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. 2015 Jun;172(11):2905-17.
doi: 10.1111/bph.13099. Epub 2015 May 5.

Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864

Y Wang  1   2 , T Kunit  1   3 , A Ciotkowska  1 , B Rutz  1 , A Schreiber  1 , F Strittmatter  1 , R Waidelich  1 , C Liu  2 , C G Stief  1 , C Gratzke  1 , M Hennenberg  1
Affiliations

Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864

Y Wang et al. Br J Pharmacol. 2015 Jun.

Abstract

Background and purpose: Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the control of smooth muscle tone in human prostates and growth of prostate stromal cells.

Experimental approach: Experiments were performed using human prostate tissues from radical prostatectomy and cultured stromal cells (WPMY-1). Expression of Rac was examined by Western blot and fluorescence staining. Effects of Rac inhibitors (NSC23766 and EHT1864) on contractility were assessed in the organ bath. The effects of Rac inhibitors were assessed by pull-down, cytotoxicity using a cell counting kit, cytoskeletal organization by phalloidin staining and cell growth using an 5-ethynyl-2'-deoxyuridine assay.

Key results: Expression of Rac1-3 was observed in prostate samples from each patient. Immunoreactivity for Rac1-3 was observed in the stroma, where it colocalized with the smooth muscle marker, calponin. NSC23766 and EHT1864 significantly reduced contractions of prostate strips induced by noradrenaline, phenylephrine or electrical field stimulation. NSC23766 and EHT1864 inhibited Rac activity in WPMY-1 cells. Survival of WPMY-1 cells ranged between 64 and 81% after incubation with NSC23766 (50 or 100 μM) or EHT1864 (25 μM) for 24 h. NSC23766 and EHT1864 induced cytoskeletal disorganization in WPMY-1 cells. Both inhibitors impaired the growth of WPMY-1 cells.

Conclusions and implications: Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible.

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Figures

Figure 1
Figure 1
RT-PCR and Western blot analysis of human prostate tissues. (A) Detection of mRNA of Rac isoforms 1–3 by RT-PCR in periurethral prostate tissues from n = 7 patients. Expression of Rac is referred to as expression of 18SrRNA. Shown are values for each sample (ratios of Ct values), and the median for each target. Note: the higher the value, the lower the expression of the target. (B) Periurethral prostate tissues from n = 7 patients were subjected to Western blotting using antibodies for Rac isoforms 1–3, calponin (smooth muscle marker), pan-cytokeratin (marker for epithelium/glands), PSA (marker for BPH), and β-actin (housekeeping, loading control).
Figure 2
Figure 2
Fluorescence staining of human prostate tissue. Tissues were double-labelled using isoform-specific antibodies for Rac isoforms 1–3, and calponin (left panels) or pan-cytokeratin (right panels). Yellow colour indicates colocalization of immunoreactivities. Shown are representative stainings from a series of tissues from n = 6 patients, with similar results.
Figure 3
Figure 3
Inhibition of prostate contraction by the Rac inhibitors, NSC23766 (A) and EHT1864 (B). In an organ bath, human prostate strips were exposed for 30 min to NSC23766 (100 μM) or solvent (ethanol) as a control for NSC23766, or for 30 min to EHT1864 (100 μM) or solvent (water) as a control for EHT1864. Subsequently, concentration–response curves for noradrenaline or the α1-adrenoceptor agonist phenylephrine were constructed, as well as EFS-induced frequency–response curves. Data are means (±SEM) from experiments with prostate tissues from n = 5 (noradrenaline/NSC23766), n = 9 (phenylephrine/NSC23766), n = 8 (EFS/NSC23766), n = 10 (noradrenaline/EHT1864), n = 7 (phenylephrine/EHT1864), or n = 6 (EFS/EHT1864) patients (#P < 0.05).
Figure 4
Figure 4
WPMY-1 cells: characterization (A), and effects of Rac inhibitors on Rac1 and RhoA activity (B) and on MLC and PAK phosphorylation (C). (A) Western blot analysis of three independent samples was performed using antibodies for different markers and α1A-adrenoceptors, for Rac isoforms 1–3, for PAK1, and β-actin. (B) Cells were treated with NSC23766 (100 μM, 1 h), EHT1864 (100 μM, 1h), or solvent (DMSO, 1 h), and subsequently subjected to pull-down assays for assessment of Rac1 or RhoA activity. (C) Cells were treated with NSC23766 (100 μM, 1 h), EHT1864 (100 μM, 1h), or solvent (DMSO, 1 h), and subsequently subjected to Western blot analysis using phospho-specific and non-phospho-specific MLC and PAK antibodies. Shown are representative blots from three independent experiments, with similar results.
Figure 5
Figure 5
Assessment of cytotoxicity of Rac inhibitors in WPMY-1 cells. Cells were treated with different concentrations of NSC23766 (50, 100 μM) or EHT1864 (25, 100 μM), or solvent (DMSO) for different periods (24, 48 or 72 h), and subjected to the CCK assay for assessment of survival. Absorbance was measured at 450 nm; lower values indicate lower survival. Shown are means ± SEM from three independent experiments for each setting.
Figure 6
Figure 6
Phalloidin staining of WPMY-1 cells. Cells were treated with different concentrations of NSC23766 (50, 100 μM) or EHT1864 (25, 100 μM), or solvent (DMSO) for different periods (24 or 48 h), and subsequently stained with FITC-labelled phalloidin (red fluorescence) and DAPI (blue fluorescence). Shown are representative stainings from three independent experiments.
Figure 7
Figure 7
EdU assay in WPMY-1 cells. Cells were treated with NSC23766 (100 μM), EHT1864 (25 μM), or solvent (DMSO) for 24, 48 or 72 h, and subsequently subjected to the EdU assay. Proliferation is indicated by red nuclei (pink in overlay) in EdU staining. The quantified results (means ± SEM), and representative stainings are shown from three independent experiments.
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