Non-malignant Prostate Research Articles

Abstract 2099: Induction of tumor suppressor genes IL24 and IL32 by microRNA-205 in prostate cancer

Abstract Introduction and Objective: MicroRNAs are small noncoding RNAs that regulate the expression of approximately 30% of all human genes, either inhibiting target mRNA translation or inducing its degradation. These genes are abnormally expressed in human malignancies, making their biological importance increasingly apparent. Given the functional complexity of miRNA mediated gene regulation, it is unlikely that miRNAs are limited to gene silencing. The main objective of this study was to investigate the involvement of miRNA-205 in prostate carcinogenesis and whether miR-205 functions as a tumor suppressor by transcriptionally up-regulating tumor suppressor genes IL24 and IL32. Methods: The methods employed in this study include quantitative-real time PCR; western blot; in-situ hybridization; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viabiltity, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays; nuclear run-on, in-vitro transcription and chromatin immunoprecipitation assays were also performed. Results: Our results indicated that miR-205 expression levels are either silenced or significantly lower in prostate cancer cell lines and prostate tumors when compared to non-malignant prostate cell lines or benign prostate hyperplasia tissues respectively. By scanning gene promoters for sequences complementary to miR-205, we identified putative miR-205 target sites in the promoters of IL24 and IL32 tumor suppressor genes. Over-expression of miR-205 in prostate cancer cells (PC-3) induced the expression of both genes by targeting specific sites in their promoters. Transfection of miR-205 impaired cancer cell growth, clonability, migration, invasiveness and induced apoptosis and cell cycle arrest by interfering with the expression of genes involved in these pathways. MicroRNA-205 induced in-vitro transcription and caused enrichment of histone markers indicative of transcriptionally active promoters in IL24 and IL32 genes. Luciferase reporter and mutational assays indicated that IL24 and IL32 are the direct targets of miR-205. Conclusion: For the first time we have shown that microRNA-205 specifically activates tumor suppressor genes IL24 and IL32 by targeting complimentary binding sites in their promoters, corroborating a new function for miRNAs in regulating gene transcription. Such a function of microRNAs may contribute to the novel therapeutic approach in the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2099.

1194 CIRCULATING VITAMIN D3 AND INTRA-PROSTATIC CYP27B1 IN PROSTATE TUMORIGENESIS

You have accessJournal of UrologyProstate Cancer: Basic Research VI1 Apr 20101194 CIRCULATING VITAMIN D3 AND INTRA-PROSTATIC CYP27B1 IN PROSTATE TUMORIGENESIS Huei-Ju Ting, James Messing, Carla Beckham, Edward Messing, and Yi-Fen Lee Huei-Ju TingHuei-Ju Ting More articles by this author , James MessingJames Messing More articles by this author , Carla BeckhamCarla Beckham More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.695AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Epidemiological and preclinical studies suggest vitamin D3 is one of the nutrients effectively inhibiting prostate tumorigenesis. Unfortunately, the concern of systemic side effects from high doses of vitamin D3 limits its application in the general population. Defining the lowest amount of vitamin D3 uptake that can effectively prevent prostate tumorigenesis will establish a guideline for general healthy population. To translate the preventive effect of active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 (1,25-VD) to clinical application, it is necessary to determine the effective dosage of the circulating form of vitamin D3, 25-hydroxyvitamin D3 (25-VD) in preventing tumorigenesis. CYP27B1 is the key enzyme converting 25-VD to 1,25-VD; therefore, this autocrine synthesis of 1,25-VD by prostate cells is one profound biochemical mechanism for the vitamin D chemopreventive action. The objective of this proposal is to determine the effective concentration of 25-VD in mediating the chemopreventive effect and elucidate intra-prostate CYP27B1 level as a new biomarker to predict individualism for prostate cancer development and behavior. METHODS The protein expression level of CYP27B1 was detected in several immortalized normal prostate cell lines. The transactivity of vitamin D receptor induced by different concentrations of 25-VD was measured by a reporter assay and Q-PCR which detects target gene expression. The effective concentration of 25-VD in preventing tumorigenesis was examined in carcinogen-induced prostate epithelial cell transformation models. The role of CYP27B1 in mediating the preventive effect of 25-VD was investigated by targeting knockdown of CYP27B1 using shRNA and human prostate tissue microarray (TMA). RESULTS The equivalent concentration of 25-VD that induced the same level of VDR transactivity as 100 nM of 1,25-VD was between 250-350 nM in these cell lines. This equivalent concentration of 25-VD could effectively prevent tumorigenesis in the parental non-malignant prostate epithelial cell line, but not in the CYP27B1 knockdown cell line. Human prostate TMA detected CYP27B1 expresseion in normal prostate, but not in the prostate cancer. CONCLUSIONS Here we defined the effective concentration of 25-VD and the role of CYP27B1 in preventing malignant-transformation of prostate epithelial cells. This study provides a guideline for future administration of vitamin D3 supplement and indicates that the individual variation of CYP27B1 expression level could contribute to the efficacy of vitamin D3 supplementation in preventing prostate cancer. Rochester, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e462-e463 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Huei-Ju Ting More articles by this author James Messing More articles by this author Carla Beckham More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Differential regulation of steroid nuclear receptor coregulator expression between normal and neoplastic prostate epithelial cells

Deregulated androgen receptor (AR) action is critical for prostate cancer (PCa) progression. Aberrant expression of AR-associated coregulators contributes to AR activity in PCa. The mechanisms underlying coregulator expression in PCa are under intense investigation as they may lead to alternative means of targeting AR activity in PCa cells. We have recently shown that over 30% of coregulator expression in the PCa cell line LNCaP is subject to androgen regulation. Using multiple PCa cell lines as well as xenograft models, non-malignant prostate epithelial cell lines and androgen-responsive tissues derived from a male Wistar rat model system, we explored the effect of androgen stimulation and androgen deprivation on the expression of the core coactivators SRC1, SRC2, SRC3, CBP, and p300. Androgen stimulation of model systems representing PCa led to a decrease in the expression of SRC1, SRC2, SRC3, CBP, and p300, whereas androgen deprivation induced the expression of these coactivators. In contrast, expression of these coregulators remained largely unaffected following changes in the androgenic milieu in AR-positive models representing non-malignant prostate cells and tissues. Our data indicate differences in the regulation of coregulator expression between neoplastic and normal prostate cells. These findings emphasize the important potential of targeting the mechanisms regulating coregulator expression for therapeutic intervention in PCa.

2-(4-Chlorophenyl)benzo-1,4-quinone induced ROS-signaling inhibits proliferation in human non-malignant prostate epithelial cells

Polychlorinated biphenyls (PCBs) and their metabolites are environmental chemical contaminants which can produce reactive oxygen species (ROS) by auto-oxidation of di-hydroxy PCBs as well as the reduction of quinones and redox-cycling. We investigate the hypothesis that 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3), induced ROS-signaling inhibits cellular proliferation. Monolayer cultures of exponentially growing asynchronous human non-malignant prostate epithelial cells (RWPE-1) were incubated with 0–6 μM of 4-Cl-BQ and harvested at the end of 72 h of incubation to assess antioxidant enzyme expression, cellular ROS levels, cell growth, and cell cycle phase distributions. 4-Cl-BQ decreased manganese superoxide dismutase (MnSOD) activity, protein, and mRNA levels. 4-Cl-BQ treatment increased dihydroethidium (DHE) fluorescence, which was suppressed in cells pretreated with polyethylene glycol conjugated superoxide dismutase (PEG-SOD). The increase in ROS levels was associated with a decrease in cell growth, and an increase in the percentage of S-phase cells. These effects were suppressed in cells pretreated with PEG-SOD. 4-Cl-BQ treatment did not change the protein levels of phosphorylated H2AX at the end of 72 h of incubation, suggesting that the inhibition in cell growth and accumulation of cells in S-phase at the end of the treatments were probably not due to 4-Cl-BQ induced DNA double strand break. These results demonstrate that MnSOD activity and ROS-signaling perturb proliferation in 4-Cl-BQ treated in vitro cultures of human prostate cells.

Low Levels of Phosphorylated Epidermal Growth Factor Receptor in Nonmalignant and Malignant Prostate Tissue Predict Favorable Outcome in Prostate Cancer Patients

To explore if the expression of phosphorylated epidermal growth factor receptor (pEGFR) in nonmalignant and malignant prostate tissue is a potential prognostic marker for outcome in prostate cancer patients. We used formalin-fixed tissues obtained through the transurethral resection of the prostate from 259 patients diagnosed with prostate cancer after the transurethral resection of the prostate, and patients were then followed with watchful waiting. Tissue microarrays of nonmalignant and malignant prostate tissue were stained with an antibody against pEGFR. The staining pattern was scored and related to clinicopathologic parameters and to outcome. Low phosphorylation of EGFR in prostate epithelial cells, both in the tumor and surprisingly also in the surrounding nonmalignant tissue, was associated with significantly longer cancer-specific survival in prostate cancer patients. This association remained significant when Gleason score and local tumor stage were added together with pEGFR to a Cox regression model. Tumor epithelial pEGFR immunoreactivity was significantly correlated to tumor cell proliferation, tumor vascular density, and nonmalignant epithelial pEGFR immunoreactivity. Patients with metastases had significantly higher immunoreactivity for tumor and nonmalignant epithelial pEGFR compared with patients without metastases. Low pEGFR immunoreactivity is associated with the favorable prognosis in prostate cancer patients and may provide information about which patients with Gleason score 6 and 7 tumors that will survive their disease even without treatment. Changes in the nonmalignant tissue adjacent to prostate tumors give prognostic information.

Contrast-Enhanced Harmonic Ultrasonography for the Assessment of Prostate Cancer Aggressiveness: a Preliminary Study

ObjectiveTo determine whether contrast-enhanced harmonic ultrasonography can be used to predict the aggressiveness of prostate cancer.Materials and MethodsContrast-enhanced harmonic ultrasonography was performed in 103 patients suspected of prostate cancer before biopsy. Time intensity curves were reconstructed for systematic biopsy sites and sonographic abnormalities. The characteristics of the curves were described using hemodynamic indices including arrival time (AT), time-to-peak (TTP), and peak intensity (PI). The differences of hemodynamic indices between high-grade and low-grade cancer were analyzed and the correlations between the hemodynamic indices and biopsy Gleason score were studied.ResultsProstate cancer was detected in 41 of 103 patients and there were significant differences in the hemodynamic indices between the biopsy sites of the non-malignant patients and prostate cancer lesions (p < 0.05). The prostate biopsies revealed 154 prostate cancer lesions, including 31 low-grade lesions and 123 high-grade lesions. The hemodynamic indices AT and TTP of high-grade tumors were significantly shorter than those of low-grade tumors (p = 0.001, 0.002). In addition, high-grade peripheral zone (PZ) tumors had higher PI than low-grade PZ tumors (p = 0.009). The PZ prostate cancer Gleason score correlated with PI, AT and TTP, with Spearman correlation coefficients of 0.223, -0.335, and -0.351, respectively (p = 0.013, < 0.001 and < 0.001).ConclusionContrast-enhanced ultrasound measurements of hemodynamic indices correlate with the prostate cancer Gleason score.

Detection of polyunsaturated omega-6 fatty acid in human malignant prostate tissue by 1D and 2D high-resolution magic angle spinning NMR spectroscopy

Polyunsaturated omega-6 fatty acids (PUFAs) have been shown to promote prostate cancer. Here, we describe the use of HRMAS NMR spectroscopy to detect omega-6 PUFA species in prostate tissues. Samples originating from non-malignant (n = 54) and malignant (n = 27) prostate tissues (from 27 prostatectomized men) were studied by 1D (1)H, 2D (1)H-(1)H and (1)H-(13)C HRMAS NMR spectroscopy followed by histopathological characterization. HRMAS NMR proved to be a powerful, non-destructive method to identify and characterize PUFAs. The omega-6 PUFA was found in 15% of examined human prostate tumors. It is possible to detect PUFAs in prostate tissues using our NMR-based spectroscopic approach.

High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram as an Inhibitor of Prostate Cancer Cell Growth

To identify novel therapeutic opportunities for patients with prostate cancer, we applied high-throughput screening to systematically explore most currently marketed drugs and drug-like molecules for their efficacy against a panel of prostate cancer cells. We carried out a high-throughput cell-based screening with proliferation as a primary end-point using a library of 4,910 drug-like small molecule compounds in four prostate cancer (VCaP, LNCaP, DU 145, and PC-3) and two nonmalignant prostate epithelial cell lines (RWPE-1 and EP156T). The EC(50) values were determined for each cell type to identify cancer selective compounds. The in vivo effect of disulfiram (DSF) was studied in VCaP cell xenografts, and gene microarray and combinatorial studies with copper or zinc were done in vitro for mechanistic exploration. Most of the effective compounds, including antineoplastic agents, were nonselective and found to inhibit both cancer and control cells in equal amounts. In contrast, histone deacetylase inhibitor trichostatin A, thiram, DSF, and monensin were identified as selective antineoplastic agents that inhibited VCaP and LNCaP cell proliferation at nanomolar concentrations. DSF reduced tumor growth in vivo, induced metallothionein expression, and reduced DNA replication by downregulating MCM mRNA expression. The effect of DSF was potentiated by copper in vitro. We identified three novel cancer-selective growth inhibitory compounds for human prostate cancer cells among marketed drugs. We then validated DSF as a potential prostate cancer therapeutic agent. These kinds of pharmacologically well-known molecules can be readily translated to in vivo preclinical studies and clinical trials.

Global levels of histone modifications predict prostate cancer recurrence

Epigenetic alterations such as DNA methylation and histone modifications play important roles in carcinogenesis. It was reported that global histone modification patterns are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone lysine (H(x)K(y)) and histone acetyl (H(x)Ac) modifications in prostate tissue. A tissue microarray with 113 prostate cancer (PCA), 23 non-malignant prostate tissues was stained with antibodies against H3K4 mono-(H3K4me1), di-(H3K4me2), tri-(H3K4me3) methylation, H3K9me1, H3K9me2, H3K9me3, H3 and H4 pan-acetylation (H3Ac, H4Ac). We also analyzed H3K4 methylation in patients with advanced PCA (hormone-refractory PCA-HRPC, n = 34; hormone-dependent PCA, n = 30). Sections were scored according the staining intensity and the proportion of epithelial cells showing nuclear staining. H3K4me1, H3K9me2, H3K9me3, H3Ac, and H4Ac were significantly reduced in PCA compared to non-malignant prostate tissue. H3Ac and H3K9me2 levels allowed discrimination of PCA and non-malignant prostate tissue highly specifically (>91%) and sensitively (>78%) as determined via ROC analyses (AUC >0.91). Histone lysine methylation and histone acetylation marks were correlated with clinical-pathological parameters (i.e., digital rectal examination, preoperative PSA, pT-stage, lymph node metastasis, Gleason score). In addition, H3K4me1 was a significant predictor of PSA recurrence following radical prostatectomy. H3K4me1, H3K4me2, and H3K4me3 levels were significantly increased in HRPC. Global histone modification levels may help to identify patients with adverse prognosis, and represent a target for the future therapy of PCA.

Role of protein kinase C‐iota in transformed non‐malignant RWPE‐1 cells and androgen‐independent prostate carcinoma DU‐145 cells

Prostate cancer is one of the leading causes of death among men in the USA. In this study, we investigated the role of atypical protein kinase C-iota (PKC-iota) in androgen independent prostate DU-145 carcinoma cellscompared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-iotaisassociated with cyclin-dependent kinase activating kinase (CAK/Cdk7) in RWPE-1 cells, but not in DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-iota silencing RNA (siRNA) decreased cell viability,cell-cycle accumulation at G2/M phase, and phosphorylation of Cdk7 and Cdk2. In addition, PKC-iota siRNA treatment caused less phosphorylation ofBad at ser-155, ser-136, and greater Bad/Bcl-xL heterodimerization, leading to apoptosis. In DU-145 cells, PKC-iota was anti-apoptotic and was required for cell survival. Treatment with PKC-iota siRNA blocked increase in cell number, and inhibited G1/S transition by accumulation of cells in G0/G1phase. In addition to cell-cycle arrest, both RWPE-1 and DU-145 cells underwent apoptosis due to mitochondrial dysfunction and apoptosis cascades, such as release of cytochrome c,activation of caspase-7, and poly (ADP-ribose)polymerase (PARP) cleavage. Our results suggest that PKC-iota is required for cell survival in both transformed non-malignant prostate RWPE-1 cells and androgen-independent malignant prostate DU-145 cells, whereas suppressing PKC-iota lead to apoptosis in DU-145 prostate cells.

Anti-CTLA-4 therapy results in higher CD4+ICOShi T cell frequency and IFN-gamma levels in both nonmalignant and malignant prostate tissues.

Cytotoxic lymphocyte antigen-4 (CTLA-4) blockade is an active immunotherapeutic strategy that is currently in clinical trials in cancer. There are several ongoing trials of anti-CTLA-4 in the metastatic setting of prostate cancer patients with reported clinical responses consisting of decreases in the prostate specific antigen (PSA) tumor marker for some patients. Immunologic markers that correlate with these clinical responses are necessary to guide further development of anti-CTLA-4 therapy in the treatment of cancer patients. We recently reported that CD4(+) inducible co-stimulator (ICOS)(hi) T cells that produce interferon-gamma (IFN-gamma) are increased in the peripheral blood and tumor tissues of bladder cancer patients treated with anti-CTLA-4 antibody. Here we present data from the same clinical trial in bladder cancer patients demonstrating a higher frequency of CD4(+)ICOS(hi) T cells and IFN-gamma mRNA levels in nonmalignant prostate tissues and incidental prostate tumor tissues removed at the time of radical cystoprostatectomy. Our data suggest immunologic markers that can be used to monitor prostate cancer patients who receive anti-CTLA-4 therapy and indicate that the immunologic impact of anti-CTLA-4 antibody can occur in both tumor and nonmalignant tissues. These data should be taken into consideration for evaluation of efficacy as well as immune-related adverse events associated with anti-CTLA-4 therapy.

Genetic and Epigenetic SLC18A2 Silencing in Prostate Cancer Is an Independent Adverse Predictor of Biochemical Recurrence after Radical Prostatectomy

This study investigates SLC18A2 (vesicular monoamine transporter 2) expression in prostate adenocarcinoma and examines its potential as a predictive marker for prostate cancer patient outcome after radical prostatectomy. Expression and single nucleotide polymorphism microarray analyses identified SLC18A2 as both down-regulated and subject to common loss-of-heterozygosity in prostate cancer. Down-regulated SLC18A2 expression was validated on tissue microarrays containing benign and malignant prostate specimens from an independent patient group (n=738). Furthermore, SLC18A2 immunoreactivity in radical prostatectomy tumor specimens (n=506) was correlated to clinicopathologic characteristics and recurrence-free survival. The possibility of SLC18A2 silencing by aberrant DNA methylation in prostate cancer cells was investigated by bisulfite sequencing. Tissue microarray analysis revealed markedly lower cytoplasmic SLC18A2 staining in cancer compared with nonmalignant prostate tissue samples, confirming RNA expression profiling results. Furthermore, multivariate analysis identified cytoplasmic SLC18A2 immunoreactivity as a novel predictor of biochemical recurrence following prostatectomy (hazard ratio, 0.485; 95% confidence interval, 0.333-0.709; P<0.001) independent of prostate-specific antigen, Gleason score, tumor stage, and surgical margin status. SLC18A2 showed loss-of-heterozygosity in 23% of the tumors and was densely hypermethylated in 15 of 17 (88%) prostate cancer samples plus 6 of 6 prostate cancer cell lines. In contrast, SLC18A2 was unmethylated in 4 of 4 adjacent nonmalignant prostate and 3 of 5 benign prostatic hyperplasia tissue samples, whereas 2 of 5 benign prostatic hyperplasia samples had monoallelic hypermethylation. Methylation and histone deacetylase inhibitory agents rescued SLC18A2 expression in three prostate cancer cell lines. SLC18A2 silencing by DNA hypermethylation and/or allelic loss is a frequent event in prostate cancer and a novel independent predictor of biochemical recurrence after prostatectomy.

Hypoxia drives prostate tumour progression and impairs the effectiveness of therapy, but can also promote cell death and serve as a therapeutic target

Hypoxia is common in prostate tumours, promoting tumour progression and impairing treatment responses. Hypoxia stimulates angiogenesis but blood vessels formed in tumours are functionally abnormal so the tissue remains hypoxic. Castration treatment is the standard therapy for advanced prostate cancer. In non-malignant prostate tissue castration-induced epithelial cell death is in part mediated by vascular insult and acute hypoxia, but in prostate tumours the cell death response is less prominent and the tumours will eventually relapse. The effect of androgen ablation therapy should therefore be enhanced by additional targeting of the vasculature and hypoxic tumour cells. However if castration fails to kill a sufficiently large number of cells it could by inducing hypoxia make the situation worse. Androgen ablation treatment, may, after the initial vascular insult, result in temporary vascular normalisation and transiently increased tissue oxygen levels. During this time window, which needs to be better defined, the efficacy of cytotoxic drug and radiation treatments are probably enhanced. In order to allow development of more effective treatment strategies for advanced prostate cancer we need to understand the role of hypoxia in prostate cancer progression and treatment responses. With this knowledge we can properly tailor and time additional treatments with androgen ablation.

The involvement of altered corticotropin releasing factor receptor 2 expression in prostate cancer due to alteration of anti‐angiogenic signaling pathways

Expression of urocortin (Ucn) in the human benign prostate and prostate cancer has been reported recently. Ucn binds and activates corticotropin releasing factor (CRF) receptor 1 (CRFR1) and 2 (CRFR2). Activation of CRFR2 has been shown to inhibit tumor growth by regulation of proliferation and apoptosis as well as suppression of vascularization. However, there is no report demonstrating expression profile of CRFR2 in normal prostate versus prostate cancer. CRFR2 mRNA expression was assessed in human normal prostate and prostate cancer by reverse transcriptase PCR. CRFR2 expression on protein level has been performed using double staining immunofluorescence (IF) of tissue microarrays of 32 cases of prostatic adenocarcioma with corresponding normal tissues. Confocal microscopy was carried out to visualize the immunostaining. PCR of normal prostate lysates exhibited specific signals for CRFR2 mRNA. However PCR of lysates of prostate cancer exhibited no signal for CRFR2 mRNA. IF study exhibited that smooth muscle components of the stroma and endothelial cells of blood vessels express an extensive staining for CRFR2. In a lesser extend vascular smooth muscle cells expressed CRFR2. The tumoral neovascular system and stroma exhibited no immunopositivity for CRFR2. The present study demonstrates for the first time that human benign prostate tissue and prostate cancer specimen differentially express CRFR2. While Ucn expression in prostate cancer has been shown to be identical to non-malignant prostate tissues, we hypothesize that expression loss of CRFR2 in prostate cancer and its neovascularization contributes to prostate tumorigenesis, progression, and neoangiogenesis.

A [‐2]proPSA‐based artificial neural network significantly improves differentiation between prostate cancer and benign prostatic diseases

The aim of this study was to combine the new automated Access [-2]proPSA (p2PSA) assay with a percent free PSA (%fPSA) based artificial neural network (ANN) or logistic regression (LR) model to enhance discrimination between patients with prostate cancer (PCa) and with no evidence of malignancy (NEM) and to detect aggressive PCa. Sera from 311 PCa patients and 275 NEM patients were measured with the p2PSA, total PSA (tPSA) and free PSA (fPSA) assays on Access immunoassay technology (Beckman Coulter, Fullerton, CA) within the 0-30 ng/ml tPSA range. Four hundred seventy-five patients (264 PCa, 211 NEM) had a tPSA of 2-10 ng/ml. LR models and leave-one-out (LOO) ANN models with Bayesian regularization by using tPSA, %fPSA, p2PSA/fPSA (%p2PSA), age and prostate volume were constructed and compared by receiver-operating characteristic (ROC) curve analysis. The ANN and LR model each utilizing %p2PSA, %fPSA, tPSA and age, but without prostate volume, reached the highest AUCs (0.85 and 0.84) and best specificities (ANN: 62.1% and 45.5%; LR: 53.1% and 41.2%) compared with tPSA (22.7% and 11.4%) and %fPSA (45.5% and 26.1%) at 90% and 95% sensitivity. The %p2PSA furthermore distinguished better than tPSA and %fPSA between pT2 and pT3, and Gleason sum <7 and >or=7 PCa. The automated p2PSA assay offers a new tool to improve PCa detection, and especially aggressive PCa detection. Incorporation of %p2PSA into an ANN and LR model further enhances the diagnostic accuracy to differentiate between malignant and non-malignant prostate diseases.

PSA after Incidental Irradiation of the Nonmalignant Prostate: Long-Term Changes

Incidental irradiation of the prostate may affect serum prostate-specific antigen (PSA). However, scarce data exist on PSA changes after irradiation of noncancerous prostatic tissue. This is an update of a study on PSA after pelvic irradiation. From 1997 to 2007, blood samples of 33 men were examined who had undergone pelvic irradiation for rectal or anal cancer. The planning target volume included the prostate in all cases. No patient had clinical evidence of prostatic disease. Radiotherapy was applied in fractions of 1.8-2 Gy up to 40-50 Gy (n = 3), 50-60 Gy (n = 21), and 60-65 Gy (n = 2). Seven patients received 5 x 5 Gy. Serum PSA was measured before, during, and after radiotherapy periodically. Median log (PSA) changes were calculated according to elapsed time from starting radiotherapy. The significance was tested with chi(2)-test. 18 patients died during follow-up. For 15 patients, long-term PSA data with a median follow-up of 9 years (2,546-3,528 days) are available. PSA levels rose during the first weeks of irradiation peaking at 2-4 weeks with a significant 2.7-fold increase (p < 0.01). 1 year after radiation therapy, PSA declined below (90%) the preirradiation level, but this difference was not significant (p = 0.36). On further follow-up PSA did not change up to 8.9 years after radiotherapy (p = 0.36). Irradiation of the prostate causes transient increase of serum PSA. By 1 year, PSA has returned near the preirradiation value and stays there for at least 9 years. A major interference with prostate cancer screening or surveillance after radiotherapy is therefore unlikely.

Anatomy of Smooth Muscle Cells in Nonmalignant and Malignant Human Prostate Tissue

Differently graded areas of human prostate adenocarcinoma were examined after Masson's trichrome staining or immunohistochemistry for smooth muscle alpha-actin, type IV collagen and laminin. In addition, the ultrastructure of the prostatic smooth muscle cells (SMC) during glandular proliferation and epithelial invasion in selected tumors was studied. The SMC formed a thick layer below the epithelial structures in unaffected areas and were closely associated with each other in homotypic interactions. As the tumor grade increased, the SMC gradually lost interactions with each other and became atrophic. With the growth of the epithelial compartment, the SMC initially segregated to the tumor periphery and the intercellular spaces increased. In high grade tumors, the epithelial cancer cells invaded the spaces between the SMC. Immunohistochemical analysis of the basal membrane revealed increased disruption of the usually thick basal membrane, which became thinner and faintly stained with each of the antibodies used. We conclude that most SMC become atrophic following epithelial invasion in human tumors and that degradation of the basal membrane is an important factor in this process. At the ultrastructural level, different SMC phenotypes occur in prostatic tissues during epithelial invasion. Interconversion between these phenotypes is suggested and a probable relationship among them is proposed.

Deoxyribonuclease Activity and Circulating DNA Concentration in Blood Plasma of Patients with Prostate Tumors

The DNase activity and circulating DNA (cirDNA) concentration in blood plasma of healthy donors, patients with chronic prostatitis, and patients with prostate tumors were analyzed. The concentration of the cirDNA from plasma was determined by PicoGreen fluorescent assay. DNase activity in blood was measured using the immunoassay based on the cleavage of a hapten-labeled 974-bp DNA substrate. The mean cirDNA concentration in the plasma of healthy donors was low (21 +/- 4 ng/mL total blood) and was accompanied by high DNase activity (0.17 +/- 0.04 U/mL blood). The mean cirDNA concentration was 90 ng/mL blood (10-234 ng/mL) in the patients with nonmalignant prostate tumors and 115 ng/mL blood (13-339 ng/mL) in those with prostate cancer. The mean DNase activity in blood plasma of the patients with prostate tumors was 0.06 U/mL blood (0-0.12 U/mL). The results obtained demonstrate that increased concentrations of cirDNA in blood of the patients with prostate tumors is accompanied by a decreased DNase activity, confirming our previous data that a low DNase activity in blood plasma of cancer patients is one reason for a high cirDNA concentration.

Extracellular Redox State Regulates Features Associated with Prostate Cancer Cell Invasion

We have examined the possible role of extracellular reduction-oxidation (redox) state in regulation of biological/biochemical features associated with prostate cancer cell invasion. DU145, PC-3, and RWPE1-derived human prostate cancer (WPE1-NB26) cell lines were used for the present in vitro analysis. Increasing levels of nitric oxide using S-nitroso-N-acetylpenicillamine resulted in a decrease in cell invasion ability, whereas increasing levels of extracellular superoxide radical (O(2)(*-)) using xanthine/xanthine oxidase resulted in an increase in cell invasion ability in these three cell lines. WPE1-NB26 cells exhibited an increased glutathione/glutathione disulfide ratio in the medium in comparison with RWPE1 cells (immortalized but nonmalignant prostate epithelial cells), suggesting an alteration of extracellular redox state of WPE1-NB26 cells. We hypothesized that O(2)(*-) production at or near the plasma membrane or in the adjacent extracellular matrix at least partially regulated prostate cancer cell invasion. Using adenovirus-mediated extracellular superoxide dismutase (EC-SOD) gene transduction to enzymatically decrease O(2)(*-) levels, we showed that in the presence of heparin, adenovirus EC-SOD gene transduction resulted in an increase in the expression of EC-SOD outside the cells with resultant inhibition of cell invasion ability. This inhibition correlated with reduced metalloproteinase [matrix metalloproteinase (MMP) 2/membrane type 1-MMP] activities and increased levels of extracellular nitrite. Our results suggest a prominent role of extracellular redox status in regulation of cell invasion, which may provide opportunities for therapeutic interventions.

Atypical protein kinase C phosphorylates IKKαβ in transformed non-malignant and malignant prostate cell survival

Mechanistic pathways involving atypical protein kinase C-ι (aPKC-ι) have been targeted in various cancer cells such as lung cancer, brain and prostate due to PKCι’s antiapoptotic function, and role in cell proliferation and cell survival. In the current study, we examined the involvement of PKC-ι in the NF-κB pathway following treatment of prostate cells with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Results demonstrated that androgen-independent DU-145 prostate carcinoma is insensitive to TNFα while transformed non-tumorigenic prostate RWPE-1 cells showed a slight sensitivity to TNFα. However, androgen-dependent LNCaP prostate cells are more sensitive to TNFα treatment and undergo apoptosis. Results demonstrated that in DU-145 cells, TNFα-induced PKC-ι in phosphorylation of IKKαβ. In RWPE-1 cells, PKC-ζ phosphorylates IKKαβ. Degradation of IκBα was observed in all three cell lines, allowing NF-κB/p65 translocation to the nucleus. Although, IKKα is weakly activated in LNCaP cells, the upstream kinase phosphorylation of IKKαβ via aPKCs was not observed. Hence, aPKCs may play a role in activation of NFκB pathway in prostate cancer cells.