Silencing of CDC42 inhibits contraction and growth-related functions in prostate stromal cells, which is mimicked by ML141.

BACKGROUND: Prostate smooth muscle contraction and stromal growth may contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia, but are incompletely understood. A role of the monomeric GTPase CDC42 for smooth muscle contraction and proliferation appears possible, but is unknown for the prostate. Here, we silenced CDC42 expression in prostate stromal cells (WPMY-1), and examined contractility, growth-related functions and responses to the presumed CDC42 inhibitor, ML141. METHODS: WPMY-1 cells were transfected with scrambled or CDC42-specific siRNA, and characterized for GTPase activities, contraction, proliferation, colony formation, apoptosis, cell death and viability. Effects of ML141 were examined in cells with and without silencing. RESULTS: CDC42 silencing was confirmed by reduced mRNA and protein expression, and reduced CDC42 activity. Silencing impaired contraction (23-47 %), actin organization (25 %), proliferation (17-63 %), colony formation and viability (64-89 %), and increased the percentage of dead cells (2.6-fold). ML141 mimicked the phenotype of silencing in scrambled siRNA-transfected controls, and in non-transfected WPMY-1 cells, including inhibition of contraction, proliferation, colony formation and viability, breakdown of actin organization and increased cell death. In CDC42-silenced cells, ML141 still affected phalloiding organization, proliferation and cell death, with effect sizes resembling controls without silencing. ML141 inhibited RhoA activity in CDC42-silenced cells, but not in cells without silencing. CONCLUSIONS: CDC42 promotes contraction of prostate stromal cells, and drives stromal growth by CDC42-mediated proliferation and suppression of apoptosis-independent cell death. ML141 mimicks all effects of CDC42 silencing, but its specificity may be limited and depends on GTPase phenotypes of cells.

Antagonism of α1-adrenoceptors by ß3-adrenergic agonists: Structure-function relations of different agonists in prostate smooth muscle contraction.

Effects of ß3-adrenergic agonists on prostate smooth muscle contraction are poorly characterized, although mirabegron is used for treatment of lower urinary tract symptoms. Off-target effects of several ß3-adrenergic agonists include antagonism of α1-adrenoceptors. Proposed, but unconfirmed explanations include phenylethanolamine backbones, found in some ß3-adrenergic agonists and imparting interaction with catecholamine binding pockets of adrenoceptors. Here, we examined effects of ß3-adrenergic agonists on contractions of human prostate tissues, including ZD7114 (without phenylethanolamine moiety), ZD2079 (phenylethanolamine backbone), BRL37344 and CL316243 (chloride-substituted phenylethanolamine deriatives). Prostate tissues were obtained from radical prostatectomy. Contractions by α1-adrenergic agonists and electric field stimulation (EFS) were studied in an organ bath. ZD7114 (10 µM) right-shifted concentration responses curves for α1-adrenergic agonists, resulting in increased EC50 values for phenylephrine, methoxamine and noradrenaline up to one magnitude, without affecting Emax values. ZD7114 (10 µM) inhibited EFS-induced contractions, resulting in reduced Emax values. All effects of ZD7114 were resistant to the ß3-adrenergic antagonist L-748337, including increases in EC50 values for α1-adrenergic agonists, up to more than two magnitudes. Using 10 µM, neither ZD2079, BRL37344 or CL316243 affected α1-adrenergic or EFS-induced contractions. At escalated concentrations, BRL37344 (200 µM) right-shifted concentration response curves for phenylephrine, increased EC50 values for phenylephrine, and inhibited EFS-induced contractions, while CL316243 (300 µM) did not affect phenylephrine- or EFS-induced contractions. In conclusion, phenylethanolamine backbones are not decisive to impart α1-adrenoceptor antagonism to ß3-agonists. Effects of ß3-adrenergic agonists on prostate smooth muscle contraction are limited to off-target effects, including α1-adrenoceptor antagonism by ZD7114 and BRL37344.