Abstract: BACKGROUND. Extended transrectal ultrasound guided biopsies (TRUSB) of the prostate may not accurately convey true morphometric information and Gleason score (GS) of prostate cancer (PCa) and the clinical use of template-guided (5-mm grid) transperineal mapping biopsies (TPMBs) remains controversial. METHODS. We correlated the clinical-pathologic results of 1,403 TPMB cores obtained from 25 men diagnosed with PCa with 64 cancer lesions found in their corresponding radical prostatectomy (RP) specimens. Special computer models of three-dimensional, whole-mounted radical prostatectomy (3D-WMRP) specimens were generated and used as gold standard to determine tumor morphometric data. Between-sample rates of upgrade and downgrade (highest GS and a novel cumulative GS) and upstage and downstage (laterality) were determined. Lesions � 0.5 cm 3 or GS � 7 were considered clinically significant. RESULTS. From 64 separate 3D-WMRP lesions, 25 had significant volume (mean 1.13 cm 3 ) and 39 were insignificant (mean 0.09 cm 3 )( P < 0.0001); 18/64 lesions were missed by TPMB, but only one was clinically significant with GS-8 (0.02 cm 3 ). When comparing the cumulative GS of TPMB versus RP, 72% (n ¼ 18) had identical scores, 12% (n ¼ 3) were upgraded, and only 16% (n ¼ 4) were downgraded. Laterality of TPMB and RP was strongly correlated, 80% same laterality, 4% were up-staged, and 16% down-staged. CONCLUSIONS. Our clinical-pathology correlation showed very high accuracy of TPMB with a 5-mm grid template to detect clinically significant PCa lesions as compared with 3D-WMRP, providing physicians and patients with a reliable assessment of grade and stage of disease and the opportunity to choose the most appropriate therapeutic options.

Abstract: BACKGROUND Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH)/low urinary tract symptoms (LUTS) are often comorbid. Chronic inflammation is one of the putative links between these diseases. Phosphodiesterase type 5 inhibitors (PDE5i) are recognized as an effective treatment of BPH-related LUTS. One proposed mechanism of action of PDE5 is the inhibition of intraprostatic inflammation. In this study we investigate whether PDE5i could blunt inflammation in the human prostate. METHODS Evaluation of the effect of tadalafil and vardenafil on secretion of interleukin 8 (IL-8, a surrogate marker of prostate inflammation) by human myofibroblast prostatic cells (hBPH) exposed to different inflammatory stimuli. We preliminary evaluate histological features of prostatic inflammatory infiltrates in BPH patients enrolled in a randomized, double bind, placebo controlled study aimed at investigating the efficacy of vardenafil (10 mg/day, for 12 weeks) on BPH/LUTS. RESULTS In vitro treatment with tadalafil or vardenafil on hBPH reduced IL-8 secretion induced by either TNFα or metabolic factors, including oxidized low-density lipoprotein, oxLDL, to the same extent as a PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. These effects were reverted by the PKG inhibitor KT5823, suggesting a cGMP/PKG-dependency. Treatment with tadalafil or vardenafil significantly suppressed oxLDL receptor (LOX-1) expression. Histological evaluation of anti-CD45 staining (CD45 score) in prostatectomy specimens of BPH patients showed a positive association with MetS severity. Reduced HDL-cholesterol and elevated triglycerides were the only MetS factors significantly associated with CD45 score. In the MetS cohort there was a significant lower CD45 score in the vardenafil-arm versus the placebo-one. Prostate 73: 1391–1402, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Prostatic inflammation has been linked to a number of prostatic diseases such as benign prostatic hyperplasia (BPH), prostatitis syndromes, and prostate cancer. Major unanswered questions include what pathogenic mechanisms, such as bacterial infections, may drive the accumulation of inflammatory infiltrates in the human prostate, and how inflammation might contribute to disease. To study this potential link in an in vivo system, we developed a mouse model of long-term bacteria-induced chronic inflammation of the prostate using a human prostatectomy-derived strain of Propionibacterium acnes. METHODS C57BL/6J mice were inoculated, via urethral catheterization, with vehicle control or a prostatectomy-derived strain of P. acnes (PA2). Animals were assessed at 2 days, 1, 2, or 8 weeks post-inoculation via histology and immunohistochemistry (IHC). RESULTS PA2 inoculation resulted in severe acute and chronic inflammation confined to the dorsal lobe of the prostate. Chronic inflammation persisted for at least 8 weeks post-inoculation. Inflammatory lesions were associated with an increase in the Ki-67 proliferative index, and diminished Nkx3.1 and androgen receptor (AR) production. Interestingly, the observed response required live bacteria and both IHC and in situ hybridization assays for P. acnes indicated a potential intracellular presence of P. acnes in prostate epithelial cells. CONCLUSIONS To our knowledge, this is the first mouse model of long-term prostatic inflammation induced by P. acnes, and more generally, any prostatectomy-derived bacterial isolate. This model may serve as a valuable preclinical model of chronic prostatic inflammation that can be used to mechanistically study the link between inflammation and prostatic disease. Prostate 73: 1007–1015, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Accumulating evidence indicates that microRNAs play a pivotal role in the development and progression of prostate cancer. The present study was aimed at clarifying the biological functions of miR-153, one of the upregulated microRNAs in prostate cancers, and the signaling transduction induced by miR-153. METHODS miR-153 was identified to be overexpressed in prostate cancers. The probable biological function of miR-153 in cellular proliferation was then examined by diverse assays, such as MTT, colony formation and BrdUrd incorporation assay. Moreover, real-time PCR and western blotting analysis were applied to investigate the underlying molecular mechanism induced by miR-153. Luciferase assays were used to determined the FOXO1 transactivity and the direct regulation of PTEN-3′-UTR by miR-153. RESULTS High-throughput method identified miR-153 to be upregulated in prostate cancers, which is further confirmed by the upregulated expression in four paired prostate tumor/adjacent non-cancerous tissues from the same patients. Further studies revealed that overexpression of miR-153 promoted cell cycle transition and cell proliferation, while inhibition of miR-153 reduced this effect. Moreover, miR-153 overexpression in prostate cancer cells increased the G1/S transitional promoter, cyclin D1 expression, and decreased cyclin-dependent kinase (CDK) inhibitor, p21Cip1expression. In addition, we demonstrated that miR-153 directly targeted the PTEN tumor suppressor gene, activated the AKT signaling and downregulated FOXO1 transcriptional activity. CONCLUSIONS Taken together, our results suggest that miR-153 play an important role in promoting proliferation of human prostate cancer cells and present a novel mechanism of microRNA-mediated direct suppression of PTEN expression in prostate cancer cells. Prostate 73: 596–604, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Enzalutamide (formerly MDV3100 and available commercially as Xtandi®), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR-dependent gene expression and cell apoptosis. METHODS The expression of AR target gene prostate-specific antigen (PSA) was measured in LnCaP and C4-2 cells. AR nuclear translocation was assessed in HEK-293 cells stably transfected with AR-yellow fluorescent protein. The in vivo effects of enzalutamide were determined in a mouse xenograft model of CRPC. Differential gene expression in LNCaP cells was measured using Affymetrix human genome microarray technology. RESULTS We found that unlike bicalutamide, enzalutamide lacked AR agonistic activity at effective doses and did not induce PSA expression or AR nuclear translocation. Additionally, it is more effective than bicalutamide at inhibiting agonist-induced AR nuclear translocation. Enzalutamide induced the regression of tumor volume in a CRPC xenograft model and apoptosis in AR-over-expressing prostate cancer cells. Finally, gene expression profiling in LNCaP cells indicated that enzalutamide opposes agonist-induced changes in genes involved in processes such as cell adhesion, angiogenesis, and apoptosis. CONCLUSIONS These results indicate that enzalutamide efficiently inhibits AR signaling, and we suggest that its lack of AR agonist activity may be important for these effects. Prostate 73:1291–1305, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND The treatment of prostate cancer has been impeded by the lack of both clinically relevant disease models and metabolic markers that track tumor progression. Hyperpolarized (HP) 13C MR spectroscopy has emerged as a new technology to investigate the metabolic shifts in prostate cancer. In this study, we investigate the glucose reprogramming using HP 13C pyruvate MR in a patient-derived prostate tissue slice culture (TSC) model. METHODS The steady-state metabolite concentrations in freshly excised human prostate TSCs were assessed and compared to those from snap-frozen biopsy samples. The TSCs were then applied to a perfused cell (bioreactor) platform, and the bioenergetics and the dynamic pyruvate flux of the TSCs were investigated by 31P and HP 13C MR, respectively. RESULTS The prostate TSCs demonstrated steady-state glycolytic and phospholipid metabolism, and bioenergetics that recapitulate features of prostate cancer in vivo. 13C spectra following injection of HP 13C pyruvate showed significantly increased pyruvate to lactate flux in malignant as compared to the benign prostate TSCs. This increased flux in the malignant prostate TSCs correlated with both increased expression of monocarboxylate transporters (MCT) and activity of lactate dehydrogenase (LDH). CONCLUSIONS We provide the first mechanistic evidence for HP 13C lactate as a prostate cancer biomarker in living human tissues, critical for the interpretation of in vivo studies. More broadly, the clinically relevant metabolic model system in combination with HP MR can facilitate the identification of clinically translatable biomarkers of prostate cancer presence, aggressiveness, and treatment response. Prostate 73: 1171–1181, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: Purpose This study was conducted to investigate the effect of Se supplementation on prostate cancer incidence in men at high risk for prostate cancer.

Abstract: BACKGROUND A rare mutation G84E in HOXB13 was recently identified to be associated with prostate cancer (PCa) in Caucasians. The goal of this study is to test association between HOXB13 genetic variants and PCa risk in Chinese men.

Abstract: INTRODUCTION Active surveillance (AS) is increasingly accepted as appropriate management for low-risk prostate cancer (PC) patients. It is unknown whether delaying radical prostatectomy (RP) is associated with increased risk of biochemical recurrence (BCR) for men with intermediate-risk PC. METHODS We performed a retrospective analysis of 1,561 low and intermediate-risk men from the Shared Equal Access Regional Cancer Hospital (SEARCH) database treated with RP between 1988 and 2011. Patients were stratified by interval between diagnosis and RP (≤3, 3–6, 6–9, or >9 months) and by risk using the D'Amico classification. Cox proportional hazard models were used to analyze BCR. Logistic regression was used to analyze positive surgical margins (PSM), extracapsular extension (ECE), and pathologic upgrading. RESULTS Overall, 813 (52%) men were low-risk, and 748 (48%) intermediate-risk. Median follow-up among men without recurrence was 52.9 months, during which 437 men (38.9%) recurred. For low-risk men, RP delays were unrelated to BCR, ECE, PSM, or upgrading (all P > 0.05). For intermediate-risk men, however, delays >9 months were significantly related to BCR (HR: 2.10, P = 0.01) and PSM (OR: 4.08, P 9 months were associated with BCR in subsets of intermediate-risk men with biopsy Gleason score ≤3 + 4 (HR: 2.51, P < 0.01), PSA ≤ 6 (HR: 2.82, P = 0.06), and low tumor volume (HR: 2.59, P = 0.06). CONCLUSIONS For low-risk men, delayed RP did not significantly affect outcome. For men with intermediate-risk disease, delays >9 months predicted greater BCR and PSM risk. If confirmed in future studies, this suggests delayed RP for intermediate-risk PC may compromise outcomes. Prostate 73: 409–417, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND. Resveratrol (Res) is recognized as a promising cancer chemoprevention dietary polyphenol with antioxidative, anti-inflammatory, and anticancer properties. However, the role of its analogues in prostate cancer (PCa) chemoprevention is unknown. METHODS. We synthesized several natural and synthetic analogues of Res and characterized their effects on PCa cells in vitro using a cell proliferation assay. A colony formation assay and in vitro validation of luciferase (Luc) activity was done for LNCaP-Luc cells that were consequently used for in vivo studies. The efficacy of Res, trimethoxy-resveratrol (3MRes) and piceatannol (PIC) was studied in a subcutaneous (s.c.) model of PCa using oral gavage. Tumor progression was monitored by traditional caliper and bioluminescent imaging. The levels of cytokines in serum were examined by ELISA, and the levels of compounds in serum and tumor tissues were determined by gas chromatography-mass spectrometry. RESULTS. We examined the anti-proliferative activities of Res/analogues in three PCa cell lines. We further compared the chemopreventive effects of oral Res, 3M-Res, and PIC in LNCaP-Luc-xenografts. We found that 2 weeks pretreatment with the compounds diminished cell colonization, reduced tumor volume, and decreased tumor growth in the xenografts. Both 3M-Res and PIC demonstrated higher potency in inhibiting tumor progression compared to Res. Notably, 3M-Res was the most active in inhibiting cell proliferation and suppressing colony formation, and its accumulation in both serum and tumor tissues was the highest. CONCLUSIONS. Our findings offer strong pre-clinical evidence for the utilization of dietary stilbenes, particularly 3M-Res, as novel, potent, effective chemopreventive agents in PCa. Prostate # 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Genome-wide association studies have established a number of replicated single nucleotide polymorphisms (SNPs) for susceptibility to prostate cancer (CaP), but it is unclear whether these susceptibility SNPs are also associated with disease aggressiveness. This study evaluates whether such replication SNPs or other candidate SNPs are associated with CaP aggressiveness in African-American (AA) and European-American (EA) men.

Abstract: BACKGROUND Detection of fusion gene TMPRSS2:ERG transcripts in urine have been recently described in order to refine urine-based detection of prostate cancer (PCa), but data its clinical impact remain scarce. We aimed at investigating the correlation of TMPRSS2:ERG, prostate cancer antigen 3 (PCA3), prostate specific antigen (PSA) density, genetic variants, and androgenic status with outcome and pathological findings at prostatic biopsy. METHODS Between 2007 and 2011, 291 patients at risk of PCa because of PSA > 3.0 ng/ml (55%) or candidate to active surveillance protocol justifying restaging biopsy management (45%) were recruited. TMPRSS2:ERG was detected by urine assay (Progensa™). PCA3-score, PSA level, bioavailable testosterone level, prostate volume, rs1447295 and rs6983267 genotypes were prospectively assessed. Univariate and multivariate analysis by logistic regression model (logit) were conducted to study the correlation of TMPRSS2:ERG status, PCA3, and PSA density with biopsy results, and Gleason score. RESULTS Of 291 patients, 173 had PCa and 118 had negative biopsy. PCA3 score, PSA density and TMPRSS2:ERG-score were correlated with presence of PCa (P < 0.0001, P = 0.046, and P < 0.0001, respectively). This correlation remained strong on multivariable analysis model (area under curve 0.743). PCA3 score and PSA density were significantly associated with presence of Grade 4 through multivariable analysis. PCA3 score was also correlated to the percentage of positive cores at biopsy (P = 0.008). CONCLUSIONS Integration of levels TMPRSS2:ERG transcripts in urine, with PCA3-score, androgenic status, genetic status and traditional clinical variables could significantly increase detection of high risk localized PCa. Prostate 73: 242–249, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Evidence suggests that high-heat cooking methods may increase the risk of prostate cancer (PCa). The addition of oil/fat, as in deep-frying, may be of particular concern, and has not specifically been investigated in relation to PCa. Potential mechanisms include the formation of potentially carcinogenic agents such as aldehydes, acrolein, heterocyclic amines, polycyclic aromatic hydrocarbons, and acrylamide. METHODS We estimated odds ratios (OR) and 95% confidence intervals (CI) for the association between tertiles of intake of deep-fried foods from a food frequency questionnaire (French fries, fried chicken, fried fish, doughnuts and snack chips) and PCa risk, adjusted for potential confounders, among 1,549 cases and 1,492 controls. We additionally examined associations with more aggressive PCa (defined as regional/distant stage, elevated Gleason score or prostate-specific antigen level). RESULTS Compared with <1/week, there was a positive association with PCa risk for intake ≥1/week of French fries (OR = 1.37; 95% CI, 1.11–1.69), fried chicken (OR = 1.30; 95% CI, 1.04–1.62), fried fish (OR = 1.32; 95% CI, 1.05–1.66), and doughnuts (OR = 1.35; 95% CI, 1.11–1.66). There was no association for snack chips (OR = 1.08; 95% CI, 0.89–1.32). Most of the estimates were slightly stronger for more aggressive disease (OR = 1.41; 95% CI, 1.04–1.92 for fried fish). CONCLUSION Regular consumption of select deep-fried foods is associated with increased PCa risk. Whether this risk is specific to deep-fried foods, or whether it represents risk associated with regular intake of foods exposed to high heat and/or other aspects of the Western lifestyle, such as fast food consumption, remains to be determined. Prostate 73: 960–969, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Lipocalin-2 (LCN2) is a member of the lipocalin superfamily, and it has an important role in the regulation of cellular oncogenesis and apoptosis. However, the role for LCN2 in prostate cancer remains unclear. METHOD LCN2 expression has been determined by Western blotting, qRT-PCR, and immunohistochemistry in the human prostate cell lines PC3, DU145, LNCaP, and 22Rv, and in human prostate tissue array. In this study, we identified shRNA-LCN2 to determine the role of LCN2 in prostate-cancer cell proliferation, migration, and invasion. Cell proliferative ability was measured by MTT, colony-formation, and cell-cycle analysis. The role of LCN2 in prostate-cancer cell migration and invasion was analyzed by cell-migration assay and Matrigel invasion assay. The effect of LCN2 knockdown on prostate tumor growth was assessed in a subcutaneous xenograft model. RESULTS LCN2 protein and mRNA expression are higher in PC3 and DU145 cells than in LNCaP and 22Rv cells, and prostate cancer tissue correlated significantly with tumor differentiation (P < 0.017) and Gleason's grade (P < 0.02). LCN2 knockdown in PC3 and DU145 cells decreased cell proliferation, colony formation, cell cycle arrest, migration, and invasion. Conversely, LCN2 overexpression in 22Rv cells produced the opposite effect. Subcutaneous xenografts in mice models showed decreased tumor growth in the LCN2-knockdown mice. CONCLUSIONS Our results suggest that LCN2 might play an important role in regulation of proliferation and invasion of human prostate cancer, and that it can be a valuable marker of prostate cancer progression. Prostate 73:1281–1290. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Resistance to chemotherapy represents a significant obstacle in prostate cancer therapeutics. Novel mechanistic understandings in cancer cell chemotherapeutic sensitivity and resistance can optimize treatment and improve patient outcome. Molecular alterations in the metabolic pathways are associated with cancer development; however, the role of these alterations in chemotherapy efficacy is largely unknown. METHODS In a bed-side to bench-side reverse translational approach, we used cDNA microarray and qRT-PCR to identify genes that are associated with biochemical relapse after chemotherapy. Further, we tested the function of these genes in cell proliferation, metabolism, and chemosensitivity in prostate cancer cell lines. RESULTS We report that the gene encoding mitochondrial malate dehydrogenase 2 (MDH2) is overexpressed in clinical prostate cancer specimens. Patients with MDH2 overexpression had a significantly shorter period of relapse-free survival (RFS) after undergoing neoadjuvant chemotherapy. To understand the molecular mechanism underlying this clinical observation, we observed that MDH2 expression was elevated in prostate cancer cell lines compared to benign prostate epithelial cells. Stable knockdown of MDH2 via shRNA in prostate cancer cell lines decreased cell proliferation and increased docetaxel sensitivity. Further, MDH2 shRNA enhanced docetaxel-induced activations of JNK signaling and induced metabolic inefficiency. CONCLUSION Taken together, these data suggest a novel function for MDH2 in prostate cancer development and chemotherapy resistance, in which MDH2 regulates chemotherapy-induced signal transduction and oxidative metabolism. Prostate 73: 1028–1037, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND. Progressive aging- and inflammation-associated fibrosis effectively remodels the extracellular matrix (ECM) to increase prostate tissue stiffness and reduce urethral flexibility, resulting in urinary flow obstruction and lower urinary tract symptoms (LUTS). In the current study, we sought to test whether senescence-accelerated mouse prone (SAMP)6 mice, which were reported to develop prostatic fibrosis, would also develop LUTS, and whether these symptoms would be exacerbated by diet-induced obesity and concurrent Type 2 Diabetes Mellitus (T2DM). METHODS. To accomplish this, SAMP6 and AKR/J background strain mice were fed regular mouse chow, low fat diet chow, or high fat diet chow for 8 months, then subjected to glucose tolerance tests, assessed for plasma insulin levels, evaluated for urinary voiding function, and assessed for lower urinary tract fibrosis. RESULTS. The results of these studies show that SAMP6 mice and AKR/J background strain mice develop diet-induced obesity and T2DM concurrent with urinary voiding dysfunction. Moreover, urinary voiding dysfunction was more severe in SAMP6 than AKR/J mice and was associated with pronounced prostatic and urethral tissue fibrosis. CONCLUSIONS. Taken together, these studies suggest that obesity, T2DM, lower urinary tract fibrosis, and urinary voiding dysfunction are inextricably and biologically linked. Prostate 73: 1123–1133, 2013. # 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Matrix metalloproteinase-2 (MMP2) has been shown to play an important role in cancer cell invasion and the expression of MMP2 is associated with the poor prognosis of prostate cancer; however, the mechanism of MMP2 expression is largely unknown. SIRT1 is a nicotinamide adenine dinucleotide-dependent histone deacetylase (class III HDAC) that has recently been shown to have implications in regulating cancer cell growth and apoptosis. The purpose of this study is to determine the role of SIRT1 in regulating MMP2 expression and tumor invasion in prostate cancer cells. METHODS The interfering RNAi was used to knockdown SIRT1 from prostate cancer cells. Immunoblots, RT-PCR, zymographic assays, co-immunoprecipitation, analysis and transwell assays were used to examine the effects of SIRT1 silencing on MMP2 expression and activity, on SIRT1 and MMP2 interaction, and on prostate cancer cell invasion. The immuno-histochemical assay was performed to study SIRT1 expression in prostate cancer tissues. RESULTS We show that SIRT1 associates and deacetylates MMP2 and SIRT1 regulates MMP2 expression by controlling MMP2 protein stability through the proteosomal pathway. Thus, we demonstrated a novel mechanism in that MMP2 expression can be regulated at the posttranslational level by SIRT1. Furthermore, we determined that SIRT1 inhibition reduced prostate cancer cell invasion and SIRT1 is highly expressed in advanced prostate cancer tissues. CONCLUSIONS SIRT1 is an important regulator of MMP2 expression, activity, and prostate cancer cell invasion. Overexpressed SIRT1 in advanced prostate cancer may play an important role in prostate cancer progression. Prostate 73: 522–530, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Prostate health index (phi) and prostate cancer antigen 3 (PCA3) have been recently proposed as novel biomarkers for prostate cancer (PCa). We assessed the diagnostic performance of these biomarkers, alone or in combination, in men undergoing first prostate biopsy for suspicion of PCa. METHODS One hundred sixty male subjects were enrolled in this prospective observational study. PSA molecular forms, phi index (Beckman coulter immunoassay), PCA3 score (Progensa PCA3 assay), and other established biomarkers (tPSA, fPSA, and %fPSA) were assessed before patients underwent a 18-core first prostate biopsy. The discriminating ability between PCa-negative and PCa-positive biopsies of Beckman coulter phi and PCA3 score and other used biomarkers were determined. RESULTS One hundred sixty patients met inclusion criteria. %p2PSA (p2PSA/fPSA × 100), phi and PCA3 were significantly higher in patients with PCa compared to PCa-negative group (median values: 1.92 vs. 1.55, 49.97 vs. 36.84, and 50 vs. 32, respectively, P ≤ 0.001). ROC curve analysis showed that %p2PSA, phi, and PCA3 are good indicator of malignancy (AUCs = 0.68, 0.71, and 0.66, respectively). A multivariable logistic regression model consisting of both the phi index and PCA3 score allowed to reach an overall diagnostic accuracy of 0.77. Decision curve analysis revealed that this “combined” marker achieved the highest net benefit over the examined range of the threshold probability. CONCLUSIONS phi and PCA3 showed no significant difference in the ability to predict PCa diagnosis in men undergoing first prostate biopsy. However, diagnostic performance is significantly improved by combining phi and PCA3. Prostate 73: 227–235, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: Background—We investigated associations between statin use begun before PCa diagnosis and prostate cancer (PCa) recurrence/progression and PCa-specific mortality (PCSM) in a prospective, population-based cohort study. Methods—The analysis included 1,001 PCa patients diagnosed in 2002–2005 in King County, Washington. Statin use was assessed at baseline using a detailed in-person interview. Prostate cancer recurrence/progression events and cause-specific survival were ascertained from a followup survey and the SEER registry. Multivariable competing risk and Cox proportional hazards regression models were used to assess the risk of PCa outcomes according to categories of statin use. Results—Of the 1,001 PCa patients in our study, 289 men were ever users of statin drugs. During follow-up, we identified 151 PCa recurrence/progression events and 123 total deaths, including 39 PCa-specific deaths. In unadjusted analysis, the risk of PCa-specific mortality (PCSM) was significantly lower for statin users compared to non-users (1% versus 5% at 10 years; P <0.01). In multivariable analysis, the adjusted hazard ratio of PCSM for statin users versus nonusers was 0.19 (95% CI: 0.06, 0.56). Statin use was not associated with overall PCa recurrence/ progression and other-cause mortality. Conclusions—Statin use begun before PCa diagnosis was unrelated to PCa recurrence/ progression but was associated with a decrease in risk of PCSM.

Abstract: BACKGROUND. Androgen receptor (AR) signaling is regarded as the driving force in prostate carcinogenesis, and its modulation represents a logical target for prostate cancer (PC) prevention and treatment. Natural products are the most consistent source of small molecules for drug development. In this study, we investigate the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum), on AR expression in PC cells and delineate its mechanism of action. METHODS. Expression and transcriptional activity of AR was examined by western blotting and luciferase reporter assay, respectively. CellTiter Blue assay was utilized to quantify cell proliferation. Reactive oxygen species (ROS) generation was examined by staining cells with a ROS indicator CM-H2DCFDA, followed by flow cytometry analysis. RESULTS. The results of our experiments demonstrate that PL rapidly reduces AR protein levels in PC cells via proteasome-mediated ROS-dependent mechanism. Moreover, PL effectively depletes a modified AR lacking the ligand-binding domain, shedding light on a new paradigm in the treatment approach to prostatic carcinoma that expresses mutated constitutively active AR. Importantly, PL effectively depletes AR in PC cells at low micromolar concentrations, while concurrently exerting a significant inhibitory effect on AR transcriptional activity and proliferation of PC cells. CONCLUSIONS. Our investigation demonstrates for the first time that PL induces rapid depletion of the AR in PC cells. As such, PL may afford novel opportunities for both prevention and treatment of prostatic malignancy. Prostate # 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND The recent recommendation of the U.S. Preventive Services Task Force against PSA-based screening for prostate cancer was based, in part, on the lack of demonstrated diagnostic utility of serum PSA values in the low, but detectable range to successfully predict prostate cancer. Though controversial, this recommendation reinforced the critical need to develop, validate, and determine the utility of other serum and/or urine transcript and protein markers as diagnostic markers for PCa. The studies described here were intended to determine whether inflammatory cytokines might augment serum PSA as a diagnostic marker for prostate cancer. METHODS Multiplex ELISA assays were performed to quantify CCL1, CCL2, CCL5, CCL8, CCL11, CCL17, CXCL1, CXCL5, CXCL8, CXCL10, CXCL12, and IL-6 protein levels in the serum of 272 men demonstrating serum PSA values of <10 ng/ml and undergoing a 12 core diagnostic needle biopsy for detection of prostate cancer. Logistic regression was used to identify the associations between specific chemokines and prostate cancer status adjusted for prostate volume, and baseline PSA. RESULTS Serum levels for CCL1 (I-309) were significantly elevated among all men with enlarged prostates (P < 0.04). Serum levels for CCL11 (Eotaxin-1) were significantly elevated among men with prostate cancer regardless of prostate size (P < 0.01). The remaining 10 cytokines examined in this study did not exhibit significant correlations with either prostate volume or cancer status. CONCLUSIONS Serum CCL11 values may provide a useful diagnostic tool to help distinguish between prostatic enlargement and prostate cancer among men demonstrating low, but detectable, serum PSA values. Prostate 73: 573–581, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Akt/mTOR/S6K1 signaling cascades play an important role both in the survival and proliferation of tumor cells. METHODS In the present study, we investigated the effects of embelin (EB), identified primarily from the Embelia ribes plant, on the Akt/mTOR/S6K1 activation, associated gene products, cellular proliferation, and apoptosis in human prostate cancer cells. RESULTS EB exerted significant cytotoxic and suppressive effects on Akt and mTOR activation against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, EB suppressed the constitutive activation of Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of anti-apoptotic (Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2) and proliferative (cyclin D1) proteins, activation of caspase-3, and cleavage of PARP. We also observed that EB can significantly enhance the apoptotic effects of a specific pharmacological Akt inhibitor when used in combination and also caused broad inhibition of all the three kinases in Akt/mTOR/S6K1 signaling axis in PC-3 cells. CONCLUSIONS EB inhibits multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of prostate cancer. Prostate 73: 296–305, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. The purpose of this study was to determine the anti-proliferative and anti-angiogenic efficacy of angiotensin-(1-7) [Ang-(1-7)], an endogenous peptide hormone, in human prostate cancer xenografts. METHODS Human LNCaP prostate cancer cells were injected into the flank of athymic mice and tumors were treated with Ang-(1-7) for 54 days. Tumor growth and angiogenesis were determined by immunohistochemistry and western blot hybridization. RESULTS Ang-(1-7) markedly reduced the volume and wet weight of LNCaP xenograft tumors. Histological analysis of tumor sections from saline-treated mice showed increased Ki67 immunoreactivity and enhanced phosphorylation of the MAP kinases ERK1/2 compared to tumors from Ang-(1-7)-treated mice, suggesting that the heptapeptide reduces cell proliferation. Intratumoral vessel density was decreased in Ang-(1-7)-treated mice with an associated reduction in vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), suggesting that the heptapeptide attenuates vascularization by reducing angiogenic factors. Ang-(1-7) administration markedly increased the soluble fraction of VEGF receptor 1 (sFlt-1), with a concomitant reduction in VEGF receptors 1 and 2. sFlt-1 serves as a decoy receptor that traps VEGF and PlGF, making the ligands unavailable to membrane-bound VEGF receptors and preventing activation of pro-angiogenic signaling. CONCLUSIONS The decrease in PlGF and VEGF coupled with the increase in sFlt-1 suggests that Ang-(1-7) may serve as a novel anti-angiogenic therapy for prostate cancer. Further, the pleiotropic mechanisms of action by Ang-(1-7) may limit angiogenic resistance that occurs with VEGF inhibitors or receptor blockers. Prostate 73: 60–70, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND We report the 30-year institutional experience of radical prostatectomy (RP) for men with clinically localized prostate cancer (PC) found to have lymph node (LN) metastases at surgery. METHODS The Johns Hopkins RP Database (1982–2011) was queried for 505 (2.5%) men with node-positive (N1) PC. Survival analysis was completed using the Kaplan–Meier method and proportional hazard regression models. RESULTS The proportion of men with N1PC was 8.3%, 3.5%, and 1.4% in the pre- (1982–1990), early- (1991–2000), and contemporary-PSA eras (2001–2011), respectively. A trend toward decreasing PSA, less palpable disease but more advanced Gleason sum was noted in the most contemporary era. Median total and positive nodes were 13.2 (1–41) and 1.7 (1–12), respectively. Of 135 patients with a unilateral tumor, 80 (59.3%), 28 (20.7%), and 15 (11.1%) had ipsilateral, contralateral, and bilateral positive LN. 15-year biochemical-recurrence free, metastases-free and cancer-specific survival was 7.1%, 41.5%, and 57.5%, respectively. Predictors of biochemical-recurrence, metastases and death from PC in multivariate analysis included Gleason sum at RP, the number and percent of positive LN; notably total number of LN dissected did not predict outcome. CONCLUSIONS In this highly-selected RP cohort, men found to have N1PC disease at RP can experience a durable long-term metastases-free and cancer-specific survival. Predictors of survival include Gleason sum, number, and percentage of positive LN. While total number of LN dissected was not predictive, approximately 30% of men with N1PC will have positive LN contralateral to the primary prostatic lesion highlighting the importance of a thorough, bilateral pelvic LN dissection. Prostate 73: 1673–1680, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Taxanes, including docetaxel, are currently the only cytotoxic chemotherapeutic agents proven to confer survival benefit in patients with castration-resistant prostate cancer (CRPC). However, the merits of taxanes remain modest, and efforts are needed to improve their therapeutic efficacy. METHODS We evaluated the sensitivity of prostate cancer cells to various agents using cytotoxicity assays. Gene and protein expression levels were evaluated by quantitative real-time polymerase chain reaction and Western blotting analysis, respectively. RESULTS Hydrogen peroxide-resistant and castration-resistant cells that overexpressed Twist1 and Y-box binding protein-1 (YB-1) were cross-resistant to cytotoxic agents, including docetaxel. Twist1 regulated YB-1 expression in prostate cancer cells, supported by the induction of Twist1 and YB-1 by transforming-growth factor-β, which is critical for taxane resistance. Twist1 and/or YB-1 were activated in docetaxel-resistant prostate cancer cells, and YB-1 was activated by docetaxel treatment. Conversely, Twist1 and YB-1 knockdown sensitized prostate cancer cells to cytotoxic agents, including docetaxel. In addition, androgen receptor (AR) knockdown increased cellular sensitivity to docetaxel, though AR expression in docetaxel-resistant LNCaP cells was paradoxically lower than in parental cells. Intriguingly, androgen deprivation treatment was more effective in docetaxel-resistant LNCaP cells compared with parental cells. CONCLUSIONS Twist1/YB-1 and AR signaling promote docetaxel resistance in CRPC cells. However, docetaxel-resistant cells were collaterally sensitive to androgen deprivation because of down-regulation of AR expression, suggesting that the therapeutic effect of initial taxane treatment in hormone-naive prostate cancer may be superior to that of salvage taxane treatment in CRPC. Prostate 73:1336–1344. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND Prostate-specific membrane antigen (PSMA) is a highly specific biological marker and treatment target for prostate cancer. So ultrasound molecular imaging using PSMA antibody-loaded targeted nanoscale microbubbles (MBs) may contribute to the early diagnosis of prostate cancer. METHODS PSMA monoclonal antibody-loaded targeted nanoscale MBs were prepared using biotin–avidin technology. Antibody binding was evaluated with immunofluorescence. Using MKN45 gastric cancer cells as controls, the targeting capability of the targeted MBs was observed in prostate cancer cells (LNCaP and C4-2) under optical microscope. Contrast enhancement was monitored by an ultrasound system in C4-2, LNCaP, and MKN45 transplanted tumors in nude mice. The arrival time, time to peak, peak intensity, and duration of contrast enhancement of targeted and blank nanoscale MBs were compared and analyzed. RESULTS Targeted PSMA monoclonal antibody-loaded nanoscale MBs were successfully synthesized. These MBs were stable and could specifically bind to LNCaP and C4-2 cells in vitro but did not bind to MKN45 cells. There were significant differences in peak intensity and duration of contrast enhancement between targeted and blank nanoscale MBs in both transplanted prostate tumors (P < 0.05). Among the three types of transplanted tumors with targeted nanoscale MBs, the peak intensity was significantly higher in prostate tumors (LNCaP and C4-2) than in gastric tumors (MKN45) (P < 0.05). CONCLUSIONS PSMA monoclonal antibody-loaded targeted nanoscale MBs can target and bind to prostate cancer cells specifically and allow for obvious contrast enhancement in vivo. Therefore, this study lays a foundation for early diagnosis and targeted therapy for prostate cancer. Prostate 73: 1147–1158, 2013. © 2013 Wiley Periodicals, Inc.

Abstract: BACKGROUND The treatment of non-localized prostate cancer involves androgen deprivation (AD) therapy which results in tumor regression. Apoptosis has been implicated in the tumor response to AD, but constitutes a small fraction of the total tumor at any time. Cellular senescence is a response to sub-lethal stress in which cells are persistently growth arrested and develop distinct morphological and biochemical characteristics. The occurrence of senescence in prostate tumor tissue after AD therapy has not previously been investigated. METHODS Phenotypic and molecular characteristics of senescence were examined in models of androgen-sensitive prostate cancer after AD and compared with androgen-intact controls. RESULTS In vitro in LNCaP cells, AD induced elevated senescence-associated β-galactosidase (SA-β-gal) staining, decreased proliferation, and increased flow cytometric side scatter while minimally affecting cell viability. The increased expression of the senescence-related proteins Glb1, the cyclin-dependent kinase inhibitor p27Kip1 and chromatin-regulating heterochromatin protein 1γ (HP1γ) were detected in LNCaP cells after AD in vitro by immunoblot and immunofluorescence microscopy. In mice bearing LuCaP xenograft tumors in vivo, surgical castration similarly increased SA-β-gal staining, increased expression of p27Kip1 and HP1γ, and decreased expression of the proliferation marker KI-67, with minimal induction of apoptosis identified by detection of cleaved caspase 3 and TUNEL. Immunohistochemical analysis of human prostate tumors removed after AD shows similar induction of Glb1, HP1γ and decreased KI-67. CONCLUSIONS We conclude that AD induces characteristics consistent with cellular senescence in androgen-sensitive prostate cancer cells. This finding may explain incomplete tumor regression in response to AD. Prostate 73: 337–345, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Estrogen receptor beta (ERβ) has been demonstrated to be expressed in prostate carcinoma cells and estrogen signals through ERβ to act as a tumor suppressor in prostate cancer patients. ERβ is thought to regulate the cell cycle of prostate carcinoma cells by controlling the expression of cell cycle regulators including cyclin D1 (CCND1). This interaction is of particular interest as CCND1 has been implicated in the development of prostate cancer. METHODS We evaluated ERβ and CCND1 immunoreactivity in human prostate cancer (n = 112, surgical specimens), and correlated the findings with clinicopathological features of the patients. Subsequent in vitro experiments using PC-3 prostate carcinoma cells were also performed to examine whether estradiol (E2) could change the expression level of CCND1 mRNA. RESULTS CCND1 immunoreactivity was detected in 78/112 cases (70%), and was significantly correlated with incidence of perineural invasion and ERβ immunoreactivity (P < 0.05). Forty-eight hours incubation with E2 (10 nM) increased the expression level of CCND1 mRNA as well as c-jun (JUN) and c-fos (FOS) in PC-3 cells, and PHTPP (ERβ antagonist) suppressed E2-induced expression of those mRNAs. CONCLUSIONS These findings suggest that CCND1 expression is possibly regulated by estrogens via ERβ and that this signaling pathway may influence prostate cancer development. Prostate 73: 590–595, 2013. © 2012 Wiley Periodicals, Inc.

Abstract: BACKGROUND Chronic inflammation promotes prostate cancer formation and progression. Furthermore, alterations in energy metabolism are a hallmark of prostate cancer cells. However, the actions of inflammatory factors on the energy metabolism of prostate epithelial cells have not been previously investigated. This is the first study to report on the effect of the inflammatory cytokine tumor necrosis factor alpha (TNFα) on the glycolytic and oxidative metabolism, and the mitochondrial function of widely used prostate epithelial cells. METHODS Pre-malignant RWPE-1 and cancerous LNCaP and PC-3 cells were treated with low-dose TNFα. Glycolytic and oxidative metabolism was quantified by measuring extracellular acidification and oxygen consumption rates, respectively. ATP content and lactate export were measured by luminescence and fluorescence, respectively. Mitochondrial content and the expression of glucose transporter 1 (GLUT1), peroxisome proliferator-activated receptor co-activator 1 alpha (PGC-1α), and Cytochrome C were measured by flow cytometry. RESULTS Our data suggest that TNFα increases glycolysis, ATP production, and lactate export, while it reduces oxidative metabolism and mitochondrial function in prostate epithelial cells. The highly aggressive PC-3 cells tend to be less responsive to the actions of TNFα than the pre-malignant RWPE-1 and the non-aggressive LNCaP cells. CONCLUSIONS Cellular energetics, that is, glycolytic and oxidative metabolism is significantly influenced by low-level inflammation in prostate epithelial cells. In widely used prostate epithelial cell models, the micro-environmental inflammatory cytokine TNFα induces aerobic glycolysis while inhibiting oxidative metabolism. This supports the hypothesis that low-level inflammation can induce Warburg metabolism in prostate epithelial cells, which may promote cancer formation and progression. Prostate 73: 1538–1546, 2013. © 2013 Wiley Periodicals, Inc.