Estrogen Induces Metastatic Potential of Papillary Thyroid Cancer Cells through Estrogen Receptor α and β

Abstract

Estradiol (E2) promotes metastatic propensity. However, the detailed mechanism remains largely unknown. E-cadherin, vimentin, and MMP-9 play a dominant role in the metastatic process. We aimed to investigate the effects of E2 on metastatic potential of PTC cell line BCPAP and on E-cadherin, vimentin, and MMP-9 protein expression. PTC cell line BCPAP was evaluated for the presence of estrogen receptor (ER) by western blot analysis. The effects of E2, PPT (a potent ER α -selective agonist), and DPN (a potent ER β -selective agonist) on modulation of metastatic phenotype were determined by using in vitro scratch wound assay and invasion assay. In addition, the effects on E-cadherin, vimentin, and matrix metalloproteinase-9 (MMP-9) protein expression were evaluated by Western blot analysis. We found that BCPAP cells expressed ER α and ER β . E2 and PPT enhanced, but DPN inhibited, the migration and invasion of BCPAP cells in an in vitro experimental model system that is modulated by E-cadherin, vimentin, and MMP-9. These findings indicate that E2 induces the metastatic potential of BCPAP cells through ER α and ER β . The two ER subtypes play differential roles in modulation of BCPAP cell metastasis and the related molecule expressions including E-cadherin, vimentin, and MMP-9.

Figure 1

BCPAP cells express ERα and ERβ. Whole cell protein was resolved by SDS-PAGE followed by western blot analysis for ERα, ERβ, and β-actin. MCF-7 and ER-positive cells were used as positive control.

Figure 2

E2 enhances migration of BCPAP cells. Confluent monolayers of BCPAP cells were wounded with a uniform scratch, washed to remove cell debris, and cultured in the presence of vehicle (DMSO) alone, 10⁻⁸M E2, 10⁻⁶M PPT, or 10⁻⁶M DPN for 24 h. Images of cell cultures were captured at 0 and 24 h after scratching; representative pictures are shown in upper panel. The amount of wound repair was expressed as uncovered area at the indicated time compared with initial uncovered area of vehicle-treated control at time zero (lower panel). Values are the mean ± SD of three separate experiments (normalized to the untreated control). *P < 0.05 compared with control.

Figure 3

E2 enhances invasion of BCPAP cells. Cells (2.0 × 10⁴ cells per well) in the phenol-red-free RPMI medium (100 μL) with 1% charcoal-dextran stripped FBS containing vehicle (DMSO) alone, 10⁻⁸M E2, 10⁻⁶M PPT, or 10⁻⁶M DPN were loaded onto the upper chamber, and 500 μL of phenol-red-free RPMI medium with 10% charcoal-dextran stripped FBS was loaded onto the bottom chamber as a chemoattractant. After 24 h, the invaded cells were counted microscopically in five random fields of view at 200x magnification and expressed as the mean number of cells per field of view. Values are the mean ± SD of three separate experiments (normalized to the untreated control). *P < 0.05 compared with control.

Figure 4

E2 induces metastasis via downregulation of E-cadherin and upregulation of vimentin and MMP-9. BCPAP cells were treated with vehicle (DMSO) alone, 10⁻⁸M E2, 10⁻⁶M PPT, or 10⁻⁶M DPN. Whole cell lysates were extracted, and E-cadherin, vimentin, and MMP-9 protein were detected by western blot analysis. β-actin was used as a loading control. *P < 0.05 compared with control.