| Objective:This study aims to investigate the effect of the deregulated expression of several antioxidant defense enzymes,including peroxiredoxin(Prx) 1,2, 5,6 and copper/zinc superoxide dismutase(Cu/Zn-SOD),on the antioxidant defense ability and basal levels of reactive oxygen species (ROS),lipid peroxidation and oxidative DNA damage in PTEN-deleted mouse embryonic fibroblasts(PTEN-/- MEFs).The regulation mechanism of PTEN on the protein and mRNA expression of Prx1,2,5,6 and Cu/Zn-SOD was also explored to supplement and perfect the anti-tumor action principles of PTEN which may have potential in prevention and therapy of cancer.Methods:(1) SOD Assay Kit-WST was used to test the superoxide dismutase (SOD) enzyme activities in control group(PTEN+/+ MEFs) and PTEN-/- MEFs.The comet assay was applied for the detection of various concentrations of H2O2-induced DNA double-strand breaks(DSBs).The expression of phosphorylation of histone H2AX(γH2AX),a widely used marker for DNA DSBs,was detected by western blot treated by H2O2 with different dose.(2) Using 2',7'-dichlorofluorescein diacetate(DCHF-DA) and dihydroethidium(DHE),the intracellular generation of H2O2 and superoxide anion(O2·-) was monitored by flow cytometry and fluorescence microscope in PTEN+/+ MEFs and PTEN-/- MEFs.(3) To detect the effect of PTEN on lipid peroxide,the levels of malonaldehyde(MDA) in PTEN+/+ MEFs and PTEN-/- MEFs was tested by MDA Assay Kit.To analyze the influence of PTEN deletion on oxidative DNA damage,immunostaining assay was used to detect the expression of 8-OH-dG.The expression ofγH2AX in PTEN+/+ MEFs and PTEN-/- MEFs was monitored by western blot and immunofluorescence.The comet assay was also applied to further detect the levels of DNA DSBs.(4) To explore whether PTEN regulate the expression of antioxidant enzymes through inhibiting PI3K/AKT signal pathway,western blot and northern blot were used to test the protein and mRNA expression of P-AKT,Prx1,2,5,6 and Cu/Zn-SOD in PTEN-/- MEFs treated by LY294002,a inhibitor of phosphatidylinositol-3-kinase(PI3K).Western blot was further applied to analyze the basal expression of FOXO3a in PTEN+/+ MEFs and PTEN-/- MEFs and the expression levels of FOXO3a treated by LY294002 with various time in PTEN-/- MEFs to identify if PTEN regulate the phosphorylation of the transcription factor FOXO3a through inhibiting PI3K/AKT signaling pathway.Results:(1) Decreased antioxidant defense ability in PTEN-deleted MEFs:SOD enzyme activity data revealed a 2.09-fold decrease in PTEN-/- MEFs(p＜0.01).The comet assay and western blot showed that exposure to each concentration of H2O2 resulted in a highly statistically significant increase in the frequency of DNA DSBs in PTEN-/- MEFs(p＜0.01) compared to H2O2-untreated control at each concentration,whereas this increase was seen only in the 0.1 mmol/L H2O2-treated PTEN+/+ MEFs (p＜0.01 compared to H2O2-untreated control),indicating the decreased antioxidant defense ability in PTEN-/- MEFs.(2) Increased ROS in PTEN-deleted MEFs:A flow-cytometry-based DCHF-DA and DHE analysis revealed that the levels of both DCF and Eth fluorescence in PTEN-/- MEFs were higher than in control wild-type cells during 15-to 60-min incubation.(p＜0.05 and p＜0.01).Fluorescence photomicrographs also suggested an increased H2O2 and O2·- in PTEN-/- MEFs.(3) Increased lipid peroxide and oxidative DNA damage in PTEN-deleted MEFs: The level of MDA in PTEN-/- MEFs was higher than in PTEN+/+ MEFs(1.75±0.05 nmol/mgprot vs 1.3±0.1 nmol/mgprot,p＜0.01). Immunostaining assay data revealed that PTEN-deleted MEFs displayed a significant increase in the levels of 8-OH-dG(p＜0.01).The frequency of DNA DSBs was estimated by immunofluorescence assay or western blotting forγH2AX.Compared to PTEN+/+ MEFs,intenseγH2AX staining or increasedγH2AX level was observed in PTEN-/- MEFs(p＜0.01).The comet assay showed that mean tail moment in PTEN-/- MEFs was significantly higher than in control(22.03 vs 15.48,p＜0.01),indicating that there is extensive spontaneous DNA DSBs in PTEN-/- MEFs.(4) PTEN may increase the expression of Prx1,2,5,6 and Cu/Zn-SOD and decrease the phosphorylation of FOXO3a through inhibiting PI3K/AKT signaling pathway:Western blot and northern blot displayed that LY294002 successfully inhibited the activation of PI3K/AKT signaling pathway and the protein and mRNA expression of Prx1,2,5,6 and Cu/Zn-SOD up-regulated in PTEN-/- MEFs treated by LY294002,indicating that PTEN may increase the expression of Prx1,2,5,6 and Cu/Zn-SOD through inhibiting PI3K/AKT signal pathway.Meanwhile,western blot also showed that the protein expression of P-FOXO3a in PTEN-/- MEFs was higher than in PTEN+/+ MEFs and deregulated in PTEN-/- MEFs treated by LY294002, indicating that PTEN may decrease the phosphorylation of FOXO3a through inhibiting PI3K/AKT signaling pathway.Conclusion:(1) PTEN,as an important tumor suppressor gene,may up-regulate the expression of several antioxidant enzymes,including Prx1,2,5,6 and Cu/Zn-SOD,which results in increased antioxidant defense ability and decreased ROS levels,lipid peroxide and oxidative DNA damage.These findings suggest an essential role for PTEN in maintaining normal redox state and genomic stability against oxidative damage.(2) PTEN may up-regulate the expression of Prx1,2,5,6 and Cu/Zn-SOD through inhibiting the PI3K/AKT signaling pathway.Its mechanism may be to deregulate the phosphorylation of the PI3K/AKT downstream Forkhead transcription factors(FOXOs). |
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