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bph  cancer  cap  cells  proliferation  prostate cancer  prostate  smoking  stromal cells  stromal  telomere length  telomere  traf 
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The Prostate

Wiley Online Library : The Prostate

Published: 2017-12-01T00:00:00-05:00


TRAF6 regulates proliferation of stromal cells in the transition and peripheral zones of benign prostatic hyperplasia via Akt/mTOR signaling


Background Increased prostatic smooth muscle tone and hyperplastic growth contribute to urethral obstruction and voiding symptoms in benign prostatic hyperplasia (BPH). It has been suggested that different proliferative potential of stromal cells between transition zone (TZ) and adjoining regions of the prostate plays a significant role in the development of BPH. However, the molecular mechanisms of this hyperplastic process remain unclear. We found tumor necrosis factor receptor-associated factor 6 (TRAF6) highly expressed in TZ stromal cells compared to peripheral zone (PZ) stromal cells by gene array analyzes. Therefore, we aim to study the potential mechanisms of stromal TRAF6 in promoting BPH progression. Methods Stromal cells obtained from BPH-derived primary cultures. The TRAF6-siRNA vector were constructed and transfected into cultured human BPH primary TZ stromal cells, and TRAF6-overexpressing vector were constructed and transfected into cultured human BPH primary PZ stromal cells. Stromal cells were recombined with BPH-1 cells then subcutaneously inoculated into the kidney capsule of male nude mice. Cell proliferation was evaluated by CCK-8 assay. Multiple proteins in the Akt/mTOR pathway were assessed using western blot. Results TRAF6 levels were increased in TZ stroma compared with PZ stroma of BPH. The in vitro cell culture and in vivo cell recombination revealed that selective downregulation of TRAF6 in TZ stromal cells led to suppression of the proliferation, while upregulation of TRAF6 in PZ stromal cells enhanced the proliferation. We found that the Phosphorylation and Ubiquitination of Akt as well as the Phosphorylation of mTOR, P70S6K were decreased when TRAF6 was downregulated in primary cultured TZ stromal cells of BPH. Conclusions TRAF6 can promote the proliferation of stromal cells of BPH via Akt/mTOR signaling. Our results may make stromal TRAF6 responsible for zonal characteristic of BPH and as a promising therapeutic strategy for BPH treatment.

Capillarisin blocks prostate-specific antigen expression on activation of androgen receptor in prostate carcinoma cells


Background Capillarisin (Cap), an active ingredient of Artemisia capillaris extracts, has known for its anti-inflammatory, antioxidant, and anticancer properties. Functions of Cap in prostate cancer are not clear. We investigate effects of Cap on downregulation of prostate specific antigen (PSA) via modulation of androgen receptor (AR) in prostate carcinoma cells. Methods Cell proliferation was measured by water-soluble tetrazolium-1 (WST-1) cell proliferation assays. The PSA and AR expressions were assessed by immunoblotting and RT-qPCR assays. Effects of Cap on PSA expressions were determined by ELISA, immunoblotting, and reporter assays. Co-immunoprecipitation and immunoblotting assays were used to define the effects of Cap on dissociation of AR-heat shock protein 90 (Hsp90) interaction. Results Cap inhibited LNCaP cell growth in a dose- and/or time-dependent way without inducing poly ADP-Ribose Polymerase (PARP) cleavage. Cap not only effectively suppressed AR and PSA protein expressions, but also attenuated activations of synthetic androgen (R1881) on PSA promoter activity dose- and time-dependently. The Cap pretreatment abrogated effects of R1881 on AR activity by reducing AR translocation to the nucleus. Immunoblotting assays indicated that Cap promoted a degradation of AR proteins dose-dependently in either cycloheximide pretreated-LNCaP cells or AR-ectopic expressed PC-3 cells. Pretreatment of MG132, a proteasome inhibitor, attenuated effect of Cap on AR degradation. Cap lessened AR stability by dissociation of AR-Hsp90 interaction. Conclusions Our results indicated that Cap inhibited growth of LNCaP cells. Cap effectively suppressed androgen activation on AR-mediated transactivation, which is AR-dependent through AR degradation and dissociation of AR-Hsp90 in prostate carcinoma cells.

Current or recent smoking is associated with more variable telomere length in prostate stromal cells and prostate cancer cells


Background Current and recent smoking have been associated with a greater risk of prostate cancer recurrence and mortality, though the underlying mechanism is unknown. Methods To determine if telomere shortening, which has been associated with poor outcomes, may be a potential underlying mechanism, we prospectively evaluated the association between smoking status and telomere length in 567 participants in the Health Professionals Follow-up Study, who were surgically treated for prostate cancer. Using tissue microarrays (TMA), we measured telomere length in cancer and benign tissue, specifically stromal cells in the same TMA spot using a telomere-specific fluorescence in situ hybridization assay. Smoking status was collected via questionnaire 2-years before diagnosis. Adjusting for age, pathologic stage and grade, the median and standard deviation of the per-cell telomere signals were determined for each man for stromal cells and cancer cells by smoking categories. In sub-analyses, we restricted to men without major co-morbidities diagnosed before prostate cancer. Results Overall, there were no associations between smoking status and telomere length or variability in stromal cells or cancer cells. However, among men without comorbidities, current smokers and former smokers who quit <10 years ago had the most variable telomere length in stromal cells (29.3% more variable than never smokers; P-trend = 0.0005) and in cancer cells (27.7% more variable than never smokers; P-trend = 0.05). Among men without comorbidities, mean telomere length did not differ by smoking status in stromal cells or cancer cells. Conclusion Telomere variability in prostate cells may be one mechanism through which smoking influences poor prostate cancer outcomes.

Long-term oral exposure to safe dose of bisphenol A in association with high-fat diet stimulate the prostatic lesions in a rodent model for prostate cancer


Background Studies have shown that exposure to environmental chemicals known as endocrine disruptors can cause permanent changes in genital organs, such as the prostate. Among these environmental chemicals stands out bisphenol A (BPA). Another factor associated with prostate changes is the consumption of a high-fat diet. Although the relationship between the consumption of a high-fat diet and an increased risk of prostate cancer is well established, the mechanisms that lead to the establishment of this disease are not completely understood, nor the simultaneous action of BPA and high-fat diet. Methods Adult gerbils (100 days old) were divided in four groups (n = 6 per group): Control (C): animals that received a control diet and filtered water; Diet (D): animals that received a high-fat diet and filtered water; BPA: animals that received a control diet and BPA − 50 µg kg−1 day−1 in drinking water; BPA + Diet (BPA + D): animals that received a high-fat diet + BPA − 50 µg kg−1 day−1 in drinking water. After the experimental period (6 months), the dorsolateral and ventral prostate lobes were removed, and analyzed by several methods. Results Histological analysis indicated premalignant and malignant lesions in both prostatic lobes. However, animals of the D, BPA, and BPA + D groups showed a higher incidence and larger number of prostatic lesions; inflammatory foci were also common. Markers to assess prostate lesions, such as increased activation of the DNA repair system (PCNA-positive cells), androgen receptor (AR), and number of basal cells, confirmed the histology. However, serum levels of testosterone did not change under the experimental conditions. Conclusions The results indicated that the methodology used was effective in generating metabolic changes, which directly compromised prostatic homeostasis. Diet and BPA appear to modulate the activation of the AR pathway and thereby optimize tumor establishment in the gerbil prostate.

Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration


Background Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis (LAP) is involved and contribute to prevent inflammation. Methods Flow cytometry, RT-PCR and immunohistochemistry were used to characterize the macrophage subpopulation residing in the epithelial layer of the rat ventral prostate (VP) after castration. Stereology was employed to determine variations in the number of ED1 and ED2. Mice were treated with either chloroquine or L-asparagine to block autophagy. Results M1 (iNOS-positive) and M2 macrophages (MRC1+ and ARG1+) were not found in the epithelium at day 5 after castration. The percentage of CD68+ (ED1) and CD163+(ED2) phenotypes increased after castration but only CD68+ cells were present in the epithelium. RT-PCR showed increased content of the autophagy markers Bcl1 and LC3 after castration. In addition, immunohistochemistry showed the presence of LC3+ and ATG5+ cells in the epithelium. Double immunohistochemistry showed these cells to be CD68+/LC3+, compatible with the LAP phenotype. LC3+ cells accumulate significantly after castration. Chloroquine and L-asparagine administration caused inflammation of the glands at day 5 after castration. Conclusions CD68+ macrophages phagocytose apoptotic cell corpses and activate the LAP pathway, thereby contributing to the preservation of a non-inflammed microenvironment. Marked inflammation was detected when autophagy blockers were administered to castrated animals.

Enhanced radiosensitization of enzalutamide via schedule dependent administration to androgen-sensitive prostate cancer cells


Background Prostate cancer (PCa) is a progressive disease and the most diagnosed cancer in men. The current standard of care for high-risk localized PCa is a combination of androgen deprivation therapy (ADT) and radiation (XRT). The majority of these patients however become resistant due to incomplete responses to ADT as a result of selective cells maintaining androgen receptor (AR) activity. Improvement can be made if increasing radiosensitivity is realized. Therefore, the aim of this study is to investigate the efficacy of the next-generation PCa drug Enzalutamide (ENZA), as a radiosensitizer in XRT therapy. Methods Using a number of androgen-dependent (LNCaP, PC3-T877A) and androgen-independent (C4-2, 22RV1, PC3, PC3-AR V7) cell lines, the effect of ENZA as a radiosensitizer was studied alone or in combination with ADT and/or XRT. Cell viability and cell survival were assessed, along with determination of cell cycle arrest, DNA damage response and repair, apoptosis and senescence. Results Our results indicated that either ENZA alone (in AR positive, androgen-dependent PCa cells) or in combination with ADT (in AR positive, hormone-insensitive PCa cells) potentiates radiation response [Dose enhancement factor (DEF) of 1.75 in LNCAP and 1.35 in C4-2] stronger than ADT + XRT conditions. Additionally, ENZA sensitized androgen dependent PCa cells to XRT in a schedule-dependent manner, where concurrent administration of ENZA and radiation lead to a maximal radiosensitization when compared to either drug administration prior or after XRT. In LNCaP cells, ENZA treatment significantly prolonged the presence of XRT-induced phospho-γH2AX up to 24 h after treatment; suggesting enhanced DNA damage. It also significantly increased XRT-induced apoptosis and senescence. Conclusions Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ADT. We have also observed that its efficacy is schedule dependent and related to increased levels of DNA damage and a delay of DNA repair processes. Finally, the initial abrogation of DNA-PKcs activity by AR inhibition and its subsequent recovery might represent an important mechanism by which PCa cells acquire resistance to combined anti-androgen and XRT treatment. This work suggests a new use of ENZA in combination with XRT that could be applicable in clinical trial settings for patients with early and intermediate hormone responsive disease.

Altered mitochondrial genome content signals worse pathology and prognosis in prostate cancer


Background Mitochondrial genome (mtDNA) content is depleted in many cancers. In prostate cancer, there is intra-glandular as well as inter-patient mtDNA copy number variation. In this study, we determine if mtDNA content can be used as a predictor for prostate cancer staging and outcomes. Methods Fresh prostate cancer biopsies from 115 patients were obtained at time of surgery. All cores underwent pathological review, followed by isolation of cancer and normal tissue. DNA was extracted and qPCR performed to quantify the total amount of mtDNA as a ratio to genomic DNA. Differences in mtDNA content were compared for prostate cancer pathology features and disease outcomes. Results We showed a significantly reduced mtDNA content in prostate cancer compared with normal adjacent prostate tissue (mean difference 1.73-fold, P-value <0.001). Prostate cancer with increased mtDNA content showed unfavorable pathologic characteristics including, higher disease stage (PT2 vs PT3 P-value = 0.018), extracapsular extension (P-value = 0.02) and a trend toward an increased Gleason score (P-value = 0.064). No significant association was observed between changes in mtDNA content and biochemical recurrence (median follow up of 107 months). Conclusions Contrary to other cancer types, prostate cancer tissue shows no universally depleted mtDNA content. Rather, the change in mtDNA content is highly variable, mirroring known prostate cancer genome heterogeneity. Patients with high mtDNA content have an unfavorable pathology, while a high mtDNA content in normal adjacent prostate tissue is associated with worse prognosis.

Influence of the neural microenvironment on prostate cancer


Background Nerves are key factors in prostate cancer (PCa), but the functional role of innervation in prostate cancer is poorly understood. PCa induced neurogenesis and perineural invasion (PNI), are associated with aggressive disease. Method We denervated rodent prostates chemically and physically, before orthotopically implanting cancer cells. We also performed a human neoadjuvant clinical trial using botulinum toxin type A (Botox) and saline in the same patient, before prostatectomy. Result Bilateral denervation resulted in reduced tumor incidence and size in mice. Botox treatment in humans resulted in increased apoptosis of cancer cells in the Botox treated side. A similar denervation gene array profile was identified in tumors arising in denervated rodent prostates, in spinal cord injury patients and in the Botox treated side of patients. Denervation induced exhibited a signature gene profile, indicating translation and bioenergetic shutdown. Nerves also regulate basic cellular functions of non-neoplastic epithelial cells. Conclusion Nerves play a role in the homeostasis of normal epithelial tissues and are involved in prostate cancer tumor survival. This study confirms that interactions between human cancer and nerves are essential to disease progression. This work may make a major impact in general cancer treatment strategies, as nerve/cancer interactions are likely important in other cancers as well. Targeting the neural microenvironment may represent a therapeutic approach for the treatment of human prostate cancer.

Hyperlipidemia is associated with an increased risk of clinical benign prostatic hyperplasia


Background A high fat diet is associated with risk of benign prostatic hyperplasia (BPH). However, whether hyperlipidemia is associated with BPH remains unclear. This population-based cohort study elucidated whether hyperlipidemia is associated with an increased risk of BPH. Methods We used a new-exposure design and analyzed data retrieved from the Taiwan National Health Insurance Database between January 1, 2000 and December 31, 2013. The cohort of men with newly diagnosed hyperlipidemia and the age- and index-date-matched (1:3) nonhyperlipidemia cohort were tracked for incidence of BPH during a 1- to 14-year follow-up. Diagnosis of BPH using the International Classification of Diseases, Ninth Revision, Clinical Modification codes, and the occurrence of BPH diagnosis plus the use of alpha-blockers or 5-alpha reductase inhibitors or receipt of transurethral resection of the prostate were the primary and secondary endpoints, respectively. The confounders in this study were diabetes mellitus, hypertension, coronary heart disease, obesity, liver cirrhosis, nonsteroidal anti-inflammatory drugs, metformin, aspirin, and number of urologist visits. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using a multivariate Cox proportional hazards regression model adjusted for the propensity score. Results A total of 35 860 subjects (aged 40-99 years)—including the hyperlipidemia cohort (n = 8,965) and nonhyperlipidemia cohort (n = 26 895)—were identified. Our data revealed that the hyperlipidemia cohort had significantly higher incidences of developing BPH (24.6% vs 12.3%, P < 0.001) and treated BPH (13% vs 5.7%, P < 0.001) compared with the nonhyperlipidemia cohort. The risk of developing BPH in the hyperlipidemia cohort was significantly higher than that in the nonhyperlipidemia cohort (HR = 1.73, 95% CI = 1.63-1.83, P < 0.001) after adjustment for the propensity score. Conclusions Hyperlipidemia is associated with an increased risk of clinical BPH.

Milk and other dairy foods in relation to prostate cancer recurrence: Data from the cancer of the prostate strategic urologic research endeavor (CaPSURE™)


Background High-fat dairy, particularly whole milk, in healthy men may increase risk of aggressive prostate cancer. However, data are limited regarding dairy after prostate cancer diagnosis. Method We conducted a prospective study among 1334 men with non-metastatic prostate cancer in the Cancer of the Prostate Strategic Urologic Research Endeavor. Men answered a food frequency questionnaire in 2004-2005 (median 2 years after diagnosis) and were followed until 2016 for recurrence, defined as: prostate cancer death, bone metastases, biochemical recurrence, or secondary treatment. Multivariate Cox proportional hazards regression was used to calculate hazards ratios (HR) and 95% confidence intervals (CI) for associations between whole and low-fat milk; total, high-fat, and low-fat dairy; and other dairy items and risk of recurrence. Results During a median follow-up of 8 years, we observed 137 events. Men who consumed >4 servings/week versus 0-3 servings/month of whole milk had an 73% increased risk of recurrence (HR: 1.73; 95%CI: 1.00, 2.98; P-value = 0.04). Body mass index (BMI) modified the association (P-interaction = 0.01). Among men with a BMI ≥27 kg/m2, >4 servings/week versus 0-3 servings/month of whole milk was associated with a 3-fold higher risk of recurrence (HR: 2.96; 95%CI: 1.58, 5.54; P-value < 0.001). No association was seen in men with BMI <27 kg/m2. Low-fat milk and other dairy foods were not associated with recurrence. Conclusion In conclusion, whole milk consumption after prostate cancer diagnosis was associated with increased risk of recurrence, particularly among very overweight or obese men. Men with prostate cancer who choose to drink milk should select non-fat or low-fat options.

99mTc-MIP-1404-SPECT/CT for the detection of PSMA-positive lesions in 225 patients with biochemical recurrence of prostate cancer


Background 99mTc-MIP-1404 (Progenics Pharmaceuticals, Inc., New York, NY) is a novel, SPECT-compatible 99mTc-labeled PSMA inhibitor for the detection of prostate cancer. We present results of its clinical use in a cohort of 225 men with histologically confirmed prostate cancer referred for workup of biochemical relapse. Methods From April 2013 to April 2017, 99mTc-MIP1404-scintigraphy was performed in 225 patients for workup of PSA biochemical relapse of prostate cancer. Whole-body planar and SPECT/CT images of the lower abdomen and thorax were obtained 3-4 h p.i. of 710 ± 64 MBq 99mTc-MIP-1404. Images were visually analyzed for presence and location of abnormal uptake. In addition, quantitative analysis of the SPECT/CT data was carried out on a subset of 125 patients. Follow-up reports of subsequent therapeutic interventions were available for 59% (139) of all patients. Results Tracer-positive lesions were detected in 77% (174/225) of all patients. Detections occurred at the area of local recurrence in the prostate in 25% of patients (or a total of 56), with metastases in lymph nodes in 47% (105), bone in 27% (60), lung in 5% (12), and other locations in 2% (4) of patients. Detection rates were 90% at PSA levels ≥2 ng/mL and 54% below that threshold. Lesional SUVmax values were, on average, 32.2 ± 29.6 (0.8–142.2), and tumor-to-normal ratios 146.6 ± 160.5 (1.9–1482.4). The PSA level correlated significantly with total uptake of MIP-1404 in tumors (P < 0.001). Furthermore, total tumor uptake was significantly higher in patients with Gleason scores ≥8 compared to those with Gleason scores ≤7 (P < 0.05). In patients with androgen deprivation therapy, the detection rate was significantly higher compared to patients without androgen deprivation therapy (86% vs 71%, P < 0.001). Based on 99mTc-MIP-1404-imaging and other information, an interdisciplinary tumor board review recommended changes to treatment plans in 74% (104/139) of those patients for whom the necessary documentation was available. Conclusion SPECT/CT with 99mTc-labeled MIP-1404 has a high probability in detecting PSMA-positive lesions in patients with elevated PSA. Statistical analysis disclosed significant relationship between quantitative 99mTc-MIP-1404 uptake, PSA level, and Gleason score.

Penetration and pharmacokinetics of non-steroidal anti-inflammatory drugs in rat prostate tissue


Background Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) involves inflammation of the prostate and affects the quality of life of men of all ages. It is well reported in clinical studies that the treatment for CP/CPPS using nonsteroidal anti-inflammatory drugs (NSAIDs) produced favorable outcomes. However, currently, there are no guidelines on choice of the NSAIDs for the treatment of CP/CPPS. Therefore, in the current research study, we evaluated the prostate tissue penetration of four NSAIDs in rats to provide guidance on choice of NSAIDs for the treatment of CP/CPPS. Methods Male Sprague-Dawley rats were administered orally with four NSAIDs viz. celecoxib, diclofenac, ibuprofen, and naproxen at 500 mg/kg dose. The animals were then sacrificed at various time points, and their prostate tissues were harvested. The NSAIDs were then extracted from the prostate tissues using liquid extraction technique, and their concentration in prostate tissue was quantified using high-performance liquid chromatography (HPLC). The prostate tissue penetration and related pharmacokinetic parameters were evaluated by non-compartmental analysis. Results The HPLC method for quantifying NSAIDs in prostate tissue resulted in single, sharp peaks without any interference and all validation parameters were within limits. Celecoxib showed the highest area under the curve (AUC) [146.50 ± 2.75 μg/mL*h] of all NSAID's. A two-factor analysis of variance (ANOVA) with replication indicated an overall statistically significant difference in the pharmacokinetic parameters for celecoxib, diclofenac, ibuprofen, and naproxen. Conclusions This study for the first time reported the relative prostate tissue penetration of four NSAIDs. The pharmacokinetic data indicated that celecoxib has the highest penetration and retention in rat prostate tissues. Therefore, celecoxib may be considered as a better choice for the treatment CP/CPPS involving NSAIDs.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes


Background Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. Methods To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. Results We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. Conclusions Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets.

FOXP3+ regulatory T cells in normal prostate tissue, postatrophic hyperplasia, prostatic intraepithelial neoplasia, and tumor histological lesions in men with and without prostate cancer


Background The tumor promoting or counteracting effects of the immune response to cancer development are thought to be mediated to some extent by the infiltration of regulatory T cells (Tregs). In the present study we evaluated the prevalence of Treg populations in stromal and epithelial compartments of normal, post atrophic hyperplasia (PAH), prostatic intraepithelial neoplasia (PIN), and tumor lesions in men with and without prostate cancer. Methods Study subjects were 102 men consecutively diagnosed with localized prostate cancer undergoing radical prostatectomy and 38 men diagnosed with bladder cancer undergoing cystoprostatectomy without prostate cancer at the pathological examination. Whole mount sections from all patients were evaluated for the epithelial and stromal expression of CD4+ Tregs and CD8+ Tregs in normal, PAH, PIN, and tumor lesions. A Friedmańs test was used to investigate differences in the mean number of Tregs across histological lesions. Logistic regression was used to estimate crude and adjusted odds ratios (OR) for prostate cancer for each histological area. Results In men with prostate cancer, similarly high numbers of stromal CD4+ Tregs were identified in PAH and tumor, but CD4+ Tregs were less common in PIN. Greater numbers of epithelial CD4+ Tregs in normal prostatic tissue were positively associated with both Gleason score and pT-stage. We observed a fourfold increased risk of prostate cancer in men with epithelial CD4+ Tregs in the normal prostatic tissue counterpart. Conclusions Our results may suggest a possible pathway through which PAH develops directly into prostate cancer in the presence of CD4+ Tregs and indicate that transformation of the anti-tumor immune response may be initiated even before the primary tumor is established.

GLUT1 regulates cell glycolysis and proliferation in prostate cancer


Background Glucose transporter 1 (GLUT1) plays a critical role in tumorigenesis and tumor progression in multiple cancer types. However, the specific function and clinical significance of GLUT1 in prostate cancer (PCa) are still unclear. Therefore, in this study, we investigated the role of GLUT1 in PCa. Methods GLUT1 protein levels in prostate cancer tissue and tumor-adjacent normal tissues were measured and compared. Furthermore, real-time PCR and Western blot analysis were both used to detect GLUT1 expression levels in different PCa cell lines. Flow cytometry and cell-based assays, such as a glucose uptake and lactate secretion assay, CCK-8 assay, and transwell migration and wound healing assay, were used to monitor cancer cell cycle distribution, glycolysis, proliferation, and motility, respectively. Moreover, a mouse tumor xenograft model was used to investigate the role of GLUT1 in tumor progression in vivo. Results GLUT1 expression levels are higher in PCa tissues than in tumor-adjacent normal tissues. The results from real-time PCR and Western blot analysis revealed a similar increase in the GLUT1 expression levels in PCa cell lines. Moreover, knockdown of GLUT1 inhibits cell glycolysis and proliferation and leads to cell cycle arrest at G2/M phase in the 22RV1 cell line but not in the PC3 cell line. In vivo experiments further confirmed that GLUT1 knockdown inhibits the growth of tumors derived from the 22RV1 cell line. In addition, we also showed that GLUT1 knockdown has no effect on cell migration in vitro. Conclusions GLUT1 may play an important role in PCa progression via mediating glycolysis and proliferation. Our study also indicated a potential crosstalk between GLUT1-mediated glycolysis and androgen sensitivity in PCa.

The orally active pterocarpanquinone LQB-118 exhibits cytotoxicity in prostate cancer cell and tumor models through cellular redox stress


Background The targeted induction of reactive oxygen species (ROS) is a developing mechanism for cancer therapy. LQB-118 is a pterocarpanquinone and ROS-inducing agent with proven antineoplastic activity. Here, LQB-118 efficacy and mechanism of activity, were examined in Prostate Cancer (PCa) cell and tumor models. Methods PC3, LNCaP, and LAPC4 PCa cells were applied. Dicoumarol treatment was used to inhibit quinone reductase activity. N-acetylcysteine (NAC) was applied as a ROS scavenger. ROS production was quantified by H2DCFDA flow cytometry. LQB-118 treated cells were evaluated for changes in lipid peroxidation, viability, and apoptosis. Treatment-induced gene expression was measured by RT-qPCR and Western Blot. SOD1 knockdown was achieved with siRNA or miRNA mimic transfection. MicroRNA specificity was determined by 3′UTR reporter assay. Oral LQB-118 treatment (10 mg/kg/day) efficacy was determined in athymic male nude mice bearing subcutaneous PC3 xenograft tumors. Results LQB-118 treatment triggered PCa cell death and apoptosis. Therapeutic activity was at least partially dependent upon quinone reduction and ROS generation. LQB-118 treatment caused an increase in cellular ROS and lipid peroxidation. Treated cells exhibited elevated levels of NQO1, Nrf2, and SOD1. The miRNAs miR-206, miR-1, and miR-101 targeted and reduced SOD1 expression. The knockdown of SOD1, by siRNA or miRNA, enhanced LQB-118 cytotoxicity. Orally administered LQB-118 treatment significantly reduced the growth of established PCa xenograft tumors. Conclusion LQB-118 is a developing and orally active pterocarpanquinone agent that effectively kills PCa cells through quinone reduction and ROS generation. The inhibition SOD1 expression enhances LQB-118 activity, presumably by impairing the cellular antioxidant response.

Circulating microRNAs and treatment response in the Phase II SWOG S0925 study for patients with new metastatic hormone-sensitive prostate cancer


Background Previous studies suggest circulating, blood-based microRNAs (miRNAs) may serve as minimally invasive prostate cancer biomarkers, however there is limited data from prospective clinical trials. Here, we explore the role of candidate plasma miRNAs as potential biomarkers in the SWOG 0925 randomized phase II study of androgen deprivation combined with cixutumumab versus androgen deprivation alone in patients with new metastatic hormone-sensitive prostate cancer. Methods Correlative biospecimens, including circulating tumor cells (CTCs) and plasma for miRNA analysis, were collected at baseline and after 12 weeks on treatment from 50 patients enrolled on SWOG 0925. Circulating microRNAs were quantified using real-time RT-PCR microRNA array that allowed specific analysis of previously identified candidate miRNAs (miR-141, miR-200a, miR-200b, miR-210, and miR-375) as well as discovery analysis to identify new candidate miRNAs. MiRNA levels were correlated to previously reported CTC counts using CellSearch® (Veridex) and with the primary study outcome of 28-week PSA response (≤0.2, 0.2 to ≤4.0, or >4.0 ng/mL), previously shown to correlate with overall survival. Results We observed a correlation between baseline circulating miR-141, miR-200a, and miR-375 levels with baseline CTCs. Baseline miR-375 levels were associated with 28-week PSA response (≤0.2, 0.2 to ≤4.0, or >4.0 ng/mL, P = 0.007). Using ROC curve analysis, there was no significant difference between baseline miR-375 and baseline CTC in predicting 28-week PSA response (≤0.2 vs >0.2 ng/mL). To discover novel candidate miRNAs, we analyzed 365 miRNAs for association with the 28-week PSA response endpoint and identified new candidate miRNAs along with the existing candidates miR-375 and miR-200b (P = 0.0012, P = 0.0046, respectively. Conclusions Baseline plasma miR-141, miR-200a, and miR-375 levels are associated with baseline CTC count. Baseline miR-375 was also associated with the trial endpoint of 28-week PSA response. Our results provide evidence that circulating miRNA biomarkers may have value as prognostic biomarkers and warrant further study in larger prospective clinical trials.

Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression


Background The androgen receptor (AR) is the classical target for prostate cancer prevention and treatment, but more recently estrogens and their receptors have also been implicated in prostate cancer development and tumor progression. Methods Recent experimental and clinical data were reviewed to elucidate pathogenetic mechanisms how estrogens and their receptors may affect prostate carcinogenesis and tumor progression. Results The estrogen receptor beta (ERβ) is the most prevalent ER in the human prostate, while the estrogen receptor alpha (ERα) is restricted to basal cells of the prostatic epithelium and stromal cells. In high grade prostatic intraepithelial neoplasia (HGPIN), the ERα is up-regulated and most likely mediates carcinogenic effects of estradiol as demonstrated in animal models. The partial loss of the ERβ in HGPIN indicates that the ERβ acts as a tumor suppressor. The tumor promoting function of the TMPRSS2-ERG fusion, a major driver of prostate carcinogenesis, is triggered by the ERα and repressed by the ERβ. The ERβ is generally retained in hormone naïve and metastatic prostate cancer, but is partially lost in castration resistant disease. The progressive emergence of the ERα and ERα-regulated genes (eg, progesterone receptor (PR), PS2, TMPRSS2-ERG fusion, and NEAT1) during prostate cancer progression and hormone refractory disease suggests that these tumors can bypass the AR by using estrogens and progestins for their growth. In addition, nongenomic estrogen signaling pathways mediated by orphan receptors (eg, GPR30 and ERRα) has also been implicated in prostate cancer progression. Conclusions Increasing evidences demonstrate that local estrogen signaling mechanisms are required for prostate carcinogenesis and tumor progression. Despite the recent progress in this research topic, the translation of the current information into potential therapeutic applications remains highly challenging and clearly warrants further investigation.

Prognostic parameter for high risk prostate cancer patients at initial presentation


Background High-risk prostate cancer can be defined by a patient's Gleason score (GS), prostate-specific antigen (PSA) level, and clinical T (cT) stage, but a novel marker is needed due to heterogeneity of the disease. In this study, we evaluated whether intraductal carcinoma of the prostate (IDC-P) confirmed by needle biopsy is an adverse prognostic parameter for progression-free survival (PFS) and cancer-specific survival (CSS) in patients with high-risk prostate cancer. Methods We retrospectively evaluated 204 patients with high-risk prostate cancer treated by radical prostatectomy from 1991 to 2005 at Nagoya University and its affiliated hospitals. Data on each patient's PSA level, biopsy GS, cT stage, presence of Gleason pattern 5, presence of IDC-P, percentage of the core involved with cancer, and maximum percentage of the core involved with cancer were analyzed. Results The median follow-up period was 108 months (range, 11-257 months). Forty-eight patients (24%) showed disease progression. Thirty-four patients (17%) died of the disease during follow-up. The IDC-P component was detected in 74 (36%) needle biopsy samples. The 5-, 10-, and 15-year CSS rates of the IDC-P-negative cases were 3.2%, 9.0%, and 23.7%; the corresponding rates of the IDC-P-positive cases were 23.9%, 33.7%, and 52.7%, respectively (P = 0.0001). In the Fine and Gray's model for PFS, IDC-P, maximum percentage of the core involved with cancer, and cT stage were significantly associated (P = 0.013, P = 0.003, P = 0.007). In the Fine and Gray's model for CSS, only IDC-P was significant (P = 0.027). In a multivariate Cox regression analysis, IDC-P (P = 0.04; hazard ratio [HR], 1.95) and maximum percentage of the core involved with cancer (P = 0.021; HR, 0.43) were significant factors in predicting overall survival (OS). Conclusions The presence of IDC-P in a needle biopsy was a prognostic factor for PFS, CSS, and OS in patients with high-risk prostate cancer who underwent radical prostatectomy. Multimodal pre-and/or post- surgical therapy may be needed when IDC-P is found in a needle biopsy specimen.

Platelets harbor prostate cancer biomarkers and the ability to predict therapeutic response to abiraterone in castration resistant patients


Background Novel therapies for castration resistant prostate cancer (CRPC) have been introduced in the clinic with possibilities for individualized treatment plans. Best practice of those expensive drugs requires predictive biomarker monitoring. This study used circulating biomarker analysis to follow cancer-derived transcripts implicated in therapy resistance. Method The isolated platelet population of blood samples and digital-PCR were used to identify selected biomarker transcripts in patients with CRPC prior chemo- or androgen synthesis inhibiting therapy. Results Fifty patients received either docetaxel (n = 24) or abiraterone (n = 26) therapy, with therapy response rates of 54% and 48%, respectively. Transcripts for the PC-associated biomarkers kallikrein-related peptidase-2 and -3 (KLK2, KLK3), folate hydrolase 1 (FOLH1), and neuropeptide-Y (NPY) were uniquely present within the platelet fraction of cancer patients and not detected in healthy controls (n = 15). In the abiraterone treated cohort, the biomarkers provided information on therapy outcome, demonstrating an association between detectable biomarkers and short progression free survival (PFS) (FOLH1, P < 0.01; KLK3, P < 0.05; and NPY, P < 0.05). Patients with biomarker-negative platelets had the best outcome, while FOLH1 (P < 0.05) and NPY (P = 0.05) biomarkers provided independent predictive information in a multivariate analysis regarding PFS. KLK2 (P < 0.01), KLK3 (P < 0.001), and FOLH1 (P < 0.05) biomarkers were associated with short overall survival (OS). Combining three biomarkers in a panel (KLK3, FOLH1, and NPY) made it possible to separate long-term responders from short-term responders with 87% sensitivity and 82% specificity. Conclusion Analyzing tumor-derived biomarkers in platelets of CRPC patients enabled prediction of the outcome after abiraterone therapy with higher accuracy than baseline serum PSA or PSA response.

Molecular correlates in urine for the obesity and prostatic inflammation of BPH/LUTS patients


Purpose Benign prostatic hyperplasia (BPH) is strongly associated with obesity and prostatic tissue inflammation, but the molecular underpinning of this relationship is not known. Here, we examined the association between urine levels of chemokines/adipokines with histological markers of prostate inflammation, obesity, and lower urinary tract symptoms LUTS in BPH patients. Methods Frozen urine specimens from 207 BPH/LUTS patients enrolled in Nashville Men's Health Study were sent for blinded analysis of 11 analytes, namely sIL-1RA, CXC chemokines (CXCL-1, CXCL-8, CXCL-10), CC chemokines (CCL2, CCL3, CCL5), PDGF-BB, interleukins IL-6, IL-17, and sCD40L using Luminex™ xMAP® technology. After adjusting for age and medication use, the urine levels of analytes were correlated with the scales of obesity, prostate inflammation grade, extent, and markers of lymphocytic infiltration (CD3 and CD20) using linear regression. Results sIL-1RA levels were significantly raised with higher BMI, waist circumference and waist-hip ratio in BPH patients after correction for multiple testing (P = 0.02). Men with greater overall extent of inflammatory infiltrates and maximal CD3 infiltration were marginally associated with CXCL-10 (P = 0.054) and CCL5 (P = 0.054), respectively. CCL3 in 15 patients with moderate to severe grade inflammation was marginally associated with maximal CD20 infiltration (P = 0.09), whereas CCL3 was undetectable in men with mild prostate tissue inflammation. There was marginal association of sCD40L with AUA-SI scores (P = 0.07). Conclusions Strong association of sIL-1RA in urine with greater body size supports it as a major molecular correlate of obesity in the urine of BPH patients. Increased urine levels of CXCL-10, CCL5, and CCL3 were marginally associated with the scores for prostate tissue inflammation and lymphocytic infiltration. Overall, elevated urinary chemokines support that BPH is a metabolic disorder and suggest a molecular link between BPH/LUTS and prostatic inflammation.





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Plumbagin improves the efficacy of androgen deprivation therapy in prostate cancer: A pre-clinical study


Background Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use. Methods Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin. Results Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR. Conclusion Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

PSCA promotes prostate cancer proliferation and cell-cycle progression by up-regulating c-Myc


Background The Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored protein. Increasing evidence has indicated PSCA plays an important role in tumorigenesis. However, its function and the underlying molecular mechanisms in prostate cancer (PCa) are still not fully elucidated. In this study, we aimed to explore the effect of PSCA on cell cycle of PCa cells and its mechanism research. Methods Immunohistochemistry, quantitative reverse transcription-PCR (qRT-PCR) and Western blotting were used to quantify PSCA expression pattern in PCa tissues and cell lines. The association of PSCA expression with the biochemical recurrence (BCR)-free survival and overall survival (OS) of PCa patients were analyzed using Kaplan-Meier method. The roles of PSCA in PCa were confirmed based on both in vitro and in vivo systems. Results Immunohistochemistry results showed that PSCA was upregulated in PCa tissue. PSCA overexpression were significantly associated with high Gleason score (GS) (P = 0.028), positive BCR (P = 0.002), and poor OS (P = 0.032) and high c-Myc expression (P = 0.019). PSCA promoted PCa cell cycle progression and tumor growth via increased c-Myc expression. Additional, PI3K/AKT signaling pathways was involved in PSCA-mediated c-Myc expression and cell proliferation. Conclusions PSCA is a novel cell cycle regulator with a key role in mediating c-Myc-induced proliferation. PSCA may be a potential diagnostic marker and therapeutic target for patients with PCa.

Circulating and intraprostatic sex steroid hormonal profiles in relation to male pattern baldness and chest hair density among men diagnosed with localized prostate cancers


Background Prospective cohort studies of circulating sex steroid hormones and prostate cancer risk have not provided a consistent association, despite evidence from animal and clinical studies. However, studies using male pattern baldness as a proxy of early-life or cumulative androgen exposure have reported significant associations with aggressive and fatal prostate cancer risk. Given that androgens underlie the development of patterned hair loss and chest hair, we assessed whether these two dermatological characteristics were associated with circulating and intraprostatic concentrations of sex steroid hormones among men diagnosed with localized prostate cancer. Methods We included 248 prostate cancer patients from the NCI Prostate Tissue Study, who answered surveys and provided a pre-treatment blood sample as well as fresh frozen adjacent normal prostate tissue. Male pattern baldness and chest hair density were assessed by trained nurses before surgery. General linear models estimated geometric means and 95% confidence intervals (95%CIs) of each hormone variable by dermatological phenotype with adjustment for potential confounding variables. Subgroup analyses were performed by Gleason score (<7 vs ≥7) and race (European American vs. African American). Results We found strong positive associations of balding status with serum testosterone, dihydrotestosterone (DHT), estradiol, and sex hormone-binding globulin (SHBG), and a weak association with elevated intraprostatic testosterone. Conversely, neither circulating nor intraprostatic sex hormones were statistically significantly associated with chest hair density. Age-adjusted correlation between binary balding status and three-level chest hair density was weak (r = 0.05). There was little evidence to suggest that Gleason score or race modified these associations. Conclusions This study provides evidence that balding status assessed at a mean age of 60 years may serve as a clinical marker for circulating sex hormone concentrations. The weak-to-null associations between balding status and intraprostatic sex hormones reaffirm differences in organ-specific sex hormone metabolism, implying that other sex steroid hormone-related factors (eg, androgen receptor) play important roles in organ-specific androgenic actions, and that other overlapping pathways may be involved in associations between the two complex conditions.

Predictive value of epithelial-mesenchymal-transition (EMT) signature and PARP-1 in prostate cancer radioresistance


Introduction Epithelial-mesenchymal-transition (EMT) has been previously identified as a contributor to prostate cancer progression to metastasis and therapeutic resistance to antiandrogens and radiotherapy. In this study we conducted a retrospective analysis to investigate the significance of radiation-induced EMT and consequential changes to the tumor microenvironment in biochemical recurrence and response to radiotherapy in prostate cancer patients. Methods Expression profiling and localization for EMT effectors, E-Cadherin, N-Cadherin, β-catenin and Vimentin was assessed in human prostate tumor specimens pre- and post-radiotherapy and correlated with biochemical recurrence. In addition, immunoreactivity of the DNA repair enzyme, polymerase (PARP-1) and the cytoskeletal-remodeling regulator, cofilin was evaluated in prostate tumor specimens pre- and post-radiotherapy and correlated with pre-treatment prostate-specific antigen levels (PSA). Results Our findings identified that characteristic changes associated with the EMT phenotype and its reversal to mesenchymal-epithelial-transition (MET) within the tumor microenvironment correlate with biochemical recurrence and resistance to radiotherapy among prostate cancer patients. Moreover, elevated PARP-1 expression among the tumor cells undergoing EMT implicates that DNA repair mechanisms may potentially reverse the cytotoxic effects of radiotherapy-induced DNA breaks. Conclusions Our results suggest that EMT programming effectors, integrated with the actin cytoskeleton regulator cofilin and mesenchymal PARP-1 expression profile provide a signature of potential predictive significance of therapeutic response to radiotherapy in a subset of prostate cancer patients.

Modified risk stratification grouping using standard clinical and biopsy information for patients undergoing radical prostatectomy: Results from SEARCH


Introduction Prostate cancer is a heterogeneous disease, and risk stratification systems have been proposed to guide treatment decisions. However, significant heterogeneity remains for those with unfavorable-risk disease. Methods This study included 3335 patients undergoing radical prostatectomy without adjuvant radiotherapy in the SEARCH database. High-risk patients were dichotomized into standard and very high-risk (VHR) groups based on primary Gleason pattern, percentage of positive biopsy cores (PPBC), number of NCCN high-risk factors, and stage T3b-T4 disease. Similarly, intermediate-risk prostate cancer was separated into favorable and unfavorable groups based on primary Gleason pattern, PPBC, and number of NCCN intermediate-risk factors. Results Median follow-up was 78 months. Patients with VHR prostate cancer had significantly worse PSA relapse-free survival (PSA-RFS, P < 0.001), distant metastasis (DM, P = 0.004), and prostate cancer-specific mortality (PCSM, P = 0.015) in comparison to standard high-risk (SHR) patients in multivariable analyses. By contrast, there was no significant difference in PSA-RFS, DM, or PCSM between SHR and unfavorable intermediate-risk (UIR) patients. Therefore, we propose a novel risk stratification system: Group 1 (low-risk), Group 2 (favorable intermediate-risk), Group 3 (UIR and SHR), and Group 4 (VHR). The c-index of this new grouping was 0.683 for PSA-RFS and 0.800 for metastases, compared to NCCN-risk groups which yield 0.666 for PSA-RFS and 0.764 for metastases. Conclusions Patients classified as VHR have markedly increased rates of PSA relapse, DM, and PCSM in comparison to SHR patients, whereas UIR and SHR patients have similar prognosis. Novel therapeutic strategies are needed for patients with VHR, likely involving multimodality therapy.

The 22Rv1 prostate cancer cell line carries mixed genetic ancestry: Implications for prostate cancer health disparities research using pre-clinical models


Background Understanding how biological factors contribute to prostate cancer (PCa) health disparities requires mechanistic functional analysis of specific genes or pathways in pre-clinical cellular and animal models of this malignancy. The 22Rv1 human prostatic carcinoma cell line was originally derived from the parental CWR22R cell line. Although 22Rv1 has been well characterized and used in numerous mechanistic studies, no racial identifier has ever been disclosed for this cell line. In accordance with the need for racial diversity in cancer biospecimens and recent guidelines by the NIH on authentication of key biological resources, we sought to determine the ancestry of 22RV1 and authenticate previously reported racial identifications for four other PCa cell lines. Methods We used 29 established Ancestry Informative Marker (AIM) single nucleotide polymorphisms (SNPs) to conduct DNA ancestry analysis and assign ancestral proportions to a panel of five PCa cell lines that included 22Rv1, PC3, DU145, MDA-PCa-2b, and RC-77T/E. Results We found that 22Rv1 carries mixed genetic ancestry. The main ancestry proportions for this cell line were 0.41 West African (AFR) and 0.42 European (EUR). In addition, we verified the previously reported racial identifications for PC3 (0.73 EUR), DU145 (0.63 EUR), MDA-PCa-2b (0.73 AFR), and RC-77T/E (0.74 AFR) cell lines. Conclusions Considering the mortality disparities associated with PCa, which disproportionately affect African American men, there remains a burden on the scientific community to diversify the availability of biospecimens, including cell lines, for mechanistic studies on potential biological mediators of these disparities. This study is beneficial by identifying another PCa cell line that carries substantial AFR ancestry. This finding may also open the door to new perspectives on previously published studies using this cell line.