31. Antioxidant and hypoxia induce the human antioxidant response element-mediated expression of NAD(P) H: quinone oxidoreductase1 gene
Background: NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in protection of cells against redox cycling, oxidative stress, and neoplasia. and can also bioactivate certain antitumor quinones. Many antioxidants are cancer chemopreventive agents, and tumour hypoxia are now being exploited in cancer treatment which shows considerable promise to overcome the resistance to cancer chemotherapy. Antioxidant response element (ARE) is sensitive to perturbations of cellular redox states. Our previous studies have shown that β-tyrosol (β-TY), as a phenolic antioxidant, can protects cells against DNA damage resulting from toxic H2O2. Aim: We take the present study with the goal of whether antioxidants such as β-tyrosol, butylated hydroxyanisole(BHA) and β-Naphthooflavone(β-NF) and hypoxia (pO2 0.1% -0.5%) can induce gene expression of NQO1, inhibit proliferation of human hepatoma cells SMMC-7721 and the relationship between them; whether ARE can mediate gene expression in response to antioxidans and hypoxia. Methods: SMMC-7721 human hepatoma cells are planted in plates, grown for 24h. and exposed to antioxidants and hypoxia, each alone or in combination for another 24h. The enzyme activity was determinied by spectrophotometric assay using direct measurement of NQO1 from cells cultured in Microtiter wells. Semi-quantitative reverse transcription-PCR (RT-PCR) technique was used to measure NQO1 mRNA levets. Proliferation was estimated using the crystal violet staining technique. Electrophoretic mobility shift assay (EMSA) was employed to assess protein binding to the ARE under all of These conditions. Hypoxia cells were harvested in an anaerobic chamber at the end of the incubation period. Results: Antioxidant (90ug/ml β-TY、60μmol/L BHA、80μmol/L β-NF) potently induce an increase in the activity of NQO1. From 60μg/ml to 90μg/ml, β-TY caused NQO1 activity enhancement in a dose-dependent manner The NQO1 activity induced by 80μg/ml β-TY was approximately equal to that induced by 80μmol/L β-NF; β-TY also caused NQO1 mRNA expression enhancement in a dose-dependent manner(r=0.824, P<0.05). A statistically significant correlation between NQO1 mRNA levels and enzyme activities induced by β-tyrosol was evident (r=0.951, P<0.01). In the almost same concentration range from 70 μg/ml to 100μg/ml, β-tyrosol caused a dose-dependent inhibition of cell proliferation which well inversely correlated with NQO1 enzyme activity(r=-0.410, P<0.01). 90 μg/ml β-TY and 60μmol/L BHA induced NQO1 activity were significantly increased in hypoxia Than in air (1. 168511-and 1.37859-fold respectively). Electrophoretic mobility shift assays (EMSA) of nuclear extracts of SMMC-7721 cells exposed to antioxidants (β-TY and BHA) and hypoxia, or antioxidants combined with hypoxia, showed specific and constitutive protein binding to the ARE, but not to the mutated ARE under all of these conditions. The same concentration of NF-κ B affinity recognition oligonucleotide as that of -32P probed ARE partially compete the ARE mediated binding to nuclear extracts of cells exposed to β-TY in hypoxia. Couclutions: Antioxidant β-TY、BHA、β-NF potently induce an increase of NQO1 activity in hepatoma cells SMMC-7721 in air. Hypoxia further enhance NQO1 activity induced by β-TY and BHA. In air β-TY induced enzyme activity of NQO1 correlated its mRNA expression increase, and inhibited cell proliferation correlated with NQO1 enzyme activity. ARE is a common cis-element that mediate NQO1 gene expression in response to antioxidants and hypoxia.
NAD(P)H:quinone oxidoreductasel、antioxidant response element、antioxidant、hypoxia
13
R730(肿瘤学)
国家自然科学基金39970649
2004-03-26（万方平台首次上网日期，不代表论文的发表时间）
共2页
227-228
