WPMY-1 Cells Research Articles

Astaxanthin Alleviates Chronic Prostatitis via the ERK1/2 Signaling Pathway: Evidence from Network Pharmacology and Experimental Validation.

Astaxanthin (AST) has been widely recognized for its therapeutic potential in chronic inflammatory ailments. This study investigates the therapeutic efficacy and underlying mechanisms of AST in the management of chronic prostatitis (CP). Male Sprague-Dawley (SD) rats were randomly divided into control, complete Freund's adjuvant (CFA), and CFA + AST groups. CFA was used to induce the CP model, and saline was used for the control group. Inflammation of the prostate was detected 28 days after oral administration of AST. qRT-PCR and ELISA were used to detect pro-inflammatory factors in RWPE-1 and WPMY-1 cells. Potential targets of AST for CP were explored by network pharmacology, and related proteins were detected by Western blotting. Oral administration of AST alleviated the increase in prostate stroma and reduced inflammatory cell infiltration in CP rats. The IC50 of AST-treated RWPE-1 and WPMY-1 cells for 48 h were 171 and 212.1 μM, respectively. AST pretreatment reduced IL-6 and IL-8 expression in these cells. PPI network, GO, and KEGG enrichment analyses suggested that the antiinflammatory effect of AST was associated with the ERK1/2 pathway. Western blotting showed that AST inhibited ERK1/2 phosphorylation. In addition, AST and ERK1/2 pathway inhibitors (U0126) synergistically inhibited LPS-induced inflammation in prostate cells. Our study identified the potential of AST in the treatment of CP. However, subsequent randomized controlled trials are needed to validate its clinical efficacy.

Cucurbitacin B Inhibits the Proliferation of WPMY-1 Cells and HPRF Cells via the p53/MDM2 Axis.

Modern research has shown that Cucurbitacin B (Cu B) possesses various biological activities such as liver protection, anti-inflammatory, and anti-tumor effects. However, the majority of research has primarily concentrated on its hepatoprotective effects, with limited attention devoted to exploring its potential impact on the prostate. Our research indicates that Cu B effectively inhibits the proliferation of human prostate stromal cells (WPMY-1) and fibroblasts (HPRF), while triggering apoptosis in prostate cells. When treated with 100 nM Cu B, the apoptosis rates of WPMY-1 and HPRF cells reached 51.73 ± 5.38% and 26.83 ± 0.40%, respectively. In addition, the cell cycle assay showed that Cu B had a G2/M phase cycle arrest effect on WPMY-1 cells. Based on RNA-sequencing analysis, Cu B might inhibit prostate cell proliferation via the p53 signaling pathway. Subsequently, the related gene and protein expression levels were measured using quantitative real-time PCR (RT-qPCR), immunocytochemistry (ICC), and enzyme-linked immunosorbent assays (ELISA). Our results mirrored the regulation of tumor protein p53 (TP53), mouse double minute-2 (MDM2), cyclin D1 (CCND1), and thrombospondin 1 (THBS1) in Cu B-induced prostate cell apoptosis. Altogether, Cu B may inhibit prostate cell proliferation and correlate to the modulation of the p53/MDM2 signaling cascade.

Diospyros rhodocalyx Kurz induces mitochondrial-mediated apoptosis via BAX, Bcl-2, and caspase-3 pathways in LNCaP human prostate cancer cell line.

Prostate cancer (PCa) is one of the causes of death in men worldwide. Although treatment strategies have been developed, the recurrence of the disease and consequential side effects remain an essential concern. Diospyros rhodocalyx Kurz, a traditional Thai medicine, exhibits diverse therapeutic properties, including anti-cancer activity. However, its anti-cancer activity against prostate cancer has not been thoroughly explored. This study aims to evaluate the anti-cancer activity and underlying mechanisms of the ethyl acetate extract of D. rhodocalyx Kurz (EADR) related to apoptosis induction in the LNCaP human prostate cancer cell line. Ethyl acetate was employed to extract the dried bark of D. rhodocalyx Kurz. The cytotoxicity of EADR on both LNCaP and WPMY-1 cells (normal human prostatic myofibroblast cell line) was evaluated using MTS assay. The effect of EADR on the cell cycle, apoptosis induction, and alteration in mitochondrial membrane potential (MMP) was assessed by the staining with propidium iodide (PI), Annexin V-FITC/PI, and JC-1 dye, respectively. Subsequent analysis was conducted using flow cytometry. The expression of cleaved caspase-3, BAX, and Bcl-2 was examined by Western blotting. The phytochemical profiling of the EADR was performed using gas chromatography-mass spectrometry (GC-MS). EADR exhibited a dose-dependent manner cytotoxic effect on LNCaP cells, with IC50 values of 15.43 and 12.35 µg/mL after 24 and 48 h, respectively. Although it also exhibited a cytotoxic effect on WPMY-1 cells, the effect was comparatively lower, with the IC50 values of 34.61 and 19.93 µg/mL after 24 and 48 h of exposure, respectively. Cell cycle analysis demonstrated that EADR did not induce cell cycle arrest in either LNCaP or WPMY-1 cells. However, it significantly increased the sub-G1 population in LNCaP cells, indicating a potential induction of apoptosis. The Annexin V-FITC/PI staining indicated that EADR significantly induced apoptosis in LNCaP cells. Subsequent investigation into the underlying mechanism of EADR-induced apoptosis revealed a reduction in MMP as evidenced by JC-1 staining. Moreover, Western blotting demonstrated that EADR treatment resulted in the upregulation of BAX, downregulation of BCL-2, and elevation of caspase-3 cleavage in LNCaP cells. Notably, the epilupeol was a prominent compound in EADR as identified by GC-MS. The EADR exhibits anti-cancer activity against the LNCaP human prostate cancer cell line by inducing cytotoxicity and apoptosis. Our findings suggest that EADR promotes apoptosis by upregulating pro-apoptotic BAX, whereas downregulation of anti-apoptotic Bcl-2 results in the reduction of MMP and the activation of caspase-3. Of particular interest is the presence of epilupeol, a major compound identified in EADR, which may hold promise as a candidate for the development of therapeutic agents for prostate cancer.

Hispidin Increases Cell Apoptosis and Ferroptosis in Prostate Cancer Cells Through Phosphatidylinositol-3-Kinase and Mitogen-activated Protein Kinase Signaling Pathway.

Chemotherapy is mainly used in the clinical treatment of prostate cancer. Different anticancer mechanisms can induce cell death in various cancers. Reactive oxygen species (ROS) play crucial roles in cell proliferation, differentiation, apoptosis, and signal transduction. It is widely accepted that ROS accumulation is closely related to chemical drug-induced cancer cell death. We utilized the MTT assay to detect changes in cell proliferation. Additionally, colony formation and wound healing assay were conducted to investigate the effect of hispidin on cell colony formation and migration ability. Fluorescence microscopy was used to detect intracellular and mitochondrial ROS levels, while western blot was used for detection of cell apoptosis. Hispidin treatment significantly decreased viability of PC3 and DU145 cancer cells but exhibited no cytotoxicity in WPMY-1 cells. Furthermore, hispidin treatment inhibited cell migration and colony formation and triggered cellular and mitochondrial ROS accumulation, leading to mitochondrial dysfunction and mitochondrion-dependent apoptosis. Moreover, hispidin treatment induced ferroptosis in PC3 cells. Scavenging of ROS with N-acetyl cysteine significantly inhibited hispidin-induced apoptosis by altering the expression of apoptosis-related proteins, such as cleaved caspase-3, 9, Bax, and Bcl2. Furthermore, hispidin treatment dramatically up-regulated MAPK (involving p38, ERK, and JNK proteins) and NF-kB signaling pathways while down-regulating AKT phosphorylation. Hispidin treatment also inhibited ferroptosis signaling pathways (involving P53, Nrf-2, and HO-1 proteins) in PC3 cells. In addition, inhibiting these signaling pathways via treatment with specific inhibitors significantly reversed hispidin-induced apoptosis, cellular ROS levels, mitochondrial dysfunction, and ferroptosis. Hispidin may represent a potential candidate for treating prostate cancer.

Abstract 1475: Investigating the carcinogenic effects and the mechanism of cadmium exposure on WPMY-1 prostate myofibroblast cell line

Abstract Prostate cancer (PCa) is the second most common cancer among males with an incidence rate of PCa is 288,300 new cases annually. Risk factors include age, genetics, and lifestyle, but the underlying stimuli remain largely uncertain. One potential emerging cause of PCa is exposure to metal carcinogens as cadmium (Cd) which is now classified as a group I human carcinogen. Chronic exposure to Cd on epithelial cells has been extensively shown to induce carcinogenesis, making it a likely contributor to PCa development. In this study, we expand this information by examining how chronic Cd exposure affects prostate-derived myofibroblast (WPMY-1) cells transforming into a cancer-associated fibroblast like phenotype. To study the transformation, WPMY-1 cells were treated with 500nM of Cd for a period of 11 months establishing the WPMY-1 Cd model. Fibroblasts are a crucial tumor microenvironment (TME) component and potentially contribute to PCa progression when transformed and activated into cancer-associated fibroblasts (CAFs). To begin, we used an MTT assay to measure the effect of Cd toxicity on normal prostate myofibroblasts versus WPMY-1 Cd. The data showed that 10µM Cd solution was toxic to WPMY-1 cells, but WPMY-Cd cells depicted high cell viability at 50 µM dose, conferring to chronic Cd exposure improved resiliency to Cd toxicity. We analyzed the expression of CAF-specific markers in WPMY-Cd cells, such as vimentin, FAP, α-SMA, and PDGFR-β. WPMY-Cd cells showed a distinct upregulation for these markers when compared to untreated WPMY-1 cells, suggesting that chronic exposure to Cd promotes CAF-like phenotypic transformation in WPMY-Cd. To further ascertain the cancer-promoting properties of WPMY-Cd, an in vitro 3-D spheroid assay was performed by growing the cells on a Matrigel scaffold. The WPMY-Cd cells formed many more tumor-like spheroid structures than WPMY-1 and exhibited sprouting. The sprouting spheroids displayed lamellipodium and filopodium-like structures, demonstrating the initiation of migratory phenotype in WPMY-Cd. Additionally, previous research suggests that CAFs may play a role in PCa progression by promoting tumor growth via the secretion of cytokines and growth factors. We tested this theory by exposing Cd-treated epithelial (BPH-Cd) cells to conditioned media containing secretions from CAFs and WPMY-Cd. WPMY-1 Cd conditioned media in conferring cell migration on epithelial BPH-Cd cells was confirmed by a wound healing assay wherein BPH-Cd cells treated with WPMY-1 Cd conditioned media led to complete wound closure in 24 hours as compared to WPMY-1 and CAF conditioned media. Overall, there is considerable evidence that Cd exposure in fibroblasts may play a crucial role in PCa disease progression and is a promising area for researching PCa treatment strategies against a crucial component of the tumor microenvironment. Citation Format: Sakshi V. Bansod, Gnanasekar Munarithinam. Investigating the carcinogenic effects and the mechanism of cadmium exposure on WPMY-1 prostate myofibroblast cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1475.

New fusidane-type nortriterpenoids from the Arctic marine-derived fungus Simplicillium lamellicola culture medium with their inhibitory effect on benign prostatic hyperplasia

Three new fusidane-type nortriterpenoids, simplifusinolide A, 24-epi simplifusinolide A, and simplifusidic acid L (1–3), were isolated from the EtOAc extract of the Arctic marine-derived fungus Simplicillium lamellicola culture medium, together with fusidic acid (4) and 16-O-deacetylfusicid acid (5). The structures of the isolated compounds were elucidated by NMR and MS analyses. The absolute configurations of compounds 1–3 were established by the quantum mechanical calculations of electronic circular dichroism and gauge-including atomic orbital NMR chemical shifts, followed by DP4 + analysis.Benign prostatic hyperplasia (BPH) is a major urological disorder in men worldwide. The anti-BPH potentials of the isolated compounds were evaluated using BPH-1 and WPMY-1 cells. Treatment with simplifusidic acid L (3) and fusidic acid (4) significantly downregulated the mRNA levels of the androgen receptor (AR) and its downstream effectors, inhibiting the proliferation of BPH-1 cells. Specifically, treatment with 24-epi simplifusinolide A (2) significantly suppressed the cell proliferation of both BPH-1 and DHT-stimulated WPMY-1 cells by inhibiting AR signaling. These results suggest the potential of 24-epi simplifusinolide A (2), simplifusidic acid L (3) and fusidic acid (4) as alternative agents for BPH treatment by targeting AR signaling.

Bioassay-guided isolation of two antiproliferative metabolites from Pterocarpus indicus Willd. against TGF-β-induced prostate stromal cells (WPMY-1) proliferation via PI3K/AKT signaling pathway.

Benign prostatic hyperplasia (BPH) is the enlargement of the prostate gland, primarily occurring in aging men, in which transforming growth factor-beta (TGF-β) plays a critical role in prostate cell hyperproliferation and leads to uncomfortable urinary symptoms in BPH patients. Pterocarpus indicus Willd. is well known for its ethnopharmacological applications for treating ailments such as diuresis and bladder stones. This study aimed to examine the effect of P. indicus extract (PI extract) on TGF-β-induced WPMY-1 cell proliferation, followed by bioassay-guided fractionation to isolate the active metabolites. Angolensin (Ang) and maackiain (Mac) were isolated from bioassay-guided fractionation. Network analysis was performed to investigate the potential mechanisms. Furthermore, network analysis of the Ang-Mac combination in BPH highlighted the potential top ten pathways, including PI3K/AKT signaling pathway. Accordingly, subsequent investigation focused on evaluating the effect of PI extract, Ang, Mac, and Ang-Mac combination on the expression of PCNA, p53, and PI3K/AKT protein localization and expression. Results revealed inhibition of cell proliferation in TGF-β-induced WPMY-1 cells, correlating with downregulated PCNA expression. While PI extract and Mac induced apoptosis via p53 upregulation, Ang and Ang-Mac combination did not significantly affect apoptosis through the p53 pathway. Additionally, both metabolites exhibited potent inhibition of p-PI3K and p-AKT protein localization and expression in the nucleus of TGF-β-induced WPMY-1 cells. This study suggests that PI extract, Ang, and Mac are promising compounds for treating BPH, as evidenced by in silico and in vitro studies. Additionally, Ang and Mac could be used to standardize PI extract in future investigations.

YAP1 Recognizes Inflammatory and Mechanical Cues to Exacerbate Benign Prostatic Hyperplasia via Promoting Cell Survival and Fibrosis.

Chronic prostatic inflammation promotes cell survival and fibrosis, leading to benign prostatic hyperplasia (BPH) with aggravated urinary symptoms. It is investigated whether yes-associated protein 1 (YAP1), an organ size controller and mechanical transductor, is implicated in inflammation-induced BPH. The correlation between YAP1 expression and fibrosis in human and rat BPH specimens is analyzed. Furthermore, the effects of YAP1 activation on prostatic cell survival and fibrosis, as well as the underlying mechanism, are also studied. As a result, total and nuclear YAP1 expression, along with downstream genes are significantly upregulated in inflammation-associated human and rat specimens. There is a significant positive correlation between YAP1 expression and the severity of fibrosis or clinical performance. YAP1 silencing suppresses cell survival by decreasing cell proliferation and increasing apoptosis, and alleviates fibrosis by reversing epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in prostatic BPH-1 and WPMY-1 cells. Mechanistically, inflammatory stimulus and rigid matrix stiffness synergistically activate the RhoA/ROCK1 pathway to provoke cytoskeleton remodeling, thereby promoting YAP1 activation to exacerbate BPH development. Overall, inflammation-triggered mechanical stiffness reinforcement activates the RhoA/ROCK1/F-actin/YAP1 axis, thereby promoting prostatic cell survival and fibrosis to accelerate BPH progression.

Inhibition of integrin receptors reduces extracellular matrix levels, ameliorating benign prostate hyperplasia

Although a high prevalence of benign prostate hyperplasia (BPH) has been documented, the risk factors are poorly understood. Metabolic syndrome increases the risk of BPH. Succinylation, a type of posttranslational modification, mostly targets metabolic processes. The level of succinylation was investigated in 4 BPH patients and 4 healthy controls. Additionally, 176 patients with BPH were analyzed by using pan-antisuccinyllysine antibody blotting. TMT-labeling proteomic and sc-RNAseq Cellchat analyses were employed to identify key signaling factors involved in the development of BPH. In vivo and in vitro experiments were used to confirm the role of integrin receptors. The global succinylation level in BPH was higher than that in the healthy prostate. Positive correlations of prostate volume with IHC score sand urodynamics testing were found in large clinical cohorts. The extracellular matrix (ECM), metabolic processes and immune signaling were involved in succinylation in BPH, as indicated by using TMT-labeling proteomic analysis, and this finding was also confirmed by sc-RNAseq CellChat analysis. The proteins upregulated in SIRT5 knockout WPMY-1 cells were also enriched in the extracellular matrix and metabolic processes. More importantly, integrin receptor inhibition in a mouse model of BPH significantly ameliorated prostate hyperplasia. High levels of succinylation modifications were found in BPH, and succinylated proteins influenced the activation of the ECM. Inhibition of ECM signaling further ameliorated prostate hyperplasia in mice.

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

BackgroundProstate 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. MethodsWPMY-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. ResultsCDC42 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. ConclusionsCDC42 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.

Neferine attenuates development of testosterone-induced benign prostatic hyperplasia in mice by regulating androgen and TGF-β/Smad signaling pathways

Benign prostatic hyperplasia (BPH) is a common urinary disease among the elderly, characterized by abnormal prostatic cell proliferation. Neferine is a dibenzyl isoquinoline alkaloid extracted from Nelumbo nucifera and has antioxidant, anti-inflammatory and anti-prostate cancer effects. The beneficial therapeutic effects and mechanism of action of neferine in BPH remain unclear.A mouse model of BPH was generated by subcutaneous injection of 7.5 mg/kg testosterone propionate (TP) and 2 or 5 mg/kg neferine was given orally for 14 or 28 days. Pathological and morphological characteristics were evaluated. Prostate weight, prostate index (prostate/body weight ratio), expression of type Ⅱ 5α-reductase, androgen receptor (AR) and prostate specific antigen were all decreased in prostate tissue of BPH mice after administration of neferine. Neferine also downregulated the expression of pro-caspase-3, uncleaved PARP, TGF-β1, TGF-β receptor Ⅱ (TGFBR2), p-Smad2/3, N-cadherin and vimentin. Expression of E-cadherin, cleaved PARP and cleaved caspase-3 was increased by neferine treatment.1–100 μM neferine with 1 μM testosterone or 10 nM TGF-β1 were added to the culture medium of the normal human prostate stroma cell line, WPMY-1, for 24 h or 48 h. Neferine inhibited cell growth and production of reactive oxygen species (ROS) in testosterone-treated WPMY-1 cells and regulated the expression of androgen signaling pathway proteins and those related to epithelial-mesenchymal transition (EMT). Moreover, TGF-β1, TGFBR2 and p-Smad2/3, N-cadherin and vimentin expression were increased but E-cadherin was decreased after 24 h TGF-β1 treatment in WPMY-1 cells. Neferine reversed the effects of TGF-β1 treatment in WPMY-1 cells. Neferine appeared to suppress prostate growth by regulating the EMT, AR and TGF-β/Smad signaling pathways in the prostate and is suggested as a potential agent for BPH treatment.

Inhibition of growth and contraction in human prostate stromal cells by silencing of NUAK1 and -2, and by the presumed NUAK inhibitors HTH01-015 and WZ4003.

Background: NUAKs promote myosin light chain phosphorlyation, actin organization, proliferation and suppression of cell death in non-muscle cells, which are critical for smooth muscle contraction and growth. In benign prostatic hyperplasia (BPH), contraction and growth in the prostate drive urethral obstruction and voiding symptoms. However, a role of NUAKs in smooth muscle contraction or prostate functions are unknown. Here, we examined effects of NUAK silencing and the presumed NUAK inhibitors, HTH01-015 and WZ4003 on contraction and growth-related functions in prostate stromal cells (WPMY-1) and in human prostate tissues. Methods: Effects of NUAK1 and -2 silencing, HTH01-015 and WZ4003 on matrix plug contraction, proliferation (EdU assay, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin staining) were examined in cultured WPMY-1 cells. Effects of HTH01-015 and WZ4003 on smooth muscle contraction were assessed in organ bath experirments with human prostate tissues. Results: Effects of silencing were most pronounced on proliferation and cell death, resulting in decreases of proliferation rate by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls), decreases in Ki-67 by 75% and 77%, while numbers of dead cells after silencing of NUAK1 and NUAK2 amounted to 2.8 and 4.9 fold of scramble-transfected controls. Silencing of each isoform was paralleled by reduced viability, breakdown in actin polymerization, and partial decreases in contractility (maximally 45% by NUAK1 silencing, 58% by NUAK2 silencing). Effects of silencing were mimicked by HTH01-015 and WZ4003, with numbers of dead cells amounting up to 16.1 fold or 7.8 fold with HTH01-015 or WZ4003, compared to solvent-treated controls. Using concentrations of 500nM, neurogenic contractions of prostate tissues were inhibited partly by HTH01-015 and U46619-induced contractions were inhibited partly by HTH01-015 and WZ4003, while α1-adrenergic and endothelin-1-induced contractions remained unaffected. Using 10μM, inhibition of endothelin-1-induced contractions by both inhibitors and inhibition of α1-adrenergic contractions by HTH01-015 added to effects seen by 500nM. Conclusion: NUAK1 and -2 suppress cell death and promote proliferation in prostate stromal cells. A role in stromal hyperplasia appears possible in BPH. Effects of NUAK silencing are mimicked by HTH01-015 and WZ4003.

MP03-14 INHIBITION OF HUMAN DETRUSOR SMOOTH MUSCLE CELL GROWTH AND MODULATION OF CYTOSKELETAL ORGANIZATION BY IMMUNO-MODULATORY IMIDE DRUGS (IMIDS)

You have accessJournal of UrologyCME1 Apr 2023MP03-14 INHIBITION OF HUMAN DETRUSOR SMOOTH MUSCLE CELL GROWTH AND MODULATION OF CYTOSKELETAL ORGANIZATION BY IMMUNO-MODULATORY IMIDE DRUGS (IMIDS) Alexander Tamalunas, Amin Wendt, Florian Springer, Ru Huang, Ruixiao Wang, Yuhan Liu, Beata Rutz, Anna Ciotkowska, Giuseppe Magistro, Christian Stief, and Martin Hennenberg Alexander TamalunasAlexander Tamalunas More articles by this author , Amin WendtAmin Wendt More articles by this author , Florian SpringerFlorian Springer More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Giuseppe MagistroGiuseppe Magistro More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: While lower urinary tract symptoms (LUTS) consist of voiding (BPH) and storage disorders (OAB), new AUA and EAU guidelines take into account the mounting number of patients suffering from both, so called “mixed LUTS”. Recently, we could show that IMiDs (thalidomide, lenalidomide and pomalidomide) inhibit prostate smooth muscle contraction, modulate cytoskeletal actin organization, and reduce prostate stromal cell growth at the same time, without showing cytotoxic effects. Based on these promising data, we now investigated the effects of IMiDs on cellular functions, including cytoskeletal organization, and growth in bladder cells. METHODS: Experiments were carried out in an immortalized line of cultured human bladder detrusor smooth muscle cells (HBdSMC). Cytoskeletal organization was visualized by phalloidin staining, while cell growth was assessed using an EdU and cell colony assay. Cell viability was quantified in CCK8 assay, and FACS. RESULTS: IMiDs ([A] thalidomide 10-100 µM, [B] lenalidomide 5-30 µM, and [C] pomalidomide 2.5-10 µM) significantly reduced the number of viable WPMY-1 cells in a concentration- and time-dependent manner (Figure 1). Correspondingly, proliferation of WPMY-1 cells was significantly reduced in a concentration-dependent manner (Figure 2 and 3), without showing pro-apoptotic effects (Figure 4). In parallel, IMiDs induced cytoskeletal disorganization: while the cellular shape of control cells was characterized by many long and thin protrusions containing bundles of actin filaments, this structure collapsed after treatment with IMiDs (Figure 5). CONCLUSIONS: IMiDs impair human detrusor smooth muscle growth, which is paralleled by a breakdown of the cytoskeleton, which may inhibit exaggerated bladder smooth muscle contraction in OAB. Urodynamic effects in vivo and a possible application in LUTS appear possible. Together with our previous data, this may suggest a possible novel drug class in LUTS treatment. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e27 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander Tamalunas More articles by this author Amin Wendt More articles by this author Florian Springer More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Yuhan Liu More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Giuseppe Magistro More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

PD02-01 NUAK1 AND -2 PROMOTE CONTRACTION, PROLIFERATION AND SUPPRESSION OF CELL DEATH IN HUMAN PROSTATE STROMAL CELLS: EVIDENCE FROM ISOFORM-SPECIFIC SILENCING

You have accessJournal of UrologyCME1 Apr 2023PD02-01 NUAK1 AND -2 PROMOTE CONTRACTION, PROLIFERATION AND SUPPRESSION OF CELL DEATH IN HUMAN PROSTATE STROMAL CELLS: EVIDENCE FROM ISOFORM-SPECIFIC SILENCING Yuhan Liu, Ru Huang, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Yuhan LiuYuhan Liu More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In benign prostatic hyperplasia (BPH), contraction and growth in the prostate contribute to urethral obstruction and voiding symptoms. NUAK1 and -2 are serine/threonine kinases and promote myosin light chain phosphorlyation, actin organization, proliferation and suppression of cell death in non-muscle cells, which are critical for smooth muscle contraction and growth. Here, we examined effects of NUAK silencing on contraction and growth-related functions of human prostate stromal cells (WPMY-1). METHODS: Effects of NUAK1 and -2 silencing on matrix plug contraction, proliferation (EdU, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin) were examined in cultured WPMY-1 cells. Effects of NUAK inhibitors were examined in matrix contraction and in colony formation assays. RESULTS: Silencing of NUAK1 and -2 by transfection of WPMY-1 cells with isoform-specific siRNAs was confirmed by RT-PCR. Effects of silencing were most pronounced on proliferation and cell death. Proliferation rates were decreased by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls, 48 h after transfection), paralleled by decreases in Ki-67 mRNA by 75% and 77%. Numbers of dead cells amounted to 2.8 fold of scramble-transfected controls after silencing of NUAK1, and 4.9 fold of scramble controls after NUAK2 silencing, while no effect on apoptosis was observed. Viability was reduced by 31% and 49%, respectively, 24 h after transfection with NUAK1 and NUAK2 siRNA, compared to scramble controls. Polymerized actin was decreased by 66% and 70% by silencing of NUAK1 and -2. Time-dependent contractions in matrix contraction assays were reduced up to 45% by silencing of NUAK1, and up to 58% by silencing of NUAK2. The presumed NUAK inhibitors HTH01-015 and WZ4003 (both 10 µM) reduced contractions of WPMY-1 cells by up to 54% and 57%, and reduced the number of colonies in colony formation assays up to 78% and 64%, respectively. CONCLUSIONS: NUAK1 and -2 suppress cell death, and promote proliferation and contraction in prostate stromal cells. A role of NUAKs for BPH-relevant prostate functions driving voiding symptoms appears possible. NUAK inhibitors mimic effects of silencing on contraction, and inhibit the growth of stromal cells. Source of Funding: Deutsche Forschungsgemeinschaft (DFG), grant HE 5825/9-1; China Scholarship Council (CSC), grant 201908440478. Conflicts of Interest: Nothing to disclose © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e69 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuhan Liu More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Regulatory mechanism of DHRS2-modified human umbilical cord mesenchymal stem cells-derived exosomes in prostate cancer cell proliferation and apoptosis

Prostate cancer (PCa) is a prevalent cause of morbidity and mortality. DHRS2-modified human umbilical cord mesenchymal stem cells-derived exosomes (hUC-MSCs-derived exos) function in PCa. We explored the mechanism of DHRS2-modified hUC-MSCs-derived exos in PCa cell malignant behaviors. DHRS2 expression levels in WPMY-1 cells and 4 PCa cell lines were detected by RT-qPCR and Western blot. 22Rv1/DU145 cells with high/low DHRS2 expression were selected to establish the low/high DHRS2 expression models by transfection. Cell proliferation and apoptosis were detected by CCK-8, colony formation assays, and flow cytometry. hUC-MSCs were identified by oil red O, alizarin staining, and flow cytometry. Exos were extracted from hUC-MSCs by ultracentrifugation and identified by transmission electron microscopy, Nano series-Nano-ZS, and Western blot. DU145 cells were selected for in vitro study to further study the effects of DHRS2-modified exos on cell proliferation and apoptosis. The effect of DHRS2-modified exos on cell cycle distribution was detected by flow cytometry. DHRS2 was repressed in PCa cells. DHRS2 overexpression suppressed PCa cell proliferation and promoted apoptosis. Exos were successfully isolated from hUC-MSC. DHRS2-modified hUC-MSCs-derived exos carried DHRS2 into PCa cells and blocked malignant behaviors. Briefly, DHRS2 was repressed in PCa cells. DHRS2-modified hUC-MSCs-derived exos blocked PCa cell proliferation and enhanced apoptosis.

Hesperidin ameliorates benign prostatic hyperplasia by attenuating cell proliferation, inflammatory response, and epithelial-mesenchymal transition via the TGF-β1/Smad signaling pathway

Excessively activated transforming growth factor-beta 1 (TGF-β1) exacerbates benign prostatic hyperplasia (BPH) by triggering epithelial-mesenchymal transition (EMT) as well as epithelial and stromal cell differentiation. Hesperidin (HSP), a flavanone rich in citrus peels, exhibits a safe anti-cancer activity with few side effects. Although HSP reportedly inhibits cell growth in prostate cancer, studies on BPH have not yet been reported. Thus, this study aimed to figure out the therapeutic effect of HSP and its underlying mechanisms in BPH models in vivo and in vitro. To evaluate the anti-BPH effect of HSP in vivo, rats were injected with testosterone propionate (TP; 10 mg/kg, s.c.), finasteride (5 mg/kg, p.o.), and HSP (50 and 100 mg/kg, i.p.) for four weeks. The in vitro efficacy of HSP was evaluated using two prostate cell models, BPH-1 and dihydrotestosterone-stimulated WPMY-1 cells, for studying the interaction between epithelial and stromal cells. Both in vivo and in vitro, HSP inhibited prostate cell proliferation by suppressing the expression of androgen receptor-related markers. In addition, HSP reduced the expression levels of inflammatory and mesenchymal markers by blocking TGF-β1 activation. Collectively, HSP alleviated BPH by attenuating prostate cell proliferation, the inflammatory response, and EMT by regulating the TGF-β1/Smad signaling pathway. Thus, these results provide evidence for a new therapeutic approach against BPH.

Inhibition of α1-Adrenergic, Non-Adrenergic and Neurogenic Human Prostate Smooth Muscle Contraction and of Stromal Cell Growth by the Isoflavones Genistein and Daidzein.

Isoflavone-rich legumes, including soy, are used for food production, as dietary supplements and in traditional medicine. Soy consumption correlates negatively with benign prostatic hyperplasia (BPH) and voiding symptoms. However, isoflavone effects on the prostate are hardly known. Here, we examined the effects on human prostate smooth muscle contractions and stromal cell growth, which are driving factors of voiding symptoms in BPH. Smooth muscle contractions were induced in prostate tissues from radical prostatectomy. Growth-related functions were studied in cultured stromal cells (WPMY-1). Neurogenic, α1-adrenergic and non-adrenergic contractions were strongly inhibited with 50 µM and by around 50% with 10 µM genistein. Daidzein inhibited neurogenic contractions using 10 and 100 µM. Agonist-induced contractions were inhibited by 100 µM but not 10 µM daidzein. A combination of 6 µM genistein with 5 µM daidzein still inhibited neurogenic and agonist-induced contractions. Proliferation of WPMY-1 cells was inhibited by genistein (>50%) and daidzein (<50%). Genistein induced apoptosis and cell death (by seven-fold relative to controls), while daidzein induced cell death (6.4-fold) without apoptosis. Viability was reduced by genistein (maximum: 87%) and daidzein (62%). In conclusion, soy isoflavones exert sustained effects on prostate smooth muscle contractions and stromal cell growth, which may explain the inverse relationships between soy-rich nutrition, BPH and voiding symptoms.

Permixon®, hexane-extracted Serenoa repens, inhibits human prostate and bladder smooth muscle contraction and exerts growth-related functions in human prostate stromal cells

AimsRecently, the European Association of Urology recommended hexane-extracted fruit of Serenoa repens (HESr) in their guidelines on management of non-neurogenic male lower urinary tracts symptoms (LUTS). Despite previously lacking recommendations, Permixon® is the most investigated HESr in clinical trials, where it proved effective for male LUTS. In contrast, underlying mechanisms were rarely addressed and are only marginally understood. We therefore investigated effects of Permixon® on human prostate and detrusor smooth muscle contraction and on growth-related functions in prostate stromal cells. Main methodsPermixon® capsules were dissolved using n-hexane. Contractions of human prostate and detrusor tissues were induced in organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). Key findingsPermixon® inhibited α1-adrenergic and thromboxane-induced contractions in prostate tissues, and methacholine-and thromboxane-induced contractions in detrusor tissues. Endothelin-1-induced contractions were not inhibited. Neurogenic contractions were inhibited in both tissues in a concentration-dependent manner. In WPMY-1 cells, Permixon® caused concentration-dependent breakdown of actin polymerization, inhibited colony formation, reduced cell viability, and proliferation, without showing cytotoxic or pro-apoptotic effects. SignificanceOur results provide a novel basis that allows, for the first time, to fully explain the ubiquitous beneficial effects of HESr in clinical trials. HESr may inhibit at least neurogenic, α1-adrenergic and thromboxane-induced smooth muscle contraction in the prostate and detrusor, and in parallel, prostate stromal cell growth. Together, this may explain symptom improvements by Permixon® in previous clinical trials.

Inhibition of Human Prostate and Bladder Smooth Muscle Contraction, Vasoconstriction of Porcine Renal and Coronary Arteries, and Growth-Related Functions of Prostate Stromal Cells by Presumed Small Molecule Gαq/11 Inhibitor, YM-254890.

Introduction: Lower urinary tract symptoms (LUTS) involve benign prostatic hyperplasia (BPH) and overactive bladder (OAB). Standard-of-care medical treatment includes α1-blockers and antimuscarinics for reduction of prostate and detrusor smooth muscle tone, respectively, and 5α-reductase inhibitors (5-ARI) to prevent prostate growth. Current medications are marked by high discontinuation rates due to unfavourable balance between efficacy and treatment-limiting side effects, ranging from dry mouth for antimuscarinics to cardiovascular dysregulation and a tendency to fall for α1-blockers, which results from hypotension, due to vasorelaxation. Agonist-induced smooth muscle contractions are caused by activation of receptor-coupled G-proteins. However, little is known about receptor- and organ-specific differences in coupling to G-proteins. With YM-254890, a small molecule inhibitor with presumed specificity for Gαq/11 became recently available. Here, we investigated effects of YM-254890 on prostate, bladder and vascular smooth muscle contraction, and on growth-related functions in prostate stromal cells. Methods: Contractions of human prostate and detrusor tissues, porcine renal and coronary arteries were induced in an organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). Results: Contractions by α1-adrenergic agonists, U46619, endothelin-1, and neurogenic contractions were nearly completely inhibited by YM-254890 (30 nM) in prostate tissues. Contractions by cholinergic agonists, U46619, endothelin-1, and neurogenic contractions were only partly inhibited in detrusor tissues. Contractions by α1-adrenergic agonists, U46619, endothelin-1, and neurogenic contractions were strongly, but not fully inhibited in renal arteries. Contractions by cholinergic agonists were completely, but by U46619 and endothelin-1 only strongly inhibited, and neurogenic contractions reduced by half in coronary arteries. YM-254890 had no effect on agonist-independent contractions induced by highmolar (80 mM) potassium chloride (KCl). Neurogenic detrusor contractions were fully sensitive to tetrodotoxin. In WPMY-1 cells, YM-254890 caused breakdown of actin polymerization and organization, and obvious, but clearly limited decreases of proliferation rate, colony formation and viability, and slightly increased apoptosis. Conclusion: Intracellular post-receptor signaling pathways are shared by Gαq-coupled contractile receptors in multiple smooth muscle-rich organs, but to different extent. While inhibition of Gαq/11 causes actin breakdown, anti-proliferative effects were detectable but clearly limited. Together this may aid in developing future pharmaceutical targets for LUTS and antihypertensive medication.

The Prostate-Associated Gene 4 (PAGE4) Could Play a Role in the Development of Benign Prostatic Hyperplasia under Oxidative Stress.

Benign prostatic hyperplasia (BPH) is a common disease in elderly men with uncertain molecular mechanism, and oxidative stress (OS) has also been found associated with BPH development. Recently, we found that prostate-associated gene 4 (PAGE4) was one of the most significantly changed differentially expressed genes (DEGs) in BPH, which can protect cells against stress stimulation. However, the exact role of PAGE4 in BPH remains unclear. This study is aimed at exploring the effect of PAGE4 in BPH under OS. Human prostate tissues and cultured WPMY-1 and PrPF cells were utilized. The expression and localization of PAGE4 were determined with qRT-PCR, Western blotting, and immunofluorescence staining. OS cell models induced with H2O2 were treated with PAGE4 silencing or PAGE4 overexpression or inhibitor (N-acetyl-L-cysteine (NAC)) of OS. The proliferation activity, apoptosis, OS markers, and MAPK signaling pathways were detected by CCK-8 assay, flow cytometry analysis, and Western blotting. PAGE4 was shown to be upregulated in human hyperplastic prostate and mainly located in the stroma. Acute OS induced with H2O2 increased PAGE4 expression (which was prevented by OS inhibitor), apoptosis, cell cycle arrest, and reactive oxygen species (ROS) accumulation in WPMY-1 and PrPF cells. siPAGE4 plus H2O2 potentiated H2O2 effect via reducing the p-ERK1/2 level and increasing p-JNK1/2 level. Consistently, overexpression of PAGE4 offset the effect of H2O2 and partially reversed the PAGE4 silencing effect. However, knocking down and overexpression of PAGE4 alone determined no significant effects. Our novel data demonstrated that augmented PAGE4 promotes cell survival by activating p-ERK1/2 and decreases cell apoptosis by inhibiting p-JNK1/2 under the OS, which could contribute to the development of BPH.