Figure 3

Oxidation of PTPs and ROS-induced phosphorylation of ERK and AKT in E2-treated MCF-7 cells. Changes in the oxidation state of PTPs, PTEN, and CDC25A were determined by western blot analysis. (A) Comparison of PTEN oxidation in MCF-7 cells treated with oxidant H₂O₂ and reductant dithiothreitol (DTT) (5 mM) for 30 min. Higher doses of H₂O₂ (0.5 and 1.0 mM) showed higher levels of oxidised PTEN (bottom band) compared with 0.2 mM dose of H₂O₂ and DTT. (B) Comparison of PTEN oxidation in E2-treated MCF-7 cells when pretreated with 20 μ M of ROS scavenger ebselen (Eb) for 4 h. Western blot showed increased PTEN oxidation by both E2 (367 nM) and 1 μ M tamoxifen (TAM) treatment in MCF-7 cells compared with control (CTRL) (dimethyl sulfoxide (DMSO)) and this was suppressed by Eb. Values in graph represent % oxidised/reduced PTEN from three independent experiments±s.d. (C) Comparison of CDC25A sulphydryl labelling by 5-iodoacetamidofluorescein (5-IAF) reagent in MCF-7 cells treated with E2 (10 ng ml⁻¹) for 30 min in the presence or absence of 10 mM of ROS scavenger NAC. Decreased 5-IAF labelling indicated a decrease in free sulphydryl (−SH) groups present in CDC25A. (D) Analysis of CDC25A phosphatase activity in MCF-7 cells treated with E2 and H₂O₂ as described previously. Values in the graph represent CDC25A phosphatase activity from IP lysate of MCF-7 cells treated with NAC. Enzyme activity was tested in vitro using OMFP as a substrate. (E) Comparison of CDC25A serine phosphorylation in E2- and H₂O₂-treated MCF-7 cells when pretreated with NAC as described previously. (F) Comparison of CDC25A tyrosine phosphorylation in E2- and H₂O₂-treated MCF-7 cells when pretreated with NAC as described previously. Cell lysates were IP with CDC25A antibody and immunoblots were detected for anti-phosphotyrosine (p-Tyr) or -serine (p-Ser). IgG bands served as a loading CTRL (n=2). (G) Western blot analysis of the effects of NAC (10 mM) and mitochondrial ROS blocker rotenone (Rot) (5 μ M) on CDC25A protein levels in E2-treated MCF-7 cells. (H) Western blot analysis of the effects of MnSOD and CAT (MOI=200) overexpression on E2 (367 pM)-induced ERK phosphorylation in MCF-7 cells. Values in the graph represent a ratio of levels of phospho-ERK1/2 compared with total ERK1/2 or β-actin from three independent experiments. (I) Western blot analysis of the effect of ROS modifiers on E2-induced AKT phosphorylation in MCF-7 cells. Experimental conditions are the same as described previously. Treatment with 10 μ M of PI3K/AKT pathway inhibitor LY294002 (Ly) served as a positive CTRL of p-AKT inhibition. Values in the graph are the densitometry data of p-AKT normalised to total AKT expressed as fold change compared with CTRL. The quantitative values are mean±s.d. Data shown in this figure are representative of three independent experiments. *P<0.05, significant difference from CTRL. **P<0.05, significant difference from E2. SOD, superoxide dismutase.