Malignant Prostate Cell Lines Research Articles

Antioxidant, cytotoxicity, and anti-prostate carcinoma effects of CuO nanoparticles containing Thymus vulgaris

In this work, we have described the green supported of CuO NPs over Thymus vulgaris (CuNPs) as a reducing/stabilizing nanocomposite in alkaline medium. In the cellular and molecular part of the recent study, the treated cells with CuNPs were assessed to determine the cytotoxicity and anti-human prostate carcinoma properties on prostate carcinoma cell lines i.e., LNCaP clone FGC-Luc2, 22Rv1, and NCI-H660. The morphological and physicochemical features of the prepared nanocomposite were determined using several advanced techniques as Field Emission Scanning Electron Microscopes (FE-SEM), Ultraviolet–visible spectroscopy (UV-Vis), and fourier transform infrared spectroscopy (FTIR) studies. In the antioxidant test, the IC50 of CuNPs and butylated hydroxytoluene (BHT) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals were 94 and 88 μg/mL, respectively. The IC50 of CuNPs were 191, 275, and 250 μg/mL against LNCaP clone FGC-Luc2, 22Rv1, and NCI-H660 cell lines, respectively. In conclusion, our data suggest that the malignant prostate cell lines viability decreased in the CuNPs presence.

Rhenium(I) conjugates as tools for tracking cholesterol in cells.

Cholesterol is vital to control membrane integrity and fluidity, but is also a precursor to produce steroid hormones, bile acids, and vitamin D. Consequently, altered cholesterol biology has been linked to many diseases, including metabolic syndromes and cancer. Defining the intracellular pools of cholesterol and its trafficking within cells is essential to understand both normal cell physiology and mechanisms of pathogenesis. We have synthesized a new cholesterol mimic (ReTEGCholestanol), comprising a luminescent rhenium metal complex and a cholestanol targeting unit, linked using a tetraethylene glycol (TEG) spacer. ReTEGCholestanol demonstrated favourable imaging properties and improved water solubility when compared to a cholesterol derivative, and structurally related probes lacking the TEG linker. A non-malignant and three malignant prostate cell lines were used to characterize the uptake and intracellular distribution of ReTEGCholestanol. The ReTEGCholestanol complex was effectively internalized and mainly localized to late endosomes/lysosomes in non-malignant PNT1a cells, while in prostate cancer cells it also accumulated in early endosomes and multivesicular bodies, suggesting disturbed cholesterol biology in the malignant cells. The ReTEGCholestanol is a novel imaging agent for visualizing endosomal uptake and trafficking, which may be used to define cholesterol related biology including membrane integration and altered lipid trafficking/processing.

Enhancing cisplatin anticancer effectivity and migrastatic potential by modulation of molecular weight of oxidized dextran carrier

The molecular weight (Mw) of dextran derivatives, such as regioselectively oxidized dicarboxydextran (DXA), is greatly influencing their faith in an organism, which could be possibly used to improve anticancer drug delivery. Here we present a modified method of sulfonation-induced chain scission allowing direct and accurate control over the Mw of DXA without increasing its polydispersity. Prepared DXA derivatives (Mw = 10–185 kDa) have been conjugated to cisplatin and the Mw of the carrier found to have a significant impact on cisplatin release rates, in vitro cytotoxicity, and migrastatic potential. Conjugates with the high-Mw DXA showed particularly increased anticancer efficacy. The best conjugate was four times more effective against malignant prostatic cell lines than free cisplatin and significantly inhibited the ovarian cancer cell migration. This was traced to the characteristics of spontaneously formed cisplatin-crosslinked DXA nanogels influenced by Mw of DXA and amount of loaded cisplatin.

MiR-10a-5p and miR-29b-3p as Extracellular Vesicle-Associated Prostate Cancer Detection Markers.

Extracellular vesicles (EVs) are shed by many different cell types. Their nucleic acids content offers new opportunities for biomarker research in different solid tumors. The role of EV RNA in prostate cancer (PCa) is still largely unknown. EVs were isolated from different benign and malignant prostate cell lines and blood plasma from patients with PCa (n = 18) and controls with benign prostatic hyperplasia (BPH) (n = 7). Nanoparticle tracking analysis (NTA), Western blot, electron microscopy, and flow cytometry analysis were used for the characterization of EVs. Non-coding RNA expression profiling of PC3 metastatic PCa cells and their EVs was performed by next generation sequencing (NGS). miRNAs differentially expressed in PC3 EVs were validated with qRT-PCR in EVs derived from additional cell lines and patient plasma and from matched tissue samples. 92 miRNAs were enriched and 48 miRNAs were depleted in PC3 EVs compared to PC3 cells, which could be confirmed by qRT-PCR. miR-99b-5p was significantly higher expressed in malignant compared to benign EVs. Furthermore, expression profiling showed miR-10a-5p (p = 0.018) and miR-29b-3p (p = 0.002), but not miR-99b-5p, to be overexpressed in plasma-derived EVs from patients with PCa compared with controls. In the corresponding tissue samples, no significant differences in the miRNA expression could be observed. We thus propose that EV-associated miR-10a-5p and miR-29b-3p could serve as potential new PCa detection markers.

Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells.

BackgroundProstate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated.MethodsTo verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (GAPDH, YWHAZ, HPRT1) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry.ResultsMedian normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-).ConclusionsWe report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens.

CD38 is methylated in prostate cancer and regulates extracellular NAD+

BackgroundCancer cell metabolism requires sustained pools of intracellular nicotinamide adenine dinucleotide (NAD+) which is maintained by a balance of NAD+ hydrolase activity and NAD+ salvage activity. We recently reported that human prostate cancer can be initiated following oncogene expression in progenitor-like luminal cells marked by low expression of the NAD+-consuming enzyme CD38. CD38 expression is reduced in prostate cancer compared to benign prostate, suggesting that tumor cells may reduce CD38 expression in order to enhance pools of NAD+. However, little is known about how CD38 expression is repressed in advanced prostate cancer and whether CD38 plays a role in regulating NAD+ levels in prostate epithelial cells.MethodsCD38 expression, its association with recurrence after prostatectomy for clinically localized prostate cancer, and DNA methylation of the CD38 promoter were evaluated in human prostate tissues representing various stages of disease progression. CD38 was inducibly over-expressed in benign and malignant human prostate cell lines in order to determine the effects on cell proliferation and levels of NAD+ and NADH. NAD+ and NADH were also measured in urogenital tissues from wild-type and CD38 knockout mice.ResultsCD38 mRNA expression was reduced in metastatic castration-resistant prostate cancer compared to localized prostate cancer. In a large cohort of men undergoing radical prostatectomy, CD38 protein expression was inversely correlated with recurrence. We identified methylation of the CD38 promoter in primary and metastatic prostate cancer. Over-expression of wild-type CD38, but not an NAD+ hydrolase-deficient mutant, depleted extracellular NAD+ levels in benign and malignant prostate cell lines. However, expression of CD38 did not significantly alter intracellular NAD+ levels in human prostate cell lines grown in vitro and in urogenital tissues isolated from wild-type and CD38 knockout mice.ConclusionsCD38 protein expression in prostate cancer is associated with risk of recurrence. Methylation results suggest that CD38 is epigenetically regulated in localized and metastatic prostate cancer tissues. Our study provides support for CD38 as a regulator of extracellular, but not intracellular, NAD+ in epithelial cells. These findings suggest that repression of CD38 by methylation may serve to increase the availability of extracellular NAD+ in prostate cancer tissues.

PO-221 Prostate cancer cells are able to use fructose as a metabolic source

IntroductionThe clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET) is based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-d-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilise hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells.Material and methodsWe determined the glucose and fructose concentration in serum from benign and CaP patients, the protein expression of Glut1, Glut5 and Glut9 and mRNA expression of Glut1, Glut5, Glut7, Glut9 and Glut11 in clinical specimens of benign and malignant prostate tissue using immunostaining and qRT-PCR. Moreover, mRNA and protein expression for the glucose transporters was analysed in vitro in benign (RWPE-1) and malignant (LNCaP, LNCaP-C4-2, DU-145, and PC3) CaP cell lines using qRT-PCR and western blot. Fructose and glucose uptake was measured in vitro in prostate cell lines using radiolabelled d-[U-14C]-fructose or 2-[1,2–3 hour]-deoxy-d-[3H]-glucose, respectively. The effect of fructose and glucose on mitochondrial metabolism was analysed in prostate cell lines using seahorse analysis and the effect of fructose on tumour growth was analysed in vivo using a PC3 cell line xenograft model using immunosuppressed NSG mice.Results and discussionsFructose concentration was elevated in CaP patients compared to benign and no difference in glucose levels was observed. The expression of the fructose transporters, Glut5 and Glut9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels, basal and maximal respiration in bening and CaP cells were similar in the presence of fructose or glucose. Lastly, fructose was able to increase the growth rate, size and weight of the tumours in the NSG mice compared to controls (without fructose).ConclusionOur results indicate that fructose may represent an alternative energy source for CaP cells and may promote CaP tumour growth of CaP cells in vivo.

Identification and Quantification of Heterogeneously-methylated DNA Fragments Using Epiallele-sensitive Droplet Digital Polymerase Chain Reaction (EAST-ddPCR).

DNA methylation plays an important role in the initiation and propagation of carcinogenesis; however, the role of heterogeneously methylated epialleles is currently not well studied, also due to the lack of sensitive, unbiased and high throughput methods. Here, a newly developed droplet digital PCR (ddPCR)-based method was evaluated regarding its ability to quantify such heterogeneously methylated epialleles with sufficient analytical sensitivity and specificity. Genomic DNA from blood leukocytes and bone marrow aspirate of an 8-year old male with B-cell acute lymphoblastic leukemia (B-ALL) and from normal and malignant prostate cell lines were analysed using ddPCR. By using these DNA samples, the specificity of an applied set of fluorescence-labeled probes was demonstrated as a proof of concept. All individual heterogeneously-methylated epialleles were quantifiable by a set of fluorescence-labeled probes with complementary sequences to epialleles in a closed-tube and high-throughput manner. The new method named epiallele-sensitive droplet digital PCR (EAST-ddPCR) may give new insights in the generation and regulation of epialleles and may help in finding new biomarkers for the diagnosis of benign und malignant diseases.

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

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

Abstract 448: Role of fructose in prostate cancer

Abstract The elevated level of glucose uptake and metabolism in cancers is the basis for the clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET scanning), based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilize hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. mRNA and protein expression for the glucose transporter Glut-1 and fructose transporters Glut-2, Glut-5, Glut-7, Glut-9 and Glut-11 was analyzed in benign (PWR-1E, RWPE-1) and malignant (LNCaP, vCaP, LNCaP-C4-2, DU-145, and PC-3) human prostate cell lines using qRT-PCR and western blot, respectively. In addition, Glut(s) protein expression was analyzed on a tissue microarray containing 200 formalin-fixed paraffin-embedded benign and malignant human prostate tissues using immunohistochemistry. Fructose and glucose uptake was measured in vitro in benign and malignant human prostate cell lines using radiolabelled D-[U-14C]-fructose or 2-[1,2-3H]-deoxy-D-[3H]-glucose, respectively. Lastly, the effect of fructose or glucose on the levels of ATP, mitochondrial metabolism, and expression of the enzymes hexokinase-2 (HK2), type-C fructokinase (KHK-C), pyruvate kinase M2 (PKM2) and type-A lactate dehydrogenase (LDH-A) was analyzed in benign and malignant human prostate cell lines using chemiluminescence, seahorse, and qRT-PCR analyses, respectively. Our results indicated that expression of the fructose transporters, Glut-5 and Glut-9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut-1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels in CaP cells were similar in the presence of fructose or glucose. Fructose, but not glucose, significantly altered mRNA expression of HK2, KHK-C, PKM2, and LDH-A in malignant human prostate cells. Taken together, our results suggest that fructose may represent an alternative energy source and may reprogram hexose metabolism in CaP cells. Citation Format: Daniela Carreño, Nestor Corro, Marcia Arredondo, Carmen Navarro, Verónica Torres, Viviana Montecinos, Paula Sotomayor, Francisco Nualart, Julio Cesar Cárdenas, Alejandro S. Godoy. Role of fructose 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 448. doi:10.1158/1538-7445.AM2017-448

The increased expression of fatty acid-binding protein 9 in prostate cancer and its prognostic significance.

In contrast to numerous studies conducted to investigate the crucial role of fatty acid binding protein 5 (FABP5) in prostate cancer, investigations on the possible involvement of other FABPs are rare. Here we first measured the mRNA levels of 10 FABPs in benign and malignant prostate cell lines and identified the differentially expressed FABP6 and FABP9 mRNAs whose levels in all malignant cell lines were higher than those in the benign cells. Thereafter we assessed the expression status of FABP6 and FABP9 in both prostate cell lines and in human tissues. FABP6 protein was overexpressed only in 1 of the 5 malignant cell lines and its immunostaining intensities were not significantly different between benign and malignant prostate tissues. In contrast, FABP9 protein was highly expressed in highly malignant cell lines PC-3 and PC3-M, but its level in the benign PNT-2 and other malignant cell lines was not detectable. When analysed in an archival set of human prostate tissues, immunohistochemical staining intensity for FABP9 was significantly higher in carcinomas than in benign cases and the increase in FABP9 was significantly correlated with reduced patient survival times. Moreover, the increased level of staining for FABP9 was significantly associated with the increased joint Gleason scores (GS) and androgen receptor index (AR). Suppression of FABP9 expression in highly malignant PC3-M cells inhibited their invasive potential. Our results suggest that FABP9 is a valuable prognostic marker to predict the outcomes of prostate cancer patients, perhaps by playing an important role in prostate cancer cell invasion.

Human Prostate Tissue-derived Extracellular Matrix as a Model of Prostate Microenvironment

BackgroundClinical experience highlights the wide heterogeneity of primary prostate cancer (PPCa), even when potentially related to the same grade and stage. Currently available prediction tools and biomarkers do not always allow for early recognition of PPCa aggressive phenotype, sometimes making it impossible to distinguish among men harbouring indolent tumours or life-threatening disease. ObjectiveTo establish a novel ex vivo/in vitro model suitable to estimate the invasive phenotype of PPCa cells (PPCaC). Design, setting, and participantsThe ability of PPCaC to infiltrate the prostate extracellular matrix (ECM) was used as an index of invasion. ECM was obtained by decellularising 24 NT-prostate specimens from radical prostatectomy. PPCaC were obtained from six tumours with different Gleason patterns and pathological stages. Invasion ability was estimated in direct-cocolture experiments. Outcome measurements and statistical analysisThe extent of ECM invasion by PPCaC was quantified by counting the number of infiltrated cells. Mann–Whitney test was utilised for statistical comparisons. Results and limitationsSamples of ECM resulted to be free of cells and DNA and with a preserved three-dimensional structure and stromal protein content. The system resulted to be reliable since well characterised normal-, benign-, and malignant-prostate cell lines either re-epitheliased or invaded the matrices, according to their specific nature. Similarly, PPCaC invaded the ECMs consistently with their stage and biochemical recurrence. Of notice, this model was able to identify a different invasive phenotype even among tumours with equal Gleason patterns and pathological stages. The small sample size represents a limitation. ConclusionsWe developed an ex vivo/in vitro model able to reproduce the original PPCa-microenvironment and suitable to recognise the inherent invasive behaviour of PPCaC. Patient summaryWe developed a novel ex vivo/in vitro system which enables us to uncover which prostate tumours host potentially aggressive cancer cells. The identification of cancer cells with different invasive abilities will likely lead to the identification of new biomarkers to safely predict disease progression.

Relation of exposure to amino acids involved in sarcosine metabolic pathway on behavior of non-tumor and malignant prostatic cell lines

Sarcosine (N-methylglycine) was previously delineated as a substantial oncometabolite of prostate cancer (PCa) and its metabolism seems to be significantly involved in PCa development and behavior. We focused on investigation whether the exposure of prostate cells (PNT1A, 22Rv1, and PC-3) to sarcosine-related amino acids (glycine, dimethylglycine, and sarcosine) affects their aggressiveness (cell mobility and division rates, using real-time cell based assay). The effect of supplementation on expression of glycine-N-methyltransferase (GNMT) mRNA was examined using qRT-PCR. Finally, post-treatment amino acids patterns were determined with consequent statistical processing using the Ward's method, factorial ANOVA and principal component analysis (P < 0.05). The highest migration induced sarcosine and glycine in metastatic PC-3 cells (a decrease in relative free area about 53% and 73%). The highest cell division was achieved after treatment of 22Rv1 and PC-3 cells with sarcosine (time required for division decreased by 65% or 45%, when compared to untreated cells). qRT-PCR revealed also significant effects on expression of GNMT. Finally, amino acid profiling shown specific amino acid patterns for each cell line. In both, treated and untreated PC-3 cells significantly higher levels of serine, glutamic acid, and aspartate, linked with prostate cancer progression were found. Sarcosine-related amino acids can exceptionally affect the behavior of benign and malignant prostate cells.

Determination of cellular glutathione:glutathione disulfide ratio in prostate cancer cells by high performance liquid chromatography with electrochemical detection

A validated method has been developed for the simultaneous measurement of reduced and oxidized glutathione in de-proteinised cellular extracts. This has been used to compare models of malignant and non-malignant human prostate cell lines. Analysis of LNCaP and DU145 cells showed a glutathione to glutathione disulfide ratio of 8:1 and 32:1 respectively, whilst the control cell line, PZ-HPV7 displayed a ratio of 93:1. Results indicate that the more aggressive phenotype displays adaptation to increased oxidative stress via up regulation of glutathione turnover. It was also noted that in the LNCaP and DU145 cell line, glutathione was only responsible for ca. 60% and 79% respectively, of the total cellular reduced thiol; indicating the presence of other biological thiols.

MP61-09 PROMOTER-TARGETED DOUBLE-STRAND RNA DUPLEX ENHANCES DPYSL3 GENE EXPRESSION IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2015MP61-09 PROMOTER-TARGETED DOUBLE-STRAND RNA DUPLEX ENHANCES DPYSL3 GENE EXPRESSION IN PROSTATE CANCER CELLS William Parker, Qingting Hu, Jiang Wencong, J. Brantley Thrasher, and Benyi Li William ParkerWilliam Parker More articles by this author , Qingting HuQingting Hu More articles by this author , Jiang WencongJiang Wencong More articles by this author , J. Brantley ThrasherJ. Brantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2190AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES CRMP4, which is derived from the gene DPYSL3, has been recognized as a tumor metastasis suppressor gene in prostate cancer. We sought to characterize DPYSL3 gene expression profiles in prostate cancer cell lines and human prostate cancer specimens and then to develop a gene therapy for DPYSL3 enhancement utilizing a double-strand small activating RNA-based approach (saRNA). METHODS Cellular gene expression profiles were assessed using SYBR Green-based PCR and correlated to protein expression of CRMP4 using Western blot in 2 benign and 6 malignant prostate cell lines as well as in thirty-one de-identified pathology specimens containing prostate cancer and individually matched benign prostate tissues from patients who underwent radical prostatectomy. Genomic sequences and promoter locations of DPYSL3 gene were extracted from UCSD Genome Browser database. Small double-strand RNA duplexes (21nt) were chemically synthesized and were transiently transfected into prostate cancer cells with these small RNA molecules using RNAiMAX reagent. RESULTS Two isoforms of DPYSL3 gene exist in the human genome. Expression profiles showed that isoform-1 is the dominant isoform expressed in DU145 and LAPC-4 cells and isoform-2 is the dominant form in LNCaP, C4-2 and PC-3 cells. Both isoforms were similarly expressed in prostate cancer 22RV1 and benign prostate epithelial BPH1 cell, and are expressed at an extremely low level in benign RWPE-1 cells. The mRNA levels correlated with protein levels as evaluated by Western blot. Analysis of patient specimens revealed that isoform-2 expression levels are 2-fold higher than isoform-1 in both cancer and matched benign tissues. However, isoform-1 expression levels within the malignant tissue are significantly lower (2-fold, p = 0.01313, paired t-Test) than that in matched benign tissue. The saRNAs targeting the isoform-1 promoter but not the isoform-2 promoter significantly enhanced DPYSL3 gene expression in the mRNA and protein levels at day 3 after transfection in 22RV1, DU145 and PC-3 cells. CONCLUSIONS Our data reveal that isoform-1 of tumor suppressor gene DPYSL3 expression is significantly reduced in malignant tissues and is responsive to up-regulation by a saRNA-based approach. Further animal based in vivo testing of the saRNAs is being undertaking by our group to evaluate their clinical significance as a novel gene therapy strategy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e749-e750 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information William Parker More articles by this author Qingting Hu More articles by this author Jiang Wencong More articles by this author J. Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

EGFR and IGF-1R in regulation of prostate cancer cell phenotype and polarity: opposing functions and modulation by T-cadherin.

T-cadherin is an atypical glycosylphosphatidylinsoitol-anchored member of the cadherin superfamily of adhesion molecules. We found that T-cadherin overexpression in malignant (DU145) and benign (BPH-1) prostatic epithelial cell lines or silencing in the BPH-1 cell line, respectively, promoted or inhibited migration and spheroid invasion in collagen I gel and Matrigel. T-cadherin-dependent effects were associated with changes in cell phenotype: overexpression caused cell dissemination and loss of polarity evaluated by relative positioning of the Golgi/nuclei in cell groups, whereas silencing caused formation of compact polarized epithelial-like clusters. Epidermal growth factor receptor (EGFR) and IGF factor-1 receptor (IGF-1R) were identified as mediators of T-cadherin effects. These receptors per se had opposing influences on cell phenotype. EGFR activation with EGF or IGF-1R inhibition with NVP-AEW541 promoted dissemination, invasion, and polarity loss. Conversely, inhibition of EGFR with gefitinib or activation of IGF-1R with IGF-1 rescued epithelial morphology and decreased invasion. T-cadherin silencing enhanced both EGFR and IGF-1R phosphorylation, yet converted cells to the morphology typical for activated IGF-1R. T-cadherin effects were sensitive to modulation of EGFR or IGF-1R activity, suggesting direct involvement of both receptors. We conclude that T-cadherin regulates prostate cancer cell behavior by tuning the balance in EGFR/IGF-1R activity and enhancing the impact of IGF-1R.

Abstract 618: Implications of inhibition of steroid receptor co-activator-1 in human prostate cancer

Abstract Background: Prostate cancer (PCa) is still the most commonly diagnosed cancer and 2nd leading cause of death in the USA and the 3rd leading cause of death behind lung and colorectal cancers in Europe, respectively. Depending on the stage of the tumor different therapy options exist. Initially, those therapies show success and lead to tumor regression but eventually castration-resistant PCa (CRPC) develops. Androgen receptor (AR) is still active in CRPC through various mechanisms. One possibility of targeting the AR axis is inhibition of its co-regulators such as SRC-1, needed for efficient transcription of AR downstream genes. SRC-1, a member of the p160 coactivator family, is known to promote the transcriptional activity of steroid receptors like the AR as well as that of other non-steroidal transcription factors. Due to the fact that SRC-1 staining is increased in metastatic tissue of patients samples compared to clinically localized PCa our goal is to investigate the specific role of SRC-1 in PCa progression and metastatogenesis. Moreover, the question whether SRC-1 could be a target for a novel therapeutic approach will be addressed. Methods: Knock-down cell lines were generated using lentiviral-based RNAi technology. Western Blotting and qRT-PCR analysis were performed to verify knock-down efficiency. The proliferation of the different cell lines was addressed by 3H thymidine incorporation assays. Scratch (wound healing) and Boyden chamber assays were used to investigate the migratory and invasive behavior of the cell lines. Results: Immortalized benign and malignant prostate cell lines were screened for SRC-1 expression on mRNA and protein level, revealing that SRC-1 is expressed in all tested cell lines. Furthermore, knock down of SRC-1 in LNCaP and MDA PCa 2b (AR+) led to a markedly reduced AR transcriptional activity, which was verified by a reduced transcription of a downstream target gene prostate specific antigen. Whereas SRC-1 knockdown in MDA PCa 2b and PC3 cells did not show any effect on proliferation, depletion of SRC-1 negatively influenced cell proliferation of LNCaP cells. Moreover, we investigated the role of SRC-1 in the migratory and invasive behavior. While depletion of SRC-1 in PC3 and MDA PCa 2b cells significantly diminished migration, it had no effect on invasiveness. Conclusion: Together these findings demonstrate that SRC-1 is not only an important regulator of AR transcriptional activity but also affects other signaling pathways. Furthermore, SRC-1 seems to play a crucial role in migration but not in invasion and proliferation, depending on the cell lines used. Thus, SRC-1 may be a possible therapeutic target in metastatic prostate cancer, although its specific role has to be further investigated. Citation Format: Birgit Luef, Florian Handle, Frédéric R. Santer, Zoran Culig. Implications of inhibition of steroid receptor co-activator-1 in human prostate cancer. [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 618. doi:10.1158/1538-7445.AM2014-618

The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

NeuroD1 regulation of migration accompanies the differential sensitivity of neuroendocrine carcinomas to TrkB inhibition

The developmental transcription factor NeuroD1 is anomalously expressed in a subset of aggressive neuroendocrine tumors. Previously, we demonstrated that TrkB and neural cell adhesion molecule (NCAM) are downstream targets of NeuroD1 that contribute to the actions of neurogenic differentiation 1 (NeuroD1) in neuroendocrine lung. We found that several malignant melanoma and prostate cell lines express NeuroD1 and TrkB. Inhibition of TrkB activity decreased invasion in several neuroendocrine pigmented melanoma but not in prostate cell lines. We also found that loss of the tumor suppressor p53 increased NeuroD1 expression in normal human bronchial epithelial cells and cancer cells with neuroendocrine features. Although we found that a major mechanism of action of NeuroD1 is by the regulation of TrkB, effective targeting of TrkB to inhibit invasion may depend on the cell of origin. These findings suggest that NeuroD1 is a lineage-dependent oncogene acting through its downstream target, TrkB, across multiple cancer types, which may provide new insights into the pathogenesis of neuroendocrine cancers.

Bisphenol A: An endocrine and metabolic disruptor

Bisphenol A (BPA), initially designed, like diethylstilbestrol, as a synthetic estrogen, has been rapidly and widely used for its cross-linking properties in the manufacture of polycarbonate plastics and epoxy resins. Because of incomplete polymerization and degradation of the polymers by exposure to higher than usual temperatures, BPA leaches out from food and beverage containers, as well as from dental sealants. In humans, free active unconjugated BPA is metabolized by rapid glucurono- or sulfo-conjugation and eliminated via renal clearance. However, exposure to environmental nanomolar concentrations of BPA is ubiquitous and continuous via different routes: oral, air, skin. In rodents, fetal and perinatal exposure to such environmentally relevant doses of BPA has been shown to affect the brain, liver, gut, adipose tissue, endocrine pancreas, mammary gland and reproductive tract and function. Similar concentrations are also able in vitro to impact human malignant breast, prostate, male germ or adipocyte cell lines (with a promoting effect and by interfering with chemotherapy drugs), or to stimulate pancreatic β cell insulin secretion. High levels of BPA have recently been correlated with obesity, diabetes, cardiovascular diseases, polycystic ovarian disease or low sperm count. However, before the real impact of BPA on human health can be clearly assessed, prospective longitudinal epidemiological studies are needed as well as characterization of selective biomarkers to verify long-term exposure and selective imprinting.