detect the cell cycle;Using western blot to detect the protein level of cyclin D1,cyclin E,cyclin A and CDK2.2.Detection of Nrf2-NQO1 signaling pathways related indicators in different passages of malignant transformed HBE and HaCaT cell models induced by NaAsO₂?HBE including 0,1,8,15,22,29,36,43passages,HaCaT cells including 0,1,7,14,21,28,35 passages?:Using western blot to detect cell protein level of Nrf2,NQO1.After adding different doses of hydrogen peroxide,antioxidant capacity was determined by MTT test?the 43 passage of HBE cells,the 35 passage of HaCaT cells?.3.Detection of several indicators after Nrf2-siRNA treatment in malignant transformed HBE and HaCaT cells?the 43 passage of HBE cells,the 35 passage of HaCaT cells?:Using western blot to detect protein level of Nrf2,NQO1 protein,antioxidant capacity was determined by MTT test after adding different doses of hydrogen peroxide,using soft agar cloning to test clone forming ability,migration assay detected cell migration ability,using MTT experiment to test cell proliferation,using flow cytometry to detect the cell cycle;Using western blot to detect cell protein level of cyclin E and CDK2.4.Detection of several indicators after NQO1-siRNA treatment in malignant transformed HBE and HaCaT cells?the 43passage of HBE cells,the 35 passage of HaCaT cells?:Using western blot to detect protein level of Nrf2,NQO1 protein,antioxidant capacity was determined by MTT test after adding different doses of hydrogen peroxide,using soft agar cloning to test clone forming ability,migration assay detected cell migration ability,using MTT experiment to test cell proliferation,using flow cytometry to detect the cell cycle;Using western blot to detect cell protein level of cyclin E and CDK2.Results:Detection of proliferation-related indicators in different passages of malignant transformed HBE and HaCaT models induced by NaAsO₂,the results showed that:The relative proliferation rate of HBE cells after the 36 passage of arsenic rose in1.0?M NaAsO₂;and the proliferation rate of HaCaT cells rose after the 21 passage of arsenic.Besides,the cell proportion in the G1 phase reduce in the 22,43 passages of HBE cells,G2/M phase change in the 29,36 passages of HBE cells,the cell proportion of S phase increased significantly in the 8,29,36,43 passages after exposure to 1.0?M NaAsO₂.Compared with the passage control group,HaCaT cells in G1 phase decreased significantly and the cells in S phase increased.Cyclin E protein expression increased in HBE cells after the 8 passages of arsenic,and maintained high level after the 22passages.While it increased in HaCaT cells after the 7 passage and hold continuous rise after the 14 passages of arsenic;However,the protein expression of cyclin A and cyclin D1 in HBE and HaCaT cells have no significant change.CDK2 protein expression in HBE cells begun to show significant rise in the 8 passages of arsenic,and maintained the trend of rising after the 29 passages;and it begun increase significantly after the 14passages in HaCaT cells.2.Detection of Nrf2-NQO1 signaling pathways related indicators in different passages of malignant transformed HBE and HaCaT cell models induced by NaAsO₂,the results found that:Nrf2 protein expression in HBE cells begun to increase in the 8 passages,and maintained high level after the 29 passages;while its expression showed rising trend in the HaCaT cells after the 7 passages,and occurred obvious increase trend after the 21 passages of arsenic.NQO1 protein expression in HBE cells obviously increased after the 8 passage;while its expression in HaCaT cells rose in the 1 passage,and showed a trend of rising after the 21 passage.After H₂O₂pretreatment in the concentration of 0.4,0.6,0.8 mmol/L in 48h,malignant-transformed HBE cells increased in survival rate significantly;And the survival rate of malignant-transformed HaCaT cells increased obviously after the H₂O₂ pretreatment in the concentration of 0.6mmol/L.3.Detection of several indicators after Nrf2-siRNA treatment in malignant transformed HBE and HaCaT cells,the results showed that:Nrf2,NQO1 protein expression of transfected HBE and HaCaT cells which add Nrf2-siRNA treatment are reduced,compared by control cells;After the H₂O₂ pretreatment in the concentration of 0.4,0.6mmol/L in 48 h,the survival rate of transfected HBE cells was lower than the control cells;while the same things happened in HaCaT cells after H₂O₂pretreatment in the tendency of 0.2,0.4mmol/L;The migration rate,relative proliferation rate,anchoring independence of transfected HBE and HaCaT cells were lower than control group;At the same time,cells which add Nrf2-siRNA treatment in G0/G1 phase increased,and cells in S phase reduced,cyclin E and CDK2 protein expression were reduced in transfected cells.4.Detection of several indicators after Nrf2-siRNA treatment in malignant transformed HBE and HaCaT cells,the results found that:NQO1 protein expression of transfected HBE and HaCaT cells which add Nrf2-siRNA treatment are reduced,compared by control cells,while Nrf2 has no change;After the H₂O₂ pretreatment of 48h in the tendency of 0.4,0.6mmol/L,the survival rate of transfected HBE cells was lower than the control cells;while the same things happened in HaCaT cells after H₂O₂ pretreatment in the tendency of 0.2,0.4mmol/L;The migration rate,relative proliferation rate,anchoring independence of transfected HBE and HaCaT cells were lower than control group;At the same time,cells which add Nrf2-siRNA treatment in G0/G1 phase increased,and cells in S phase reduced,cyclin E and CDK2 protein expression were reduced in the transfected cells.Conclusions:1.Exposure of sodium arsenite induced HBE and HaCaT cells malignant transformation with increased cell proliferation,accelerated cell cycle progression from G0/G1 phase to S phase,rising expression of cyclin E-CDK2 complex.2.Exposure of sodium arsenite induced HBE and HaCaT cells malignant transformation with activation of Nrf2-NQO1 signaling pathway and enhancement of anti-oxidation ability.3.Inhibition of Nrf2 or NQO1 gene expression can reduce the expression of cyclin E-CDK2 complex,S phase cells decline,reduce the speed of cell proliferation,and eventually reduce the malignant degree.4.Nrf2-NQO1 signaling pathway may be the cause that sodium arsenite induced HBE and HaCaT cells malignant transformation,its possible mechanism is by raising the expression of cyclin E-CDK2,accelerating the cell cycle and cell proliferation.