Human BPH Research Articles

Dihydroartemisinin ameliorates chronic nonbacterial prostatitis and epithelial cellular inflammation by blocking the E2F7/HIF1α pathway.

Chronic nonbacterial prostatitis (CNP) has remained one of the most prevalent urological diseases, particularly in older men. Dihydroartemisinin (DHA) has been identified as a semi-synthetic derivative of artemisinin that exhibits broad protective effects. However, the role of DHA in inhibiting CNP inflammation and prostatic epithelial cell proliferation remains largely unknown. CNP animal model was induced by carrageenan in C57BL/6 mouse. Enzyme linked immunosorbent assay (ELISA), Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to examine inflammatory cytokines and proliferation genes expression. Immunofluorescence and immunochemistry staining were used to detect and E2F7 expression. Human prostatic epithelial cells (HPECs) and RWPE-1 was induced by lipopolysaccharide (LPS) to mimic CNP model in vitro. Cell proliferation was determined using MTS assay. DHA significantly alleviated the rough epithelium and inhibited multilamellar cell formation in the prostatic gland cavity and prostatic index induced by carrageenan. In addition, DHA decreased the expression of TNF-α and IL-6 inflammatory factors in prostatitis tissues and in LPS-induced epithelial cells. Upregulation of transcription factor E2F7, which expression was inhibited by DHA, was found in CNP tissues, human BPH tissues and LPS-induced epithelial cells inflammatory response. Mechanically, we found that depletion of E2F7 by shRNA inhibited epithelial cell proliferation and LPS-induced inflammation while DHA further enhance these effects. Furthermore, HIF1α was transcriptional regulated by E2F7 and involved in E2F7-inhibited CNP and cellular inflammatory response. Interestingly, we found that inhibition of HIF1α blocks E2F7-induced cell inflammatory response but does not obstruct E2F7-promoted cell growth. The results revealed that DHA inhibits the CNP and inflammation by blocking the E2F7/HIF1α pathway. Our findings provide new evidence for the mechanism of DHA and its key role in CNP, which may provide an alternative solution for the prevention and treatment of CNP.

Human Tissue Kallikrein 1 Is Downregulated in Elderly Human Prostates and Possesses Potential In Vitro Antioxidative and Antifibrotic Effects in Rodent Prostates.

Objective The aim of the present study was to investigate the protective effects and mechanisms of KLK1 on aging-related prostate alterations and search clues about the application of KLK1 to the treatment of human BPH. Methods Thirty-six rats including 26 male wild-type SD rats and 10 transgenic rats were fed to 3- or 18-month-old and divided into three groups: young WTR (yWTR) as the control (n = 16), aged WTR (aWTR) (n = 10), and aged TGR (aTGR) (n = 10). The prostates of the three groups of rats (10 rats per group) were harvested to evaluate the levels of KLK1 expression, oxidative stress, fibrosis, and involved signaling pathways, such as NO/cGMP, COX-2/PTGIS/cAMP, and TGF-β1/RhoA/ROCK1, via quantitative PCR, Western blot, histological examinations, and ELISA. Moreover, the remaining 6 yWTRs were sacrificed to obtain primary prostate fibroblast and aortic endothelial cells, and a coculture system was built with the cells for the verification of above signaling pathways in vitro. And the direct effects of bradykinin on prostate cells were detected by MTT experiment. Prostate specimens of 47 patients (age from 48 to 92 years) undergoing BPH surgery were collected after approval. Histological examinations and KLK1 IHC were preformed to analyze the relationship between KLK1 expression and age and prostate fibrosis. Results The human KLK1 gene only existed and was expressed in aTGR. The prostate of young rats expressed more KLK1 than the aged and the expression of KLK1 in prostate decreased with age in humans (r = −0.347, P = 0.018). Compared to the aWTR group, the yWTR and aTGR groups showed milder fibrosis, less oxidative stress, upregulated NO/cGMP, and COX-2/PTGIS/cAMP signaling pathways and inhibited TGF-β1/RhoA/ROCK1 signaling pathway. In the coculture system, KLK1 suppressed TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation via cleaving LMWK to produce the BK which upregulate eNOS expression and NO production in endothelial cells. BK not only slightly stimulated the proliferation ability of prostatic stromal cells but also upregulated iNOS and inhibited TGF-β1 expression in them. Conclusion KLK1 protects prostate from oxidative stress and fibrosis via amplified NO/cGMP signal in aged rats. The decrease of KLK1 expression with aging is laying the groundwork for the application of KLK1 to the treatment of human BPH. The current experimental data showed that the side effects of KLK1 on the prostate cell were not obvious.

Prostate-Specific Deletion of Cdh1 Induces Murine Prostatic Inflammation and Bladder Overactivity.

Benign prostatic hyperplasia (BPH) is an age-related debilitating prostatic disease that is frequently associated with prostatic inflammation and bothersome lower urinary tract symptoms (LUTS). Animal models have shown that formalin- and bacterial-induced prostatic inflammation can induce bladder dysfunction; however, the underlying mechanisms contributing to prostatic inflammation in BPH and bladder dysfunction are not clear. We previously reported that E-cadherin expression in BPH is downregulated in hyperplastic nodules compared with expression in adjacent normal tissues. Here, we explored the potential consequences of prostatic E-cadherin downregulation on the prostate and bladder in vivo using an inducible murine model of prostate luminal epithelial-specific deletion of Cdh1. The prostate-specific antigen (PSA)-CreERT2 transgenic mouse strain expressing tamoxifen-inducible CreERT2 recombinase driven by a 6-kb human PSA promoter/enhancer was crossed with the B6.129-Cdh1tm2Kem/J mouse to generate bigenic PSA-CreERT2/Cdh1-/- mice. Deletion of E-cadherin was induced by transient administration of tamoxifen when mice reached sexual maturity (7 weeks of age). At 21 to 23 weeks of age, the prostate, bladder, and prostatic urethra were examined histologically, and bladder function was assessed using void spot assays and cystometry. Mice with Cdh1 deletion had increased prostatic inflammation, prostatic epithelial hyperplasia, and stromal changes at 21 to 23 weeks of age, as well as changes in bladder voiding function compared with age-matched controls. Thus, loss of E-cadherin in the murine prostate could result in prostatic defects that are characteristic of BPH and LUTS, suggesting that E-cadherin downregulation could be a driving force in human BPH development and progression.

TRIM52 positively mediates NF-κB to promote the growth of human benign prostatic hyperplasia cells through affecting TRAF2 ubiquitination

AimsBenign prostatic hyperplasia (BPH) is a progressive disease, which severely affects men's health. Here, we sought to analyze the functions and mechanism of action of the tripartite motif protein 52 (TRIM52), a novel prostate basal cell biomarker in BPH. Materials and methodsImmunohistochemistry assay was performed in sectioned human BPH tissues, BPH-1 cells, and prostate RWPE-1 cells, to detect the expressions of TRIM52 and NF-κB. Western blotting and qRT-PCR analyses were conducted to measure the relative protein and mRNA expression levels, respectively. Further, lentiviral transfection was performed in BPH-1 and RWPE-1 cells to study the overexpression and siRNA knockdown of TRIM52. Dual-luciferase reporter assay was applied to evaluate the relationship between NF-κB and TRIM52. Furthermore, CCK-8 assay and flow cytometry were employed to analyze cell proliferation and apoptosis. Key findingsTRIM52 and NF-κB levels were elevated in BPH tissues, and TRIM52 expression positively correlated with NF-κB expression. TRIM52 silencing suppressed the growth of BPH-1 cells and decreased the promoter activity of NF-κB. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed TRIM52-induced proliferation of RWPE-1 cells and inhibited NF-κB promoter activity in oeTRIM52 transfected RWPE-1 cells. Silencing TRIM52 also inhibited TRAF2 ubiquitination in BPH-1 cells. Further, NF-κB promoter activity in siNC transfected cells was enhanced by the recombinant protein TNF-α and inhibited by siTRIM52. SignificanceTRIM52 accelerated the growth of BPH-1 cells by upregulating NF-κB, and TRIM52 could promote TRAF2 ubiquitination. These findings might contribute to the understanding of the biological functions and action mechanisms of TRIM52 in BPH.

Oncogenic transformation of human benign prostate hyperplasia with chronic cadmium exposure

Experimentally, it has been proved that cadmium served as an effective carcinogen and able to induce tumors in rodents in a dose-specific manner. However, systemic evaluation of cadmium exposure for the transformation of prostatic hyperplasia into prostate cancer (PCa) is still unclear. In the present study, an attempt has been made to establish cadmium-induced human prostate carcinogenesis using an in vitro model of BPH cells. Wide range of cadmium concentrations, i.e., 1 nM, 10 nM, 100 nM and 1μM, were chronically exposed to the human BPH cells for transformation into PCa and monitored using cell and molecular biology approaches. After eight weeks of exposure, the cells showed subtle morphological changes and shifts of cell cycle in the G2M phase. Significant increase in expression of prostatic genes AR, PSA, ER-β, and 5αR with increased nuclear localization of AR and pluripotency markers Cmyc, Klf4 indicated the carcinogenic effect of Cd. Further, the BPH cells exposed to Cd showed a substantial increase in the secretion of MMP-2 and MMP-9, influencing migratory potential of the cells along with decreased expression of the p63 protein which further strengthen the progression towards carcinogenesis and aggressive tumor studies. Data from the present study state that Cd exhibited marked invasiveness in BPH cells. These observations established a connecting link of BPH towards PCa pathogenesis. Further, the study will also help in investigating the intricate pathways involved in cancer progression.

MP06-17 THE EFFECT OF INTRAVESICAL INSTILLATION OF LIPOSOME-CONJUGATED NGF ANTISENSE ON BLADDER HYPERSENSITIVITY AND NGF EXPRESSION IN A RAT MODEL OF PROSTATIC INFLAMMATION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-17 THE EFFECT OF INTRAVESICAL INSTILLATION OF LIPOSOME-CONJUGATED NGF ANTISENSE ON BLADDER HYPERSENSITIVITY AND NGF EXPRESSION IN A RAT MODEL OF PROSTATIC INFLAMMATION Taro Igarashi*, Pradeep Tyagi, Shinsuke Mizoguchi, Masahiro Kurobe, Daisuke Gotoh, Tetsuichi Saito, Zhou Wang, Akira Furuta, Yasuyuki Suzuki, Shin Egawa, and Naoki Yoshimura Taro Igarashi*Taro Igarashi* More articles by this author , Pradeep TyagiPradeep Tyagi More articles by this author , Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Masahiro KurobeMasahiro Kurobe More articles by this author , Daisuke GotohDaisuke Gotoh More articles by this author , Tetsuichi SaitoTetsuichi Saito More articles by this author , Zhou WangZhou Wang More articles by this author , Akira FurutaAkira Furuta More articles by this author , Yasuyuki SuzukiYasuyuki Suzuki More articles by this author , Shin EgawaShin Egawa More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Local upregulation of nerve growth factor (NGF) in the bladder has been implicated in lower urinary tract (LUT) dysfunction due to afferent sensitization. Also, male LUT symptoms are known to be associated with prostatic inflammation (PI) in human BPH tissues. Thus, we examined the effect of liposomes conjugated with NGF antisense oligonucleotide (OND) on bladder hypersensitivity and NGF expression in PI rats. METHODS: Male SD rats were divided into 4 groups: (1) Sham, (2) Sham-NS; with intravesical instillation of normal saline (NS, 0.5ml), (3) PI-NS; with PI and instillation of NS, (4) PI-OND; with PI and instillation of liposome-conjugated NGF antisense. PI was induced by intraprostatic formalin injection (5%, 50 μl per each ventral lobe). On day 14, phosphorothioated NGF antisense OND (0.5ml, 12μM) complexed with liposomes (PI-OND) or NS (Sham-NS and PI-NS) was instilled directly into the bladder with a 30G needle after draining urine, and kept for 60 minutes. On day 28, we evaluated awake cystometry (CMG) and harvested tissues to measure mRNA expressions of NGF in the bladder and C-fiber afferent markers (TRPV1 & TRPA1) in L6-S1 dorsal root ganglia (DRG). RESULTS: In CMG, PI-OND group had significantly longer intercontraction intervals (ICIs) vs. PI-NS group while ICIs were significantly reduced in PI-NS group vs. Sham group (Fig. 1A, 1B). mRNA expressions of NGF in bladder mucosa and TRPV1 & TRPA1 in DRG in PI-OND group was significantly lower vs. PI-NS group while there were no significant differences between PI-OND and Sham (Fig. 1C). CONCLUSIONS: These results demonstrate that bladder NGF is an important mediator to induce bladder overactivity following PI and that liposome-based, local NGF-targeting therapy could be effective for bladder hypersensitivity induced by prostate-to-bladder cross-sensitization after prostatic inflammation. Source of Funding: NIH U54DK112079 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e57-e58 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taro Igarashi* More articles by this author Pradeep Tyagi More articles by this author Shinsuke Mizoguchi More articles by this author Masahiro Kurobe More articles by this author Daisuke Gotoh More articles by this author Tetsuichi Saito More articles by this author Zhou Wang More articles by this author Akira Furuta More articles by this author Yasuyuki Suzuki More articles by this author Shin Egawa More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

Mesenchymal stem cells and the embryonic reawakening theory of BPH.

The prostate is the only organ in a man that continues to grow with age. John McNeal proposed, 40 years ago, that this BPH is characterized by an age-related reinitiation of benign neoplastic growth selectively in developmentally abortive distal ducts within the prostate transition-periurethral zone (TPZ), owing to a reawakening of inductive stroma selectively within these zones. An innovative variant of this hypothesis is that, owing to its location, the TPZ is continuously exposed to urinary components and/or autoantigens, which produces an inflammatory TPZ microenvironment that promotes recruitment of bone marrow-derived mesenchymal stem cells (MSCs) and generates a paracrine-inductive stroma that reinitiates benign neoplastic nodular growth. In support of this hypothesis, MSCs infiltrate human BPH tissue and have the ability to stimulate epithelial stem cell growth. These results provide a framework for defining both the aetiology of BPH in ageing men and insights into new therapeutic approaches.

Abstract 1293: A high through approach to delineate protein complexes in adenocarcinoma tissues

Abstract Although it is well known that prostate cancer (PCa) is a progressive disease involving multiple gene alterations, little is known at the proteome level. Most of the functional information of the cancer-associated genes relies in the proteome, an exceptionally complex biological system involving several proteins that function through dynamic protein-protein interactions and post-translational modifications. To identify potential PCa protein biomarkers, we carried out an in depth proteomic analysis (ESI-MS/MS) using human PCa and BPH tissue. Samples were obtained using phase-transfer surfactant-aided extraction/tryptic digestion of formalin-fixed and paraffin-embedded sections mounted on microscope slides. We identified 1331 and 1239 proteins in PCa and BPH tissue proteomes respectively. 71 proteins were present in at least 50% of PCa samples and not in BPH samples, while 122 proteins where present in at least 50% of BPH samples and not in carcinoma samples. In order to prioritize candidate markers for PCa, we compared the differential protein expression based on normalized spectral counts between tissue samples. We set as cut-offs proteins that were found with a minimum of three peptides within our PCa proteomes. This filter resulted in the selection of 11 proteins. The list contained proteins that were previously studied in the context of prostate cancer progression, including SSBP1, GDF15, NDRG1, C4A & APOE, thus providing further confirmation for the robustness of our quantification method. Bioinformatics analysis (Oncomine) showed that the proteins aforementioned were significant up-regulated (fold change >1.5, P≤0.05) in prostate adenocarcinoma vs. normal prostate gland. Whole exome analysis (cBioportal), revealed amplification as the most frequent genetic alteration and RNASeq data also confirmed a significant up-regulation for these proteins (P≤0.05). To contextualize the proteomic data, we utilized protein complexes information from CORUM database. Utilizing biological complexes as a cluster vector, we calculate a Proteomics Signature Profile (PSP) for each sample based on the hit rates of its reported proteins, against the cluster vector. We identified 25 complexes differentially expressed between PCa and BPH. Among those, the top ranked cluster upregulated in PCa was EIF3 (p-value= 4,37x10-5), a protein complex that functions during the initiation phase of eukaryotic translation, composed of EIF3B, EIF3J and EIF3I. We hereby report and offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis. Citation Format: Sofia Lage Vickers, Juan Bizzotto, Emiliano Ortiz, Alejandra Paez, Nicolás Anselmino, Javier Brandani, Mercedes Abbate, Javier Cotignola, Maria Pía Valacco, Elba Vazquez, Geraldine Gueron. A high through approach to delineate protein complexes in adenocarcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1293.

Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells

Ethnopharmacological relevanceBenign prostatic hyperplasia (BPH), also called benign enlargement of the prostate, is a progressive disease that is observed in most elderly men. Yongdamsagan-tang, a traditional herbal formula, is used commonly for the treatment of inflammation-related diseases. Although the therapeutic efficacy of Yongdamsagan-tang against BPH in vivo was reported previously, its underlying mechanisms are not clearly understood. Aim of the studyIn this study, we investigated the effect of Yongdamsagan-tang water extract (YSTE) and its mechanism on the growth of human BPH epithelial BPH-1 cells. Materials and methodsYSTE was extracted from 11 herbaceous plants and its chemical composition was analyzed by High-performance liquid chromatography (HPLC). YSTE was treated in the epithelial BPH-1 cell line and then cell lysates or supernant were used to evaluate cell viability, cell cycle, proliferation and cytokine production. ResultsHPLC revealed that Baicalin and gentiopicroside were involved as the major compounds of YSTE. YSTE treatment in BPH-1 cells repressed cell viability in a dose-dependent manner. Regarding the inhibitory mechanisms of YSTE on cell growth, YSTE inhibited cell proliferation via a decrease in endogenous cyclin D1 protein levels and arrest at the S phase during cell-cycle progression. Furthermore, YSTE treatment in BPH-1 cells suppressed prostaglandin E2 production and cyclooxygenase-2 (COX-2) protein levels. The secretion of the proinflammatory cytokines, interleukin-8 and interleukin-6, was also reduced by YSTE treatment. ConclusionsYSTE in BPH-1 cells showed antiproliferative and anti-inflammatory activities via cell-cycle arrest and downregulation of COX-2 expression, respectively. Taken together, the results of the present study will enhance our understanding of the mechanisms underlying the effect of YSTE in BPH.

Abstract 4950: Collagen, type I, alpha 1 (COL1A1): a potential urinary biomarker that can distinguish between benign prostate hyperplasia and localized prostate cancer

Abstract Benign prostate hyperplasia (BPH) is the most frequent benign disease among men worldwide and its incidence increases with age. Prostate cancer (PCa) and BPH share similar symptoms and an elevated serum prostate-specific antigen (PSA) can be observed with either benign or malignant growth of the prostate. It is the demographic overlap of BPH and PCa, and the lack of discrimination between these two prostate diseases by PSA, that defines the diagnostic dilemma faced by clinicians when treating prostate disease. The goal of the current study was to identify novel non-invasive, biomarkers that can distinguish between BPH and PCa. We utilized iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry techniques to sensitively and accurately identify the urinary proteome of men with BPH vs. men with PCa. We then performed functional enrichment analysis and pathways enrichment analysis of these proteins using Ingenuity Pathway Analysis (IPA) tools to determine differentially expressed pathways and functions in PCa as compared to BPH. Network analysis identified differences in a number of signaling molecules including ERK1/2, TGFβ, PI3K, p38 MAPK and NFκB, with a high degree of interactivity. Protein expression was validated by immunoblot analyses using monospecific antibodies. Here, we focused on collagen, type I, alpha 1 (COL1A1), one of a number of extracellular matrix-associated proteins detected. We found that COL1A1 was significantly (P < 0.001) elevated in the urine of patients diagnosed with early or localized PCa but not in the urine of men with BPH. Complementary studies were conducted on human BPH and PCa cells. Our data suggest that COL1A1 may represent a novel non-invasive biomarker that can discriminate between BPH and early PCa. (Supported by The Ellison Foundation) Citation Format: Andrej Jedinak, Camille Vuichoud, Andrew El-Hayek, Kevin R. Loughlin, Marsha A. Moses. Collagen, type I, alpha 1 (COL1A1): a potential urinary biomarker that can distinguish between benign prostate hyperplasia and localized prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4950.

Abstract 1052: Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation

Abstract BACKGROUND: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by finasteride for patients with bladder outlet obstrution secondary to BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter would be regulated by DNA methyltransferases leading to suppression of SRD5A2. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, 96 prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cell line, BPH-1. To determine the effect of CpG methylation, SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. Statistical analyses were performed with JMP Pro version 11 (SAS Institute Inc., Cary, NC). RESULTS: We analyzed 96 human BPH samples from transurethral resection of prostate (TURP) surgeries and found that absence of SRD5A2 was closely associated with hypermethylation of the SRD5A2 promoter region. We found that methylation of SRD5A2 was regulated by DNA methytransferase 1 (DNMT1) and the cytokines, TNF-alpha, NF-κB and IL-6, regulated DNMT1protein expression and thereby indirectly affected SRD5A2 promoter methylation and gene expression. Suppression of cytokines inhibited activity of DNMT1, while over-expression of p65 or treatment with TNF-alpha or IL-6 up-regulated DNMT1. In addition, we found that methylation of the SRD5A2 promoter and concomitant decrease in protein expression were closely associated with patient's increasing age (P<0.05). Finally, in murine prostate tissues, cytokine levels, DNMT1 and global methylation were all increased in older mice. Induction of inflammation with lipopolysaccharide (LPS) stimulated the TNFR1/NF-κB/IL-6/DNMT1 pathway, leading to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, while treatment with both LPS and TNF-alpha inhibitor reversed this pathway and reactivated SRD5A2. CONCLUSIONS: Expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. We show that SRD5A2 expression is lacking in many benign human adult prostate tissues. Methylation of SRD5A2 promoter region, which is regulated by DNMT1, accounts for absence of SRD5A2 expression in many adult human prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. Citation Format: Ge Rongbin, Zongwei Wang, Seth Bechis, Alexander Otsetov, Shengyu Hua, Shulin Wu, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2015-1052

Clinical significance of CUL4A in human prostate cancer.

Aberrant expression of the Cullin 4A (CUL4A) is found in many tumor types, but the functions and mechanism of CUL4A in prostate cancer (PCa) development and progression remain largely unknown. The aim of this study was to investigate the possible role of CUL4A in prostate tumorigenesis. Immunohistochemistry was used to examine CUL4A expression in human PCa tissues and BPH tissues. Cell proliferation was assessed by MTT, and migration and invasion were analyzed by Transwell and Matrigel assays after CUL4A knockdown in PCa in vitro. The results showed that CUL4A protein was overexpressed in 86.21% of PCa tissues. CUL4A knockdown with siRNA in PCa cells decreased cell proliferation, migration, and invasion. Mechanistically, CUL4A could modulate the expression of P53 in PCa cells. Our results indicate that CUL4A overexpression play an oncogenic role in the pathogenesis of PCa, and CUL4A may be a potential therapeutic target for PCa.

Autonomic nervous system activity assessement by heart rate variability in experimental bladder outlet obstruction

A syndrome with urgency, with or without associated urine incontinence and usually accompanied by higher urinary frequency and nocturia has been named "overactive bladder; OAB". OAB is an entity with complex pathophysiology, involving both myogenic and neurogenic (afferent / efferent bladder innervation) disturbances. OAB symptoms accompany benign prostatic hypertrophy--BPH ("obstructive OAB"). The aim of the study was to estimate the autonomic nervous system activity (ANS) in the experimental bladder outlet obstruction (BOO) which was an animal model of the human BPH. The study was conducted using 30 female rats, divided into two groups: BOO animals (n=15), with surgically induced BOO (by partial ligation of the proximal urethra) and control ones (n=15), which underwent sham procedure (without urethral ligation). Two weeks after the surgery, in both groups, ANS activity was estimated using time- and spectral analysis of the heart rate variability recordings. The bladder overactivity in BOO animals was confirmed using urodynamic recordings and bladder histological assessment, juxtaposed against the results of the control group. The key finding of our study was the development of autonomic disturbances in bladder outlet obstruction (BOO) rats. Our study revealed that BOO animals were characterised by diminished rMSSD and spectral HRV parameters: TP, LF and HF, in comparison with the control group. The normalised nLF and nHF parameters did not differ significantly in both groups, although slight changes in the nLF (increased) and nHF (decreased) were noted in BOO group. The absolute VLF value was almost the same in both studied populations, however, the percentage part of this component in the appropriate HRV spectrum differed considerably in both studied groups. In BOO animals, VLF percentage amounted to about 90%, whereas in control animals this parameter reached only about 53% of the total power spectrum. Thus, to sum up, our findings suggest autonomic imbalance with decreased global autonomic tension and diminished parasympathetic activity with relatively sympathetic overactivity.

Impact of CXCL1 overexpression on growth and invasion of prostate cancer cell

The role of CXCL1 in prostate cancer (PCa) progression has been poorly investigated. A limitation of previous studies is linked to the use of human PCa cell lines PC3 and DU145, producing CXCL8 at levels strongly exceeding CXCL1 levels. Moreover, in mouse models the sharing of CXCR2 receptor by both ligands makes the phenotype induced by CXCL8 and CXCL1 almost indistinguishable. To overcome this problem we used the murine TRAMP-C2 cell line, not expressing CXCL8 and expressing CXCL1 at low levels. The effect of CXCL1 overexpression was examined by in vivo subcutaneous tumor studies and in vitro functional assays of invasion and adhesion. Biochemical modifications were evaluated by Western blotting and antibody arrays. Our data show that the overexpression of CXCL1 in TRAMP-C2 cells represses tumor establishment and in situ invasion. In vitro, the main action of CXCL1 expression in TRAMP cells is associated with the perturbation of molecules linked to cell adhesion and migration thus explaining in vivo data. Other in vitro findings also suggest that signaling by CXCL1 might activate a secretory network limiting in vivo tumor growth by reinforcing senescence. Immunohistochemical staining of human PCa, BPH, and normal prostate biopsies strengthen our observations on the mouse model: when expressed, CXCL1 is limited to small areas with faint staining and PCa progression does not rely on CXCL1 expression. We could speculate that CXCL1 overexpression acts as a suppressor of malignancy limiting the escape of tumor cells from the primary tumor and reinforcing growth arrest.

Evidence for Steroidogenic Potential in Human Prostate Cell Lines and Tissues

Malignant prostate cancer (PCa) is usually treated with androgen deprivation therapies (ADTs). Recurrent PCa is resistant to ADT. This research investigated whether PCa can potentially produce androgens de novo, making them androgen self-sufficient. Steroidogenic enzymes required for androgen synthesis from cholesterol (CYP11A1, CYP17A1, HSD3β, HSD17β3) were investigated in human primary PCa (n = 90), lymph node metastases (LNMs; n = 8), and benign prostatic hyperplasia (BPH; n = 6) with the use of IHC. Six prostate cell lines were investigated for mRNA and protein for steroidogenic enzymes and for endogenous synthesis of testosterone and 5α-dihydrotestosterone. All enzymes were identified in PCa, LNMs, BPH, and cell lines. CYP11A1 (rate-limiting enzyme) was expressed in cancerous and noncancerous prostate glands. CYP11A1, CYP17A1, HSD3β, and HSD17β3 were identified, respectively, in 78%, 52%, 16%, and 82% of human BPH and PCa samples. Approximately 10% of primary PCa, LNMs, and BPH expressed all four enzymes simultaneously. CYP11A1 expression was stable, CYP17A1 increased, and HSD3β and HSD17β3 decreased with disease progression. CYP17A1 expression was significantly correlated with CYP11A1 (P = 0.0009), HSD3β (P = 0.0297), and HSD17β3 (P = 0.0090) in vivo, suggesting CYP17A1 has a key role in prostatic steroidogenesis similar to testis and adrenal roles. In vitro, all cell lines expressed mRNA for all enzymes. Protein was not always detectable; however, all cell lines synthesized androgen from cholesterol. The results indicate that monitoring steroidogenic metabolites in patients with PCa may provide useful information for therapy intervention.

Regulation of soluble VEGFR-2 secreted by microvascular endothelial cells derived from human BPH

Recently, it was reported that the soluble vascular endothelial growth factor receptor-2 (sVEGFR-2) is secreted by microvascular endothelial cells from human BPH (HPECs). The purpose of this study was to investigate the modulation of sVEGFR-2 by common endothelial cell stimulators. In addition, the physiological role of sVEGFR-2 with regard to the VEGF-stimulated proliferation of HPEC was investigated. HPECs were isolated and cultured from fresh BPH tissue. After the incubation of HPECs either with adenosine triphosphate (ATP), interleukin (IL)-6, IL-8 or IL-12, the secretion of sVEGFR-2 was measured by enzyme-linked immunosorbent assay. For measurement of HPEC proliferation influenced by sVEGFR-2, VEGF-stimulated HPEC was cultured with/without sVEGFR-2. Cell proliferation was assessed with the Alamar Blue method. The sVEGFR-2 secretion was increased by ATP and decreased by IL-12 and IL-8, respectively. IL-6 did not show any significant effect on sVEGFR-2 secretion of HPECs. HPEC proliferation was significantly inhibited by sVEGFR-2. In this study, our data suggest that the secretion of sVEGFR-2 by microvascular endothelial cells from prostate origin is influenced by multiple endothelial cell stimulators. Furthermore, our data suggest that sVEGFR-2 acts as an antiangiogenic factor.

Human Benign Prostatic Hyperplasia Stromal Cells As Inducers and Targets of Chronic Immuno-Mediated Inflammation

Benign prostatic hyperplasia (BPH), a highly prevalent prostatic condition, could involve an inflammatory component in disease pathogenesis. In this study, we show that human stromal prostate cells obtained from BPH tissue can actively contribute to the inflammatory process by secreting proinflammatory cytokines as well as chemokines able to recruit lymphomonuclear cells and by acting as APCs. BPH cells express all of the TLRs and their ligation leads to the secretion of CXCL8/IL-8, CXCL10, and IL-6. In addition, BPH cells express costimulatory as well as class I and class II MHC molecules, which activate alloreactive CD4(+) cells that in turn markedly up-regulate IL-12/IL-23p40 and IL-12p75 secretion by BPH cells. Alloreactive CD4(+) cells activated by BPH cells secrete IFN-gamma and IL-17. These cytokines up-regulate IL-6, IL-8, and CXCL10 production by BPH cells, creating a positive feedback loop that can amplify inflammation. IL-8 induces autocrine/paracrine proliferation of BPH cells, indicating also a growth-promoting activity of this chemokine in disease pathogenesis. These results show that human BPH cells represent nonprofessional APCs able to induce and sustain chronic inflammatory processes, supporting the relevance of inflammation in BPH pathogenesis.

Ablation of canine prostate using two-stage intraprostatic hot agarose solution and enzyme injection.

Enzyme ablation of the hyperplastic prostate may be an ideal method of management of BPH. However, the unsatisfactory ablation affects in vivo contrast with successful in vitro results limiting the enthusiasm for further research. In this study, we make efforts to solve the problems in the use of enzyme ablation of BPH in vivo and to measure satisfactory effect. A total of 18 hybrid dogs between the ages of 7 and 11 y underwent this experiment. Eight dogs were divided into four groups according to the injection formula: enzyme solution, hot D-Hanks' plus enzyme solution, hot agarose plus enzyme solution, and hot agarose solution alone. After selecting the agarose plus enzyme solution group in the first month, the remainder 10 dogs were treated with this two-stage method. Intravenous or oral antibiotics were administered perioperatively. All operations were performed directly by way of laparotomy. The prostates were observed and harvested with surrounding tissue at 24 hrs, 7 days, 14 days, 1 month and 3-5 months after treatment. Gross and microscopic examinations were performed. Only agarose plus enzyme group shows obvious cavity formation with concomitant size reduction and softening of the prostate ablation effect in the four groups. At 24 h after injection, the prostates demonstrated cavity formation containing liquefied necrotic tissue. The liquefied tissue was absorbed in 7-14 days. At 1 month, the size of most prostates decreased with a corresponding decrease in the size of the cavities. The cavities nearly disappeared within 3-5 months, and the size of prostates decreased to between 1/2 and 1/4 of the pretreatment sizes. All prostates had intact urethral mucosa and capsule. No complications directly related to enzyme ablation were identified. In the control groups there were no significant cavities or decrease in prostate size. This two-stage thermal and enzyme ablative method can significantly ablate prostate tissue without identifiable complications, and would be possibly applied to treating human BPH in the future.

In vitro assessment of the efficacy of thermal therapy in human benign prostatic hyperplasia

The successful management of BPH with minimally invasive thermal therapies requires a firm understanding of the temperature–time relationship for tissue destruction. In order to accomplish this objective, the present in vitro study assesses the cellular viability of human BPH tissue subjected to an experimental matrix of different temperature–time combinations. Hyperplastic prostate tissue was obtained from 10 radical prostatectomy specimens resected for adenocarcinoma. A portion of hyperplastic tissue from the lateral lobe of each prostate was sectioned into multiple 1 mm thick tissue strips, placed on a coverslip and thermally treated on a controlled temperature copper block with various temperatures (45–70°C) for various times (1–60 min). After heat treatment, the tissue slices were cultured for 72 h and viability was assessed using two independent assays: histology and dye uptake for stromal tissue and using histology alone for the glandular tissue. The hyperplastic human prostate tissue showed a progressive histological increase in irreversible injury with increasing temperature–time severity. The dye uptake and histology results for stromal viability were similar for all temperature–time combinations. In vitro thermal injury showed 85–90% stromal destruction (raw data) of human BPH for temperature–time combinations of 45°C for 60 min, 50°C for 30 min, 55°C for 5 min, 60°C for 2 min and 70°C for 1 min. Apoptosis was also identified in the control and milder treated tissues with the degree of glandular apoptosis (about 20%) more than that seen in the stromal regions (<5%). The Arrhenius model of injury was fitted to the data for conditions leading to a 90% drop in viability (normalized to control) obtained for stromal tissue. The activation energies (E) were 40.1 and 38.4 kcal/mole for the dye uptake study and histology, respectively, and the corresponding frequency factors (A) were 1.1 × 1024 and 7.78 × 1022/s. This study presents the first temperature–time versus tissue destruction relation for human BPH tissue. Moreover, it supports the concept that higher temperatures can be used for shorter durations to induce tissue injury comparable with the current clinically recommended lower temperature–longer time treatments (i.e. 45°C for 60 min) for transurethral microwave thermotherapy of the prostate.

Molecular characterization of prostate hyperplasia in prolactin-transgenic mice by using cDNA representational difference analysis.

Transgenic mice overexpressing the rat prolactin (PRL) gene develop a dramatic enlargement of the prostate gland. The objective of this study was to characterize the molecular mechanisms in the prostate of importance for the prostate hyperplasia seen in these transgenic mice. cDNA representational difference analysis (cDNA RDA) was used to isolate differentially expressed transcripts in the prostate hyperplasia of the transgenic mice compared with wildtype littermates. Furthermore, cDNA microarray analysis was used to verify the RDA output. Here we report 10 transcripts, some of them described to be involved in proliferation and apoptosis, which are differentially expressed in the enlarged transgenic prostates compared with controls. The identified differentially expressed transcripts presented herein supports molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human BPH that may contribute to explain the molecular basis of prostate hyperplasia.