G-protein-coupled receptor 30 and estrogen receptor-alpha are involved in the proliferative effects induced by atrazine in ovarian cancer cells

Abstract

Background: Atrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein-coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17beta-estradiol and environmental estrogens.

Objectives: Given the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells.

Methods and results: Atrazine did not transactivate the endogenous ERalpha in different cancer cell contexts or chimeric proteins encoding the ERalpha and ERbeta hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERalpha nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30-epidermal growth factor receptor transduction pathway and the involvement of ERalpha.

Conclusions: Our results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action.

Keywords: 17β-estradiol; GPR30; atrazine; estrogen receptor; ovarian cancer cells.

Figure 1

Structures of E₂ and atrazine.

Figure 2

ERα transactivation in BG-1 (A), MCF-7 (B), and Ishikawa (C) cells transfected with the ER luciferase reporter plasmid XETL (ERE-luc) and treated with 100 nmol/L E₂ or 1 μmol/L atrazine (Atr), with and without 10 μmol/L ER antagonist ICI. Luciferase activities were normalized to the internal transfection control, and values of cells receiving vehicle (−) were set as 1-fold induction, from which the activity induced by treatments was calculated. (D–F) SkBr3 cells were transfected with ER luciferase reporter gene XETL and ERα expression plasmid (D) and with Gal4 reporter gene (GK1) and the Gal4 fusion proteins encoding the HBD of ERα (GalERα; E) and or ERβ (GalERβ; F) and treated with 100 nmol/L E₂ or 1 μmol/L atrazine, with and without 10 μmol/L ICI. Values shown are mean ± SD of three independent experiments performed in triplicate. *p< 0.05 compared with vehicle.

Figure 3

mRNA expression and binding of ERα in BG-1 cells treated for 24 hr with vehicle (−), 100 nmol/L E₂, or 1 μmol/L atrazine (Atr). (A) mRNA expression of ERα was evaluated by semiquantitative RT-PCR; the values of housekeeping gene 36B4 were determined as a control. (B) Immunoblot of ERα from BG-1 cells, with 100 nmol β-actin serving as a loading control. Results in (A) and (B) are representative of three independent experiments. (C) ERα binding assay using increasing concentrations of atrazine.

Figure 4

Aromatase activity assessed by tritiated water release in BG-1 and H295R cells treated with vehicle (−) or 1 μmol/L atrazine (Atr). Results are expressed as percentages of untreated cells (100%). Values are mean ± SD of three independent experiments, each performed in triplicate. *p < 0.05 compared with vehicle.

Figure 5

ERK1/2 phosphorylation (pERK1/2) in BG-1 cells exposed to increasing concentrations of E2 or atrazine (Atr) for 20 min.

Figure 6

BG-1 (A–C) and 2008 (D–F) cells treated with vehicle (−) or 100 nmol/L E₂ with or without 1 μmol/L atrazine (Atr) for 5, 10, 20, or 30 min (A,D), or for 20 min with vehicle E₂ (B and E), or 1 μmol Atr in combination with 10 μmol/L ICI, AG, PD, GFX, H89, or WM, inhibitors of ER, EGFR, MEK (MAP/ERK kinase), PKC (protein kinase C), PKA (protein kinase A), and PI3K (phosphoinositide 3-kinase), respectively. pERK1/2, phosphorylated ERK 1/2.

Figure 7

ERK1/2 phosphorylation (pERK1/2) in SkBr3 cells treated for 20 min with vehicle (−) or increasing concentrations of ICI.

Figure 8

Immunoblots of c-fos from BG-1 (A,B) and 2008 (C,D) cells treated for 2 hr with vehicle (−), 100 nmol/L E₂, or 1 μmol/L atrazine (Atr) in combination with 10 μmol/L ICI, AG, PD, GFX, H89, or WM, inhibitors of ER, EGFR, MEK, PKC, PKA, and PI3K, respectively. β-Actin served as a loading control.

Figure 9

Immunoblots of c-fos from BG-1 (A,B) and 2008 (C,D) cells after silencing ERα and GPR30 expression. Cells were transfected with control siRNA or siRNA-ERα (A,C) or with vector or shGPR30 (B,D) and treated for 2 hr with vehicle (−) or 100 nmol/L E₂ or 1 μmol/L atrazine (Atr). Efficacy of ERα and GPR30 silencing was ascertained by immunoblots, as shown in side panels. β-Actin served as a loading control.

Figure 10

ERK1/2 phosphorylation (A) and c-fos expression (B) after silencing GPR30 in SkBr3 cells treated with vehicle (−) or 1 μmol/L atrazine (Atr). (C) The efficacy of GPR30 silencing was ascertained by immunoblots. β-Actin served as a loading control.

Figure 11

Proliferation of BG-1 (A–D) and 2008 (E–H) cells exposed to E₂ or atrazine (Atr). (A,D) Proliferation of cells in response to increasing concentrations of E₂ or Atr. (B–H) Proliferation of cells treated with vehicle (−), 100 nmol/L E₂, or 1 μmol/L Atr with or without 10 μmol/L AG or PD (B,F) (C,D, G, H) or transfected with vector or shGPR30 (C,G) or with control siRNA or siRNA-ERα(D,H). See “Materials and Methods” for details of experiments. Proliferation of cells receiving vehicle was set as 100%, and the cell growth induced by treatments was calculated. Values shown are mean ± SD of three independent experiments performed in triplicate; Efficacy of ERα and GPR30 silencing was ascertained by immunoblots (Figure 9). *p< 0.05 compared with treated cells.