J Korean Med Sci.  2020 Dec;35(50):e420. 10.3346/jkms.2020.35.e420.

Expression of Estrogen Receptor-alpha in Nasal Polyps and the Effects of Dexamethasone on Estrogen Receptoralpha Expression in RPMI 2650 Cells

• Ban WW
• Lee YJ
• Lee SH
• Jung JY
• Baek BJ

• KMID: 2509754
• DOI: http://doi.org/10.3346/jkms.2020.35.e420

Abstract

Background
Studies have reported that epithelial cell proliferation may be involved in the pathogenesis of nasal polyps (NPs). Estrogen receptor (ER)-α, one type of ER, is related to antiinflammatory action and cell survival in certain tissues. In this study, we examined the presence or absence of ER-α in NPs and healthy inferior turbinate mucosae. We also investigated the effect of dexamethasone on ER-α expression, cell viability, and apoptosis in RPMI 2650 cells.
Methods
Immunohistochemical staining and Western blot analysis were conducted to determine the expression of ER-α in 15 NPs and 15 healthy inferior turbinate mucosae. After treating RPMI 2650 cells with dexamethasone, ER-α expression was analyzed using Western blot analysis and cell viability was determined using the MTT assay. Western blot analysis and annexin V-phycoerythrin (PE) staining were used to examine apoptotic cell death.
Results
Western blot analysis showed that ER-α expression was upregulated in 13 of the 15 NP tissues. Immunohistochemical staining for ER-α confirmed the results of the Western blot analysis. When RPMI 2650 cells were treated with dexamethasone, both ER-α expression and cell viability were decreased. Furthermore, the treatment of RPMI 2650 cells with dexamethasone increased apoptotic cell death, as shown by increased levels of BAX and cleaved caspase-3, decreased levels of Bcl-2, and an increased percentage of positive annexin V-PE stained cells.
Conclusion
ER-α expression was higher in NPs than in healthy inferior turbinate mucosae. When RPMI 2650 cells were treated with dexamethasone, ER-α expression was downregulated, cell viability decreased, and apoptosis increased. The decreased cell viability may be related, at least in part, to the decreased ER-α protein levels, which likely contributed to the induction of apoptotic cell death in RPMI 2650 cells.

Keyword

Estrogen Receptor-α; Nasal Polyp; Apoptosis; RPMI 2650 Cells; Cell Viability

Figure

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  Fig. 1 Immunohistochemical staining of ER-α expression in nasal polyps (A, B) and healthy inferior turbinate mucosa (C, D). Immunohistochemical reactions for ER-α in the nuclei and cytoplasm of the respiratory epithelium, submucous glands, venules, and inflammatory cells of the lamina propria (original magnification: 400×).ER = estrogen receptor, P = plasma cells, F = fibroblast, GL = submucous gland, V = venule.

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  Fig. 2 ER-α protein expression in healthy inferior turbinate mucosa and nasal polyp tissues.ER = estrogen receptor, Normal = healthy inferior turbinate mucosa, Polyp = nasal polyp.*P < 0.05 vs. normal.

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  Fig. 3 The effect of ICI 182780 on RPMI 2650 cell viability. The cells were treated with increasing concentrations of ICI 182780 (0, 1.25, 2.5, 5, 10, and 20 μM) for 24, 48, and 72 hours. The percentage of viable cells was then determined using the MTT assay. The error bars represent the mean ± standard deviation of three independent experiments.ICI = ICI 182780.*P < 0.05 vs. untreated controls.

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  Fig. 4 Expression of ER-α protein in RPMI 2650 cells. The cells were treated with various concentrations of dexamethasone for 72 hours. The levels of ER-α protein were determined by Western blot analysis.ER = estrogen receptor.*P < 0.05 vs. untreated controls.

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  Fig. 5 The effect of dexamethasone on RPMI 2650 cell viability. The cells were treated with increasing concentrations of dexamethasone (0, 6, 12.5, 25, 50, and 100 μM) for 24, 48, and 72 hours. The percentage of viable cells was then determined using the MTT assay. The error bars represent the mean ± standard deviation of three independent experiments.*P < 0.05 vs. untreated controls.

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  Fig. 6 The effects of combined treatment with dexamethasone and ICI 182780 on RPMI 2650 cell viability. The cells were treated with combinations of ICI 182780 (2.5 and 5 μM) and increasing concentrations (0, 6, 12.5, and 25 μM) of dexamethasone for the indicated times. The percentage of viable cells was then determined using the MTT assay. The error bars represent the mean ± standard deviation of three independent experiments. ICI, ICI 182780.*P < 0.05 vs. the respective untreated controls.

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  Fig. 7 The expression of apoptosis-related proteins in nasal epithelial RPMI 2650 cells. The cells were treated with various concentrations of dexamethasone for 72 hours. The levels of apoptosis-regulating proteins were determined by Western blot analysis.

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  Fig. 8 The effects of dexamethasone and ICI 182780 treatment on RPMI 2650 cell death. The cells were treated with increasing concentrations of dexamethasone (0, 6, 12.5, and 25 μM) and ICI 182780 (0, 2.5, 5, and 20 μM), alone or in combination, for 48 hours. The percentage of apoptotic cells after annexin V-phycoerythrin binding was determined using a Muse cell analyzer.ICI = ICI 182780, PE = phycoerythrin.

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