Dorsal Prostate Lobes Research Articles

PD11-04 LOSS OF OSTEOPONTIN LEADS TO THE RESOLUTION OF E. COLI-INDUCED PROSTATIC INFLAMMATION AND FIBROSIS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (PD11)1 Sep 2021PD11-04 LOSS OF OSTEOPONTIN LEADS TO THE RESOLUTION OF E. COLI-INDUCED PROSTATIC INFLAMMATION AND FIBROSIS Petra Popovics, Asha Jain, Francesca Van, Hannah Ruetten, Mark Cadena, Kristen S. Uchtmann, Chad M. Vezina, and William A. Ricke Petra PopovicsPetra Popovics More articles by this author , Asha JainAsha Jain More articles by this author , Francesca VanFrancesca Van More articles by this author , Hannah RuettenHannah Ruetten More articles by this author , Mark CadenaMark Cadena More articles by this author , Kristen S. UchtmannKristen S. Uchtmann More articles by this author , Chad M. VezinaChad M. Vezina More articles by this author , and William A. RickeWilliam A. Ricke More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001986.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men, but the molecular mechanism is unknown. We previously established that osteopontin (OPN), a pro-fibrotic cytokine, is more abundant in the prostate of men with LUTS compared to prostate tissue with normal histology. We also identified that OPN secretion is stimulated by inflammatory cytokines in prostate stromal cells and hypothesized that OPN exacerbates the prostatic inflammatory environment. The current study investigates whether the lack of OPN ameliorates inflammation-induced prostatic fibrosis and urinary dysfunction in mice following transurethral instillation of E. coli. METHODS: Uropathogenic Escherichia coli (UTI89) or saline (control) was instilled via a transurethral catheter (OD 0.8, 100 ul) two times, 3 days apart to C57BL/6J (WT) or B6.129S6(Cg)-Spp1tm1Blh/J (OPN-KO) mice. Urinary function was assessed by weekly void spot assays (VSA). Prostatic collagen was visualized with picrosirius red (PSR) staining and inflammation by immunohistochemistry of the pan-leukocyte marker CD45. RNA-Seq analysis was performed from the ventral prostate lobe. RESULTS: Urinary frequency significantly increased (2.05-fold, p=0.0097) 33 days after E. coli instillation in WT but not in OPN-KO mice. One week after infection, the ventral and dorsal prostate lobes were significantly inflamed (more CD45 cells) and collagen fibers were thicker in both WT and OPN-KO mice. In contrast, 2 months after instillation, OPN-KO mouse prostates contained significantly less total collagen and inflammation compared to WT mice. RNAseq analysis identified that E. coli-instilled WT mice expressed more inflammatory and fibrotic marker RNAs, such as Col3a1, Il1b, MMP3, Mmp7 and Cxcl12 than OPN-KO mice. CONCLUSIONS: Our results show that by preventing the inflammation-related increase in OPN levels, mice undergo accelerated recovery from fibrosis and do not develop chronic inflammation. This suggests that drugs targeting OPN signaling could be beneficial for LUTS patients whose symptoms are primarily linked to fibrosis. Source of Funding: The study was supported by an NIH NIDDK K01 (K01DK127150) and a K12 award (K12DK100022-06, both to PP) and by U54 DK104310 (to WAR and CMV) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e198-e199 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Petra Popovics More articles by this author Asha Jain More articles by this author Francesca Van More articles by this author Hannah Ruetten More articles by this author Mark Cadena More articles by this author Kristen S. Uchtmann More articles by this author Chad M. Vezina More articles by this author William A. Ricke More articles by this author Expand All Advertisement Loading ...

Abstract 1146: Development of CRISPR human Skp2 knock-in in the prostate of mouse and the associated prostate organoids for testing Skp2 targeting agents

Abstract S-phase kinase associated protein 2 (Skp2) is a promising drug target as studies have demonstrated that Skp2 is required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb), Pten or p53 deficient mice. We therefore have aimed to establish CRISPR human Skp2 (hSkp2) knock-in in the prostate of mouse to examine its role in prostate carcinogenesis. In addition, Skp2 overexpressing organoids are being developed for convenient and efficient screening of specific Skp2 inhibitors. cDNA of hSkp2 and porcine teschovirus-1 (P2A) was introduced into immediate upstream of mouse probasin coding sequencing using CRISPR knock-in targeting approach to establish transgenic mice specifically overexpressing hSkp2 in the prostate glands. Quantitative PCR, western blot and immunohistochemistry (IHC) were used to identify the expression levels of Skp2 and histopathological analyses were performed after H&E staining. Prostate glands were dissected and digested for organoid culture, then morphological features and viability of organoids were evaluated following treatments of potential Skp2 inhibitors. Prostate glands and other tissues from mice of three transgenic lines which were genotyped positive were dissected and hSkp2 was detected in ventral, dorsal and lateral prostate lobes, but not in testis, kidney, spleen, and liver. Line 10092 was found to have the highest expression of hSkp2 among these three lines. Prostate weight slightly increased in transgenic mice compared to that of wild-type control mice. Hyperplasia, prostatic intraepithelial neoplasia (PIN), and carcinoma were noted, which suggested that overexpression of hSkp2 in the prostate of mice promoted proliferation and prostate tumorigenesis. Organoids that were derived from the prostate of hSkp2 transgenic mice recapitulated prostatic phenotypic features and maintained a high level of hSkp2. A novel Flavokawain A derivative was found to selectively alter the morphology of hSkp2 organoids and decrease the viability. Prostatic lesions found in the hSkp2 knock-in mice demonstrated a potential role of hSkp2 in early prostate carcinogenesis and organoids overexpressing hSkp2 have the utility for screening out compounds targeting Skp2 in prostate cancer. Citation Format: Liankun Song, Shan Xu, Kia Arabzadehkaffash, Ali Fazelpour, Dongjun Fu, Matthew Tippin, Xiaolin Zi. Development of CRISPR human Skp2 knock-in in the prostate of mouse and the associated prostate organoids for testing Skp2 targeting agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1146.

STAT5a/b Deficiency Delays, but does not Prevent, Prolactin-Driven Prostate Tumorigenesis in Mice.

The canonical prolactin (PRL) Signal Transducer and Activator of Transcription (STAT) 5 pathway has been suggested to contribute to human prostate tumorigenesis via an autocrine/paracrine mechanism. The probasin (Pb)-PRL transgenic mouse models this mechanism by overexpressing PRL specifically in the prostate epithelium leading to strong STAT5 activation in luminal cells. These mice exhibit hypertrophic prostates harboring various pre-neoplastic lesions that aggravate with age and accumulation of castration-resistant stem/progenitor cells. As STAT5 signaling is largely predominant over other classical PRL-triggered pathways in Pb-PRL prostates, we reasoned that Pb-Cre recombinase-driven genetic deletion of a floxed Stat5a/b locus should prevent prostate tumorigenesis in so-called Pb-PRLΔSTAT5 mice. Anterior and dorsal prostate lobes displayed the highest Stat5a/b deletion efficiency with no overt compensatory activation of other PRLR signaling cascade at 6 months of age; hence the development of tumor hallmarks was markedly reduced. Stat5a/b deletion also reversed the accumulation of stem/progenitor cells, indicating that STAT5 signaling regulates prostate epithelial cell hierarchy. Interestingly, ERK1/2 and AKT, but not STAT3 and androgen signaling, emerged as escape mechanisms leading to delayed tumor development in aged Pb-PRLΔSTAT5 mice. Unexpectedly, we found that Pb-PRL prostates spontaneously exhibited age-dependent decline of STAT5 signaling, also to the benefit of AKT and ERK1/2 signaling. As a consequence, both Pb-PRL and Pb-PRLΔSTAT5 mice ultimately displayed similar pathological prostate phenotypes at 18 months of age. This preclinical study provides insight on STAT5-dependent mechanisms of PRL-induced prostate tumorigenesis and alternative pathways bypassing STAT5 signaling down-regulation upon prostate neoplasia progression.

Dimethylarsinic acid may promote prostate carcinogenesis in rats.

Arsenic is a known human carcinogen, inducing tumors of the lung, urinary bladder, skin, liver and prostate. However, there are no reports of prostate tumors induced by arsenicals in in vivo animal models. In a previous study, we found that HMGB2 expression was a predictive marker for prostate carcinogens in the rat 4-week repeated dose test. In this study, six-week-old male F344 rats were orally treated with a total of six chemicals (2-acetylaminofluorene (2-AAF), p-cresidine, dimethylarsinic acid (DMA), glycidol, N-nitrosodiethylamine and acrylamide) for four weeks. Animals were sacrificed at the end of the study, and HMGB2 and Ki-67 immunohistochemistry was performed. The numbers of HMGB2- and Ki-67- positive cells in all prostate lobes were significantly increased by DMA, one of the arsenicals, compared with the controls. Meanwhile, the number of Ki-67-positive cells in lateral and dorsal prostate lobes was significantly decreased by 2-AAF with the reduction of body weight, but HMGB2 expression was not. The other chemicals did not change HMGB2 and Ki-67 expression. These data indicate that DMA may have an ability to enhance prostate carcinogenesis.

Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat

Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10–14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5–3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26–30kDa Ca2+-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not leak into or is inactivated in the blood. The role of this proteinase remains to be determined, but its possible interaction with extracellular glycosaminoglycans could focus its proteolytic activity in the tumor microenvironment and affect tumor growth.

MMP-2 and MMP-9 activities and TIMP-1 and TIMP-2 expression in the prostatic tissue of two ethanol-preferring rat models.

We investigated whether chronic ethanol intake is capable of altering the MMP-2 and MMP-9 activities and TIMP-2 and TIMP-1 expression in the dorsal and lateral prostatic lobes of low (UChA) and high (UChB) ethanol-preferring rats. MMP-2 and MMP-9 activities and TIMP-1 and TIMP-2 expression were significantly reduced in the lateral prostatic lobe of the ethanol drinking animals. Dorsal prostatic lobe was less affected showing no significant alterations in these proteins, except for a reduction in the TIMP-1 expression in UChA rats. These important findings demonstrate that chronic ethanol intake impairs the physiological balance of the prostate extracellular matrix turnover, through downregulation of MMPs, which may contribute to the development of prostatic diseases. Furthermore, since these proteins are also components of prostate secretion, the negative impact of chronic ethanol intake on fertility may also involve reduction of MMPs and TIMPs in the seminal fluid.

Suppression of Prostate Epithelial Proliferation and Intraprostatic Progrowth Signaling in Transgenic Mice by a New Energy Restriction-Mimetic Agent

Cells undergoing malignant transformation often exhibit a shift in cellular metabolism from oxidative phosphorylation to glycolysis. This glycolytic shift, called the Warburg effect, provides a mechanistic basis for targeting glycolysis to suppress carcinogenesis through the use of dietary caloric restriction and energy restriction-mimetic agents (ERMA). We recently reported the development of a novel class of ERMAs that exhibits high potency in eliciting starvation-associated cellular responses and epigenetic changes in cancer cells though glucose uptake inhibition. The lead ERMA in this class, OSU-CG5, decreases the production of ATP and NADH in LNCaP prostate cancer cells. In this study, we examined the effect of OSU-CG5 on the severity of preneoplastic lesions in male transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Daily oral treatment with OSU-CG5 at 100 mg/kg from 6 to 10 weeks of age resulted in a statistically significant decrease in the weight of urogenital tract and microdissected dorsal, lateral, and anterior prostatic lobes relative to vehicle controls. The suppressive effect of OSU-CG5 was evidenced by marked decreases in Ki67 immunostaining and proliferating cell nuclear antigen (PCNA) expression in the prostate. OSU-CG5 treatment was not associated with evidence of systemic toxicity. Microarray analysis indicated a central role for Akt, and Western blot analysis showed reduced phosphorylation and/or expression levels of Akt, Src, androgen receptor, and insulin-like growth factor-1 receptor in prostate lobes. These findings support further investigation of OSU-CG5 as a potential chemopreventive agent.

1088 ESCHERICHIA COLI STRAINS FROM CHRONIC PROSTATITIS PATIENTS INVADE PROSTATE EPITHELIAL CELLS AND INDUCE SECRETION OF INFLAMMATORY MEDIATORS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Prostate & Genitalia1 Apr 20121088 ESCHERICHIA COLI STRAINS FROM CHRONIC PROSTATITIS PATIENTS INVADE PROSTATE EPITHELIAL CELLS AND INDUCE SECRETION OF INFLAMMATORY MEDIATORS Li-Lin Cheng, Laura E. Goeser, Alexandra E. Burleigh, Justin J. Lemke, Rodney A. Welch, and Walter J. Hopkins Li-Lin ChengLi-Lin Cheng Madison, WI More articles by this author , Laura E. GoeserLaura E. Goeser Madison, WI More articles by this author , Alexandra E. BurleighAlexandra E. Burleigh Madison, WI More articles by this author , Justin J. LemkeJustin J. Lemke Madison, WI More articles by this author , Rodney A. WelchRodney A. Welch Madison, WI More articles by this author , and Walter J. HopkinsWalter J. Hopkins Madison, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1195AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Category IIIA chronic prostatitis is characterized by the presence of leukocytes in prostatic secretions without culturable bacteria. One possible explanation is that infecting bacteria are harbored within prostate epithelial cells and their intracellular presence induces local secretion of inflammatory mediators. We examined the ability of uropathogenic E. coli to invade mouse prostate epithelial cells in vitro and to induce production of cytokines and chemokines by these cells. METHODS Twenty-four E. coli isolates from chronic prostatitis patients were genotyped for urovirulence factor genes associated with adherence, toxin production, iron acquisition, secretion, and outer membrane proteins. Motility was measured by a swimming assay. Isolates were tested for their invasion of primary epithelial cells derived from the dorsal and lateral prostate lobes of C3H/HeOuJ mice using a gentamicin protection assay. A multiplex assay was used to quantify cytokines/chemokines secreted by cells exposed to E. coli for two hours before addition of gentamicin. RESULTS Among the 24 strains, 10 were intermediate to highly invasive and 14 were low to non-invasive. There was no significant link between any individual gene and invasion intensity; however, a significant positive correlation between motility and epithelial cell invasion was noted. An initial study with eight isolates showed strong responses of IL-6, monocyte chemotactic protein (MCP-1) and granulocyte-macrophage colony stimulating factor (GM-CSF) at 24 hours post exposure, while IL-1β and TNF-α were undetectable. The mediators were induced by seven invasive strains but not by a non-invasive isolate. In addition, strains with cnf-1, cdt, and hlyA toxin genes appeared to induce the least amounts of mediators in prostate epithelial cells. CONCLUSIONS E. coli isolates from chronic prostatitis patients are able to invade prostate epithelial cells with a wide range of intensities. Inflammatory mediators are produced by prostate epithelial cells when exposed to invasive, chronic prostatitis E. coli strains. The secretion of MCP-1, GM-CSF, and IL-6 is associated with recruitment and production of mononuclear inflammatory cells, implying their roles in promoting chronic inflammation. These results thus support a model of chronic prostatic inflammation in the absence of extracellular bacteria, which is the primary characteristic of Category IIIA prostatitis. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e441 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Li-Lin Cheng Madison, WI More articles by this author Laura E. Goeser Madison, WI More articles by this author Alexandra E. Burleigh Madison, WI More articles by this author Justin J. Lemke Madison, WI More articles by this author Rodney A. Welch Madison, WI More articles by this author Walter J. Hopkins Madison, WI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Rat dorsal prostate is necessary for vaginal adhesion of the seminal plug and sperm motility in the uterine horns.

The rat prostate comprises dorsal, ventral and lateral lobes that are morphologically and biochemically distinct. Lesions to these structures are expected to affect the quality of the ejaculate and male fertility. In experiment 1, we analyzed ejaculate parameters of males that had chemical lesions of the dorsal or ventral lobes. At pre-lesion and at 5 and 20 days post-lesion males were mated, and after ejaculation, seminal fluid and seminal plug were obtained from the mated females. In experiment 2, the ventral lobes were ablated, and the ejaculate was analyzed. In experiment 3, the fertility of males with chemically-lesioned dorsal lobes or ablation of the ventral lobes was evaluated. Chemical lesion of the dorsal lobe prevented the adhesion of the seminal plug to vaginal walls. When these males were tested at 5-days postlesion, no sperm were found in uterus, and at 20-days post-lesion, the few sperm encountered showed slow progressive motility. None of the females that mated with dorsal lobe-lesioned males became pregnant. However, chemical lesion or ablation of the ventral lobes did not affect ejaculate or fertility. Our results indicate that the dorsal prostatic lobes are indispensable for reproductive success in males, and define parameters of ejaculate with which fertility can be estimated.

Rat dorsal prostate is necessary for vaginal adhesion of the seminal plug and sperm motility in the uterine horns

The rat prostate comprises dorsal, ventral and lateral lobes that are morphologically and biochemically distinct. Lesions to these structures are expected to affect the quality of the ejaculate and male fertility. In experiment 1, we analyzed ejaculate parameters of males that had chemical lesions of the dorsal or ventral lobes. At pre-lesion and at 5 and 20 days post-lesion males were mated, and after ejaculation, seminal fluid and seminal plug were obtained from the mated females. In experiment 2, the ventral lobes were ablated, and the ejaculate was analyzed. In experiment 3, the fertility of males with chemically-lesioned dorsal lobes or ablation of the ventral lobes was evaluated. Chemical lesion of the dorsal lobe prevented the adhesion of the seminal plug to vaginal walls. When these males were tested at 5-days postlesion, no sperm were found in uterus, and at 20-days post-lesion, the few sperm encountered showed slow progressive motility. None of the females that mated with dorsal lobe-lesioned males became pregnant. However, chemical lesion or ablation of the ventral lobes did not affect ejaculate or fertility. Our results indicate that the dorsal prostatic lobes are indispensable for reproductive success in males, and define parameters of ejaculate with which fertility can be estimated.

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue-specific transgene expression in the prostate epithelium has previously been achieved using short prostate-specific promoters, rendering transgenic mouse lines susceptible to integration site-dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre-induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre-ERT2 protein from small prostate-specific promoters, these mouse lines will be useful in research focused on prostate-specific disorders such as benign hyperplasia or cancer.

Effects of In Utero Exposure to Vinclozolin on the Adult Prostate.

Embryonic and prepubertal exposure to vinclozolin, a dicarboximide fungicide, has been shown to promote early onset atrophy in the adult prostate lobes. Vinclozolin has the potential to act as an androgen receptor antagonist. The androgen receptor is essential for proper development and growth of the prostate. Inhibition of the androgen receptor via vinclozolin exposure during pre- and postpubertal rats results in reduced prostate weights and an increase in apoptotic cells in the adult prostate. Previously we have shown that an embryonic exposure to vinclozolin via intraperitoneal administration affects the adult prostate in a similar manner as pre- or postpubertal exposures. However, it is unclear if an embryonic vinclozolin exposure via oral administration would affect the adult prostate lobes. Furthermore, it is unknown if the embryonic exposure to vinclozolin affects the expression of the androgen receptor in the adult prostate. In this study, we investigated the affects of an embryonic exposure to vinclozolin on the expression of the androgen receptor in the adult prostate lobes as well as gene candidates that are indicative of proliferative or apoptotic pathways. Pregnant female rats were administered vinclozolin orally at 350mg/kg of body weight/day from embryonic day (E)8-14. The pups were then raised to postnatal day 50, sacrificed and the ventral, lateral and dorsal prostate lobes were collected. Immunohistochemistry was used to identify androgen receptor localization in prostate cells. The androgen receptor was localized to the nucleus of the epithelial and stromal cells in both the control and vinclozolin treated lobes. Real time PCR was used to quantify relative mRNA levels of the androgen receptor, clusterin, Bcl2, and bax in each lobe of the prostate. There was an increase in androgen receptor levels in the ventral and dorsal lobes. Clusterin was decreased in the ventral lobe and increased in the dorsal in the vinclozolin treated animals. Both the Bcl2 and bax were decreased in the ventral and increased in the dorsal. The number of apoptotic cells was unchanged in the vinclozolin treated animals. This study demonstrates that the androgen receptor is affected from an oral embryonic exposure to vinclozolin. (poster)

Retinoid metabolism and ALDH1A2 (RALDH2) expression are altered in the transgenic adenocarcinoma mouse prostate model

Retinoids, which include vitamin A (retinol) and metabolites such as retinoic acid, can inhibit tumor growth and reverse carcinogenesis in animal models of prostate cancer. We analyzed retinoid signaling and metabolism in the TRAMP (transgenic adenocarcinoma mouse prostate) model. We detected increased retinol and retinyl esters in prostates pooled from 24 to 36 week TRAMP transgenic positive mice compared to nontransgenic littermates by HPLC. We used quantitative RT-PCR to measure transcripts for genes involved in retinoid signaling and metabolism, including ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, LRAT, and RARβ 2, in prostate tissue from TRAMP positive (+) and age-matched littermate control mice ranging from 18 to 36 weeks. Transcript levels of ALDH1A1, a putative stem cell marker, were decreased in ventral and lateral lobes of prostates from TRAMP mice compared to age-matched, nontransgenic mice. ALDH1A2 (RALDH2) mRNA levels in dorsal and anterior lobes of TRAMP+ mice were lower than in age-matched (24 week) nontransgenic mice. We detected lower RARβ 2 mRNA levels in dorsal prostate lobes of 36 week TRAMP mice relative to nontransgenic mice. We detected high levels of ALDH1A2 protein in the cytoplasm and nucleus in nontransgenic murine prostate paraffin sections, and lower ALDH1A2 protein levels in all prostate lobes of TRAMP mice compared to nontransgenic mice by immunohistochemistry. We also detected much lower cytoplasmic ALDH1A2 protein levels in all human prostate cancer paraffin sections stained (19 total) relative to normal human prostate tissue on the same sections. Our data indicate that this reduction in ALDH1A2 protein is an early event in human prostate cancer.

Differential age‐associated regulation of clusterin expression in prostate lobes of brown norway rats

Serum androgen concentrations decline with age in male Brown Norway rats and castration induces apoptosis of luminal secretory epithelial cells in the ventral but not in the dorsal and lateral prostate lobes. Clusterin has been described as an androgen-repressed gene and a protein with either anti- or pro-apoptotic actions. We measured clusterin mRNA and protein levels, the effects of aging and castration on clusterin protein levels and clusterin immunolocalization within the prostatic ductal network in the prostate lobes of young and aged rats. Whereas levels of clusterin mRNA and protein expression measured by RT-PCR and Western blot, respectively, were higher in the ventral and lateral lobes of aged (24 months) compared to young (4 months) rats, no age-dependent differences were observed in the dorsal lobe. Clusterin expression was localized by immunohistochemistry exclusively to the proximal duct segment of young rats, but extended to the distal segment of the ventral and lateral lobes of aged rats. Despite an age-related decrease in serum testosterone concentration, clusterin gene expression was not altered in the dorsal lobe. After castration, levels of clusterin expression increased significantly in the ventral and lateral lobes despite the absence of epithelial cell apoptosis in the latter. In castrated rats, clusterin expression extended throughout the proximal-distal duct regions of the prostate lobes of young and aged rats. Regulation of clusterin expression in the prostate lobes of aging rats appears complex and is neither directly repressed by androgen nor dependent on apoptotic-induced stress.

Loss of Nkx3.1 expression in the transgenic adenocarcinoma of mouse prostate model

The transgenic adenocarcinoma of mouse prostate (TRAMP) model has been extensively characterized at the histological and molecular levels, and has been shown to mimic significant features of human prostate cancer. However, the status of Nkx3.1 expression in the TRAMP model has not been elucidated. Immunohistochemical analyses were performed using dorsal, lateral, and ventral prostate (VP) lobes from ages 6 to 30 weeks. Quantitative RT-PCR analyses were performed to determine relative mRNA expression. Heterogeneous loss of Nkx3.1 was observed in hyperplastic lesions of the ventral, dorsal, and lateral lobes. At 6 weeks of age, the ventral lobe displayed profound loss of Nkx3.1. Diminished Nkx3.1 protein was observed in well- to moderately-differentiated cancer lesions of all lobes. Poorly differentiated (PD) tumors stained negatively for Nkx3.1. Quantitative RT-PCR analyses revealed the presence of Nkx3.1 mRNA in each lobe at all ages, albeit reduced to variable levels. These data suggest that disease progression in the TRAMP model may be driven by loss of function of Nkx3.1, in addition to p53 and Rb. Lobe-specific disease progression in the TRAMP model correlates with the reduction of Nkx3.1 protein. Regulation of Nkx3.1 expression during tumorigenesis appears to occur by post-transcriptional and post-translational mechanisms.

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP>AP>DP>VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma.

Protective effects of citrus nobiletin and auraptene in transgenic rats developing adenocarcinoma of the prostate (TRAP) and human prostate carcinoma cells

Dietary phytochemicals, including nobiletin and auraptene, have been shown to exert inhibiting effects in several chemically induced carcinogenesis models. We here investigated the influence of nobiletin and auraptene on prostate carcinogenesis using transgenic rats developing adenocarcinoma of the prostate (TRAP) bearing the SV40 T antigen transgene under control of the probasin promoter and human prostate cancer cells. Starting at 5 weeks of age, male TRAP rats received powder diet containing 500 p.p.m. nobiletin or auraptene, or the basal diet for 15 weeks and then were sacrificed for analysis of serum testosterone levels and histological changes. The body and relative prostate weights and serum testosterone levels did not differ among the groups. Since all animals developed prostate carcinomas, these were semiquantitatively measured and expressed as relative areas of prostate epithelial cells. Nobiletin caused significant reduction in the ventral (P<0.01), lateral (P<0.001) and dorsal (P<0.05) prostate lobes, while decreasing high grade lesions (P<0.05) in the ventral and lateral lobes. Feeding of auraptene also effectively reduced the epithelial component (P<0.05) and high grade lesions (P<0.05), in the lateral prostate. A further experiment demonstrated that growth of androgen sensitive LNCaP and androgen insensitive DU145 and PC3 human prostate cancer cells, was suppressed by both nobiletin and to a lesser extent auraptene in a dose-dependent manner, with significant increase in apoptosis. In conclusion, these compounds, particularly nobiletin, may be valuable for prostate cancer prevention.

Superoxide dismutase in the prostate lobes of aging brown Norway rats

Spontaneous age-dependent epithelial cell hyperplasia occurs in the lateral and dorsal, but not the ventral, lobes of aging Brown Norway (BN) rats. Diminished antioxidant enzyme activities and increased formation of reactive oxygen species (ROS) promote the pathology of many aging disorders. We investigated the hypothesis that prostatic epithelial cell hyperplasia in the BN rat was related to age-dependent and/or lobe-specific changes in superoxide dismutase (SOD). Using Western blots, immunohistochemistry and enzyme activity assays we determined the levels of protein expression, subcellular localization, and activities, respectively, of the three SOD isoforms, cytoplasmic SOD1, mitochondrial SOD2, and extracellular SOD3 in the ventral, lateral, and dorsal prostate lobes of 4-month-old rats with normal prostate morphology, in 24-month-old rats with lobe-specific hyperplasia and in older 30-month-old rats. We observed little change in SOD activities as a function of age, although expression of SOD3 increased in the prostatic lobes of older rats. SOD2 levels were higher in the lateral lobe of 4- and 24-month-old rats, but declined by 30 months of age to levels in the ventral and dorsal lobes. SOD1 was localized by immunohistochemistry to the nuclei of epithelial cells in all lobes, but the number of immunopositive nuclei increased in the lateral and dorsal lobes of 24-month-old animals. The concentration of zinc was highest in the prostate lobes of 24-month-old animals. Based upon our data, superoxide dismutase is not significantly altered in the rat prostate during aging and thus is unlikely to be an important factor in the evolution of epithelial cell hyperplasia.

Effects of castration on the expression of brain-derived neurotrophic factor (BDNF) in the vas deferens and male accessory genital glands of the rat

Brain-derived neurotrophic factor (BDNF) is a growth factor belonging to the family of neurotrophins. Although neurotrophins in the male genital organs have been well documented, their role in the biology of these organs is far from clear. In particular, little is known about the influence of sex hormones on neurotrophin expression. In the present study, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), we investigated the distribution and tissue concentration of BDNF in the vas deferens and accessory male genital glands in normal and castrated rats. The expression of BDNF mRNA was also investigated. In normal rats, BDNF immunoreactivity was localized in the musculature of the vas deferens and vesicular gland and in the fibromuscular stromal cells of the prostate. In the ventral prostatic lobes, BDNF immunoreactivity was localized in basal, secretory and neuroendocrine epithelial cells. Innervating ganglia and nerves were immunoreactive in all the examined tracts. After castration, BDNF immunoreactivity increased in the musculature of the vesicular gland and in the fibromuscular stromal cells of both dorsal and ventral prostatic lobes. BDNF immunoreactivity also increased in the nerves. ELISA and reverse transcription/real-time polymerase chain reaction confirmed the findings of the immunohistochemical study. In the accessory glands, castration induced an increase of both BDNF tissue concentration and mRNA expression. These results suggest that BDNF is expressed in the internal male genital organs of the rat and that its expression is downregulated by androgen hormones. We hypothesize that the observed BDNF increases are related to the castration-induced regression of the sympathetic nerves.

Sonic hedgehog-patched Gli signaling in the developing rat prostate gland: lobe-specific suppression by neonatal estrogens reduces ductal growth and branching

While prostate gland development is dependent on androgens, other hormones including retinoids and estrogens can influence this process. Brief exposure to high-dose estrogen during the neonatal period in rats leads to permanent, lobe-specific aberrations in the prostate gland, a phenomenon referred to as developmental estrogenization. We have previously shown that this response is mediated through alterations in steroid receptor expression; however, further downstream mechanisms remain unclear. Herein, we examined Sonic hedgehog ( Shh)-patched ( ptc)- gli in the developing rat prostate gland, its role in branching morphogenesis, and the effects of neonatal estrogens on its expression and localization to determine whether a disturbance in this signaling pathway is involved in mediating the estrogenized phenotype. Shh was expressed in epithelial cells at the distal tips of elongating ducts in discreet, heterogeneous foci, while ptc and gli1–3 were expressed in the adjacent mesenchymal cells in the developing gland. The addition of Shh protein to cultured neonatal prostates reduced ductal growth and branching, decreased Fgf10 transcript, and increased Bmp4 expression in the adjacent mesenchyme. Shh-induced growth suppression was reversed by exogenous Fgf10, but not noggin, indicating that Fgf10 suppression is the proximate cause of the growth inhibition. A model is proposed to show how highly localized Shh expression along with regulation of downstream morphogens participates in dichotomous branching during prostate morphogenesis. Neonatal exposure to high-dose estradiol suppressed Shh, ptc, gli1, and gli3 expressions and concomitantly blocked ductal branching in the dorsal and lateral prostate lobes specifically. In contrast, ventral lobe branching and Shh-ptc-gli expression were minimally affected by estrogen exposure. Organ culture studies with lateral prostates confirmed that estradiol suppressed Shh-ptc-gli expression directly at the prostatic level. Taken together, the present findings indicate that lobe-specific decreases in Shh-ptc-gli expression are involved in mediating estradiol-induced suppression of dorsal and lateral lobe ductal growth and branching during prostate morphogenesis.