LNCaP-LN3 Cells Research Articles

A Combination of Natural Products, BenPros (Green Tea Extract, Soybean Extract and Camellia Japonica Oil), Ameliorates Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is one of the most common diseases in elderly men and causes lower urinary tract symptoms due to excessive proliferation of prostate stromal and epithelial cells. The present study investigated the improving effect of BenPros, an edible natural product mixture (green tea extract, soybean extract and camellia japonica oil), against the development of BPH in vitro and in vivo. BenPros treatment showed inhibitory ability on testosterone-induced androgen receptor, prostate-specific antigen (PSA), and 5α-reductase protein expression in LNCap-LN3 cells and anti-inflammatory effects on LPS-induced increases in interleukin-6 and tumor necrosis factor-α in RAW264.7 cells. In a testosterone propionate (TP)-induced BPH rat model, BenPros decreased the up-regulated serum 5α-dihydrotestosterone and PSA levels. Moreover, BenPros also significantly reduced PSA protein expression in prostate tissue. Furthermore, TP-induced increased expression of cyclooxygenase 2 and B-cell lymphoma 2 (Bcl-2) were reduced by BenPros, resulting in an increase in the Bcl-2/BCL2-related X ratio. These regulatory abilities of BenPros on BPH inducing markers also reduced prostate size and epithelial thickness based on histological analysis. These results indicate that BenPros has a protective ability against BPH in vitro and in vivo, and it may be a promising candidate as a functional food in regulating BPH.

Moiety modeling framework for deriving moiety abundances from mass spectrometry measured isotopologues

BackgroundStable isotope tracing can follow individual atoms through metabolic transformations through the detection of the incorporation of stable isotope within metabolites. This resulting data can be interpreted in terms related to metabolic flux. However, detection of a stable isotope in metabolites by mass spectrometry produces a profile of isotopologue peaks that requires deconvolution to ascertain the localization of isotope incorporation.ResultsTo aid the interpretation of the mass spectroscopy isotopologue profile, we have developed a moiety modeling framework for deconvoluting metabolite isotopologue profiles involving single and multiple isotope tracers. This moiety modeling framework provides facilities for moiety model representation, moiety model optimization, and moiety model selection. The moiety_modeling package was developed from the idea of metabolite decomposition into moiety units based on metabolic transformations, i.e. a moiety model. The SAGA-optimize package, solving a boundary-value inverse problem through a combined simulated annealing and genetic algorithm, was developed for model optimization. Additional optimization methods from the Python scipy library are utilized as well. Several forms of the Akaike information criterion and Bayesian information criterion are provided for selecting between moiety models. Moiety models and associated isotopologue data are defined in a JSONized format.By testing the moiety modeling framework on the timecourses of 13C isotopologue data for uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc) in human prostate cancer LnCaP-LN3 cells, we were able to confirm its robust performance in isotopologue deconvolution and moiety model selection.ConclusionsSAGA-optimize is a useful Python package for solving boundary-value inverse problems, and the moiety_modeling package is an easy-to-use tool for mass spectroscopy isotopologue profile deconvolution involving single and multiple isotope tracers. Both packages are freely available on GitHub and via the Python Package Index.

Angiotensin II receptor blockers induce autophagy in prostate cancer cells.

Angiotensin II receptor blockers (ARBs) are anti-hypertensive drugs that competitively inhibit the binding of angiotensin II to its receptor, resulting in blood vessel dilation and the reduction of blood pressure. These antagonists are also known as sartans, and are a group of pharmaceuticals that possess tetrazole or imidazole groups. In the present study, the anticancer and antimetastatic effects of the ARBs fimasartan, losartan, eprosartan and valsartan on the human prostate cancer PC-3, DU-145 and LNCap-LN3 cell lines were investigated in vitro. The proliferation of the prostate cancer cells was inhibited following treatment with 100 µM ARB. In particular, treatment with fimasartan resulted in marked anti-proliferative activity compared with the other ARBs. With respect to the molecular mechanism of the growth inhibition exhibited by the ARBs, 3-methyladenin (3-MA), an autophagy inhibitor, was revealed to increase the survival rate of PC-3 cells when cell death inhibitors were pretreated with fimasartan. In addition, the ARBs induced autophagy with increased expression levels of autophagy protein (Atg) 5–12, Atg 16-like-1, beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3). Notably, the enhanced expression of LC3-II (a 6.7-fold increase at 72 h) was observed in PC3 cells treated with fimasartan. This was supported by the observation of the time-dependent accumulation of LC3-positive foci in PC-3. In addition, a migration assay indicated that the ARBs induced anti-metastatic effects in PC-3 and DU-145 cells. The aforementioned results suggest that ARBs may induce autophagy-associated cell death and anti-metastatic activity in prostate cancer cells. Thus, ARBs may be a potential medication for patients with prostate cancer and hypertension.

6-(3,4-Dihydro-1H-isoquinoline-2-yl)-N-(6-methoxypyridine-2-yl) nicotinamide-26 (DIMN-26) decreases cell proliferation by induction of apoptosis and downregulation of androgen receptor signaling in human prostate cancer cells

Previously, we reported that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl) nicotinamide (DIMN) analogues inhibited the growth of prostate cancer cells as an anti-androgenic compound. In the present study, we evaluated cytotoxic effects of these DIMN derivatives and found that DIMN-26 most potently inhibited the proliferation of the LNCap-LN3 androgen-dependent and DU145 androgen-independent prostate cancer cells through induction of G2/M phase cell cycle arrest and subsequent apoptosis. The G2/M phase arrest was found due to increases in the activation of cdc2 (also known as cyclin-dependent kinase 1, CDK1)/cyclin B1 complex. DIMN-26 also induced apoptosis in LNCap-LN3 and DU145 prostate cancer cells through activation of caspase-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). In addition, DIMN-26 caused the dephosphorylation and mitochondrial accumulation of Bad protein and induced the loss of mitochondria membrane potential, consequently releasing cytochrome c into the cytosol of the cell. Furthermore, overexpression of AKT protein significantly reduced DIMN-26-induced PARP-1 cleavage and p-Bad decrease and cdc2 activation. In addition, DIMN-26 inhibited the 5α-dihydrotestosterone (DHT)-induced cell growth and proliferation and nuclear translocation and transcriptional activities of androgen receptor (AR) in LNCap-LN3 prostate cancer cells. Consistent with these findings, DIMN-26 significantly inhibited the DHT-induced expression of AR-response genes (ARGs), such as prostate-specific antigen (PSA), AR, β2-microglobulin (B2M), selenoprotein P (SEPP1), and ste20-related proline-alanine-rich kinase (SPAK) in LNCap-LN3 prostate cancer cells. Taken together, these results suggest that DIMN-26 plays a therapeutic role not only in induction of G2/M arrest and apoptosis but also in suppression of androgen receptor signaling in androgen-dependent and androgen-independent prostate cancer cells.

CACUL1 functions as a negative regulator of androgen receptor in prostate cancer cells

The androgen receptor (AR) plays a critical role in the initiation and progression of prostate cancer (PCa), and thus its regulation is an important tool in PCa therapy. Here, we report that CDK2-associated cullin 1 (CACUL1) directly associates with AR and suppresses AR transcriptional activity. In addition, CACUL1 represses histone demethylase LSD1-mediated AR transactivation by competing with LSD1 for AR binding. Depletion of CACUL1 enhances the LSD1 occupancy of the AR-target promoter, accompanied by decreased accumulation of H3K9me2, a repressive transcriptional marker. CACUL1 and LSD1 oppositely regulate CDX-induced cell death in AR-positive LNCaP and metastatic castrate-resistant LNCaP-LN3 cells. These data suggest that CACUL1 impairs LSD1-mediated activation of AR, thereby implicating it as a potential antitumor target in PCa.

Embelin-Induced Apoptosis of Human Prostate Cancer Cells Is Mediated through Modulation of Akt and β-Catenin Signaling.

There is increasing evidence that embelin, an active component of Embelia ribes, induces apoptosis in human cancer cells, but the detailed mechanisms are still unclear. Here, we have investigated the effect of embelin on the growth of human prostate cancer cells. Embelin strongly inhibited cell growth especially in human prostate cancer cell lines, including PC3, DU145, LNCaP-LN3 and normal prostate epithelial cell, RWPE-1 compared to breast cancer (MDA-MB-231, MCF-7, and T47D), hepatoma (HepG2, Hep3B, and HuH-7), or choriocarcinoma (JEG-3). We observed that embelin induced apoptosis of PC3 cells in a time-dependent manner correlated with decreased expression of Bcl-2, Bcl-xL, and Mcl-1, increased translocation of Bax into mitochondria, and a reduction in the mitochondrial membrane potential. Furthermore, embelin induced voltage-dependent anion channel (VDAC) 1 expression and oligomerization, which may promote cytochrome c and AIF release. Because embelin was able to inhibit Akt activation and cyclooxygenase-2 expression, the effects on Wnt/ β-catenin signaling were determined. Embelin activated glycogen synthase kinase (GSK)-3β by preventing phosphorylation and suppressed β-catenin expression. Attenuation of β-catenin-mediated TCF transcriptional activity and gene transcription, such as cyclin D1, c-myc, and matrix metalloproteinase (MMP)-7, were shown in embelin-treated cells. The changes in β-catenin levels in response to embelin were blocked by lithium chloride, a GSK-3 inhibitor, indicating that embelin may decrease β-catenin expression via GSK-3β activation. Furthermore, exposure of PC3 cells to embelin resulted in a significant decrease in cell migration and invasion. In conclusion, these findings suggest that inhibition of Akt signaling and activation of GSK-3β partially contributes to the pro-apoptotic effect of embelin in prostate cancer cells.

Plexin-B1 signalling promotes androgen receptor translocation to the nucleus.

Semaphorins and their receptors plexins have diverse roles in many cancers affecting tumour growth, metastasis and angiogenesis. Plexin-B1, the receptor for semaphorin4D (Sema4D), has been implicated in prostate cancer where mutation of the gene and overexpression of the protein occur. It is not clear, however, as to which of the several Sema4D-activated signalling pathways downstream of plexin-B1 function in prostate cancer progression. We show here that Sema4D/plexin-B1 increases the expression of androgen-responsive genes and activates the transcriptional activity of the androgen receptor (AR). Activation of plexin-B1 results in phosphorylation of AR at Serine 81, a site that is phosphorylated by nuclear kinases. Cell fractionation and immunocytochemistry studies demonstrated that the proportion of cells with AR in the nucleus increases significantly upon Sema4D treatment. The N-terminal (AF-1) domain of AR, which contains binding sites for transcription regulators, is not required for this response. Depletion of AR suppressed Sema4D-induced anchorage-independent growth of LNCaP and LNCaP-LN3 cells, demonstrating the functional significance of these findings. These results show that Sema4D/plexin-B1 signalling promotes the translocation of AR to the nucleus and thereby enhances AR transcriptional activity. Plexin-B1 is therefore a promising target for cancer therapy, especially in low androgen situations such as those imposed by androgen deprivation therapy.

NMR-based evaluation of the metabolic profile and response to dichloroacetate of human prostate cancer cells

The aim of this study was to evaluate the metabolic profile of human prostate cancer cells that have different metastatic potential and to determine their response to dichloroacetate (DCA) using NMR technology. Two isogenic human prostate cancer cell lines, differing in their metastatic potential [LNCaP (poorly metastatic) and LNCaP-LN3 (highly metastatic)], were studied. Metabolite ratios from NMR spectral integrals acquired at a field strength of 9.4 T using a 5-mm broadband probe with an NMR-compatible bioreactor were compared in the presence and absence of the pyruvate dehydrogenase kinase inhibitor DCA. Lactate dehydrogenase (LDH) isoenzymes were assessed by zymography. Following the treatment of cells with 50 mm DCA, there was a significant reduction in the lactate/choline, lactate/creatine, lactate/alanine and the combined lactate/(choline + creatine + alanine) ratios in LNCaP-LN3 cells relative to LNCaP cells. No significant changes in metabolite ratios were found in LNCaP cells following DCA treatment. As expected, LDH zymography assays showed an absence of the LDH-B subunit in LNCaP-LN3 cells, whereas both LDH-A and LDH-B subunits were present in LNCaP cells. DCA was shown to significantly modify the metabolite ratios in highly metastatic LNCaP-LN3 cells, but not in poorly metastatic LNCaP cells. This effect was probably related to the absence of the LDH-B subunit in LNCaP-LN3 cells, and could have a bearing on cancer treatment with DCA and related compounds. © 2014 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.

IGF-1R inhibition enhances radiosensitivity and delays double-strand break repair by both non-homologous end-joining and homologous recombination

Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) enhances tumor cell sensitivity to ionizing radiation. It is not clear how this effect is mediated, nor whether this approach can be applied effectively in the clinic. We previously showed that IGF-1R depletion delays repair of radiation-induced DNA double-strand breaks (DSBs), unlikely to be explained entirely by reduction in homologous recombination (HR) repair. The current study tested the hypothesis that IGF-1R inhibition induces a repair defect that involves non-homologous end-joining (NHEJ). IGF-1R inhibitor AZ12253801 blocked cell survival and radiosensitized IGF-1R over-expressing murine fibroblasts but not isogenic IGF-1R null cells, supporting specificity for IGF-1R. IGF-1R inhibition enhanced radiosensitivity in DU145, PC3 and 22Rv1 prostate cancer cells, comparable to effects of ATM inhibition. AZ12253801-treated DU145 cells showed delayed resolution of γH2AX foci, apparent within 1hr of irradiation and persisting for 24hr. In contrast, IGF-1R inhibition did not influence radiosensitivity or γH2AX focus resolution in LNCaP-LN3 cells, suggesting that radiosensitization tracks with the ability of IGF-1R to influence DSB repair. To differentiate effects on repair from growth and cell survival responses, we tested AZ12253801 in DU145 cells at sub-SF50 concentrations that had no early (≤48hr) effects on cell cycle distribution or apoptosis induction. Irradiated cultures contained abnormal mitoses, and after 5 days IGF-1R inhibited cells showed enhanced radiation-induced polyploidy and nuclear fragmentation, consistent with the consequences of entry into mitosis with incompletely repaired DNA. AZ12253801 radiosensitized DNA-PK proficient but not DNA-PK deficient glioblastoma cells, and did not radiosensitize DNA-PK-inhibited DU145 cells, suggesting that in the context of DSB repair, IGF-1R functions in the same pathway as DNA-PK. Finally, IGF-1R inhibition attenuated repair by both NHEJ and HR in HEK293 reporter assays. These data indicate that IGF-1R influences DSB repair by both major DSB repair pathways, findings that may inform clinical application of this approach.

Effects of the monoamine oxidase inhibitors pargyline and tranylcypromine on cellular proliferation in human prostate cancer cells

Chemotherapy is one of the therapeutic strategies that has been used for the inhibition of cancer cell proliferation in several types of cancer, including prostate cancer. Although monoamine oxidase (MAO) inhibitors, phytoestrogen and antioxidants used in chemotherapy have been systematically studied, their effects on cancer cell growth remain to be fully understood. The purpose of this study was to investigate the effects of the MAO inhibitors, pargyline and tranylcypromine on cell survival in human prostate carcinoma (LNCaP-LN3) cells. After treating LNCaP-LN3 cells with pargyline or tranylcypromine, we examined cell proliferation, cell cycle pattern, apoptosis and the expression levels of apoptosis-related genes. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner, while tranylcypromine-treated cells showed the opposite results. Treatment with pargyline significantly induced cell cycle arrest at the G1 phase compared to the control and tranylcypromine-treated cells. In addition, pargyline induced an increase in the cell death rate by promoting apoptosis; however, tranylcypromine had no effect on LNCaP-LN3 cells. Based on our results, we suggest that pargyline is more powerful than tranylcypromine for the treatment of human prostate cancer.

Marked improvement of cytotoxic effects induced by docetaxel on highly metastatic and androgen-independent prostate cancer cells by downregulating macrophage inhibitory cytokine-1

Background:Overexpression of macrophage inhibitory cytokine-1 (MIC-1) frequently occurs during the progression of prostate cancer (PC) to androgen-independent (AI) and metastatic disease states and is associated with a poor outcome of patients.Methods:The gain- and loss-of-function analyses of MIC-1 were performed to establish its implications for aggressive and chemoresistant phenotypes of metastatic and AI PC cells and the benefit of its downregulation for reversing docetaxel resistance.Results:The results have indicated that an enhanced level of secreted MIC-1 protein in PC3 cells is associated with their acquisition of epithelial–mesenchymal transition features and higher invasive capacity and docetaxel resistance. Importantly, the downregulation of MIC-1 in LNCaP-LN3 and PC3M-LN4 cells significantly decreased their invasive capacity and promoted the antiproliferative, anti-invasive and mitochrondrial- and caspase-dependent apoptotic effects induced by docetaxel. The downregulation of MIC-1 in PC3M-LN4 cells was also effective in promoting the cytotoxic effects induced by docetaxel on the side population (SP) endowed with stem cell-like properties and the non-SP cell fraction from PC3M-LN4 cells.Conclusion:These data suggest that the downregulation of MIC-1 may constitute a potential therapeutic strategy for improving the efficacy of current docetaxel-based chemotherapies, eradicating the total mass of PC cells and thereby preventing disease relapse and the death of PC patients.

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

BackgroundThe molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.MethodsPC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.ResultsKU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.ConclusionsOverall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

BackgroundStable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes.ResultsWe utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits against experimental data. The reproducibility and robustness of the deconvolution were verified by replicate experiments, extensive statistical analyses, and cross-validation against NMR data.ConclusionsThis computational approach revealed the relative fluxes through the different biosynthetic pathways of UDP-GlcNAc, which comprises simultaneous sequential and parallel reactions, providing new insight into the regulation of UDP-GlcNAc levels and O-linked protein glycosylation. This is the first such analysis of UDP-GlcNAc dynamics, and the approach is generally applicable to other complex metabolites comprising distinct metabolic subunits, where sufficient numbers of isotopologues can be unambiguously resolved and accurately measured.

696 CHARACTERIZATION OF A C-TERMINAL HEAT SHOCK PROTEIN 90 INHIBITOR IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2010696 CHARACTERIZATION OF A C-TERMINAL HEAT SHOCK PROTEIN 90 INHIBITOR IN PROSTATE CANCER CELLS Heidi A. Penn, Craig A. Manthe, Shawna B. Comer, George A. Vielhauer, Roger J. Rajewski, Brian S.J. Blagg, and Jeffrey M. Holzbeierlein Heidi A. PennHeidi A. Penn Kansas City, KS More articles by this author , Craig A. MantheCraig A. Manthe Kansas City, KS More articles by this author , Shawna B. ComerShawna B. Comer Kansas City, KS More articles by this author , George A. VielhauerGeorge A. Vielhauer Kansas City, KS More articles by this author , Roger J. RajewskiRoger J. Rajewski Lawrence, KS More articles by this author , Brian S.J. BlaggBrian S.J. Blagg Lawrence, KS More articles by this author , and Jeffrey M. HolzbeierleinJeffrey M. Holzbeierlein Kansas City, KS More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1095AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Heat shock protein 90 (Hsp90) assists in folding, activation, and maintenance of proteins, as well as protects from degradation in stressed cells. In cancer, it has been found to be constitutely over-expressed and involved in various signaling pathways to promote tumor survival. Because of this, HSP 90 is a viable drug target. Traditionally, Hsp90 inhibitors have targeted the N-terminal ATP-binding site and 17-AAG has demonstrated activity both in vitro and in vivo and clinical trials are currently ongoing. Unfortunately, significant toxicity and difficult pharmacokinetic properties have limited their use. A second ATP-binding site was identified in the carboxyl terminus of Hsp90 and the antibiotic novobiocin has been demonstrated to weakly bind to and inhibit, however it has very low potency. We evaluate the in vitro efficacy of one of our analogues, KU-174, in hormone dependent (LNCaP-LN3) and independent (PC3-MM2) prostate cancer cell lines and normal renal proximal tubule epithelial cells (RPTEC). METHODS Three cell lines, LNCaP-LN3, PC3-MM2, and RPTEC, were treated with KU-174, 17-AAG, and novobiocin and the effects compared. MTS anti-proliferation assays were used to determine potency. Western blot experiments determined the effect on Hsp90, Hsp70 and client protein expression and trypan blue exclusion assays assessed cell death. Additionally, KU-174 was analyzed in flow cytometry experiments for the induction of apoptosis and arrest of cell cycle. RESULTS KU-174 demonstrated anti-proliferative activity in both cancer cell lines and was more potent at earlier time points compared to novobiocin and 17-AAG. KU-174 induced the degradation of client proteins without inducing a heat-shock response. The percentage of viable cells was decreased significantly in both PC3–MM2 and LNCaP-LN3 lines with no effect on normal cells and KU-174 appears to be cytotoxic. In cell cycle experiments, KU-174 induced significant G2/M and S-phase arrest with the LNCaP-LN3 cell line demonstrating an increasing percentage of cells in the sub-G0/apoptotic fraction. In both cancer cell lines, the induction of apoptosis and cell cycle arrest increased over time in a dose-dependent manner with less of an effect on RPTEC cells. CONCLUSIONS KU-174 is specific for cancer cells and differs from N-terminal inhibitors in that it is cytotoxic versus cystostatic, it does not induce a heat-shock response, and is more effective at earlier times. KU-174 represents a new class of Hsp-90 inhibitors that may prove to be superior to N-terminal inhibitors for the treatment of prostate cancer. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e271-e272 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Heidi A. Penn Kansas City, KS More articles by this author Craig A. Manthe Kansas City, KS More articles by this author Shawna B. Comer Kansas City, KS More articles by this author George A. Vielhauer Kansas City, KS More articles by this author Roger J. Rajewski Lawrence, KS More articles by this author Brian S.J. Blagg Lawrence, KS More articles by this author Jeffrey M. Holzbeierlein Kansas City, KS More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Downmodulation of Bcl‐2 sensitizes metastatic LNCaP‐LN3 cells to undergo apoptosis via the intrinsic pathway

We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3). LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined. LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect. Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.

ITRAQ-FACILITATED PROTEOMIC ANALYSIS OF HUMAN PROSTATE CANCER CELLS IDENTIFIES PROTEINS ASSOCIATED WITH PROGRESSION

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (≥95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (≥1.50 fold), while 4 proteins were significantly down-regulated (≥−1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol- O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann–Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

ITRAQ-Facilitated Proteomic Analysis of Human Prostate Cancer Cells Identifies Proteins Associated with Progression

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer

In order to identify novel candidates associated with prostate cancer metastasis, we compared the proteomic profile of the poorly metastatic human prostate cancer cell line LNCaP, with its highly metastatic variant LNCaP-LN3, by two-dimensional gel electrophoresis. A major protein spot (pI of 5.9 and molecular weight of 37 kDa) was seen in LNCaP cells, but not in LNCaP-LN3 cells and was identified as lactate dehydrogenase-B (LDHB), by tandem mass spectrometry. Furthermore, enzyme kinetic assays and zymography showed a higher LDH enzyme activity in LNCaP cells compared with LNCaP-LN3. Bisulphite-modified DNA sequencing showed promoter hypermethylation in LNCaP-LN3 cells but not in LNCaP, Du145, PC3, CWR22 or BPH45 cells. Treatment of LNCaP-LN3 cells with 5'-azacytidine caused re-expression of LDHB transcripts. In tissues, LDHB promoter hypermethylation occurred at a higher frequency in prostate cancer, 14/ 31 (45%), compared to adjacent nonmalignant or benign tissue, 2/19 (11%) (P < 0.025). Immunohistochemistry showed a higher frequency of LDHB expression in benign or non-malignant tissues, 59/ 73 (81%), compared to cancer cases, 3/53 (6%) (P < 0.001). Absent LDHB expression was also seen in 7/7 (100%) cases of metastatic cancer in bone. Our data are the first to show loss of LDHB expression in prostate cancer, the mechanism of which appears to involve promoter hypermethylation.

Promoter hyper-methylation of calcium binding proteins S100A6 and S100A2 in human prostate cancer

S100A6 and S100A2 are members of the S100 family of calcium binding proteins, which are down regulated in prostate cancer, however the molecular mechanism(s) underlying their loss of expression is unknown. The promoter and exon 1 region of the S100A6 and S100A2 genes was sequenced in bisulfite modified DNA from non-malignant, benign prostatic hyperplasia (BPH), malignant and metastatic prostate tissues and in cell lines. Immunohistochemistry was performed to correlate S100A2 expression with methylation status. S100A6 methylation was absent or occurred at isolated sites in 14/14 cases of non-malignant epithelium and 5/5 cases of BPH tissues, whereas methylation was seen in 14/27 (52%) cases of prostatic cancer (P<0.0001), 2/2 cases of metastatic cancer and in the CWR22 prostatic cancer xenograft. Critical CpG sites within the S100A2 promoter were methylated in LNCaP, LNCaP-LN3, and CWR22 cells but not in Du145, PC3 or BPH45 cells. In tissues, S100A2 methylation was seen in 32/34 (94%) cases of adenocarcinoma and 5/5 cases of metastatic cancer. However, S100A2 methylation was also seen in 9/12 (75%) cases of non-malignant tissues and in 5/5 cases of BPH. Immunostaining, showed absent S100A2 expression all 41 cases of prostatic cancer, whereas staining was seen in the basal cells of non-malignant epithelium. Loss of S100A6 and S100A2 proteins is frequent in human prostatic cancer. A major mechanism underlying the loss of S100A6 expression appears to involve promoter hyper-methylation. However, mechanisms other than methylation of the known promoter are involved in silencing S100A2 in the prostate.

Antigenic expression of human metastatic prostate cancer cell lines for in vitro multiple-targeted α-therapy with 213Bi-conjugates

Purpose Control of metastatic prostate cancer (CaP) is an elusive objective. Some 30% of patients with clinically localized CaP will develop micrometastatic disease. Defining the expression of tumor-associated antigens on CaP will enable appropriate selection of therapeutic targets. Methods and materials The expression of tumor-associated antigens on CaP cell lines (PC-3, DU 145, and LNCaP-LN3) was detected by immunohistochemistry and flow cytometry. Test and control α-conjugates were prepared using monoclonal antibodies, an inhibitor, plasminogen activator inhibitor type 2, that binds to the cell-membrane-bound protease, urokinase plasminogen activator, and a control protein labeled with 213Bi using standard methods. These were used singly or together against three different CaP cell lines in vitro. The cytotoxicity of the α-conjugates was assessed using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay. Results The PC-3 and DU 145 cancer cell lines expressed antigens that bind monoclonal antibodies BLCA-38 and #394 (mouse anti-human urokinase plasminogen activator B-chain) but not J591. The LNCaP-LN3 cells bound J591 but not #394 or BLCA-38. For the PC-3, DU 145, and LNCaP-LN3 cell lines, multiple-targeted α-therapy combining four α-conjugates (one-quarter doses of each) gave D 0 (37% cell survival) values of 15, 17, and 27 μCi/mL compared with those of the controls of 272, 289, and 281 μCi/mL, respectively. Conclusion Metastatic prostate cancer-associated antigens recognized by multiple monoclonal antibodies are potential targets for α-therapy. Multiple-targeted α-therapy produced cytotoxicity specific to three CaP cell lines and may form the basis of treatment for micrometastatic CaP, overcoming the heterogeneity of expression of the targeted antigens.