| PTEN (phosphatase and tensin homologue deleted on chromosome 10) is the first phosphatase identified as a tumor suppressor. Reduced expression or loss-of-function mutations of the PTEN gene are found at high frequency in a wide variety of human tumors. In early studies, using two-dimensional gel electrophoresis, combined with Western and Northern blot analyses, we observed that PTEN deficiency in mouse embryonic fibroblasts (MEFs) showed deregulated expression of several antioxidant enzymes, including peroxiredoxins 1, 2, 5, and 6 and Cu, Zn superoxide dismutase.The present study aimed to demonstrate the mechanism of PTEN for regulation the protein expression of peroxiredoxin(Prx)1, 2, 5, 6 and copper/zinc superoxide dismutase (Cu/Zn-SOD) and the effect of the deregulated expression of Prx1, 2, 5, 6 and Cu/Zn-SOD on the basal levels of reactive oxygen species (ROS), antioxidant defense ability and DNA DSBs damage in PTEN+/+ MEFs transfected stably with plxsp-HAGFP -AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP - PTEN-WT/AKT-DN. In this study, we show for the first time that mechanism of PTEN for regulation the protein expression of Prx1,2,5,6 and Cu/Zn-SOD.Methods:1. To explore whether PTEN can regulate the expression of antioxidant enzymes through inhibiting PI3K/AKT/FoxOs signaling pathway, the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD was measured with Western blot in PTEN+/+MEFs transfected transiently with pIRES2-EGFP-AKT-WT /AKT-AC and stably with plxsp-HAGFP-AKT-WT/AKT-AC and PTEN-/- MEFs transfected transiently with pIRES2-EGFP-PTEN-WT/AKT-DN and stably with plxsp-HAGFP-PTEN-WT/AKT-DN. Western blot was further applied to detect the basal expression of FoxOs in PTEN+/+ MEFs and PTEN-/- MEFs and the expression levels of FoxO1 in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT/AKT-DN to identify if PTEN regulate the phosphorylation of the transcription factor FoxO1 through inhibiting PI3K/AKT signaling pathway.2. To detect the effect of PTEN on the basal levels of ROS and antioxidant defense ability through inhibiting PI3K/AKT signaling pathway. Using 2',7'-dichlorofluorescein diacetate (DCHF-DA) and dihydroethidium (DHE), the intracellular generation of H2O2 and superoxide anion(O2·ˉ) was determined by flow cytometry in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT/AKT-DN. The comet assay was used for the evaluation of various concentrations of H2O2-induced DNA double-strand breaks (DSBs) in PTEN-/- MEFs and PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT.3. To determine the effect of PTEN on DNA DSBs through inhibiting PI3K/AKT signaling pathway. The comet assay was applied to detect the levels of DNA DSBs. The expression ofγH2AX was further detected by immunofluorescence in PTEN+/+ MEFs transfected stably with plxsp-HAGFP- AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP- PTEN-WT/AKT-DN that were cultured for different hours after being treated with 0.01u mol/L H2O2 for 15 minutes.Results:1. PTEN can increase the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD through inhibiting PI3K/AKT signaling pathway: western blot displayed the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD down-regulated in PTEN+/+MEFs transfected transiently and stably with AKT-WT/AKT-AC and up-regulated in PTEN-/- MEFs transfected transiently and stably with PTEN-WT/AKT-DN, indicating that PTEN can increase the expression of Prx1, 2, 5, 6 and Cu/Zn-SOD through inhibiting PI3K/AKT signaling pathway.2. PTEN can decrease the phosphorylation level of FoxO1 through inhibiting PI3K/AKT signaling pathway: western blot displayed that the protein expression of P-FoxO1 and P-FoxO3a in PTEN-/- MEFs was higher than that in PTEN+/+ MEFs(p<0.01). Western blot further showed that the protein expression of P-FoxO1 was up-regulated in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC (p<0.05) and down-regulated in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT/AKT-DN(p<0.01),suggesting that PTEN can decrease the phosphorylation level of FoxO1 through inhibiting PI3K/AKT signaling pathway.3. PTEN can decrease ROS levels in MEFs through inhibiting PI3K/AKT signaling pathway: A flow-cytometry-based DCHF-DA and DHE analysis indicated that the levels of both DCF and Eth fluorescence were higher in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC (p<0.05) and lower in PTEN-/- MEFs transfected stably with plxsp-HAGFP- PTEN-WT/AKT-DN(p<0.01).4. Increased antioxidant defense ability in PTEN-reexpression MEFs: The comet assay displayed 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 with H2O2-untreated control at each concentration, whereas this increase was seen only in the 0.05 mmol/L H2O2-treated PTEN-reexpression MEFs(p<0.01 compared with H2O2- untreated control), indicating the increased antioxidant defense ability in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT.5. PTEN can decrease DNA DSBs damage in MEFs through inhibiting PI3K/AKT signaling pathway: The comet assay showed that mean tail moment in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT was significantly lower than that in PTEN-/- MEFs(20.34 vs 27.23, p<0.01). The frequency of DNA DSBs was measured by immunofluorescence assay forγH2AX. The result displayed that a statistically significant decrease in the frequency of DNA DSBs in PTEN+/+ MEFs at 30 minuates was detected (p<0.05), whereas this decrease was found only at 4h in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC (p<0.05). And this decrease in the frequency of DNA DSBs was seen at 4h in PTEN-/- MEFs(p<0.05), whereas a higly statistically significant decrease was showed at 4h in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT /AKT-DN(p<0.01). These results suggest that PTEN can promote DNA damage repair through inhibiting PI3K/AKT signaling pathway.Conclusion:1. PTEN regulates the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD through PI3K/AKT/FoxOs signaling pathway.2. PTEN can reduce ROS levels, increase antioxidant defense ability and decrease DNA DSBs damage in MEFs through inhibiting PI3K/AKT signaling pathway. These findings show that PTEN plays an essential role in promoting DNA damage repair and maintaining genomic stability against oxidative damage. |
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