Figure 2

Cell proliferation and colony formation of E2-treated MCF-7 cells depend on the oxidative state of Trx and mitochondrial biogenesis genes. Changes in the oxidation state of Trx associated with impaired E2-induced colony formation. (A) Comparison of Trx oxidation in MCF-7 cells treated for 30 min with E2 (367.1 pM) or the chemical inducer of TrxR erucin (Eru) by redox western blot analysis. 17β-Oestradiol treatment showed a higher level of oxidised Trx (top band) compared with vehicle (dimethyl sulfoxide (DMSO)) and reduced the level of oxidised Trx in a 48 h pre-treatment with 10 μ M Eru. (B) Values in the graph are of the steady-state redox potential (Eh) for Trx oxidation in MCF-7 cells treated with reductant (dithiothreitol (DTT), 5 mM), oxidant (H₂O₂, 2 mM), and E2. (C) Comparison of colony formation in soft agar at 14 days of E2-treated MCF-7 cells when cotreated with Eru. Values in the graph show significant inhibition of E2-induced bromodeoxyuridine (BrdU) incorporation by Eru treatment. (D) Comparison of colony formation in E2-treated MCF-7 cells with and without overexpression of TrxR2 for 48 h. Graph indicates significant inhibition of E2-induced BrdU incorporation when MCF-7 cells overexpress TrxR2. Western blot confirmed overexpression of TrxR2. (E) Analysis of E2 effects on mitochondrial mass with MitoTracker Red. MCF-7 cells showed increased mitochondrial labelling intensity in E2 treatment compared with control (CTRL). Comparison of E2-induced NRF1 DNA-binding activity by EMSA showed increased NRF1 binding at 3 h. C=CTRL; +Comp=negative NRF1-binding CTRL. (F) Comparison of cellular protein levels of TFAM in short hairpin RNA (shRNA) Tet-off/on cells. Western blot confirmed lower protein level of TFAM by inducible shRNA in MCF-7 cells. Values are shown in the graph of protein band intensity as well as in the immunoblot of TFAM Tet/on cells (TFAM knockdown (KD)) compared with Tet/off cells (Mock). (G) Comparison of E2-induced MCF-7 colony formation in TFAM Tet/on cells (TFAM KD) compared with Tet/off cells (Mock). (H) Comparison of NRF-1 and TFAM KD effects on ROS formation, BrdU incorporation, and cell viability in E2-treated MCF-7 cells. Values are mean±s.d. Data shown in each panel are representative of three independent experiments. *P<0.05, significantly different from CTRL. **P<0.05, significantly different from E2.