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

Wang, Yiming; Gratzke, Christian; Tamalunas, Alexander; Nicolas, Wiemer; Ciotkowska, Anna; Rutz, Beata; Waidelich, Raphaela; Strittmatter, Frank; Liu, Chunxiao; Stief, Christian G.; Hennenberg, Martin

doi:10.1371/journal.pone.0153312

Cited by 30 publications

(42 citation statements)

References 73 publications

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“…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 ., ).…”

Section: Resultsmentioning

confidence: 96%

“…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 ., ).…”

Section: Discussionmentioning

confidence: 97%

“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Gratzke

Wang

et al. 2018

British J Pharmacology

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BACKGROUND AND PURPOSEIn men with benign prostatic hyperplasia, increased smooth muscle tone in the prostate may lead to bladder outlet obstruction and subsequent lower urinary tract symptoms. Consequently, medical treatment aims to inhibit prostate smooth muscle contraction. However, the efficacy of the treatment options available is limited, and improved understanding of mechanisms of prostate smooth muscle contraction and identification of new targets for medical intervention are mandatory. Several studies suggest that LIM kinases (LIMKs) promote smooth muscle contraction; however, this has not yet been examined. Here, we studied effects of the LIMK inhibitors on prostate smooth muscle contraction. EXPERIMENTAL APPROACHHuman prostate tissues were obtained from radical prostatectomy. Phosphorylation of cofilin, a LIMK substrate, was examined using a phospho-specific antibody. Smooth muscle contractions were studied in organ bath experiments. KEY RESULTSReal-time PCR, Western blot and immunofluorescence suggested LIMKs are expressed in smooth muscle cells of prostate tissues. Two different LIMK inhibitors, SR7826 (1 μM) and LIMKi3 (1 μM), inhibited contractions of prostate strips, which were induced by electrical field stimulation, α 1 -adrenoceptor agonists phenylephrine and methoxamine and the TXA 2 analogue, U46619. LIMK inhibition in prostate tissues and cultured stromal cells (WPMY-1) was confirmed by cofilin phosphorylation, which was reduced by SR7826 and LIMKi3. In WPMY-1 cells, SR7826 and LIMKi3 caused breakdown of actin filaments and reduced viability. CONCLUSIONS AND IMPLICATIONSSmooth muscle tone in the hyperplastic human prostate may underlie the effects of LIMKs, which promote contraction. Contraction of prostate strips can be inhibited by small molecule LIMK inhibitors.

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Section: Resultsmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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

Gratzke

Wang

et al. 2018

British J Pharmacology

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“…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).…”

Section: Analysis Of 3d Motility Within Extracellular Matrix Scaffoldsmentioning

confidence: 99%

Effect of PAK Inhibition on Cell Mechanics Depends on Rac1

Mierke

Puder

Aermes

et al. 2020

Front. Cell Dev. Biol.

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Besides biochemical and molecular regulation, the migration and invasion of cells is controlled by the environmental mechanics and cellular mechanics. Hence, the mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the migratory capacity in confined 3D extracellular matrices. Recently, we have shown that the migratory and invasive capacity of mouse embryonic fibroblasts depends on the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration and invasion through the polymerization of actin filaments. Since Rac1 deficient cells become mechanically softer than controls, we investigated the effect of group I PAKs and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore, we determined whether mouse embryonic fibroblasts, in which Rac1 was knockedout, and control cells, displayed cell mechanical alterations after treatment with group I PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1 inhibition decreased the stiffness and the Young's modulus of fibroblasts in the presence of Rac1 independent of their adhesive state. However, in the absence of Rac1 the effect was abolished in the adhesive cell state for both inhibitors and in their nonadhesive state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3 inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I (PAK1, 2, and 3) as major players in cell mechanics.

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“…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, ).…”

Section: Discussionmentioning

confidence: 99%

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

Wang

Gratzke

Tamalunas

et al. 2016

British J Pharmacology

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Background and Purpose In benign prostatic hyperplasia, increased prostate smooth muscle tone and prostate volume may contribute alone or together to urethral obstruction and voiding symptoms. Consequently, it is assumed there is a connection between smooth muscle tone and growth in the prostate, but any molecular basis for this is poorly understood. Here, we examined effects of Src family kinase (SFK) inhibitors on prostate contraction and growth of stromal cells. Experimental Approach SFK inhibitors, AZM475271 and PP2, were applied to human prostate tissues to assess effects on smooth muscle contraction, and to cultured stromal (WPMY‐1) and c‐Src‐deficient cells to examine effects on proliferation, actin organization and viability. Key Results SFKs were detected by real time PCR, western blot and immunofluorescence in human prostate tissues, some being located to smooth muscle cells. AZM475271 (10 μM) and PP2 (10 μM) inhibited SFK in prostate tissues and WPMY‐1 cells. Both inhibitors reduced α1‐adrenoceptor‐mediated and neurogenic contraction of prostate strips. This may result from cytoskeletal deorganization, which was observed in response to AZM475271 and PP2 in WPMY‐1 cells by staining of actin filaments with phalloidin. This was paralleled by reduced proliferation of wildtype but not of c‐Src‐deficient cells; cytotoxicity was mainly observed at higher concentrations (>50 μM). Conclusions and Implications In human prostate, smooth muscle tone and growth are both controlled by an SFK‐dependent process, which may explain their common role in bladder outlet obstruction. Targeting prostate smooth muscle tone and prostate growth simultaneously by a single compound may, in principal, be possible.

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

Cited by 30 publications

References 73 publications

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

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

Effect of PAK Inhibition on Cell Mechanics Depends on Rac1

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

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