Cultured Prostate Cancer Cells Research Articles

Thermally Induced Apoptosis, Necrosis, and Heat Shock Protein Expression in Three-Dimensional Culture

This study was conducted to compare the heat shock responses of cells grown in 2D and 3D culture environments as indicated by the level of heat shock protein 70 expression and the incidence of apoptosis and necrosis of prostate cancer cell lines in response to graded hyperthermia. PC3 cells were stably transduced with a dual reporter system composed of two tandem expression cassettes-a conditional heat shock protein promoter driving the expression of green fluorescent protein (HSPp-GFP) and a cytomegalovirus (CMV) promoter controlling the constitutive expression of a "beacon" red fluorescent protein (CMVp-RFP). Two-dimensional and three-dimensional cultures of PC3 prostate cancer cells were grown in 96-well plates for evaluation of their time-dependent response to supraphysiological temperature. To induce controlled hyperthermia, culture plates were placed on a flat copper surface of a circulating water manifold that maintained the specimens within ±0.1°C of a target temperature. Hyperthermia protocols included various combinations of temperature, ranging from 37°C to 57°C, and exposure times of up to 2 h. The majority of protocols were focused on temperature and time permutations, where the response gradient was greatest. Post-treatment analysis by flow cytometry analysis was used to measure the incidences of apoptosis (annexin V-FITC stain), necrosis (propidium iodide (PI) stain), and HSP70 transcription (GFP expression). Cells grown in 3D compared with 2D culture showed reduced incidence of apoptosis and necrosis and a higher level of HSP70 expression in response to heat shock at the temperatures tested. Cells responded differently to hyperthermia when grown in 2D and 3D cultures. Three-dimensional culture appears to enhance survival plausibly by activating protective processes related to enhanced-HSP70 expression. These differences highlight the importance of selecting physiologically relevant 3D models in assessing cellular responses to hyperthermia in experimental settings.

Effects of eicosapentaenoic acid and the GPR120 agonist TUG‐891 on lysophosphatidic acid signaling in human prostate cancer cells (1066.10)

There is a high incidence of prostate cancer in the Western world, which may be partially attributed to Western diets that are generally deficient in omega‐3 fatty acids (n‐3 FAs). Dietary n‐3 FAs have been shown to inhibit proliferation of prostate cancer cells in culture. The G protein‐coupled receptor GPR120, which is expressed in normal prostate and prostate tumors, serves as a receptor for the n‐3 FA eicosapentaenoic acid (EPA). We therefore hypothesized that the inhibitory effects of n‐3 FAs on prostate cancer cell proliferation are mediated by GPR120. To test this hypothesis, we performed proliferation and signal transduction studies, comparing the effects of lysophosphatidic acid (LPA), EPA, the GPR120 agonist TUG‐891, and the GPR40 agonist GW9508 in DU145 and PC‐3 cells. We found that EPA and TUG‐891 inhibit proliferation of human prostate cancer cells in response to LPA. In addition, brief (15 minute) pre‐incubation of serum‐starved cells with EPA or TUG‐891 suppresses subsequent activation of ERK, p70S6K, and FAK in response to LPA. This rapid effect of EPA is consistent with mediation by a GPCR. Our data suggest that GPR120 shows promise as a therapeutic target in prostate cancer.Grant Funding Source: Washington State University College of Pharmacy

Regulation of LNCaP respiration by plasma membrane and mitochondrial angiotensin II receptors (1159.11)

Prostate cancer is diagnosed in over 186,000 men in the United States and accounts for more than 28,000 deaths annually. Angiotensin II (AngII) is a major effector peptide of the renin‐angiotensin system (RAS), and is an important factor in hypertension. Correlations between ACE therapy and improved prostate cancer outcomes led to in vitro studies the action of AngII in cultured prostate cancer cell lines. Investigators have reported that exogenous application of Ang II to LNCaP cells results in the activation of Akt signaling, increased activity of SOD, and glutathione peroxidase. As the mitochondrion is a major contributor of total cellular ROS, we investigated the effect of AngII on mitochondrial respiratory function directly. AngII can bind to either of two receptors: the losartan‐sensitive AT1‐R and the PD123319‐sensitive AT2‐R. We present Western‐blot evidence of the presence of AT1‐R at the plasma membrane and both AT1‐R and AT2‐R receptor at the mitochondrion in LNCaP cells. Using fluorescent respirometry, we show AngII stimulates LNCaP whole‐cell respiration over a 24‐hour period with maximal stimulation at 5 hours post‐application. Increased respiratory rates are abolished using either losartan or PD123319 at time points up to 5 hours post‐application implicating the participation of both receptor subtypes in mediating this effect. At 24 hours post‐application respiratory rates remain elevated, though to a lesser degree, and appear to be sustained by AT1‐R activity only. Given the differential contribution of these two receptor sub‐types to modulation in respiratory activity, there may be two independent pathways activated by their respective AT‐R subtype.

Ablation of the oncogenic transcription factor ERG by deubiquitinase inhibition in prostate cancer

The transcription factor E-twenty-six related gene (ERG), which is overexpressed through gene fusion with the androgen-responsive gene transmembrane protease, serine 2 (TMPRSS2) in ∼40% of prostate tumors, is a key driver of prostate carcinogenesis. Ablation of ERG would disrupt a key oncogenic transcriptional circuit and could be a promising therapeutic strategy for prostate cancer treatment. Here, we show that ubiquitin-specific peptidase 9, X-linked (USP9X), a deubiquitinase enzyme, binds ERG in VCaP prostate cancer cells expressing TMPRSS2-ERG and deubiquitinates ERG in vitro. USP9X knockdown resulted in increased levels of ubiquitinated ERG and was coupled with depletion of ERG. Treatment with the USP9X inhibitor WP1130 resulted in ERG degradation both in vivo and in vitro, impaired the expression of genes enriched in ERG and prostate cancer relevant gene signatures in microarray analyses, and inhibited growth of ERG-positive tumors in three mouse xenograft models. Thus, we identified USP9X as a potential therapeutic target in prostate cancer cells and established WP1130 as a lead compound for the development of ERG-depleting drugs.

Feasibility of multimodal molecular characterization of the androgen receptor (AR) in circulating tumor cells (CTCs) from patients with castrate-resistant prostate cancer (CRPC).

133 Background: Despite the wealth of agents targeting the androgen receptor (AR) signaling pathway in castrate-resistant prostate cancer (CRPC), emergence of resistance is common. Mechanisms for resistance are diverse and likely include quantitative alterations in AR as well as alterations in nuclear localization. Clinical interrogation of such events is challenged by difficulties in biopsying metastatic sites and limitations in available circulating tumor cell (CTC) platforms. We set out to explore different methodologies of AR interrogation within CTCs. Methods: CTCs and cultured prostate cancer cells were isolated from nucleated blood cells using fluorescence associated cell sorting (FACS) targeting the Epithelial Cell Adhesion Molecule (EpCAM) with CD45 negative selection. In addition to standard immunofluorescence imaging, ImageStreamX spectral flow analysis was used to analyze AR protein expression and nuclear localization. Targeted DNA sequencing and TaqMan copy number evaluation were used for downstream analysis of AR alterations. Patients with highly refractory disease were enrolled for these assay development studies. Results: Thirteen of 15 (87%) patients in an initial feasibility cohort had CTCs isolated for downstream evaluation. Targeted DNA sequencing of as few as 10 C4-2 cells isolated from nucleated blood cells confirmed specificity for prostate cells by identifying a known AR mutation. TaqMan copy number evaluation revealed AR amplification in two out of four evaluable patients. Fluorescence microscopy demonstrated feasibility of AR staining, with 10 of 10 patients exhibiting AR staining above negative control and 50% staining above that of AR+ LAPC4 cells. Though the numeric variability in AR expression using ImageJ software was high, AR protein interrogation with ImageStreamX allowed for more precise evaluation of nuclear localization of the AR revealing that in CRPC patients there can be distinct CTC populations with respect to AR localization. Conclusions: Identifying mechanisms of AR derangement is crucial for the precision management of advanced prostate cancer. CTCs are a promising way to evaluate molecular characteristics of AR in patients with late stage disease.

Synthesis and DFT studies of structural and some spectral parameters of nickel(II) complex with 2-(2-hydroxybenzoyl)-N-(1-adamantyl) hydrazine carbothioamide

An efficient synthesis of the novel 2-(2-hydroxybenzoyl)-N-(1-adamantyl) hydrazine carbothioamide and its Ni(II) complex are described. Quantum chemical calculations including molecular geometry and vibrational frequencies were carried out for the structures to explain stability and geometry using both density functional (DFT/B3LYP) with 6-311+G(d,p) and LANL2DZ basis sets. 1H-NMR chemical shifts were also studied using a gauge-including atomic orbital approach (GIAO), and were found to be in good agreement with the experimental values. The Mulliken charges and HOMO–LUMO orbital energies were calculated and analyzed. The calculated HOMO–LUMO energies show that the charge transfer occurs within the molecule. The maximum positive atomic charge was obtained for C11 and the presence of a large negative charge on O (46) and O (45) for the ligand suggested the bonding of the Ni(II) ion with oxygen atoms. The chemical structures of the compounds were established by elemental analysis, FT-IR, 1H-NMR, 13C-NMR, UV–Vis spectra, magnetic moments, and thermal analyses. The electronic spectra and magnetic measurement studies revealed that the structure of complex is octahedral. Additionally, the cytotoxic effects of the obtained ligand and its complex were determined by using PC3 prostate cancer cell cultures. The results indicated that the ligand showed distinctly better activity than the complex.

Morphological Differences between Circulating Tumor Cells from Prostate Cancer Patients and Cultured Prostate Cancer Cells

Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

TBLR1 as an androgen receptor (AR) coactivator selectively activates AR target genes to inhibit prostate cancer growth

Androgen receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. Transducin β-like-related protein 1 (TBLR1), a core component of the nuclear receptor corepressor complex, shows both corepressor and coactivator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and determined that the activation is dependent on both phosphorylation and 19S proteosome. We showed that TBLR1 physically interacts with AR and directly occupies the androgen-response elements of the affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared with the surrounding benign prostatic glands (P<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen-regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3-1), but not cell proliferation of the prostate cancer. Understanding the molecular switches involved in the transition from AR-dependent growth promotion to AR-dependent growth suppression will lead to more successful treatments for prostate cancer.

MicroRNA-185 and 342 Inhibit Tumorigenicity and Induce Apoptosis through Blockade of the SREBP Metabolic Pathway in Prostate Cancer Cells

MicroRNA (miRNA or miR) inhibition of oncogenic related pathways has been shown to be a promising therapeutic approach for cancer. Aberrant lipid and cholesterol metabolism is involved in prostate cancer development and progression to end-stage disease. We recently demonstrated that a key transcription factor for lipogenesis, sterol regulatory element-binding protein-1 (SREBP-1), induced fatty acid and lipid accumulation and androgen receptor (AR) transcriptional activity, and also promoted prostate cancer cell growth and castration resistance. SREBP-1 was overexpressed in human prostate cancer and castration-resistant patient specimens. These experimental and clinical results indicate that SREBP-1 is a potential oncogenic transcription factor in prostate cancer. In this study, we identified two miRNAs, miR-185 and 342, that control lipogenesis and cholesterogenesis in prostate cancer cells by inhibiting SREBP-1 and 2 expression and down-regulating their targeted genes, including fatty acid synthase (FASN) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR). Both miR-185 and 342 inhibited tumorigenicity, cell growth, migration and invasion in prostate cancer cell culture and xenograft models coincident with their blockade of lipogenesis and cholesterogenesis. Intrinsic miR-185 and 342 expression was significantly decreased in prostate cancer cells compared to non-cancerous epithelial cells. Restoration of miR-185 and 342 led to caspase-dependent apoptotic death in prostate cancer cells. The newly identified miRNAs, miR-185 and 342, represent a novel targeting mechanism for prostate cancer therapy.

Chemokine receptor expression on integrin-mediated stellate projections of prostate cancer cells in 3D culture

The chemokine receptor CXCR7 has emerged as a regulator of prostate tumor growth and invasion, along with the well-established role of its closely related receptor, CXCR4, and their shared ligand, SDF-1α. Consequently, inhibition of the CXCR7/CXCR4/SDF-1α axis may assist in controlling prostate tumor growth and progression. To facilitate the development of potential therapeutics, further clarification of CXCR7 function is required, specifically in relation to CXCR4.In this study, we report that CXCR7 and CXCR4 were co-expressed in LNCaP, DU145 and PC3 cell lines in 2D culture. When cultured in 3D using Matrigel, a marked up-regulation of both receptors was observed in PC3 cells. Interestingly, both CXCR7 and CXCR4 co-localized within radiating cellular structures, termed stellate projections, which protruded outward into the matrix. The stellate projections were rich in the expression of pro-invasive integrin β1, β-laminin and MMP-11 proteins. The development of the stellate projections was mediated by integrin β1-mediated interactions with the ECM, which also regulated the expression of CXCR7 and CXCR4. Taken together, these results demonstrate that integrin-mediated cell-ECM interactions can modulate tumor cell morphology, and regulate the expression of chemokine receptors which are associated with the invasive phenotype and progression of PCa.

Circulating microRNA Profiling Identifies a Subset of Metastatic Prostate Cancer Patients with Evidence of Cancer-Associated Hypoxia

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.

Lycopene modulates growth and survival associated genes in prostate cancer

Lycopene is a fat soluble red-orange carotenoid pigment present in tomato that reduces the risk for prostate cancer, a common malignancy among men. However, the mechanism by which lycopene attenuates prostate cancer is not fully defined. In this study we examined the effect of lycopene on proliferation, survival, and biomarker gene expression in prostate cancer (PC-3) cells in culture. WST-1 assay showed that lycopene induces a biphasic effect on PC-3 cells with a modest increase in proliferation at 1–5 μM, no change at 10–25 μM and a decrease at 50–100 μM doses in culture. Interestingly, combination treatment with lycopene induced anti-proliferative effect of Temozolomide on PC-3 cells. Lycopene also augmented the anti-proliferative effect of peroxisome proliferator-activated receptor gamma (PPARγ) agonists, but not Doxorubicin or Taxol, in prostate cancer. Flow cytometry analyses showed that lycopene, in combination with chemotherapeutic agents and PPARγ agonists, induced modest cell cycle arrest with significant increase in cell death by apoptosis and necrosis on prostate cancer. Gene array and quantitative reverse transcription polymerase chain reaction analyses showed that lycopene alters the expression of growth and apoptosis associated biomarkers in PC-3 cells. These findings highlight that lycopene attenuates prostate cancer by modulating the expression of growth and survival associated genes.

CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27kip1. The elevated p27kip1 expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients.

Abstract 3713: Chemoprevention of prostate carcinogenesis in TRAMP mice by α-santalol by causing cell cycle arrest and induction of apoptosis.

Abstract α-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. The present study was undertaken to determine the in vivo efficacy of α-santalol using TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice as a model. The i.p administration of α-santalol (50 mg/kg) did not cause any side effects or weight loss throughout the study. Administration of α-santalol (50 mg/kg) showed a trend in decrease in average wet weights of urogenital weights compared to control mice (∼34% decrease in α-santalol group compared to control). Furthermore, the dorsolateral sections of prostate from α-santalol-treated mice exhibited decreased cell proliferation in association with induction of apoptosis. Western blotting analysis of prostate tumor samples from α-santalol-treated group revealed an increase in the expression levels of cleaved PARP, phospho p53 (ser 15), cleaved caspase-3, cyclin B, p21 and a modest reduction in antiapoptotic protein Bcl-xL compared to control samples. In agreement with these results, α-santalol treatment resulted in G2/M cell cycle arrest in cultured prostate cancer cells. In conclusion, the present study indicates that α-santalol administration inhibits the development of prostate cancer in TRAMP mice by decreasing cell proliferation, causing cell cycle arrest, and inducing apoptosis. This study was supported by Wilkes University Type I grant, American Association of Colleges of Pharmacy and by Translational Cancer Research Center funded by South Dakota Governor's Office of Economic Development. Citation Format: Ajay Bommareddy, William Eggelston, Stacy Prelewicz, Andrea Antal, Adam L. VanWert, Hiral Patel, Aleona Chinikaylo, Linda S. Gutierrez, Santha Sreevidya, Chandradhar Dwivedi. Chemoprevention of prostate carcinogenesis in TRAMP mice by α-santalol by causing cell cycle arrest and induction of apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3713. doi:10.1158/1538-7445.AM2013-3713

Abstract 1692: SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines.

Abstract SR16388 is a novel amino steroid that targets estrogen-binding proteins including genomic estrogen receptors (ERs). Preclinical studies have demonstrated that SR16388 is a well-tolerated, orally active compound with compelling properties as an experimental antitumor agent: SR16388 has been demonstrated to 1) inhibit the proliferation and viability of diverse human cancer cell lines in vitro; 2) have antiangiogenic activity in vivo; and 3) reduce the growth of human tumor xenografts in mice. SR16388, which binds to and potently inhibits ERα and ERβ in breast cancer cells, also inhibits the widely expressed orphan nuclear receptor ERRα that has been implicated in the development of various human malignancies. ERRα is thought to regulate tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. We have been exploring potential mechanisms that could explain SR16388’s broad anti-proliferative effects toward human tumor cell lines with different genotypes and tissues of origin. In these studies, we observed that SR16388 can initiate a cell-cycle arrest at G1 or G2 and ultimately cause cytotoxicity (e.g., by apoptosis) even in cells that do not express ERs or ERRα. We recently observed that SR16388 can induce persistent macroautophagy (autophagy) in diverse tumor cells, including cells from hematopoietic malignancies (e.g., multiple myeloma/MM cells) in addition to carcinomas such as prostate and breast cancers. We demonstrated induction of autophagy by using established assays (e.g., LC3I/II processing; fluorescent reporters for acidic vacuole formation; long-lived protein degradation). Here we present results demonstrating that SR16388 can rapidly and potently induce autophagy in cultures of MM cells (U266, RPMI-8226) and PC3 prostate cancer cells. Induction of autophagy in PC3 cells, in particular, was detected in tumor xenografts as early as 3 hours after dosing of mice with a therapeutic dose level of SR16388. We will present data supporting the hypothesis that SR16388 exerts its anti-proliferative and anti-tumor effects in part through the induction of stress pathways modulated by perturbations of protein homeostasis. These effects could yield therapeutic benefits that derive from targets within solid tumor microenvironments. Citation Format: Lidia C. Sambucetti, Wei Zhou, Joy Calaoagan, Laurie Kara, Xiaohe Liu, Barbara Sato, Steve Samuelson, Nathan Collins, Keith Laderoute. SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1692. doi:10.1158/1538-7445.AM2013-1692

Abstract 1929: A sensitive cell-based assay for the evaluation of prostate cancer cells in urine.

Abstract Introduction: The current prostate cancer (CaP) screening in the United States includes the prostate specific antigen (PSA) test and the digital rectal exam (DRE). Abnormal results lead to a prostate biopsy, pathologic evaluation and diagnosis. The PSA test has high sensitivity, but low specificity, leading to a high percentage of unnecessary biopsies. Therefore, there remains an urgent need for a specific and sensitive, non-invasive assay for CaP detection. Our objective was to develop an assay for the detection of CaP cells in post-DRE urine. Methods: Urine CaP (UCaP) assay development was performed by spiking CaP cell lines (VCaP, LNCaP, and NCI-H660) into normal urine and optimizing cell stability, recovery, and biomarker detection by immunohistochemistry (IHC). Cells were recovered from the urine by translucent membrane filtration. Using the filter as the assay platform, retained cells were assessed for the detection of selected protein markers (ETS related gene [ERG]-CaP marker; alpha-methylacyl-CoA racemase [AMACR]-CaP marker; PSA-normal prostate marker). As a clinical assessment, post-DRE urine from patients undergoing diagnostic biopsy was analyzed for the presence of CaP cells. Results: Assay sensitivity was enhanced by maximizing the percentage of cells recovered following filtration. To assess enhanced cell recovery of the UCaP assay over currently reported methods (cytospin, centrifugation), low numbers of cultured prostate cancer cells (10 or 100 cells) were spiked into centrifuged urine samples. Cells captured on the filter membrane were stained with DAPI nuclear dye and counted. Between 70% and 85% of cells were recovered after spiking VCaP, LNCaP and NCI-H660 CaP cells. Cells were recovered 100% of the time, even when spiking in as little as a single cell. This is in contrast to published cell recovery studies that require at least 1,000 cells spiked into urine for consistent detection. Furthermore, detection of ERG, AMACR, and PSA protein expression was demonstrated by IHC in cells recovered from post-DRE urine. A feasibility test of ten patients was conducted. Our assay confirmed the presence of CaP marker positive cells in all biopsy positive patients (3 of 3). We also detected the presence of CaP cells in two patients whose biopsy results were negative. Conclusions: This novel yet simple cell capture platform for CaP cell detection and characterization yielded enhanced assay sensitivity. In a clinical feasibility test the assay confirmed positive biopsy results, and detected CaP cells in some urine specimens potentially missed by biopsy. A larger study is in progress for further evaluations of the UCaP assay. Citation Format: Kristen Nickens, Shyh-Han Tan, Lakshmi Ravindranth, Amina Ali, David G. McLeod, Shiv Srivastava, Gyorgy Petrovics. A sensitive cell-based assay for the evaluation of prostate cancer cells in urine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1929. doi:10.1158/1538-7445.AM2013-1929

Abstract 1206: Evaluation of androgen receptor function index (ARFI) in prostate cancer.

Abstract Introduction: Initially prostate cancer is driven by the male hormone androgen through the androgen receptor (AR). However, in some cases androgen receptor is functionally altered at late stages of tumorigenesis. Early knowledge of androgen receptor dysfunctions may assist in patient stratification for emerging therapeutic strategies. Although AR function can be altered by numerous mechanisms, the net effect of these changes is reflected in defective transcription factor functions of the AR. To develop readouts for AR function in prostate cancer we have evaluated the expression of a functionally relevant panel of AR-regulated genes in a prostate cancer cell culture model and in whole-mounted prostate tumor tissues. Methods: In the VCaP cell culture model of prostate cancer that endogenously express, AR, TMPRSS2-ERG fusion (ERG), KLK3 (PSA), NKX3.1, PMEPA1, ODC1 and AMD1 genes we have evaluated the dose and time kinetics of these AR regulated genes in response to androgen (R1881) treatment at protein and mRNA levels. Radical prostatectomy specimens representing favorable or poor prognostic cases were selected for the study. Each prostate was fixed, sectioned and immunohistochemical (IHC) staining on adjacent four-micron sections of the whole-mounted blocks were performed by using mouse monoclonal anti-AR, anti-ERG antibody (clone 9FY), rabbit polyclonal anti-PSA and anti-NKX3.1 antibodies. IHC intensities were scored and the final score was determined after multiplying the intensity score and percentage of positively stained area in the respective lesions. Results: Towards defining the expression levels of AR regulated genes in prostate cancer cells we have evaluated and confirmed the androgen dose and time kinetic response of endogenously expressed KLK3(PSA), PMEPA1, NKX3.1, ODC1, AMD1 and TMPRSS2-ERG (ERG) genes in VCaP cell culture model. Assessment of AR dysfunction by the immunohistochemical evaluation of AR regulated genes in whole-mounted prostate cancer specimens showed reduced expression of AR regulated genes in a sub-set of prostate cancer cases with notable inter- and intratumoral heterogeneity. Conclusions: Androgen regulation of examined genes was confirmed in VCaP prostate cancer cell culture model. Assessment of AR, ERG, NKX3.1 and PSA protein levels by IHC indicated reduced expression of AR-regulated genes in a subset of the cases. The analysis revealed inter- and intratumoral heterogeneity, as well as, challenges in the quantitative assessment of IHC. The observed readouts of AR dysfunction may provide a new tool for improved prognostic accuracy and patient stratification at early stages of prostate cancer treatment. Citation Format: Albert Dobi, Denise Young, Wei Huang, Lakshmi Ravindranth, Shashwat Sharad, Hua Li, Gyorgy Petrovics, David G. McLeod, Isabell Sesterhenn, Shiv Srivastava. Evaluation of androgen receptor function index (ARFI) in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1206. doi:10.1158/1538-7445.AM2013-1206

Photodynamic therapy induced cell death of hormone insensitive prostate cancer PC-3 cells with autophagic characteristics

The introduction of photodynamic therapy (PDT) to the treatment of advanced prostate cancer can accomplish the eradication of local neoplasm and distant metastases with minimized damage to the adjacent structures. The evidence of PDT efficacy for androgen-refractory prostate cancer will be especially meaningful for the patients resistant to hormone therapy. Pheophorbide a (PhA) as a photosensitizer was employed to evaluate the photodynamic efficacy in androgen-insensitive PC-3 prostate cancer cells in culture by cell viability assay, reactive oxygen species (ROS) measurement and cell cycle test. Characteristics of apoptosis and autophagy were investigated via DNA fragmentation electrophoresis and immune-fluorescence staining, acidic vesicle determination and detection of LC3B in puncta form by fluorescence microscopy, Western blotting of autophagy-related (Atg) proteins and detailed phenotype shown by electron microscopy. PhA exerted significant photo-cytotoxicity toward androgen-insensitive prostate cancer PC-3 cells in photosensitizer-dose and light-dose dependent manners. The photoactivation immediately initiated hyperproduction of ROS, the depolarization of mitochondrial membrane potential and the arrest of the cell cycle in the G0/G1 phase. Autophagy was revealed in PhA-PDT treated PC-3 cells by a significant high amount of acidic vesicular organelles with acridine orange staining, recruitment of LC3B on the membrane of autophagosomes by fluorescent microscopy, double membrane-bound vesicles suggesting autophagosomes by electron microscopy, significant increased Atg proteins such as beclin-1, Atg12-Atg5 conjugation, Atg7 and the conversion of LC3B-I to LC3B-II by Western blot analysis. PhA-mediated PDT induced significant autophagy in hormone-refractory prostate cancer PC-3 cells.

Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy

Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized (15)N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear (1)H-(15)N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ~17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules.

331 ANDROGEN DEPRIVATION PROMOTES INTRATUMORAL SYNTHESIS OF DIHYDROTESTOSTERONE FROM ANDROGEN METABOLITES IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research (II)1 Apr 2013331 ANDROGEN DEPRIVATION PROMOTES INTRATUMORAL SYNTHESIS OF DIHYDROTESTOSTERONE FROM ANDROGEN METABOLITES IN PROSTATE CANCER Fumio Ishizaki, Tsutomu Nishiyama, Takashi Kawasaki, Yoshimichi Miyashiro, Noboru Hara, Itsuhiro Takizawa, Makoto Naito, and Kota Takahashi Fumio IshizakiFumio Ishizaki Nagaoka, Japan More articles by this author , Tsutomu NishiyamaTsutomu Nishiyama Niigata, Japan More articles by this author , Takashi KawasakiTakashi Kawasaki Niigata, Japan More articles by this author , Yoshimichi MiyashiroYoshimichi Miyashiro Kawasaki, Japan More articles by this author , Noboru HaraNoboru Hara Niigata, Japan More articles by this author , Itsuhiro TakizawaItsuhiro Takizawa Niigata, Japan More articles by this author , Makoto NaitoMakoto Naito Niigata, Japan More articles by this author , and Kota TakahashiKota Takahashi Niigata, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1716AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Dihydrotestosterone (DHT) is reduced by aldo-keto reductase 1C2 and 1C1 (AKR1C2 and AKR1C1) and is metabolized to 5α-androstane-3α/β,17β-diol (3α/β-diol), respectively. 3α-diol can be converted back to DHT via oxidative 3α-hydroxysteroid dehydrogenase (HSD) activity. On the other hand, it has been reported that synthesis of DHT from 3β-diol is prevented. The significance of the back conversion in humans during androgen deprivation therapy (ADT) has barely been studied, despite evidence that the balance between DHT synthesis and metabolization determines intratumoral DHT concentrations. METHODS Intracellular androgen levels under incubation with the addition of 3α- or 3β-diol into LNCaP and VCaP cells were measured by the liquid chromatography tandem mass spectrometry (LC/MS). LNCaP was cultured in steroid hormone-depleted medium with 1 nM dehydroepiandrosterone (DHEA) for 70 passages (LNCaP-ADT). Transcriptome expression were analyzed by real-time PCR. Expression scores for 17β-HSD type 6 (HSD17B6) in radical prostatectomy specimens treated with or without ADT were evaluated using immunohistochemistry. We prospectively analyzed blood samples of 72 patients receiving ADT to reveal the in vivo kinetics of 3α-diol. RESULTS Our studies showed the synthesis of DHT from 3β-diol in both prostate cancer cell lines and culture medium. Different from the results of 3α-diol, we found a lot of other C19 androgens such as A-dione, A-diol, and DHEA in prostate cancer (PCa) cells and the culture medium. Additionally, long-term culture in androgen-deprived media increased mRNA expression of HSD17B6 in PCa cells. Conversely, AKR1C1·2 was down-regulated at the beginning of androgen deprivation. The expression score for HSD17B6 in prostatectomy specimens of 42 PCa patients undergoing neoadjuvant ADT was about 2-fold higher than that in tissues of 100 untreated individuals (p<0.001). In men receiving ADT, patients showing biochemical progression had a higher HSD17B6 score than those without progression (p=0.038). Transfection with HSD17B6 siRNA significantly reduced PSA by 20.6% in LNCaP-ADT cells treated with 3α-diol (P=0.014). Serum 3α-diol glucuronide levels correlated with serum adrenal androgen such as DHEA sulfate (DHEA-S) and A-dione before and after ADT. CONCLUSIONS Our study revealed that 3α- and 3β-diol has a much more significant role in intratumoral androgen metabolism during ADT than previously thought, and the back conversion of DHT from 3α- and 3β-diol can be a promising target for advanced combination hormone therapy. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e134 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Fumio Ishizaki Nagaoka, Japan More articles by this author Tsutomu Nishiyama Niigata, Japan More articles by this author Takashi Kawasaki Niigata, Japan More articles by this author Yoshimichi Miyashiro Kawasaki, Japan More articles by this author Noboru Hara Niigata, Japan More articles by this author Itsuhiro Takizawa Niigata, Japan More articles by this author Makoto Naito Niigata, Japan More articles by this author Kota Takahashi Niigata, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...