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.