Figure 4.

In vitro fulvestrant inhibits ERα expression and cytokines secretion. (A–C) PPT (100 nM), DPN (10 nM) were added to HiBECs parallel with 17β-estradiol (10 nM) or vehicle. And, fulvestrant (8.24 μM) was added to HiBEC 2 h before stimulation with 17β-estradiol (10 nM). Total RNA was isolated and analyzed by real-time PCR for IL-6mRNA, IL-8mRNA, and TNF-α mRNA expression. The resulted showed that PPT treatment significantly induced IL-6 mRNA (A), IL-8mRNA (B), and TNF-α mRNA (C) expression as well as 17β-estradiol. In contrast, DPN had no obvious effects on them. In addition, pretreatment with fulvestrant (8.24 μM) inhibited IL-6mRNA, IL-8mRNA, and TNF-α mRNA expression. *p < 0.05, **p < 0.01, ***p < 0.001, compared with controls or 17β-estradiol group by one-way analysis of variance. (D–F) ELISA analysis of IL-6 (D), IL-8 (E), and TNF-α (F) protein in cell culture supernatants. The results were consistent with RT-RCR *p < 0.05, **p < 0.01, ***p < 0.001, compared with controls or 17β-estradiol groups by one-way analysis of variance. (G–H) Immunoblotting analysis of ERα and ERβ expression levels in HiBECs that were stimulated with PPT, DPN, fulvestrant, and 17β-estradiol. (H) The expression of ERα in HiBECs was up-regulated by 17β-estradiol and PPT, and fulvestrant effectively blocked ERα expression (n = 3, mean ± SEM). *p < 0.05, determined by ANOVA with a Tukey test. (I) ERβ expression was not changed in all groups, except for the DPN groups. *p < 0.05, compared with controls. All data are representative of three independent experiments.