2-Methoxyestradiol Attenuates Testosterone-Induced Benign Prostate Hyperplasia in Rats through Inhibition of HIF-1 α/TGF- β/Smad2 Axis

Abstract

Benign prostatic hyperplasia (BPH) is a common disorder in the male population. 2-Methoxyestradiol (2ME) is an end metabolite of estrogens with pleiotropic pharmacological properties. This study aimed to explore the potential ameliorative effects of 2ME against testosterone-induced BPH in rats. 2-Methoxyestradiol (50 and 100 mg/kg, dissolved in DMSO) prevented the rise in prostatic index and weight in comparison to testosterone-alone-treated animals for 2 weeks. Histological examination indicated that 2ME ameliorated pathological changes in prostate architecture. This was confirmed by the ability of 2ME to decrease the glandular epithelial height when compared to the testosterone group. Also, 2ME improved testosterone-induced oxidative stress as it inhibited the rise in lipid peroxide content and the exhaustion of superoxide dismutase (SOD) activity. The beneficial effects of 2ME against the development of BPH were substantiated by assessing proliferation markers, preventing the rise in cyclin D1 protein expression and enhancing Bax/Bcl2 mRNA ratio. It significantly reduced prostate content of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), nuclear factor κB (NF-κB), and transforming growth factor β (TGF-β). In addition, 2ME reduced hypoxia-inducible factor 1-α (HIF-1α) and phospho-Smad2 (p-Smad2) protein expression compared to the testosterone group. In conclusion, 2ME attenuates experimentally induced BPH by testosterone in rats through, at least partly, inhibition of HIF-1α/TGF-β/Smad2 axis.

Figure 1

Histological examination of hematoxylin-eosin sections of rat prostates. (a) Section taken from control rats showing normal histoarchitecture of the ventral prostates. (b) Section taken from the ventral prostate of testosterone-treated group exhibiting hypertrophy with increased epithelial thickness and intraluminar projections. (c) Section taken from the ventral prostate of the testosterone group cotreated with 50 mg/kg 2ME showing mild reduction in hypertrophy and hyperplasia. (d) Section taken from the ventral prostates of testosterone groups cotreated with 100 mg/kg 2ME showing obvious reduction in prostate hypertrophy and hyperplasia. (e) A graph showing the effect of 2ME cotreatment on prostate glandular epithelial height. Scale bar (50 μm). Data are expressed as mean ± SD (n = 6). a or b: statistically significant from control or testosterone group, respectively, at P < 0.05 using one-way ANOVA followed by Tukey's post hoc test.

Figure 2

Immunohistochemistry photomicrographs of prostate sections showing the influence of 2ME on testosterone-induced hyperplasia of prostate expression of cyclin D1. (a) Section taken from control rats. (b) Section taken from the prostate of testosterone-treated group. (c) Section taken from the ventral prostate of the testosterone group cotreated with 50 mg/kg 2ME. (d) Section taken from the ventral prostates of testosterone groups cotreated with 100 mg/kg 2ME. (e) A graph showing the effect of 2ME cotreatment on prostate expression of cyclin D1. Scale bar (50 μm). Data are expressed as mean ± SD (n = 6). a or b: statistically significant from control or testosterone group, respectively, at P < 0.05 using one-way ANOVA followed by Tukey's post hoc test.

Figure 3

Effects of 2ME treatment on prostatic mRNA levels of Bax (a) and Bcl-2 (b) expressed as relative quantification (RQ) compared to the control group. (c) Bax/Bcl-2 ratio. Data are presented as the mean ± SD (n = 3). a or b: significantly different from control or testosterone-treated groups, respectively, at p < 0.05 using one-way ANOVA followed by Tukey's post hoc test.

Figure 4

Effect of 2ME on prostate content of IL-1β (a), TNF-α (b), NF-κB (c), and TGF-β (d) in testosterone-treated rats. Data are expressed as mean ± SD (n = 3). a or b: statistically significant from control or testosterone group, respectively, at P < 0.05 using one-way ANOVA followed by Tukey's post hoc test.

Figure 5

(a) Western blot analysis of HIF-1α and p-Smad2 expression in prostate tissues. I: untreated control; II: testosterone-induced BPH group (3 mg/kg, 5 days/week for 2 weeks, S.C.); III: testosterone-induced BPH treated with 2ME (50 mg/kg); IV: testosterone-induced BPH treated with 2ME (100 mg/kg). (b) Densitometric quantitation of HIF-1α. (c) Densitometric quantitation of p-Smad2. Data are expressed as mean ± SD (n = 3). a or b: statistically significant from control or testosterone group, respectively, at P < 0.05 using one-way ANOVA followed by Tukey's post hoc test.

Figure 6

Schematic presentation of the proposed mechanisms of the 2ME protective effects against testosterone-induced prostatic hyperplasia in rats.