P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

Abstract

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

Inhibition of human prostate smooth muscle contraction by the LIM kinase inhibitors, SR7826 and LIMKi3

“…Similarly, PSA was detected in almost all prostate samples included to these analyses, with very obvious variations of PSA content between prostates from different patients (Figure B). Together, this may reflect individual variations, and/or different degree of hyperplasia, as recently described (Kunit et al ., ; Wang et al ., ; ,b). Calponin, but not pan‐cytokeratin, was detectable in WPMY‐1 cells (Figure C), confirming the smooth muscle‐like phenotype of these cells (Wang et al ., ).…”

“…EC 50 values in organ bath experiments and in intact tissues may be higher than IC 50 values in biochemical assays, for example, due to divergent access of inhibitors to their targets resulting from barriers like membranes or connective tissue (Vauquelin et al ., ; Swinney, ; Kunit et al ., ; Wang et al ., ). In fact, EC 50 values for biological effects of LIMK inhibitors in previous studies were higher than IC 50 values in kinase assays, so that migration of prostate cancer cells was inhibited by 100 nM SR7826 by 16% and by 74% by 1 μM (Yin et al ., ).…”

“…This requires a more precise understanding of the molecular mechanisms involved in prostate smooth muscle contraction. However, the recent discovery of previously unknown mechanisms demonstrated that prostate smooth muscle contraction is still insufficiently understood (Strittmatter et al ., ; Hennenberg et al ., ; Kunit et al ., ; Wang et al ., ; Wang et al ., ,b). Thus, our present findings may contribute to a better understanding of smooth muscle contraction in the prostate.…”

Effect of PAK Inhibition on Cell Mechanics Depends on Rac1

“…It has been reported that the IPA3 inhibitor has a lower potency compared to ATP-competitive inhibitors, such as FRAX597 (Kim et al, 2016;Semenova and Chernoff, 2017). Other studies used also a 10-fold difference between these two inhibitors (Wang et al, 2016). Cells were cultured 72 h, fixated with 2.5% glutaraldehyde solution (Serva, Heidelberg, Germany) and stained with 4 µg/ml of the Hoechst 33342 dye (Serva, Heidelberg, Germany).…”

Smooth muscle contraction and growth of stromal cells in the human prostate are both inhibited by the Src family kinase inhibitors, AZM475271 and PP2

“…This link may include SFKs, which may affect contractility and growth concurrently by regulation of actin dynamics. Notably, other compounds may affect contraction and growth at once in the prostate as well, including inhibitors for Rho kinase, Rac GTPases or p21‐activated kinases, what may additionally point to this link (Rees et al, ; Wang et al, ; Wang et al, ). Together, the idea of such a connection may appear attractive, as both processes, that is contraction and growth, are important targets for medical therapy of BOO (Oelke et al, ).…”