J Breast Cancer.  2009 Sep;12(3):134-141. 10.4048/jbc.2009.12.3.134.

Reactive Oxygen Species Generated by 17beta-estradiol Play a Role in the Up-regulation of GPX4 Protein in MCF-7 Breast Cancer Cells

• Lee SH
• Kim HJ
• Kang HJ
• Lee YJ
• Nam HS
• Bae I

• KMID: 2242003
• DOI: http://doi.org/10.4048/jbc.2009.12.3.134

Abstract

PURPOSE
Estrogen is known to act as both a growth factor and a survival factor for breast cancer. The responsible molecular mechanisms remain, however, to be fully elucidated. We hypothesize that the effect of estrogen relates to its ability to induce the cellular antioxidant defense enzymes.
METHODS
In the presence study, we examined the ability of 17beta-estradiol (E2) to regulate the level of phospholipid hydroperoxide glutathione peroxidase (GPX4) protein, which is an anti-oxidative enzyme that can directly reduce both phospholipids and cholesterol-hydroperoxides located in the cell membranes and lipoproteins.
RESULTS
E2 elicited a dose- and time-dependent increase in the GPX4 expression in the MCF-7 breast cancer cells, and this up-regulation was blocked by the free radical scavenger N-acetylcysteine (NAC). Additionally, we confirmed that E2 triggered a rapid and transient increase in the intracellular reactive oxygen species (ROS) levels, and this E2-induced increase in the ROS levels was inhibited by pretreatment with NAC. Moreover, such ROS inducers as TGF-beta, TNF-alpha and insulin induced an increase in the level of GPX4 protein. However, estrogen receptor (ER)alpha knockdown by transfection with ERalpha-siRNA did not significantly change the GPX4 protein level that was induced by E2. Furthermore, pre-incubation with the ER antagonist ICI 182,780 did not inhibit E2-mediated GPX4 induction. Conversely, pretreatment of cells with LY294002, a pharmacological inhibitor of phosphatidylinositol 3-kinase inhibitor, suppressed the E2-augmented GPX4 expression.
CONCLUSION
Collectively, our data show that E2 may partly provide a survival advantage through the regulation of cellular oxidative homeostasis in MCF-7 breast cancer cells.

Keyword

17beta-estradiol; Estrogen receptors; Glutathione peroxidase; Oxidative stress; Reactive oxygen species

MeSH Terms

Acetylcysteine
Breast
Breast Neoplasms
Cell Membrane
Chromones
Estradiol
Estrogens
Glutathione Peroxidase
Homeostasis
Hydrogen Peroxide
Imidazoles
Insulin
Lipoproteins
Morpholines
Nitro Compounds
Oxidative Stress
Phosphatidylinositol 3-Kinase
Phospholipids
Reactive Oxygen Species
Receptors, Estrogen
Transfection
Transforming Growth Factor beta
Tumor Necrosis Factor-alpha
Up-Regulation
Acetylcysteine
Chromones
Estradiol
Estrogens
Glutathione Peroxidase
Hydrogen Peroxide
Imidazoles
Insulin
Lipoproteins
Morpholines
Nitro Compounds
Phosphatidylinositol 3-Kinase
Phospholipids
Reactive Oxygen Species
Receptors, Estrogen
Transforming Growth Factor beta
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Protective effect of E2 against H2O2-induced oxidative stress. MCF-7 cells were plated in 5% CCS-containing DMEM for 24 hr and then incubated with vehicle (media) or E2 (10 nM) for 24 hr prior to treatment with increasing concentrations of H2O2 for 24 hr. The percentage of viable cells was then determined by an MTT assay, as described in the Methods. Error bars represent the mean±SEM of three independent experiments. E2=17β-estradiol. *p<0.05 compared to H2O2-treated group.

• 

  Figure 2 E2-induced up-regulation of the GPX4 protein. MCF-7 cells were treated with increasing concentrations of E2 for 24 hr (A) or E2 (10 nM) for the indicated times (B). Cell lysates (40 µg) were then analyzed by Western blot analysis to determine the level of GPX proteins. β-actin was used as a loading control. E2=17β-estradiol.

• 

  Figure 3 Modulation of the GPX4 protein expression in response to E2-induced ROS. (A) Time course of ROS generation induced by E2. MCF-7 cells were treated with vehicle (media) or NAC (10 mM) for 1 hr prior to incubation with E2 (10 nM) for the indicated times. The intracellular ROS levels were then measured using the redox-sensitive dye, DCF-DA, as described in the Methods. (B) Inhibition of E2-induced GPX4 expression by the antioxidant NAC. MCF-7 cells were treated with vehicle (media) or NAC (10 mM) for 1 hr prior to incubation with E2 (10 nM) for the indicated times. (C) Effect of ROS inducers on GPX4 expression. MCF-7 cells were treated with vehicle (media), KGF (10 ng/mL), E2 (10 nM), TGFβ(1 ng/mL), TNFα(100 ng/mL), and insulin (200 nM). The level of GPX4 in the cell lysates (40 µg) was then analyzed by Western blot analysis. β-actin was used as a loading control. Error bars represent the mean±SEM for three independent experiments. E2=17β-estradiol; ROS=reactive oxygen species. *p<0.05 compared to E2-treated group.

• 

  Figure 4 Effects of ERα gene silencing and various chemical inhibitors on E2-induced GPX4 expression. (A) MCF-7 cells were transfected with ERa-specific siRNAs (siRNA-ERα; 50 nM) and control siRNA (siRNA-C; 50 nM) for 72 hr, after which they were treated with E2 (10 nM) for 24 hr. (B) MCF-7 cells were then treated with vehicle (media) or with ICI 182,780 (5 µM), PD98059 (50 µM), U0126 (20 µM), or LY294002 (25 µM) for 1 hr prior to incubation with E2 (10 nM) for the indicated times. Cell lysates (40 µg) were then analyzed for the level of GPX4 proteins by Western blot analysis. β-actin was used as a loading control. E2=17β-estradiol; ER=estrogen receptor.

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