220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

Abstract

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10^–9 M), P4 (10^–6 M), 2-ME (10^–8 M, 10^–7 M, 10^–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10^–7 M ICI 182, 780 and 10^–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg^–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg^–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg^–1 of BW) and RU 486 (10 mg kg^–1 of BW) at 30 min before injection with 2-ME (40 mg kg^–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10^–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10^–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg^–1 of BW) group, similar to the response with E2 (40 μg kg^–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.