Antagonists of growth hormone-releasing hormone inhibit proliferation induced by inflammation in prostatic epithelial cells

Abstract

The etiology of benign prostatic hyperplasia (BPH) is multifactorial, and chronic inflammation plays a pivotal role in its pathogenesis. Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that has been shown to act as paracrine/autocrine factor in various malignancies including prostate cancer. GHRH and its receptors are expressed in experimental models of BPH, in which antagonists of GHRH suppressed the levels of proinflammatory cytokines and altered the expression of genes related to epithelial-to-mesenchymal transition (EMT). We investigated the effects of GHRH antagonist on prostatic enlargement induced by inflammation. Autoimmune prostatitis in Balb/C mice was induced by a homogenate of reproductive tissues of male rats. During the 8-wk induction of chronic prostatitis, we detected a progressive increase in prostatic volume reaching 92% at week 8 compared with control (P < 0.001). Daily treatment for 1 mo with GHRH antagonist MIA-690 caused a 30% reduction in prostate volume (P < 0.05). Conditioned medium derived from macrophages increased the average volume of spheres by 82.7% (P < 0.001) and elevated the expression of mRNA for N-cadherin, Snail, and GHRH GHRH antagonist reduced the average volume of spheres stimulated by inflammation by 75.5% (P < 0.05), and TGF-β2 by 91.8% (P < 0.01). The proliferation of primary epithelial cells stimulated by IL-17A or TGF-β2 was also inhibited by 124.1% and 69.9%, respectively. GHRH stimulated the growth of BPH-1 and primary prostate spheres. This study provides evidence that GHRH plays important roles in prostatic inflammation and EMT and suggests the merit of further investigation to elucidate the effects of GHRH antagonists in prostatitis and BPH.

Keywords: chronic prostatic inflammation; experimental autoimmune prostatitis; neuropeptide; prostatic hyperplasia; targeted therapy.

Fig. 1.

GHRH antagonists MIA-690 and JV-I-38 reduce prostate enlargement in EAP. (A) GHRH and GHRHRs are localized mainly in the epithelial cells of the ventral prostate of Balb/C mice, as shown by immunohistochemistry. (Scale bar: 100 µm.) (B) Representative images showing volumetric changes in the ventral prostate detected by VEVO 1100 US imaging system. EAP was induced by two immunizations with a homogenate of rat male tissue injected 2 wk apart. Ultrasound images were recorded before the first injection (baseline) and 2 and 8 wk after the second injection. (C) Average volume of ventral prostates in EAP and control animals before and 2, 4, and 8 wk after the induction. (D) H&E staining shows leukocyte infiltrate in the ventral prostate of the EAP model (arrow) and no signs of inflammation in the control tissue. (Scale bar: 100 µm.) (E and F) GHRH antagonists reduce inflammation-induced enlargement of the ventral prostate in EAP. As depicted by the scheme, mice were treated with GHRH antagonists MIA-690 or JV-I-38 at 5 µg/d for 4 wk starting 8 wk after the induction of EAP and compared with vehicle-treated animals. Representative ultrasound images (E) and average volumes of the ventral prostates (F) are shown 8 and 12 wk after the induction of EAP. (G) Western blot analysis of the expression of phospho-NF-κβ p65, NF-κβ p65, and prepro-GHRH in ventral prostates from control, EAP, and EAP treated with MIA-690. GAPDH or β-actin were used as standard genes (*P < 0.05, **P < 0.01, and ***P < 0.01 by Student’s t test).

Fig. 2.

GHRH antagonists inhibit proliferation and EMT induced by inflammation in 3D cell culture in BPH-1 cells and in PrEp cells. (A) GHRH and GHRHRs are expressed in BPH-1 cells and in PrEp cells as detected by immunocytochemistry. (Scale bar: 100 µm.) (B) In vitro model of inflammation-induced proliferation: BPH-1 cells were treated with conditioned medium derived from differentiated THP-1 macrophages that are cocultured with BPH-1 cells (BPH/THP) for 8 d. Control cells were incubated in medium derived from BPH-1 cells only (BPH). Representative images of BPH-1 spheres are shown. (Scale bar: 100 µm.) The average volume of spheres was determined from three parallel experiments. (C) Expression of protein for GHRH and (D) the expression of mRNA for N-cadherin, Snail, and GHRH are increased in cells treated with BPH/THP medium compared with control as determined by Western blot and quantitative PCR, respectively. (E) Representative images of Western blot analysis of the expression of N-cadherin, E-cadherin, and GAPDH in 3D cultures from BPH, BPH/THP, or BPH/THP with 100 nM treatment. (F and G) GHRH antagonist MIA-690 added at 30 nM or 100 nM concentration to BPH/THP medium suppresses the stimulatory action of macrophage-conditioned medium on the growth of BPH-1 spheres. (Scale bar: 100 µm.) (H) The effect of IL-17A on the growth of PrEp cells was tested at 0.1 ng/mL, 1 ng/mL, and 10 ng/mL. (I and J) GHRH antagonist MIA-690 inhibits growth of PrEp spheres stimulated by IL-17A. Cultures were treated with 0.1 ng/mL IL-17A alone or with MIA-690 at 30-nM or 100-nM concentrations. (Scale bar 100 µm.) *P < 0.05, **P < 0.01, and ***P < 0.01 by Student’s t test.

Fig. S1.

GHRH antagonists inhibit proliferation and EMT induced by TGF-β2 in prostate epithelial cells. (A) TGF-β2, but not TGF-β1, promotes the growth of BPH-l spheres. TGF-β1 and TGF-β2 were tested at 0.1 ng/mL and 1 ng/mL for 8 d. (B and C) GHRH antagonist MIA-690 at 30-nM or 100-nM concentrations suppressed the growth of BPH-1 spheres induced by 1 ng/mL TGF-β2. (Scale bar: 100 µm.) (D) The expression of GHRH protein is up-regulated by treatment with TGF-β2 at 1 ng/mL concentration in BPH-1 cells grown in 3D culture. (E) The expression of N-cadherin increased by TGF-β2 treatment is reduced by MIA-690 as determined by Western blot. E-cadherin levels were not affected by MIA-690. (F) The effect of TGF-β2 on the growth of BPH-1 spheres is reduced when the expression of GHRHR is silenced. The levels of pituitary-type GHRHR (pGHRH) and its splice variant 1 (SV1) are down-regulated by the stable transfection of shRNA in psi-H1 vector as determined by Western blot. (G) The growth of PrEp spheres is stimulated by TGF-β1 and TGF-β2. Cells were stimulated with 0.1 ng/mL TGF-β1 or 0.1 ng/mL or 1 ng/mL TGF-β2 for 8 d. (H) GHRH antagonist MIA-690 inhibits the effect of TGF-β2 on the growth of PrEp spheres. (I) GHRH(1-29)NH₂ at concentrations of 0.01 nM and 0.1 nM significantly increased the average diameter of BPH-1 spheres, but had no effect at 1 nM. (J) The average diameter of PrEp spheres was stimulated by 0.1 nM GHRH(1-29)NH₂. (K) GHRH(1-29)NH₂ at 0.1 nM up-regulated the expression of N-cadherin, whereas it did not affect the levels of E-cadherin in BPH-1 cells. *P < 0.05, **P < 0.01, and ***P < 0.01 by Student's t test.