RWPE-1 Cells Research Articles

Abstract LB-326: A novel oncomiR negatively regulates PTEN pathway in prostate cancer

Abstract Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that a novel microRNA, microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Kaplan-Meier and ROC analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy. Note: This abstract was not presented at the meeting. Citation Format: Nadeem S. Bhat, Melissa Colden, Prerna Arora, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Soichiro Yamamura, Yuichiro Tanaka, Taku Katu, Yutaka Hashimoto, Marisa Shiina, Priyanka Kulkarni, Pritha Dasgupta, Mitsuho Imai-sumida, Shigekatsu Maekawa, Guoren Deng, Rajvir Dahiya, Shahana Majid. A novel oncomiR negatively regulates PTEN pathway in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-326. doi:10.1158/1538-7445.AM2017-LB-326

Abstract LB-167: A novel metabolite of citrus tangeretin epigenetically inhibits the growth of human prostate cancer cells

Abstract Prostate cancer is the second leading cause of cancer death in men in the United States. Polymethoxyflavonoids (PMFs) such as tangeretin which exclusively exist in citrus fruit peels have been studied to have many bioactivities. In this study, we investigated the inhibitory effect of the metabolites of tangeretin in human prostate cancer LNCaP and PC3 cells. The results showed that PMF1 is a synthetic tangereitin metabolite can significantly inhibits the growth of LNCaP and PC-3 cells (IC50 14.0 and 13.5 µM, respectively) while PMF1 at 15 µM cannot affect the growth of human normal prostate epithelial RWPE-1 cells. The results of the anchorage-independent growth analysis indicated that PMF1 can suppress the colony formation in LNCaP and PC-3 cells as well as LNCaP and PC-3 stem-like cells which were isolated when the parent cells were cultured in the serum-free medium on the ultra-low attached dishes. Furthermore, PMF1 can induce apoptosis in LNCaP cells in which PMF1 increases protein expressions of Bax and Bad and decreases protein expressions of procaspases-3 and Bcl-2. We also found that PMF1 also decreases the protein levels of DNA Methyltransferase 1 and histone deacetylases 1, 2, and 4/5/9. These results suggested that PMF1 might effectively inhibit the growth of human prostate cancer cells as well as prostate cancer stem cells, which provides new insights in the prevention and treatment of prostate cancer. Citation Format: Yen-Hsiang Chao, Zheng-Yuan Su. A novel metabolite of citrus tangeretin epigenetically inhibits the growth of human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-167. doi:10.1158/1538-7445.AM2017-LB-167

Synthesis of thiazole linked indolyl-3-glyoxylamide derivatives as tubulin polymerization inhibitors

A series of thiazole linked indolyl-3-glyoxylamide derivatives were synthesized and evaluated for their in vitro cytotoxic activity against DU145 (prostate), PC-3 (prostate), A549 (lung) and HCT-15 (colon) cancer cell lines by employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among all the synthesized compounds, compound 13d displayed cytotoxicity of IC50 = 93 nM towards DU145 cancer cell line. The most active compound 13d was also tested on RWPE-1 cells and was found to be safe compared to the DU145 cells. The target compounds were also evaluated for their inhibition activity of tubulin polymerization. Further, the treatment of compound 13d on DU145 cells led to the inhibition of cell migration ability. The detailed studies such as acridine orange/ethidium Bromide (AO/EB), DAPI, annexin V-FITC/propidium iodide staining assay suggested that the compound 13d induced apoptosis in DU145 cells. The influence of the cytotoxic compound 13d on the cell cycle distribution was assessed on the DU145 cell line, exhibiting a cell cycle arrest at the G2/M phase. Moreover, the treatment with compound 13d caused collapse of mitochondrial membrane potential and elevated intracellular ROS levels in DU145 cells. The results from molecular modelling studies revealed that these compounds bind at the colchicine binding site of the tubulin. Thus, this new molecular scaffold could be a new lead for the development of anticancer agents that target tubulin.

ETS (E26 transformation-specific) up-regulation of the transcriptional co-activator TAZ promotes cell migration and metastasis in prostate cancer

Prostate cancer is a very common malignant disease and a leading cause of death for men in the Western world. Tumorigenesis and progression of prostate cancer involves multiple signaling pathways, including the Hippo pathway. Yes-associated protein (YAP) is the downstream transcriptional co-activator of the Hippo pathway, is overexpressed in prostate cancer, and plays a vital role in the tumorigenesis and progression of prostate cancer. However, the role of the YAP paralog and another downstream effector of the Hippo pathway, transcriptional co-activator with PDZ-binding motif (TAZ), in prostate cancer has not been fully elucidated. Here, we show that TAZ is a basal cell marker for the prostate epithelium. We found that overexpression of TAZ promotes the epithelial-mesenchymal transition (EMT), cell migration, and anchorage-independent growth in the RWPE1 prostate epithelial cells. Of note, knock down of TAZ in the DU145 prostate cancer cells inhibited cell migration and metastasis. We also found that SH3 domain binding protein 1 (SH3BP1), a RhoGAP protein that drives cell motility, is a direct target gene of TAZ in the prostate cancer cells, mediating TAZ function in enhancing cell migration. Moreover, the prostate cancer-related oncogenic E26 transformation-specific (ETS) transcription factors, ETV1, ETV4, and ETV5, were required for TAZ gene transcription in PC3 prostate cancer cells. MAPK inhibitor U0126 treatment decreased TAZ expression in RWPE1 cells, and ETV4 overexpression rescued TAZ expression in RWPE1 cells with U0126 treatment. Our results show a regulatory mechanism of TAZ transcription and suggest a significant role for TAZ in the progression of prostate cancer.

Identification of endonuclease domain-containing 1 as a novel tumor suppressor in prostate cancer

BackgroundEndonuclease domain containing 1 (ENDOD1) is implicated in tumorigenesis and aggressiveness of multiple tumors. In this study, we aimed to investigate the role of ENDOD1 in prostate cancer (PCa).MethodsImmunohistochemistry were performed in 30 cases of benign prostatic hyperplasia (BPH) and 50 cases of PCa to identify its association with clinicopathological characteristics. Real-time PCR and western blot were used to detect ENDOD1 mRNA and protein expression in normal prostatic epithelial and PCa cell lines. MTT assays were employed to determine the effect of cell proliferation. Flow cytometry was used to explore the cell cycle distribution and apoptotic effects. Transwell migration and invasion assays were done to evaluate changes in the ability of cell migration and invasion.ResultsImmunoreactivity scores of ENDOD1 showed no statistical difference between BPH and low-grade PCa, whereas lower immunostaining scores were observed in high-grade compared with low-grade PCa. Real-time PCR data indicated that ENDOD1 mRNA expression was markedly increased in LNCaP and 22Rv1 cells and decreased in PC3 and DU145 cells compared to the normal epithelial cells RWPE1. Western blot showed that androgen-sensitive LNCaP cells had the highest protein expression level of ENDOD1, whereas castration-resistant PCa cell lines PC3 and DU145 had significantly lower protein levels. Meanwhile, overexpression of ENDOD1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and inhibited cell migration and invasion. Conversely, siRNA-mediated silencing of ENDOD1 promoted cell proliferation, migration and invasion. No apoptotic effects occurred upon manipulation of ENDOD1 expression.ConclusionOur results indicate that ENDOD1 is a novel tumor suppressor in PCa, which may be employed as a new drug target of preventing progression to metastatic castration-resistant prostate cancer.

MiR-221 to modulate tumor growth in vivo and as a regulator of TGF.

e23008 Background: Despite the advances in cancer therapy, when Prostate Cancer (PCa) progress to castration resistant phase, patients develop incurable bone metastases. Understanding the processes that regulate homing and survival of metastatic cancer cells in the bone is crucial for the identification of new therapies. TGF-β signaling plays a major role in bone remodeling and according to the “vicious cycle hypothesis” is a master regulator of maintenance of prostate cancer cells in lytic bone lesions. microRNAs (miRs) are a class of small non-coding RNAs that regulates many biological process. miR-221 expression has been previously associated with prostate cancer progression. Here we studied the effect of miR-221 on TGF-β signaling and the impact of miR-221 on tumor growth in vivo Methods: miR-221 was overexpressed in PC3 and RWPE-1 cells and PMEPA was monitored by RT-qPCR and western blot. Expression of miR-221 in PC3 and RWPE-1 cells was assessed by RT-qPCR. Luciferase assay was used to investigate the interaction between miR-221 and PMEPA1. For zebrafish experiment, fluorescently labelled PC-3M-Pro4 cells overexpressing miR-221 were injected in the Duct of Cuvier (DC) of zebrafish embryos. Results: miR-221 overexpression resulted in decreased PMEPA1 mRNA and protein in PC-3 cells. Luciferase reporter assay indicated that miR-221 can directly interact with PMEPA1 3’ UTR. We show an enhancement of the proliferative effect of TGF-β in PC-3 cells, following miR-221 overexpression. In conclusion, we observed increased Smad2 activation upon TGF-β treatment in miR-221 overexpressing PC-3 cells. Inoculation of PC-3M-Pro4 cells overexpressing miR-221 in the DC of zebrafish embryos resulted reduced tumor burden compared to control. Finally, we observed inverse correlation between miR-221 and PMEPA1 expression in normal vs. tumor tissue collected from PCa patients. Conclusions: Our results indicate that miR-221 is a regulator of TGF-β signaling via modulation of PMEPA1 and miR-221 overexpression can reduce tumor growth in vivo.

Precision prostate cancer chemoprevention with gamma-tocotrienol.

e13043 Background: A goal of precision chemoprevention is to prevent or delay cancer progression by using minimally toxic agents guided by knowledge of alterations in oncogenic molecular pathways and signaling cascades. Indolent prostate cancer can benefit from chemopreventive agents by delaying growth. Vitamin E is not a single compound and refers to four tocopherols and four tocotrienols. The use of all-racemic-alpha tocopheryl acetate in preventing cancer was studied in the SELECT clinical trial. This trial found it was not chemopreventive and could promote prostate cancer. We compared the abilities of RRR-alpha-tocopherol (AT), RRR-gamma-tocopherol (GT), and gamma-tocotrienol (GT3) in preventing growth of two prostate cancer cell lines (LNCaP and PC-3) and a control prostate cell line (RWPE-1) by quantifying their effects on two signaling pathways known to be pivotal in prostate carcinogenesis, AKT and MAP Kinase (pERK). Methods: LNCaP, PC-3, and RWPE-1 cell lines were treated with increasing concentrations of AT, GT and GT3 in DMEM, and cytotoxicity determined by MTS cell culture experiments. We determined the effect of GT3 on signaling pathways by analyzing B-actin, P-AKT and p-ERK in combination with AKT and/or MEK inhibitors via Western immunoblot and PCR analysis. Results: Initial experiments with LNCaP and PC-3 cells showed GT3 induced the expression of pERK and p-c-JUN, inhibited cell growth and promoted apoptotic cell death. Neither AT nor GT had these effects. AKT was not activated by AT, GT or GT3. Inhibition of AKT activation via MK-2206 did not block GT3 growth inhibition in LNCaP cells. No growth inhibition was found with RWPE-1 control cells in presence of AT, GT or GT3 yet GT3 induced pERK expression. AT did not interfere with cancer growth inhibition and did not block the anticancer effects of GT3. Conclusions: GT3 induces the activation of pERK but this effect is not sufficient to account for the in vitro chemopreventive effects of this form of vitamin E. The AKT pathway is not modulated by GT3 and does not appear to be relevant to GT3 killing of PC-3 or LNCaP. Of five signaling pathways implicated in prostate carcinogenesis, we explored AKT and pERK. Future research will explore B-catenin, mTOR and PI3K.

Low expression of microRNA-30c promotes prostate cancer cells invasion involved in downregulation of KRAS protein.

Aberrant microRNA expression is associated with tumor development. The present study aimed to elucidate the role of miR-30c in the development of prostate cancer. Quantitative polymerase chain reaction was performed to compare miR-30c expression in LNCaP, DU145, PC-3 and RWPE-1 cell lines. Lentivirus expressing miR-30c was used to create stable overexpression cell lines to investigate the effects of miR-30c overexpression on cell proliferation, migration and invasion, which were determined in the prostate cancer cell line PC-3 by MTT, colony formation, wound healing and Transwell assays. Effects of miR-30c on KRAS were examined by western blot analysis. miR-30c expression was significantly lower (P<0.05) in the PC-3 cell line compared with LNCaP, DU145 and RWPE-1 cell lines. miR-30c overexpression in PC-3 inhibited tumor cell proliferation, migration and invasion in vitro. Furthermore, KRAS protein expression was downregulated in miR-30c overexpression cell lines compared with the negative control (NC) group (P<0.05). The present results demonstrated that overexpression of miR-30c inhibits prostate cancer cell line proliferation, migration and invasion, which was possibly caused by downregulation of KRAS protein by miR-30c. The data implicate miR-30c in the prognosis and treatment of prostate cancer.

Oncogenic activity of amplified miniature chromosome maintenance 8 in human malignancies.

Miniature chromosome maintenance (MCM) proteins play critical roles in DNA replication licensing, initiation and elongation. MCM8, one of the MCM proteins playing a critical role in DNA repairing and recombination, was found to have over-expression and increased DNA copy number in a variety of human malignancies. The gain of MCM8 is associated with aggressive clinical features of several human cancers. Increased expression of MCM8 in prostate cancer is associated with cancer recurrence. Forced expression of MCM8 in RWPE1 cells, the immortalized but non-transformed prostate epithelial cell line, exhibited fast cell growth and transformation, while knocked down of MCM8 in PC3, DU145 and LNCaP cells induced cell growth arrest, and decreased tumor volumes and mortality of severe combined immunodeficiency mice xenografted with PC3 and DU145 cells. MCM8 bound cyclin D1 and activated Rb protein phosphorylation by cyclin-dependent kinase 4 in vitro and in vivo. The cyclin D1/MCM8 interaction is required for Rb phosphorylation and S phase entry in cancer cells. As a result, our study showed that copy number increase and overexpression of MCM8 may play critical roles in human cancer development.

Vasoactive intestinal peptide induced epithelial to mesenchymal transition activities in PC3 prostate cancer cells

Abstract Epithelial to mesenchymal transition (EMT) is a fundamental cellular process preceding movement. Malignant transformation of epithelial cells also undergo EMT leading to metastasis and a worse prognosis for human cancer patients. Understanding molecular pathways that are linked to EMT may provide additional drug targets to treat epithelial cancers like prostate cancer. Vasoactive intestinal peptide signaling through one of its naturally occurring G-protein coupled receptors, termed VPAC1, induces non-tumor and tumorigenic epithelial cell lines (RWPE-1, LnCaP and PC3 cells) to undergo EMT and downregulate E-cadherin, a hallmark for EMT. Curcumin, an anti-inflammatory molecule, blocked these VIP EMT effects suggesting a role for NF-kB signaling. Our group has uncovered EMT-associated molecular mRNA targets induced by VIP treatment of primary, resting CD4 T cells, including the EMT master regulator transcription factor, SNAIL1. We hypothesize that EMT transition effects by VIP signaling may also involve the upregulation of SNAIL1 protein. To this end, we plan on treating PC3 cells with varying levels of VIP over time (0–24 hr) and measure by immunoblots the extent to which Snail1 protein, and up to 10 additional EMT-associated proteins, are differentially altered. Successful completion of this research is expected to uncover important VIP protein targets that is expected to better explain its epithelial tumorigenic potential.

Anticancer properties of a defensin like class IId bacteriocin Laterosporulin10

Laterosporulin10 (LS10) is a defensin like peptide from Brevibacillus sp. strain SKDU10 that inhibited microbial pathogens. However, in this study, anticancer activity of LS10 was examined against different cancer cell lines and compared with normal cells. LS10 displayed cytotoxicity against cancer cells like MCF-7, HEK293T, HT1080, HeLa and H1299 at below 10 μM concentration, but not against prostate epithelium cells RWPE-1. Additionally, no hemolysis was observed at significantly higher concentration compared to IC50 values observed for different cancer cell lines. Release of lactate dehydrogenase from cancer cell lines at 15 μM concentration upon 120 min treatment indicated the lytic ability of LS10. Accordingly, electron microscopy experiments also confirmed the necrotic effect of LS10 at 15 μM concentration against cancer cells. Furthermore, flow cytometry analysis of treated cancer cell lines revealed that LS10 induce apoptosis even at 2.5 μM concentration. Nevertheless, RWPE-1 cells remained viable even at 20 μM concentration. These results provide evidence that LS10 is an anticancer bacteriocin, which causes apoptotic and necrotic death of cancer cells at lower and higher concentrations, respectively. Taken all results together, the present study signifies that LS10 is an anticancer peptide that could be further developed for therapeutic applications.

Interaction between Trichomonas vaginalis and the Prostate Epithelium.

Most men infected with Trichomonas vaginalis are asymptomatic and can remain undiagnosed and untreated. This has been hypothesized to result in chronic persistent prostatic infection. Adhesion of the protozoan organisms to mucosal cells is considered a first and prerequisite step for T. vaginalis infection. Adhesion of T. vaginalis to prostate epithelial cells has not yet been observed; however, there are several reports about inflammation of prostate epithelial cells induced by T. vaginalis. The aim of this study was to investigate whether adhesion and cytotoxicity of T. vaginalis are involved in inflammation of prostate epithelial cells. When RWPE-1 cells were infected with T. vaginalis (1:0.4 or 1:4), adhesion of T. vaginalis continuously increased for 24 hr or 3 hr, respectively. The cytotoxicity of prostate epithelial cells infected with T. vaginalis (RWPE-1: T. vaginalis=1:0.4) increased at 9 hr; at an infection ratio of 1:4, cytotoxicity increased after 3 hr. When the RWPE-1 to T. vaginalis ratio was 1:0.4 or 1:4, production of IL-1β, IL-6, CCL2, and CXCL8 also increased. Epithelial-mesenchymal transition (EMT) was verified by measuring decreased E-cadherin and increased vimentin expression at 24 hr and 48 hr. Taken together, the results indicate that T. vaginalis adhered to prostate epithelial cells, causing cytotoxicity, pro-inflammatory cytokine production, and EMT. Our findings suggest for the first time that T. vaginalis may induce inflammation via adhesion to normal prostate epithelial cells.

PD37-10 BIOCHEMICAL VALIDATION OF PIPERLONGUMINE AS A POTENT THERAPEUTIC AGAINST NEUROENDOCRINE PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) II1 Apr 2017PD37-10 BIOCHEMICAL VALIDATION OF PIPERLONGUMINE AS A POTENT THERAPEUTIC AGAINST NEUROENDOCRINE PROSTATE CANCER Kamlesh K Yadav, Khader Shameer, Jennifer Stockert, Cordelia Elaiho, Benjamin Readhead, Shalini S Yadav, Joel Dudley, and Ashutosh Tewari Kamlesh K YadavKamlesh K Yadav More articles by this author , Khader ShameerKhader Shameer More articles by this author , Jennifer StockertJennifer Stockert More articles by this author , Cordelia ElaihoCordelia Elaiho More articles by this author , Benjamin ReadheadBenjamin Readhead More articles by this author , Shalini S YadavShalini S Yadav More articles by this author , Joel DudleyJoel Dudley More articles by this author , and Ashutosh TewariAshutosh Tewari More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1572AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies have highlighted the existence of a highly lethal and drug-resistant variant of prostate cancer (PCa) termed neuroendocrine prostate cancer (NEPC). We have used a genomics-based drug-repositioning approach to identify piperlongumine, a natural product constituent of the fruit of the Long pepper (Piper longum), as a potential therapeutic against NEPC. The efficacy of this drug was tested in the NEPC cell line model NCI-H660 and compared to several other PCa cell lines in a modified WST-1 assay. Pre-clinical testing in mouse xenograft models of NEPC was also undertaken. Finally, the ability of piperlongumine to inhibit p-STAT3 signaling and promote apoptosis in cell lines and extracted tumors were measured by western blot analyses. METHODS PCa cell lines (LNCaP, 22Rv1, Du145, PC3, H660 and RWPE) were grown in complete media (RPM1 10%FBS, Advanced DMEM 5%FBS or Keratinocyte-SFM) and treated with piperlongumine for 3 or 7 days. IC50 values were generated from WST assay data using Prism6. Nude mice (n=5) were injected with 1.5x106 H660 cells on each flank, and intraperitoneally administered with either 2.5mg/kg piperlongumine (n=3) or DMSO (n=2) daily for 3 weeks after tumors formed 200mm3 in volume. Tumor volume was measured daily with calipers. Western blot analyses were performed on protein lysates extracted from tumors and PCa cells treated with 0-10 μM of drug. RESULTS Piperlongumine was highly effective in inhibiting the growth of H660 cells (IC50 = 0.4 μM) compared to LNCaP, 22Rv1, Du145, PC3, and RWPE cells. On average, the growth rate of untreated tumors was 2.7 times greater than those treated was piperlongumine. Treated H660 cells exhibited significant inhibition of p-STAT3 and increases in cPARP1 levels while other cell lines displayed little to no fluctuation. CONCLUSIONS Using drug-repositioning approaches we have identified a lead compound that inhibits the growth of the highly drug-resistant NEPC cell line NCI-H660. Remarkably, piperlongumine happens to be a water-soluble plant product with no known side effects. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e677-e678 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Kamlesh K Yadav More articles by this author Khader Shameer More articles by this author Jennifer Stockert More articles by this author Cordelia Elaiho More articles by this author Benjamin Readhead More articles by this author Shalini S Yadav More articles by this author Joel Dudley More articles by this author Ashutosh Tewari More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The specific killing effect of matrine on castration-resistant prostate cancer cells by targeting the Akt/FoxO3a signaling pathway.

Matrine, a Sophora alkaloid, exhibits antiproliferative and anti-carcinogenic activities through several mechanisms. In a previous study, we found that matrine could effectively inhibit the proliferation of castration-resistant prostate cancer (CRPC). In the present study, the effect of matrine and LY294002 on the expression of the Akt/FoxO3a signaling pathway was examined by western blot analyses and RT-PCR. We discovered that matrine significantly inhibited the proliferation of both prostate cancer cell line PC-3 and prostate epithelial cell line RWPE1, induced apoptosis and induced cell cycle arrest. In addition, LY294002 was found to enhance the effect of matrine. Furthermore, the effects of matrine on the inhibition of proliferation and the induction of cell cycle arrest and cell apoptosis were more effective on PC-3 than on RWPE1 cells. Compared to RWPE1 cells, matrine exerted a more powerful influence on PC-3 cells in increasing the expression of the relevant protein. Our data suggested that FoxO3a-Bim and FoxO3a-P27 may mediate matrine-inhibited proliferation of CRPC cells by activating cell apoptosis and inducing cell cycle arrest. Matrine exhibited high selectivity in killing CRPC cells. Our findings demonstrated that matrine could be used in a potential therapeutic role in the management of CRPC in humans.

Stanniocalcin 1 promotes cell proliferation via cyclinE1/cyclin‑dependent kinase 2 in human prostate carcinoma.

Stanniocalcin 1 (STC1) is a glycoprotein hormone that is involved in calcium/phosphate homeostasis. Increasing evidence suggests that STC1 is involved in carcinogenesis; however, few studies have defined the mechanisms and functional roles of STC1 activity in prostate carcinogenesis. In the present study, MTT, flow cytometry and colony formation assays, and small interfering RNA (siRNA) and overexpression in multiple cell lines were used to investigate the function of STC1 in prostate carcinoma invivo and invivo. Knockdown of endogenous STC1 using a siRNA decreased the proliferation of DU145 and LNCaP2 cells. These results were consistent with the changes in the protein levels of cyclinE1 and cyclin‑dependent kinase2. By contrast, increased expression of STC1 in RWPE-1 cells led to increased cell proliferation, suggesting that STC1 promotes prostate carcinoma cell proliferation. In summary, the present study investigated the impact of STC1 on the proliferation and growth of prostate cancer in an effort to evaluate STC1 as a predictive biomarker and as a potential target for therapy.

Testis specific Y-like 5: gene expression, methylation and implications for drug sensitivity in prostate carcinoma

BackgroundTSPYL5, a putative tumor suppressor gene, belongs to the nucleosome assembly protein family. The chromosomal location of the TSPYL5 gene is 8Q22.1, and its exact role in prostate cancer etiology remains unclear. Further TSPYL5 gene and protein expression in prostate carcinoma cells and diseased tissues including its susceptibility for epigenetic silencing is unknown. Also, not known is the variation in TSPYL5 protein expression with regards to progression of prostatic carcinoma and its possible role in drug sensitivity.MethodsTSPYL5, DNMT-1 and DNMT-B gene expression in DU145, LNCaP and RWPE-1 cells and prostate tumor tissues was analyzed by qRT-PCR and RT-PCR. Demethylation experiments were done by treating DU145 and LNCaP cells with 5-aza-2′-deoxycytidine in vitro. Methylation analysis of TSPYL5 gene was performed by methylation specific PCR and pyrosequencing. TSPYL5 protein expression in benign and diseased prostate tumor tissues was performed by immunohistochemistry and in the cells by Western blotting.ResultsTSPYL5 was differentially expressed in non-tumorigenic prostate epithelial cells (RWPE-1), androgen independent (DU145), dependent (LNCaP) prostate carcinoma cells and tissues. Methylation-specific PCR and pyrosequencing analysis identified an inverse relationship between DNA methylation and expression leading to the silencing of TSPYL5 gene. Treatment of prostate carcinoma cells in which TSPYL5 was absent or low (DU145 and LNCaP) with the demethylating agent 5-aza-2′-deoxycytidine upregulated its expression in these cells. Immunohistochemical studies clearly identified TSPYL5 protein in benign tissue and in tumors with Gleason score (GS) of 6 and 7. TSPYL5 protein levels were very low in tumors of GS ≥ 8. TSPYL5 overexpression in LNCaP cells increased the cell sensitivity to chemotherapy drugs such as docetaxel and paclitaxel, as measured by the cellular viability. Furthermore, the cells also exhibited reduced CDKN1A expression with only marginal reduction in pAKT.ConclusionsDecrease in TSPYL5 protein in advanced tumors might possibly function as an indicator of prostate tumor progression. Its absence due to methylation-induced silencing can lead to reduced drug sensitivity in prostate carcinoma.

Effects of astaxanthin on oxidative stress induced by Cu2+ in prostate cells.

Astaxanthin (AST), a carotenoid molecule extensively found in marine organisms and increasingly used as a dietary supplement, has been reported to have beneficial effects against oxidative stress. In the current paper, the effects of AST on viability of prostate cells were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay; cell apoptosis and intracellular reactive oxygen species (ROS) levels were determined by flow cytometry; the mitochondrial membrane potential (MMP) was measured by fluorospectrophotometer; and activities of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were evaluated by a detection kit. The results show that copper ion (Cu2+) induced apoptosis, along with the accumulation of intracellular ROS and MDA, in both prostate cell lines (RWPE-1 and PC-3). AST treatments could decrease the MDA levels, increase MMP, and keep ROS stable in RWPE-1 cell line. An addition of AST decreased the SOD, GSH-Px, and CAT activities in PC-3 cell line treated with Cu2+, but had a contrary reaction in RWPE-1 cell lines. In conclusion, AST could contribute to protecting RWPE-1 cells against Cu2+-induced injuries but could cause damage to the antioxidant enzyme system in PC-3 cells.

Abstract B33: Determination of protein expression of candidate prostate cancer genes from a novel region on chromosome 7 identified in African American men

Abstract In the United States, African American (AA) men are at 60% higher risk of developing prostate cancer than European American (EA) men, and AA men are 2.4 times more likely to die from prostate cancer than EA men. Although these disparities are frequently attributed to the greater aggressiveness of prostate cancer among AA men, it is not known what causes that prostate cancer aggressiveness in AA men. To identify germline genetic variation that could explain some of this disparity, our previous admixture mapping study identified a novel region on chromosome 7q31-34 where local ancestry was associated with risk of disease. The main objective of this study is to investigate whether candidate genes in this gene region show differences in protein product expression by cell line origin (AA vs. EA) and by cell line metastatic potential. We specifically examined the expression of three proteins sourced from this gene region through immunoblot analysis, filamin C (FLNC), glutamate receptor metabotropic B (GRM8), and cadherin-like and PC-esterase domain containing 1 (CPED1). The cell lines DU-145, PC3, LNCaP, RWPE1, and WPE1 were derived from biological specimens from EA men, whereas the PCa2b and the RC77T cell lines were sourced from AA men. The RWPE1 and WPE1 cell lines were derived from normal prostate cells, while the remaining cell lines were derived from metastatic prostate cancers. Using immunoblot analysis, we found that GRM8 is expressed at higher levels in the cell lines derived from AA men with prostate cancer compared to those from EA men with prostate cancer. In contrast, FLNC and CPED1 are expressed at higher levels in the cell lines from EA men with prostate cancer compared to those from AA men with prostate cancer. Interestingly, both FLNC and CPED1 expression is also low in the androgen sensitive LNCaP cell line. Importantly, these results are consistent with our fine mapping results in AA men, where the SNP with greatest evidence for association is located. Further, previous studies have demonstrated that the methylation status and mRNA expression of CPED1 and FLNC differed between prostate cancers from AA and EA men. Taken together, these results provide us with a foundation to test the relevance of these genes in prostate cancer growth and metastasis and contribute to our understanding of the molecular differences in prostate cancer between EA and AA men. Citation Format: Casey Wizner, Julie Ruterbusch, Nicole Kubinec, Gregory Dyson, Albert Levin, Julie Boerner, Cathryn Bock. Determination of protein expression of candidate prostate cancer genes from a novel region on chromosome 7 identified in African American men. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B33.

Characterization of an in vitro model to study the role of human Polyomavirus BK in prostate cancer

Prostate cancer (PCa) is one of the most common male neoplasm in the western world, being the most commonly diagnosed non-skin cancer and the second leading cause of cancer death. Various potential risk factors exist for the initial triggering events, including exposure to infectious agents, such as the human Polyomavirus BK (BKV). BKV is a good candidate as risk factor of PCa because it naturally infects the human reno-urinary tract, it establishes latency, and encodes oncoproteins that interfere with tumor suppressors pathways, thus altering the normal progression of cell cycle. Previous studies suggested a potential association between BKV and PCa, revealing that the prevalence of BKV was significantly higher in cancer than in control tissues, with a significant association between viral expression and cancer. However, this hypothesis is controversial because BKV is not restricted to tumor tissues but also infects healthy individuals in a high percentage. Moreover, an in vitro model of BKV infection in prostate cells is not available to understand the role for BKV in pathogenesis of PCa. Our aims were to determine whether BKV a) could infect normal epithelial prostate cells, b) affects cell phenotype and c) affects the phenotype of human prostate tumor cell line PC3. For this purpose normal epithelial prostate cell line RWPE-1 and prostate cancer cells PC3 were infected with BKV for 21 days. Cell proliferation, epithelial-to-mesenchymal markers (EMT) and invasion potential were analyzed by, respectively, MTT, immunofluorescence and SDS-zymography. Our results show that cell proliferation was increased or decreased by BKV, respectively, in RWPE-1 and PC3 cells. BKV induced E-cadherin downregulation and vimentin expression in both control and BKV-infected cells RWPE-1, suggesting that uninfected cells underwent EMT. Matrix metalloproteinase-2 and 9 activity was increased in RWPE-1 cells after BKV infection. By contrast, BKV did not significantly modified the phenotype of PC3 cells. These preliminary results suggest that normal epithelial prostate cells RWPE-1 and PC3 are susceptible and permissive to BKV infection. However, RWPE-1 cells exhibit some phenotype modifications related to EMT, possibly induced by the papilloma virus used to obtain their immortalization, thus suggesting that further experiments will be necessary to define if they represent a good experimental model to study prostate cancer.

MiR-141-3p Suppresses Expression of Androgen Receptors and Functions as a Tumor Suppressor Gene in Prostate Carcinogenesis

Background: Prostate cancer (PCa) is a leading cause of tumor mortality in Western societies. In China, the PCa mortality rate is increasing yearly. Androgen receptors (ARs) and microRNAs (miRNAs) play central roles in prostate carcinogenesis and progression. Methods: To characterize the underlying molecular mechanisms, we compared the miRNA profiles of early PCa (G ≤ 7), advanced PCa (G > 7) and non-tumor prostate tissues using deep-sequencing. The target genes of differentially expressed miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assays and Western blot (WB) and quantitative reverse transcription-PCR (qRT-PCR) analyses. Finally, we performed in vitro functional studies by inducing or inhibiting miR-141-3p expression using an artificial mimic or inhibitor. Results: A computational search implicated the open reading frame (ORF) of AR mRNA as a potential miR-141-3p target site. The qRT-PCR, WB and luciferase reporter assays revealed a reverse regulatory effect of miR-141-3p on AR. Mutation of the potential miR-141-3p binding site in the AR ORF resulted in a loss of responsiveness to the corresponding miRNA. Moreover, miR-141-3p expression levels were unchanged in early PCas, but were obviously increased in advanced PCas. MiR-141-3p overexpression inhibited RWPE-1 cell proliferation, mobility, and prohibited the entry of cells into the G2-S-M phase; miR-141-3p inhibition had the inverse effects. At the same time, we tested miR-141-3p’s functions in PC-3 and VCaP prostate cancer cell lines. Conclusions: Taken together, our results indicate that miR-141-3p targets AR and its downstream signaling pathways, and functions as a tumor suppressor miR in PCa carcinogenesis by suppressing cell growth and mobility, but the effect is not significant in maglinant PCas. MiR-141-3p is implicated as a novel therapeutic target for early PCa.