Normal Prostate Epithelial Cell Line Research Articles

MP48-11 THE EFFECT OF MORPHINE AND ITS INHIBITORS ON RT112 BLADDER CANCER CELL GROWTH

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2017MP48-11 THE EFFECT OF MORPHINE AND ITS INHIBITORS ON RT112 BLADDER CANCER CELL GROWTH Brian Birch, Bashir Lwaleed, Alan Cooper, Phil Harper, and Iliana Giatsidou Brian BirchBrian Birch More articles by this author , Bashir LwaleedBashir Lwaleed More articles by this author , Alan CooperAlan Cooper More articles by this author , Phil HarperPhil Harper More articles by this author , and Iliana GiatsidouIliana Giatsidou More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1492AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clinical studies suggest opioid use affects rates of metastasis in breast and prostate cancer. Results are variable and conflicting. This investigation tested the effects of morphine on bladder cancer cell growth in vitro and examined whether those effects can be reversed with receptor blockers. METHODS Cell lines: Adherent epithelial cell lines were used:RT-112 - Human urinary bladder transitional carcinoma cell lines, histological grade G2 (moderately differentiated). PNT2 - Normal (virally transformed) Human prostate epithelial cell line. These cells were grown using routine cell culture techniques supplemented with 10% foetal calf serum and antibiotics. Proliferation assay: The assay recorded residual viable biomass (RVB) 72 hours into exposure with the agent being studied RESULTS Doubling dilution of morphine produced a bell-shaped curve, with high (non-clinical)concentrations inhibiting growth, followed by stimulation between 100 and 1 microgram per ml (clinically relevant concentrations shown in Figure 1 green circle), reverting to control levels thereafter. The near-normal PNT-2 cell line showed minimal responsiveness to morphine Incubation with naloxone alone has little effect on residual biomass compared to controls Conversely, pre-incubating with a fixed concentration of Naloxone, then titrating morphine across the plate showed stimulation to decrease as the relative proportion of blocker rises (Figure 2) CONCLUSIONS Morphine in clinically relevant concentrations stimulated growth in the RT-112 transitional carcinoma cell line, as assessed by the MTT assay. This effect was minimal under the same conditions using the virally transformed normal prostate cell line (PNT2). Treating cells with the antagonists Naloxone or Naltrexone inhibited the action of morphine. The tentative implication is that using opioids in cancer patients despite their important role in pain relief may also tend to promote tumour growth. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e640-e641 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Brian Birch More articles by this author Bashir Lwaleed More articles by this author Alan Cooper More articles by this author Phil Harper More articles by this author Iliana Giatsidou More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Upregulation of long non-coding RNA PlncRNA-1 promotes proliferation and induces epithelial-mesenchymal transition in prostate cancer.

ObjectiveTo confirm that PlncRNA-1 regulates the cell cycle in prostate cancer cells and induces epithelial-mesenchymal transition (EMT) in prostate cancer through the TGF-β1 pathway.ResultsPlncRNA-1 and TGF-β1 expression levels were significantly higher in prostate cancer tissues than in normal prostate tissues (P < 0.05) and were significantly positively correlated. TGF-β1, N-cadherin and Cyclin-D1 were downregulated and E-Cadherin was upregulated in LNCAP cells after silencing of PlncRNA-1, as determined by real-time PCR and Western blot. TGF-β1, N-cadherin and Cyclin-D1 were upregulated and E-cadherin was downregulated in C4-2 cells, as determined by real-time PCR and Western blot. Overexpression of PlncRNA-1 in C4-2 cells was observed when TGF-β1 inhibitor LY2109761 was added. Western blot analysis showed that compared with their expression when TGF-β1 inhibitor LY2109761 was not added, N-Cadherin and CyclinD1 expression decreased and E-Cadherin expression increased. Transwell results showed that the invasive ability of C4-2 cells was enhanced after overexpression of PlncRNA-1, and the invasion ability was decreased after addition of TGF-β1 inhibitor LY2109761. The cell cycle was blocked by overexpression of PlncRNA-1 in C4-2 and by the addition of TGF-β1 inhibitor LY2109761, as determined by flow cytometry. In vitro experiments showed that PlncRNA-1 can regulate the growth of prostate cancer cells and EMT through the TGF-β1 pathway. In vivo experiments also confirmed the above results. Tumor growth was significantly blocked by overexpressing PlncRNA-1 in C4-2 cells and by the TGF-β1 inhibitor LY2109761 in animal experiments.Materials and MethodsThe expression levels of PlncRNA-1 and TGF-β1 were analyzed in 19 prostate cancer tissue samples and in adjacent normal tissue samples, 4 Pca cell lines, including LNCaP, C4-2, DU145, and PC3, and 1 normal prostate epithelial cell line RWPE-1. LNCAP cells were divided into the LNCAP control group and the LNCAP-PlncRNA-1-siRNA group. Cells from the prostate cancer cell line C4-2 were divided into the C4-2 control group and the C4-2-PlncRNA-1 experimental group. Changes in TGF-β1, E-cadherin and N-cadherin were detected by qPCR and Western Blot assay after silencing and overexpression of PlncRNA-1. The cell cycle, cell invasion, and levels of Cyclin-D1, E-Cadherin, and N-Cadherin were observed after adding TGF-β1 inhibitor LY2109761 in the C4-2-PlncRNA-1 group. The effects of TGF-β1 inhibitor LY2109761 on the tumorigenicity of C4-2 cells after overexpression of PlncRNA-1 was investigated in vivo.ConclusionsPlncRNA-1 is an oncogene that regulates the cell cycle, cyclin-D1 and EMT in prostate cancer cells through the TGF-β1 pathway.

Inhibitory effect of oncolytic adenovirus ZD55-special AT rich sequence binding protein 1 on proliferation and invasion of andragon-independent prostate cancer DU145 cells

Objective To investigate the suppressive effect of oncolytic adenovirus ZD55-special AT rich sequence binding protein 1 (SATB1) expressing small hairpin RNA targeting SATB1 gene on growth and invasion of prostate cancer DU145 cells. Methods The oncolytic adenovirus ZD55-SATB1 was constructed and identifies. The inhibitory effect of ZD55-SATB1 on SATB1 expression was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting analysis at 72 h after infection of DU145 cells with ZD55-SATB1 at a MOI of 10. The cytotoxicity of ZD55-SATB1 was detected by cell counting kit-8 (CCK-8) assay and crystal violet staining assay. Cell invasion was detected by Matrigel invasion assay. The expression of E1A protein in DU145 cell lines infected with viruses respectively was detected by Western blotting. Results The oncolytic adenoviruses ZD55-SATB1 were successfully constructed and identified. RT-PCR and Western blotting assay showed that ZD55-SATB1 could significantly decrease the expression of SATB1 in DU145 cells (P<0.05). The relative protein levels of E1A in DU145 cells treated with ZD55-SATB1 were significantly higher than those in human normal prostate epithelial cell line PZ-HPV-7 cells (P<0.01), which indicated cancer specificity of ZD55-SATB1 for prostate cance cells. Crystal violet staining assay showed that ZD55-SATB1 exhibited obvious cytotoxicity in DU145 cells. The cytopathic effect of ZD55-SATB1 was much stronger than that of ZD55-enhanced green fluorescent protein (EGFP) and Ad-SATB1. CCK-8 assay showed that cell viability of ZD55-SATB1 group [(31.60±3.31)%] was significantly lower than that in ZD55-EGFP group [(61.70±2.42)%, P=0.025] and Ad-SATB1 group [(82.40±3.54)%, P=0.017] for DU145 cells after 4 days. Transwell cell invasive experiment showed that the number of cells that invaded the membrane in ZD55-SATB1 group was (61.30±17.25) cells, significantly less than that in the Ad-SATB1 group [(170.10±25.44) cells, P=0.043], the ZD55-EGFP group [(295.40±23.36) cells, P=0.013] and the PBS group [(490.50±33.36) cells, P=0.009]. Conclusion Oncolytic adenovirus ZD55-SATB1 could effectively knockdown the SATB1 expression in DU145 cells, and dramatically inhibit proliferation and invasion of DU145 cells. Key words: Oncolytic adenovirus; Special AT rich sequence binding protein 1; Proliferation; Invasion

Zinc induces LPS-mediated upregulation of HBD-2 via ERK1/2 and p38MAPK signaling pathways in human prostate epithelial cells

ABSTRACTThis study aimed to clarify the role of zinc in human prostate epithelial cell defense against bacterial infection. To explore the effect of zinc on lipopolysaccharide (LPS)-mediated induction of human β-defensin-2 (HBD-2), the normal human prostate epithelial cell lines (RWPE-1) were co-treated with zinc/LPS and HBD-2 mRNA expression was quantitated by the reverse transcription polymerase chain reaction (RT-PCR). We also conducted a Western blot analysis to determine whether zinc stimulates p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To investigate the involvement of the p38MAPK and ERK1/2 signaling pathways in zinc-mediated upregulation of HBD-2, quantitative real-time PCR and immunocytochemical staining were then used to quantify HBD-2 mRNA expression and protein production, respectively, which was treated with either U0126 (ERK1/2 inhibitor) or SB203580 (p38MAPK inhibitor) prior to each analysis of HBD-2. Cotreatment of RWPE-1 cells with zinc/LPS-upregulated HBD-2 expression to an even greater extent than either LPS alone or zinc alone. Moreover, the treatment of RWPE-1 cells with zinc significantly increased both the total and phosphorylated forms of ERK1/2 and p38MAPK. ERK1/2 and p38MAPK signaling via the inhibitors U0126 and SB203580 pharmacologically inhibited zinc-mediated upregulation of HBD-2. These results strongly suggest that zinc plays an important role in the immune response of the prostate. Furthermore, we demonstrate that zinc-mediated upregulation of HBD-2 expression upon bacterial infection of prostate epithelial cells involves the ERK1/2 and p38MAPK signaling pathways.

The effects of metformin and simvastatin on the growth of LNCaP and RWPE-1 prostate epithelial cell lines

The anti-diabetic drug metformin and cholesterol-lowering statins inhibit prostate cancer cell growth in vitro and have been linked with lowered risk of prostate cancer in epidemiological studies. We evaluated the effects of these drugs on cancerous and non-cancerous prostate epithelial cell lines. Cancer (LNCaP) and normal (RWPE-1) prostate epithelial cell lines were treated with pharmacologic concentrations of metformin and simvastatin alone and in combinations. Relative changes in cell number were measured with crystal violet staining method. Drug effects on apoptosis and cell cycle were measured with flow cytometry. We also measured changes in the activation and expression of a set of reported target proteins of metformin and statins with Western blotting. Metformin decreased the relative cell number of LNCaP cells by inducing G1 cell cycle block, autophagy and apoptosis, and slightly increased cytosolic ATP levels, whereas RWPE-1 cells were resistant to metformin. However, RWPE-1 cells were sensitive to simvastatin, which induced G2 cell cycle block, autophagy and apoptosis, and increased cytosolic ATP levels in these cells. Combination of metformin and simvastatin synergistically decreased cytosolic ATP levels, increased autophagy and instead of apoptosis, induced necrosis in LNCaP cells. Synergistic effects were not observed in RWPE-1 cells. These results suggest, that prostate cancer cells may be more vulnerable to combined growth-inhibiting effects of metformin and simvastatin compared to normal cells. The data presented here provide evidence for the potency of combined metformin and statin, also at pharmacologic concentrations, as a chemotherapeutic option for prostate cancer.

S100A16 promotes cell proliferation and metastasis via AKT and ERK cell signaling pathways in human prostate cancer.

S100A16 is a member of the S100 calcium-binding protein family. It is overexpressed in many types of tumors and associated with proliferation, migration, and invasion; however, its function in human prostate cancer is unresolved. Our objective was to determine its effects and the underlying pathways of S100A16 in prostate cancer tissues and cells. We measured S100A16 expression by quantitative real-time polymerase and Western blotting in eight matched prostate cancer and adjacent normal tissues, and in three prostate cancer cell lines, DU-145, LNCaP, and PC-3, compared to a normal prostate epithelial cell line PrEC. DU-145 cells stably overexpressing S100A16 and PC-3 cells with S100A16 knockdown were established by transfection with S100A16 overexpression plasmid or shRNAs. Invasion, migration, and proliferation were analyzed by transwell assay, wound healing, and colony formation assays, respectively. Western blotting and invasion assays were performed to determine expressions and activation of AKT, ERK, p21, and p27. S100A16 was significantly overexpressed in both prostate cancer tissues and cells lines compared to normal controls (P < 0.05). Overexpression of S100A16 significantly promoted invasion, migration, and proliferation in prostate cancer cells in vitro, whereas silencing S100A16 showed the converse effects (P < 0.05). Furthermore, overexpression of S100A16 activated cell signaling proteins AKT and ERK and downregulated tumor suppressors p21 and p27. Specific inhibitors, LY294002 and PD98059, suppressed activation of AKT and ERK, which attenuated DU-145 cell clone formation and invasion induced by S100A16 overexpression. S100A16 may promote human prostate cancer progression via signaling pathways involving AKT, ERK, p21, and p27 downstream effectors. Our findings suggest that S100A16 may serve as a novel therapeutic or diagnostic target in human prostate cancer.

MiR-301a expression: A prognostic marker for prostate cancer

PurposeThe diagnosis and treatment of prostate cancer (CaP) continues to be challenging, as prostate-specific antigen (PSA) appears to be overly sensitive and biopsy is the only reliable method for confirmation. Hence, the goal of the study is to identify a biomarker that could distinguish malignant cancer from benign prostatic hyperplasia (BPH) during the early diagnosis of the disease. Materials and methodsA total of 75 formalin fixed paraffin embedded (FFPE) with matching controls, 4 paired metastatic tumors, 6 fresh tumor tissues and BPH (13 cases) with their clinical diagnosis were selected for this study. Prostate cancer cell lines and normal prostate epithelial cell lines were obtained from ATCC and subjected to phenotypic analysis. ResultsWe observed significant differential expression of miR-301a in CaP samples in comparison to BPH and adjacent benign samples. The overexpression of miR-301a activates the invasion/migration of CaP cells. In contrast, silencing miR-301a expression inhibited the colony-forming ability, adhesion, invasion and migration of CaP cells. Similarly, the overexpression of miR-301a increased cell motility in normal RWPE-1 prostate epithelial cells. Our results suggest that miR-301a is differentially expressed between BPH and CaP specimens and that the expression of miR-301a correlates with biochemical recurrence and/or metastasis in CaP patients. ConclusionsThe expression of miR-301a could be a potential marker for metastasis in CaP patients. Detecting miR-301a expression during diagnosis will avoid wait and watch timelines, thus preventing morbidity.

Hypoxia primes human normal prostate epithelial cells and cancer cell lines for the NLRP3 and AIM2 inflammasome activation.

The molecular mechanisms by which hypoxia contributes to prostatic chronic inflammation (PCI) remain largely unknown. Because hypoxia stimulates the transcriptional activity of NF-κB, which “primes” cells for inflammasome activation by inducing the expression of NLRP3 or AIM2 receptor and pro-IL-1β, we investigated whether hypoxia could activate the NLRP3 and AIM2 inflammasome in human normal prostate epithelial cells (PrECs) and cancer cell lines. Here we report that hypoxia (1% O2) treatment of PrECs, prostate cell lines, and a macrophage cell line (THP-1) increased the levels of NLRP3, AIM2, and pro-IL-1β. Further, hypoxia in cells potentiated activation of the NLRP3 and AIM2 inflammasome activity. Notably, hypoxia “primed” cells for NLRP3 and AIM2 inflammasome activation through stimulation of the NF-κB activity. Our observations support the idea that hypoxia in human prostatic tumors contributes to PCI, in part, by priming cells for the activation of NLRP3 and AIM2 inflammasome.

MiR-24 regulates CDKN1B/p27 expression in prostate cancer.

MicroRNAs (miRNAs) are small, non-coding RNA molecules with an important role in cancer. In prostate cancer, several miRNAs are expressed abnormally suggesting they may be useful markers for diagnosis, prognosis, and potential therapeutic intervention in this disease. However, the contribution of individual miRNAs to the development and progression of this disease remains poorly understood. This study investigated the role of miR-24, which has not been extensively studied in relation to prostate cancer. We used PCR to investigate the expression of miR-24 in a panel of prostate cancer cell-lines and in a series of clinical prostate biopsy specimens. The biological significance of miR-24 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets p27 (CDKN1B) and p16 (CDK2NA) was investigated. We showed that miR-24 expression was significantly lower in prostate cancer cell lines compared to a normal prostate epithelial cell line. Decreased expression of miR-24 was also more frequently observed in both needle core and prostatectomy tumor tissue relative to matched normal tissue. Low miR-24 expression correlated with high PSA serum levels and other markers of increased prostate cancer progression. Importantly, over-expression of miR-24 inhibited cell cycle, proliferation, migration, and clonogenic potential of prostate cancer cells, as well as inducing apoptosis. p27 and p16 were confirmed as targets of miR-24 in prostate cancer cells and a significant inverse correlation between miR-24 and p27 was revealed in clinical prostatectomy specimens. These findings provide evidence that miR-24 has a tumor suppressor role in prostate cancer and also targets p27 and p16 in prostate cancer cells. We propose that it may be a useful progression biomarker or focus of therapeutic intervention for this disease.

Abstract A33: CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+

Abstract In order to maintain high rates of proliferation, tumor cells must alter their metabolic machinery in favor of increased macromolecule synthesis and energy production. A key factor in tumor cell metabolism is Nicotinamide Adenine Dinucleotide, NAD+. NAD+ is used as a cofactor for catabolic reactions (NAD(H)), or after conversion to NADP(H), for anabolic reactions. In addition, NAD+ is utilized as part of the catalytic mechanism for several classes of enzymes including the sirtuins, PARPs, and CD38. Given the juxtaposition of NAD+ to cell metabolism and other critical cellular process we hypothesize a balance between synthesis and consumption of NAD+ is a primary metabolic determinant in cancer. In prostate cancer, the primary pathway by which NAD+ is synthesized is via the salvage pathway, which recycles nicotinamide into Nicotinamide Mononucleotide (NMN) via the rate-limiting enzyme Nicotinamide Phosphoribosyltransferase (Nampt). We determined the metabolic consequences of Nampt blockade in prostate cancer and glioblastoma (GBM) cell lines using the pharmacological inhibitor FK866. As expected, treatment with FK866 decreases the levels of NAD+/NADP+ and the NAD+ precursor nicotinamide. In relation to glycolysis, there were increases in metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, which requires NAD+ as a cofactor. Specifically, glucose is increased 1.6 fold in PC3 cells (p≤0.05), glucose-6-phosphate is decreased 1.7 fold SNB19s (p≤0.05), and both lines have decreased fructose-1-phosphate levels between 6 and11 fold (p≤0.05), respectively. To compliment these changes we measured the effects of Nampt inhibition on glycolysis in real time using a Seahorse XF extracellular flux analyzer. Nampt inhibition lowers basal glycolysis 40% (p&amp;lt;0.01) in prostate cancer (PCa) cells and glycolytic capacity 46% (p&amp;lt;0.01) and 64% (p&amp;lt;0.01) in PCa and glioblastoma (GBM) cells, respectively. Reactions requiring NAD+ as a cofactor in the TCA cycle were similarly perturbed. Specifically, in both cell lines fumarate and malate were increased between 1.5 and 2 fold (p&amp;lt;0.05) resulting from reduced malic enzyme activity. Corresponding to altered TCA cycle metabolites there was a 45% decrease in spare mitochondrial respiratory capacity (p&amp;lt;0.01) in PCa cells and a 90% decrease in GBM cells (p&amp;lt;0.01), relative to vehicle. Together these data suggest blockage of NAD+ synthesis is sufficient to perturb the activity of NAD+ dependent metabolic enzymes and induce energetic stress. Of the three classes of NAD+ consuming enzymes, the cADP-ribose synthase CD38 is the primary NAD'ase in cells. Western blot analysis of normal prostate epithelial cells (PrEC) and several prostate tumor cell lines demonstrates expression of CD38 is nearly non-existent in PCa. In line with this, CD38 mRNA levels are decreased 2-fold across 31 independent PCa samples when compared to mRNA from 8 normal prostate samples (p=0.007). CD38 expression levels were also determined by immunohistochemistry (IHC) in primary PCa tissue micro-arrays (TMA). In a total of 91 samples: 37 benign, 17 prostatic intraepithelial neoplasia (PIN), and 37 Cancers, CD38 staining intensity was reduced 20% in PIN samples (p≤0.001) and 50% in cancer samples (p≤0.01). In these experiments, no change in Nampt protein expression, mRNA levels, or staining intensity was detected. Induction of CD38 expression in prostate cancer cell lines resulted in blocked cell proliferation. CD38 expression also reduced NAD levels by 54% and 91% after 48 and 96 hours, respectively (p≤0.01). Concomitant with decreased NAD levels, CD38 expression also reduced spare mitochondrial respiratory capacity 24% (p=0.026), mimicking the effects of Nampt inhibition. Fatty acid synthesis was similarly inhibited following CD38 expression. When taken together, these data suggest an interplay between Nampt and CD38 regulates total NAD+ levels, ultimately supporting the metabolic reprogramming associated with prostate cancer. Citation Format: Jeffrey P. Chmielewski, Frances Wheeler, Scott Cramer, Shi Lihong, Joseph Sirintrapun, Steven J. Kridel. CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A33.

The LMO2 protein overexpressed WPMY-1 prostatic stromal cells promote proliferation and invasion of PC-3/BPH-1 prostatic epithelial cells

Objective To investigate the effect of LMO2 gene, which is overexpressed in prostate peripheral zone than transitional zone, on proliferation and invasion of prostate cancer PC-3 cell line or normal prostate epithelial BPH-1 cell line. Methods Lentivirus overexpression system was used to establish the LMO2 protein overexpressed prostate WPMY-1 stromal cell line. The LMO2 mRNA and protein expression level of those cells was evaluated by qRT-PCR and western blot. The PC-3 or BPH-1 cells was co-cultured with prostate stromal cells and the in vitro proliferation and invasion activity were detected by Cell Counting Kit-8 (CCK-8), EdU and Matrigel invasive assays. Results The stable LMO2 overexpressed prostate stromal cells WPMY-1-LMO2 was successfully established. The results of CCK-8, EdU experiments indicated the proliferation and invasion activity of PC-3 or BPH-1 cells were both enhanced through co-cultured with WPMY-1-LMO2 cells. The proportion of EdU positive cells of PC-3 cultured in WPMY-1-LMO2 supernatant was (42.67±6.03)%, and the difference was significant compared with the control group (29.33±3.51)% (P<0.05). The BPH-1 cultured in the supernatant was (35.00±2.52)%, and the difference was significant compared with the control group (23.33±2.52)% (P<0.05). The number of invaded PC-3 or BPH-1 cells after co-cultured with WPMY-1-LMO2 cells was 38±5 and 43±3, respectively, which was significantly different compared with the control group (P<0.05). Conclusions The LMO2 overexpressed prostate stromal cells could induce proliferation and invasion of PC-3 or BPH-1 cells. The propensity of benign prostatic hyperplasia and prostate cancer at different zones may probably be related to distinctive gene expression between peripheral zone and transitional zone derived stromal cells. Key words: Prostatic neoplasms; Stromal cells; Epithelial cells; LMO2 gene

Abstract LB-A18: Combination therapy of carmustine and selenite targets AR full length and AR variants: New hope for patients with castration resistant prostate cancer

Abstract The major obstacle for successful treatment of advanced prostate cancer is the development of resistance to androgen ablation therapy leading to castration resistant prostate cancer (CRPC). Evidence strongly suggests that the androgen receptor (AR) and AR variants in CRPC build resistance to anti-cancer drugs, including next-generation therapies such as abiraterone and MDV3100 that target AR signaling. Despite established AR antagonists, AR signaling remains a central mechanism of CRPC progression, involving AR gene amplification, reactivation of AR/AR variants, and generation of constitutively active AR-V7 variant. Therefore, there is an urgent need for anticancer drugs that can target these crucial signaling pathways to trigger apoptosis and growth inhibition in CRPC. We have previously shown that the combination treatment of carmustine and selenite effectively induces apoptosis in AR-independent PC-3 cells (Cancer Manag Res. 2012; 4: 383-95). Since there are no drugs available as of today to inhibit AR/AR variants, we tested whether the combination of carmustine and selenite could induce apoptosis and inhibit growth of CRPC cells (22Rv1) in in vitro and in vivo models and if this response is mediated by targeting AR/AR variants and AR-V7 in tandem with multiple signaling pathways. CRPC (22Rv1 and PC-3) and normal prostate epithelial cell lines were used to study the efficacy of carmustine and selenite in combination. Anchorage-independent growth of 22Rv1 and PC-3 cells was determined by a soft-agar assay. In vivo antitumor efficacy of the combination agent with therapeutically relevant doses was studied in a 22Rv1 xenograft mouse model. AR/AR variants, PSA and multiple signaling pathway (AKT, anti-apoptotic proteins, Bcl-2 and Mcl-1; cell cycle regulator, p27; and markers of proliferation, c-Myc and cyclin-D1) proteins were determined by western analysis. Combination of carmustine and selenite treatment completely abolished the protein expression of AR/AR variants, AR-V7, PSA in a time dependent manner, substantially decreased cell viability, growth, and induced apoptosis in 22Rv1 cells. In addition, the combination treatment dramatically induced p-H2AX, p27 while strongly inhibiting pAKT, Bcl-2, Mcl-1, c-Myc and cyclin-D1 in 22Rv1 cells without causing genotoxicity in normal prostate epithelial cells. In addition, anchorage-independent growth of 22Rv1 and PC-3 cells was completely inhibited. Consistent with in vitro results, combination treatment induced apoptosis and eradicated 22Rv1 xenograft tumors in nude mice correlating with dramatic inhibition of AR/AR variants, AR-V7, PSA, and Bcl-2 in xenograft tumors. A significant reduction in toxicity was revealed by an observed decrease in body weight loss. Whereas, individual agent treatment showed only partial effect both in vitro and in vivo. This is the first study to demonstrate that the combination of carmustine and selenite treatment completely inhibits AR/AR variants, AR-V7, and induces apoptosis in CRPC which is highly correlated with tumor growth inhibition in vitro and in-vivo. Our findings indicate that the combination of carmustine and selenite can be the next-generation therapy for successful treatment, survival, and improved quality of life of patients with castration resistant prostate cancer. Source of funding: HFHS-H10100 Citation Format: Vijayalakshmi Thamilselvan, Mani Menon, Sivagnanam Thamilselvan. Combination therapy of carmustine and selenite targets AR full length and AR variants: New hope for patients with castration resistant prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A18.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Abstract 1423: SSX-2 expression and function in metastatic prostate cancer

Abstract Background: The SSX family of proteins, whose function and aberrant expression in prostate cancer isn't fully understood, may lead to disease progression from localized to metastatic disease. Cancer-testis antigens (CTAs) are a group of proteins whose expression is restricted to germline cells among normal somatic cells. CTAs have been found to be aberrantly expressed in many different cancers. Due to their restricted expression to tumor cells among MHC class I-expressing cells, there has been interest in CTAs as tumor-specific targets for CD8+ T cell-directed therapies. Amongst the CTA genes is the Synovial Sarcoma X chromosome breakpoint (SSX) gene family. The SSX family consists of ten highly homologous members, several of which have been detected in a wide variety of cancers. We have previously shown that SSX expression is confined to metastatic prostate tissue, and is not detected in primary prostate tumors. The function of the SSX family is largely unknown, and further insights into its function may reveal avenues for future directed therapies. Methods: We determined the identity and prevalence of SSX family members in metastatic prostate cancer tissues and in peripheral blood samples of patients with prostate cancer. Specifically, cDNA from metastatic prostate tissues was examined by PCR with primers specific for each SSX family member; SSX family member expression was detected in peripheral blood samples using rt-PCR. To further understand SSX-2 function, prostate cell lines with SSX-2 expression knocked down or overexpressed were evaluated in vitro for morphological changes, tumorigenicity (soft agar colony formation), proliferation, and invasion (scratch test). Finally using qPCR, expression levels of markers of the epithelial to mesenchymal transition (EMT) were examined in the knocked down and overexpressed cell lines. Results: SSX-2 was found to be the dominant family member expressed in prostate cancer, with 47% (7/15) of metastatic cDNA samples expressing SSX-2. SSX-1 was detected in one sample (1/15), and no additional family members were detected. SSX-2 mRNA was similarly detectable in the peripheral blood of 17 of 33 (51%) patients with recurrent prostate cancer and 0% (0 of 10) control blood donors without prostate cancer. In in vitro functional studies, SSX-2 knockdown of the 22Rv1 cell line led to cell morphology changes, increased cell proliferation, and a decrease in soft agar colony formation. Knockdown was also accompanied by altered expression of genes involved in EMT (Twist-1, ZEB1/2, Slug, vimentin). Complementary changes were observed in the RWPE-1 normal prostate epithelial cell line transfected to overexpress SSX-2. Conclusions: Due to the preferential expression of SSX-2 in disseminated prostate cancer, as well as the effects of SSX knockdown and overexpression on EMT-related genes, we believe that SSX-2 is involved in the EMT process, and potentially in the progression of prostate cancer to metastatic disease. Citation Format: Jordan E. Bloom, Douglas G. McNeel. SSX-2 expression and function in metastatic prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1423. doi:10.1158/1538-7445.AM2015-1423

Abstract 3085: MicroRNA-186 inhibition alters cell proliferation and colony formation in prostate cancer

Abstract MicroRNAs (miRs), key regulators of cancer-related genes, are dysregulated in many solid tumors, including prostate cancer. However, the role of miR-186 in prostate cancer (PCA) requires further elucidation. Previously, we observed mature miR-186-5p was significantly up-regulated in the serum collected from PCA patients (n = 15) diagnosed with aggressive PCA (stage III/IV) disease compared to disease-free men (n = 5). In addition, miR-186 expression was significantly up-regulated in metastatic PCA cells (PC3) compared to a normal prostate epithelial cell line (RWPE1). Putative and predicted miR-186 gene targets in various cancers include pro-apoptotic and tumor suppression-related genes (e.g., P2×7, FOXO1, AKAP12). We hypothesized that miR-186 inhibition will reduce the aggressive PCA phenotype. To test this hypothesis, PC3 cells were transiently transfected with miR-186 inhibitor and scramble control for 24hrs. Cell viability and colony formation of transfected cells were evaluated via the ATPlite Luminescence assay and soft agar assay. We also evaluated expression profiles of predicted miR-186 gene targets in PCA cell lines and transiently transfected PC3 cells using qRT-PCR. Transient inhibition of miR-186 repressed cellular proliferation and colony formation by 48-58% in 72-96hrs and 30% after 21 days, respectively. We observed a 1.6-2.24 fold up-regulation in ROCK1 gene expression in PC3 cells treated with miR-186 inhibitor. However, additional studies are needed to assess whether stable inhibition of miR-186 will increase PCA invasiveness by up-regulating ROCK1 using in vitro assays and murine models. Moreover, predicted miR-186 gene targets require validation using qRT-PCR, western blots and luciferase reporter assays. Such efforts may lead to the identification of novel biomarkers to improve diagnostic, prognostic and clinical management strategies. Citation Format: Dominique Zilpha Jones, Katharine R. Hobbing, M. L. Schmidt, Geoffrey J. Clark, LaCreis R. Kidd. MicroRNA-186 inhibition alters cell proliferation and colony formation in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3085. doi:10.1158/1538-7445.AM2015-3085

Down-regulated CFTR During Aging Contributes to Benign Prostatic Hyperplasia.

Benign prostatic hyperplasia (BPH) is a hyper-proliferative disease of the aging prostate; however, the exact mechanism underlying the development of BPH remains incompletely understood. The present study investigated the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR), which has been previously shown to negatively regulate nuclear factor-κB (NF-κB)/cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway, in the pathogenesis of BPH. Our results showed decreasing CFTR and increasing COX2 expression in rat prostate tissues with aging. Furthermore, suppression of CFTR led to increased expression of COX2 and over-production of PGE2 in a normal human prostate epithelial cell line (PNT1A) with elevated NF-κB activity. PGE2 stimulated the proliferation of primary rat prostate stromal cells but not epithelial cells, with increased PCNA expression. In addition, the condition medium from PNT1A cells after inhibition or knockdown of CFTR promoted cell proliferation of prostate stromal cells which could be reversed by COX2 or NF-κB inhibitor. More importantly, the involvement of CFTR in BPH was further demonstrated by the down-regulation of CFTR and up-regulation of COX2/NF-κB in human BPH samples. The present results suggest that CFTR may be involved in regulating PGE2 production through its negative regulation on NF-κB/COX2 pathway in prostate epithelial cells, which consequently stimulates cell growth of prostate stromal cells. The overstimulation of prostate stromal cell proliferation by down-regulation of CFTR-enhanced PGE2 production and release during aging may contribute to the development of BPH.

Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status.

O‐GlcNAcylation Modifies the Metastatic Properties of Prostate Cancer Cells

O‐GlcNAc is a ubiquitous and dynamic post‐translational modification that occurs on serine/threonine residues of nuclear and cytoplasmic proteins. It is regulated by O‐GlcNAc transferase, which attaches O‐GlcNAc to proteins and O‐GlcNAcase, which removes O‐GlcNAc.It serves as a nutrient sensor to regulate virtually all cellular processes, as well as playing roles in various diseases, including Alzheimer's disease, diabetes and cancer. The roles of O‐GlcNAc in the regulation of prostate cancer remain unclear. Herein, we investigate how the regulation of O‐GlcNAc may drive prostate cancer progression using normal prostate epithelial cells and prostate cancer cell lines as models, and human tissue arrays. Using western blotting, we demonstrate that O‐GlcNAc, OGT and OGA are highly elevated in LNCaP and PC‐3 when compared to PrEC. LNCaP exhibited the highest levels of O‐GlcNAc, OGT and OGA. OGT and OGA are differentially localized in lines with different metastatic properties. Additionally, 2D gel electrophoresis reveals significant differences in O‐GlcNAcylated protein profiles among the three cell lines. In order to address how O‐GlcNAc affects the biological properties of these cancer cells, including shapes, proliferation, invasion and metastasis, several assays were used. The result of soft agar assays indicates that cycling of O‐GlcNAc in cells influence the anchorage independent growth of prostate cancer. Interestingly, analyses of human tissues indicate that there is a field effect in O‐GlcNAc expression with higher levels in the cytoplasm of cancer and the cytoplasm of benign tissue in close proximity to the cancer.

SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

BackgroundSULF2 is a 6-O-endosulfatase which removes 6-O sulfate residues from N-glucosamine present on heparan sulfate (HS). The sulfation pattern of HS influences signaling events mediated by heparan sulfate proteoglycans (HSPGs) located on cell surface, which are critical for the interactions with growth factors and their receptors. Alterations in SULF2 expression have been identified in the context of several cancer types but its function in cancer is still unclear where the precise molecular mechanism involved has not been fully deciphered. To further investigate SULF2 role in tumorigenesis, we overexpressed such gene in prostate cancer cell lines.MethodsThe normal prostate epithelial cell line RWPE-1 and the prostate cancer cells DU-145, and PC3 were transfected with SULF2-expressing plasmid pcDNA3.1/Myc-His(−)-Hsulf-2. Transfected cells were then submitted to viability, migration and colony formation assays.ResultsTransfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells. The effect was reverted by the knockdown of SULF2 using specific siRNAs. Furthermore, forced expression of SULF2 augmented cell migration and colony formation in both prostate cell lines. Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S). There was an increase in epithelial-mesenchymal transition markers and an increase in WNT signaling pathway.ConclusionsThese results indicate that SULF2 have a pro-tumorigenic effect in DU-145 and PC3 cancer cells, suggesting an important role of this enzyme in prostatic cancer metastasis.

Abstract 2944: Decline in arylsulfatase B (N-acetylgalactosamine-4-sulfatase) leads to increased expression of the transmembrane glycoprotein GPNMB

Abstract The enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase), which removes 4-sulfate groups from chondroitin-4-sulfate (C4S) and dermatan sulfate, is reduced in malignant human epithelial breast, colonic, and prostate cells and tissues. Lower ARSB immunohistochemical scores have been associated with shorter intervals to recurrence in paired cases of prostate cancer and with propensity for metastasis in malignant colonic cells. The transmembrane glycoprotein GPNMB has been shown to be increased in malignant melanoma, glioblastoma multiforme, and mammary carcinoma, and is a target of antibody therapy in triple-negative breast cancers. In the ARSB-deficient mouse, cDNA microarray of prostate tissue demonstrated 9.53-fold increase in expression of GPNMB, compared to age- and gender- matched C57BL/6J wild-type controls. Measurements by ELISA of GPNMB protein in the ARSB-null mouse liver and prostate tissues showed significant increases, compared to C57BL/6J wild-type controls. In the liver of female ARSB-deficient mice, the gpnmb increased to 47.1 ± 5.7 ng/mg protein from 8.5 ± 1.2 ng/mg protein in the control mouse liver (n=5 in each group), and similar six-fold increases occurred in the male ARSB-null mouse liver. Prostate gpnmb increased more that three-fold, and serum levels in female and male null mice were significantly greater. When ARSB was silenced by siRNA in the normal human prostate stromal cell line (WPMY-1), GPNMB increased from 3.6 ± 0.3 ng/mg protein to 18.3 ± 1.1 ng/mg protein, and in the normal human prostate epithelial cell line (WPE1-NB14) from 4.8 ± 0.4 to 24.7 ± 1.6 ng/mg protein. Similarly in the human hepatic cell line, HepG2, GPNMB increased greater than five-fold. Decline in ARSB leads to increased chondroitin-4-sulfation and reduced galectin-3 binding to C4S, enabling increased nuclear translocation of galectin-3. We have shown that the increase in nuclear galectin-3 following decline in ARSB impacts on transcriptional events mediated by AP-1 in human epithelial cells, including increases in HIF-1α and versican. However, in the prostate stromal and epithelial cell lines, AP-1 inhibitors, including a c-Jun mimetic peptide and an oligonucleotide inhibitor of c-Fos DNA-binding, did not reduce the increase in GPNMB expression that followed ARSB silencing. Although the mechanism for the decline in GPNMB expression is unknown at this time, the findings support a critical role for ARSB in vital cell processes and suggest that ARSB may be useful as a therapeutic target in malignancy. Citation Format: Sumit Bhattacharyya, Leo Feferman, Joanne Kramer Tobacman. Decline in arylsulfatase B (N-acetylgalactosamine-4-sulfatase) leads to increased expression of the transmembrane glycoprotein GPNMB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2944. doi:10.1158/1538-7445.AM2014-2944