Prostate Carcinoma Cell Line DU145 Research Articles

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Improved Anticancer Activities of a New Pentafluorothio-Substituted Vorinostat-Type Histone Deacetylase Inhibitor.

The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising epigenetic anticancer drugs and new HDAC inhibitors are sought for in order to obtain potent drug candidates. The new HDAC inhibitor SF5-SAHA was synthesized and analyzed for its anticancer properties. The new compound SF5-SAHA showed strong inhibition of tumor cell growth with IC50 values similar to or lower than that of the clinically applied reference compound vorinostat/SAHA (suberoylanilide hydroxamic acid). Target specific HDAC inhibition was demonstrated by Western blot analyses. Unspecific cytotoxic effects were not observed in LDH-release measurements. Pro-apoptotic formation of reactive oxygen species (ROS) and caspase-3 activity induction in prostate carcinoma and hepatocellular carcinoma cell lines DU145 and Hep-G2 seem to be further aspects of the mode of action. Antiangiogenic activity of SF5-SAHA was observed on chorioallantoic membranes of fertilized chicken eggs (CAM assay). The presence of the pentafluorothio-substituent of SF5-SAHA increased the antiproliferative effects in both solid tumor and leukemia/lymphoma cell models when compared with its parent compound vorinostat. Based on this preliminary study, SF5-SAHA has the prerequisites to be further developed as a new HDAC inhibitory anticancer drug candidate.

Functional and mutational analysis after radiation and cetuximab treatment on prostate carcinoma cell line DU145

BackgroundEpidermal Growth Factor Receptor is often overexpressed in advanced prostate carcinoma. In-vitro-studies in prostate carcinoma cell line DU145 have demonstrated increased sensibility to radiation after cetuximab treatment, but clinical data are not sufficient to date.MethodsWe analyzed effects of radiation and cetuximab in DU145 and A431 using proliferation, colony-forming-unit- and annexin-V-apoptosis-assays. Changes in protein expression of pEGFR and pERK1/2 after radiation and cetuximab treatment were analyzed. Using NGS we also investigated the impact of cetuximab long-term treatment.ResultsCell counts in DU145 were reduced by 44% after 4 Gy (p = 0.006) and 55% after 4 Gy and cetuximab (p < 0.001). The surviving fraction (SF) was 0.69 after 2 Gy, 0.41 after 4 Gy and 0.15 after 6 Gy (each p < 0.001). Cetuximab treatment did not alter significantly growth reduction in 4 Gy radiated DU145 cells, p > 0.05 or SF, p > 0.05, but minor effects on apoptotic cell fraction in DU145 were detected. Using western blot, there were no detectable pEGFR and pERK1/2 protein signals after cetuximab treatment. No RAS mutation or HER2 amplification was detected, however a TP53 gen-mutation c.820G > T was found.ConclusionsRadiation inhibits cell-proliferation and colony-growth and induces apoptosis in DU145. Despite blocking MAP-Kinase-pathway using cetuximab, no significant radiation-sensitizing-effect was detected. Cetuximab treatment did not induce resistance-mutations. Further research must clarify which combination of anti-EGFR treatment strategies can increase radiation-sensitizing-effects.

Radio-thermo-sensitivity Induced by Gold Magnetic Nanoparticles in the Monolayer Culture of Human Prostate Carcinoma Cell Line DU145.

Prostate cancer is the second cause of death in men worldwide. In this study, the cytotoxic effects of PLGA polymer-coated gold Magnetic Nanoparticles (MGNPs), as a novel treatment to enhance radiation and thermal sensitivity in the presence of hyperthermia (43°C) and electron beam, on DU145 prostate cancer cells were investigated. Nanoparticles were characterized using TEM, DLS, XRD and SAED methods. MGNPs entrance into the cells was determined using Prussian blue staining and TEM. Furthermore, the cytotoxic effects of combinatorial treatment modalities were assessed by applying colony and sphere formation assay. Our results revealed that the decrease of colony and sphere numbers after combinatorial treatment of hyperthermia and radiation in the presence of nanoparticles was significantly higher than the other treatment groups (P<0.05). This treatment method proved that it has the capability of eliminating most of the DU145 cells (80-100%), and increased the value of the linear parameter (α) to 4.86 times. According to the study, magnetic gold nanoparticles, in addition to having a high atomic number, can effectively transmit heat produced inside them to the adjacent regions under hyperthermia, which increases the effects of radio-thermosensitivity, respectively.

Sclerin, a New Cytotoxic Cyclononapeptide from Annona scleroderma.

A new cytotoxic cyclononapeptide, sclerin, cyclo(–Dab1–Ser2–Tyr3–Gly4–Thr5–Val6–Ala7– Ile8–Pro9–) (1), was isolated from the methanol extract of the seeds of Annona scleroderma, together with the known metabolite, cyclosenegalin A, cyclo(–Pro1–Gly2–Leu3–Ser4–Ala5–Val6–Thr7–) (2). The planar structures for the two compounds were established by comprehensive analysis of NMR and ESI-HRMS data, and the absolute stereochemistry was stablished by Marfey’s method. Compound 1 showed moderate cytotoxic activity against the human prostate carcinoma cell line DU-145 at µM concentration.

Abstract 3923: Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model

Abstract C-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair. In tumors, including breast, prostate, glioblastoma, and thyroid cancer, Met protein levels are often elevated and associated with disease progression and metastasis. The c-Met pathway is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). HGF is produced by mesenchymal cells and binds to the c-Met protein, leading to phosphorylation and upregulation of a variety of downstream signaling pathways that result in increased tumor cell migration, proliferation, survival, and induction of angiogenesis. C-Met is associated with disease progression and metastasis. Consequently, there has been significant interest in the development of c-Met inhibitors as anti-cancer therapeutics. The efficacy of such agents usually initially are determined in vitro utilizing HGF concentrations of 25-50 ng/mL. However, HGF serum levels in humans typically range from 0.4-0.8 ng/mL. In our studies, we examined the prostate carcinoma cell line DU145, and BMS-777607. Migration and phosphorylation of c-Met increased in a dose-dependent manner with HGF treatment. Additionally, c-Met inhibition with BMS-777607 reduced migration and invasion when in the presence of 25-50 ng/mL HGF. However, at physiologically relevant HGF concentrations the drug had no effect on migration or invasion of this cell line. In vivo models of c-Met inhibition have been limited mainly to mice genetically engineered to express HGF, on the basis that mouse HGF does not stimulate human c-Met. However, in our studies, mouse HGF did stimulate c-Met in the DU145 tumor cell line. Subsequent in vivo evaluation revealed treatment with BMS-777607 had no reduction on DU145 tumor cell-induced angiogenesis in cells pretreated with the drug, nor in mice receiving oral administration of the drug. An experimental metastasis model also resulted in no significant reduction in metastasis formation, as assessed by a tail vein injection model. To characterize HGF concentration in vivo, an ELISA was used to determine an average concentration of 7.7 ng/g of DU145 tumor tissue. Further evaluations of phospho-Met are underway utilizing immunofluorescence of FFPE samples. Preliminary results indicate low levels of phospho-Met in DU145 tumor sections that can be induced by treating tumor-bearing mice with exogenous HGF. Our results suggest that in vitro evaluation of c-Met inhibitors utilizing non-physiological concentrations of HGF may produce an abnormally high phospho-Met that can subsequently be reduced by treatment with c-Met inhibition. In vitro studies performed at high HGF concentrations may not accurately predict the in vivo drug efficacy. Further characterization of HGF concentrations within the tumor is necessary to properly conduct in vitro evaluation of c-Met inhibitors. Citation Format: Veronica S. Hughes, Mumtaz Rojiani, Dietmar Siemann. Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3923.

Involvement of apoptotic pathways in docosahexaenoic acid-induced benefit in prostate cancer: Pathway-focused gene expression analysis using RT2 Profile PCR Array System

BackgroundPresent study aimed to better understand the potential apoptotic pathways that involved in docosahexaenoic acid (DHA)-induced apoptosis of prostate cancer cells.MethodsHuman prostate cancer DU145 cells were treated with different concentrations of fish oil, omega-3 PUFA (DHA, and Eicosapentaenoic acid, EPA), or omega-6 PUFA (Arachidonic acid, AA). Cell viability and apoptosis were evaluated by MTT assay and Hoechst staining. Pathway-focused gene expression profiling of DU145 cells was analyzed with the RT2 Profile PCR Array System. The results were verified by real time quantitative polymerase chain reaction (RT-qPCR).ResultsAA exposure showed no obvious effect on viability of DU145 cells. However, exposure with fish oil, EPA, or DHA for 24 h significantly affected cell viability. The growth inhibition of DHA was more pronounced than that of EPA and showed a time-dependent increase. DHA exposure caused typical apoptotic characteristics. Ten genes were more expressed, while 5 genes were less expressed following DHA exposure. RT-qPCR confirmed the time dependent effect of DHA on the expression of these differentially expressed genes. KEGG pathway analysis showed that DHA may induce the apoptosis of cancer cells preferentially through mediating P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways.ConclusionOur study demonstrated the beneficial action of DHA on human prostate carcinoma cell line DU145. The pro-apoptotic effect of DHA on DU145 cells may involve mediation various pathways, especially P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Molecular mechanisms of DHA on apoptosis of cancer cells still need to be further clarified.

Induction of Oxidants Distinguishes Susceptibility of Prostate Carcinoma Cell Lines to p53 Gene Transfer Mediated by an Improved Adenoviral Vector.

Previously, the authors developed an adenoviral vector, Ad-PG, where transgene expression is regulated by a p53-responsive promoter. When used to transfer the p53 cDNA, a positive feedback mechanism is established. In the present study, a critical comparison is performed between Ad-PGp53 and AdRGD-PGp53, where the RGD motif was incorporated in the adenoviral fiber protein. AdRGD-PGp53 provided superior transgene expression levels and resulted in the killing of prostate carcinoma cell lines DU145 and PC3. In vitro, this effect was associated with increased production of cytoplasmic and mitochondrial oxidants, DNA damage as revealed by detection of phosphorylated H2AX, as well as cell death consistent with apoptosis. Differential gene expression of key mediators of reactive oxygen species pathways was also observed. Specifically, it was noted that induction of known p53-target genes Sestrin2 and PIG3, as well as a novel target, NOX1, occurred in PC3 cells only when transduced with the improved vector, AdRGD-PGp53. The participation of NOX1 was confirmed upon its inhibition using a specific peptide, resulting in reduced cell death. In situ gene therapy also resulted in significantly improved inhibition of tumor progression consistent with oxidant-induced DNA damage only when treated with the novel AdRGD-PGp53 vector. The study shows that the improved adenovirus overcomes limitations associated with other p53-expressing vectors and induces oxidant-mediating killing, thus supporting its further development for cancer gene therapy.

Abstract 3304: Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer

Abstract Radiotherapy is an important treatment regimen for prostate cancer. We have shown that the lymphangiogenic growth factor, Vascular Endothelial Growth Factor C (VEGF-C), induces docetaxel resistance and autophagy in prostate cancer (Muders et al., Cancer Res 2009 and Stanton et al., Cancer Res 2013). In this study we have evaluated the role of VEGF-C and VEGF-C induced autophagy in radioresistance of prostate cancer using cell culture experiments and human tissue samples. The human prostate carcinoma cell lines PC-3, LNCaP and DU145 were used in these cell culture experiments. Clonogenic survival of prostate cancer cells, after RNA interference (RNAi) of VEGF-C, autophagy related gene 5 (ATG5), and after addition of recombinant human VEGF-C following irradiation therapy, was evaluated. Autophagic flux was tested after ionizing irradiation using a Light-Chain-3 II immunoblot. To confirm our in vitro findings, human tissues of patients who underwent radical prostatectomy and adjuvant radiotherapy at Mayo Clinic were evaluated for VEGF-C expression. The quantity and intensity of VEGF-C staining was evaluated by two pathologists independently and correlated with biochemical relapse free survival (BRFS). The follow-up time is up to 23 years after radiotherapy. Multivariate Cox analysis was performed. VEGF-C levels correlated with a significantly higher radioresistance in colony formation assays and human tissue samples by multivariate analysis. The risk of biochemical recurrence increased 2.8 fold (95% confidence interval: 1.109 to 7.237; p = 0.03) when VEGF-C was highly expressed in prostatectomy patients with adjuvant radiotherapy. In line with our studies on autophagy in VEGF-C mediated docetaxel resistance of prostate cancer, autophagic flux was reduced after VEGF-C depletion during radiation. Interestingly, RNAi for ATG5 showed no effect on radiosensitivity which suggests that the decrease in autophagic flux after VEGF-C depletion is not responsible for the increased radiosensitivity. We conclude that VEGF-C is an important mediator of radiotherapy resistance in prostate cancer. In contrast to our published results on chemotherapy resistance, VEGF-C induced autophagy appears to be less important for resistance against ionizing radiation. Other VEGF-C dependent mechanisms for therapy resistance are under investigation. Citation Format: Steffi Haberlau, Pia Hönscheid, Rafael E. Jimenez, Gustavo B. Baretton, Donald J. Tindall, Mechthild Krause, Kaustubh Datta, Michael H. Muders. Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2015-3304

Design and synthesis of pH-sensitive polyamino-ester magneto-dendrimers: Surface functional groups effect on viability of human prostate carcinoma cell lines DU145

Novel pH-sensitive, biocompatible and biodegradable magneto-dendrimers with OH and/or NH2 functional groups based on poly amino-ester were synthesized for delivery of anti-cancer drugs. Magnetite nanoparticles (MNPs) were synthesized by the co-precipitation method and their surfaces were modified by 3-aminopropyl triethoxysilane. The first and second generations of the magneto-dendrimer with hydroxyl end groups were produced by sequential acrylation and Michael addition reactions using the required amounts of acryloyl chloride and diethanolamine, respectively. The dendrimer containing amino functional surface groups up to second generation was synthesized by the same method using the necessary amounts of acryloyl chloride and ethylenediamine. These dendrimers were fully characterized by the Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), dynamic light scattering (DLS) and zeta potential analysis, vibrating-sample magnetometer (VSM), scanning electron microscope (SEM), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). In-vitro release profiles of the drug-loaded magnetic nanoparticles and their cytotoxicity assay were investigated at two pHs (7.4 and 5.8). The hydrolytic degradation behavior of magneto-dendrimers was evaluated in PBS buffer. Our research suggests that magneto-dendrimers having amine or hydroxyl functional groups could be considered as the suitable nanocarriers for therapy applications.

Evidence base of Mistletoe drug -interaction in Supportive- care.

e20746 Background: Mistletoe is often used as complimentary therapy in Europe . There is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Like other Herbal extracts etc. , Viscum Album extract [VAE] is used as supportive care during chemotherapy. The anti-tumor effects of VAE are well documented . However the clinical activity of VAE treatment still remains contraversal. The effect of VAE was investigated with common chemo-drugs usings different cell lines in vitro . Methods: Human leukemia cell lines K562 and KG1a were treated with Vincistrin, Mafosfamide, Idarubicine and Cisplatin. Breast cell carcinoma cell lines HCC1937 and HCC1143were treated with Doxorubicin HCl , prostate carcinoma cell line DU145, with Docetaxel and Mitoxantrone HCl and lung carcinoma cell line NCL-H460 was treated with Docetaxal and Cispaltin . Each dose of chemotherapeutic drug was combined with VAE in clinically relavent concentrations and proliferaton and apoptosis were measured. Results: VAE inhibited colony formation and did not exert any protective activity against induced inhibition of colongenicity .VAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of experimental settings . Conclusions: The study provides evidence that VAE is capable of enhancing cytotxicity of antcancer agents in vitro. Hence no risk of safety by herb drug- interactions is to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE at the same time. The study shows moderate to high evidence and further studies are warrented for clinical trial with optimized study design and treatment conditions.

Nedd4 E3 ubiquitin ligase promotes cell proliferation and autophagy.

Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) is a member of the HECT E3 ubiquitin ligases, and is elevated in prostate, bladder and colorectal cancers, and promotes colonic cell population expansion. Up to now, molecular mechanisms of how Nedd4 functions, have not been well understood. In this study, shRNA was used to reduce expression of Nedd4 in the human prostate carcinoma cell line DU145. To analyse effects of Nedd4 on cell proliferation, MTT and colony formation assays were performed. DAPI staining and FACS analysis were used to investigate outcomes of Nedd4 activity, on apoptosis. Results of Nedd4 expression on lysosomal membrane permeabilization and autophagy were further investigated using acridine orange (AO) staining, immunofluorescence and western blot analysis. We found that in HeLa cells, expression of Nedd4 promoted cell proliferation, whereas its knockdown inhibited colony formation and induced apoptosis in DU145 cells. Furthermore, down-regulation of Nedd4 in DU145 cells promoted lysosomal membrane permeabilization. We also found that down-regulation of Nedd4 inhibited autophagy in both DU145 and A549 cells. Investigation into mechanisms involved revealed that knockdown of endogenous Nedd4 expression notably increased activated mTOR (p-mTOR) levels, which suggests that mTOR signalling was involved in the Nedd4-mediated autophagy. Our results indicate that expression of Nedd4 promoted cell proliferation and colony formation but prevented apoptosis. Moreover, Nedd4 promoted autophagy and was associated with the mTOR signalling pathway.

Anti-cancer activity of glucosamine through inhibition of N-linked glycosylation.

BackgroundWe have reported that the glucosamine suppressed the proliferation of the human prostate carcinoma cell line DU145 through inhibition of STAT3 signaling. DU145 cells autonomously express IL-6 and the IL-6/STAT3 signaling is activated. IL-6 receptor subunits are subject to N-glycosylation, a posttranslational modification which is important for protein stability and function. We speculated that the inhibition of STAT3 phosphorylation by glucosamine might be a functional consequence of the reduced N-glycosylation of gp130.MethodsThe human prostate cancer cell lines DU145 and PC-3 and human melanoma cell line A2058 were used in this study. Glucosamine effects on N-glycosylation of glycoproteins were determined by Western blot analysis. IL-6 binding to DU145 cells was analyzed by flow cytometry. The cell proliferation suppression was investigated by colorimetric Janus green staining method.ResultsIn DU145 cells glucosamine reduced the N-glycosylation of gp130, decreased IL-6 binding to cells and impaired the phosphorylation of JAK2, SHP2 and STAT3. Glucosamine acts in a very similar manner to tunicamycin, an inhibitor of protein N-glycosylation. Glucosamine-mediated inhibition of N-glycosylation was neither protein- nor cell-specific. Sensitivity of DU145, A2058 and PC-3 cells to glucosamine-induced inhibition of N-glycosylation were well correlated to glucosamine cytotoxicity in these cells.ConclusionOur results suggested that the glucosamine-induced global inhibition of protein N-glycosylation might be the basic mechanism underlying its multiple biochemical and cellular effects.

The role of iron oxide nanoparticles in the radiosensitization of human prostate carcinoma cell line DU145 at megavoltage radiation energies

Purpose: To examine the radiosensitizing effects of iron oxide nanoparticles in the presence of 6 MV (megavoltage) X-ray radiation.Materials and methods: Iron oxide nanoparticles with two different modifications – dextran coating (plain) and amino-group dextran coating – were used. The rate of iron oxide penetration was monitored using Prussian blue staining, magnetic resonance imaging and atomic adsorption spectroscopy. The effect of iron oxide on the viability of cells was determined using trypan blue dye exclusion assay followed by evaluating the cytotoxicity effect of amino-group iron oxide nanoparticles and ionizing radiation. Radiation dose enhancement studies were carried out on DU145 human prostate carcinoma cell line with 1 mg/ml amino-group iron oxide nanoparticles and different doses of 6 MV X-ray radiation.Results: The uptake of amino-group coated nanoparticles by DU145 cells was significantly more than the plain nanoparticles. In addition, cell viability was decreased with the increase of iron oxide concentration. The dose enhancement factor (DEF) is approximately 1.2 at different doses in the range of 2–8 Gy of 6 MV X-ray radiation.Conclusions: It was demonstrated that iron oxide nanoparticles with the appropriate surface modifications can enter the DU145 cells and it can be used as a cell sensitizer to megavoltage ionizing radiations in radiation therapy.

Interaction of standardized mistletoe (Viscum album) extracts with chemotherapeutic drugs regarding cytostatic and cytotoxic effects in vitro.

BackgroundGiven the importance of complementary and alternative medicine (CAM) to cancer patients, there is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Mistletoe (Viscum album L.) belongs to the medicinal herbs that are used as supportive care during chemotherapy. In the in vitro study presented here the effect of standardized mistletoe preparations on the cytostatic and cytotoxic activity of several common conventional chemotherapeutic drugs was investigated using different cancer cell lines.MethodsHuman breast carcinoma cell lines HCC1937 and HCC1143 were treated with doxorubicin hydrochloride, pancreas adenocarcinoma cell line PA-TU-8902 with gemcitabine hydrochloride, prostate carcinoma cell line DU145 with docetaxel and mitoxantrone hydrochloride and lung carcinoma cell line NCI-H460 was treated with docetaxel and cisplatin. Each dose of the respective chemotherapeutic drug was combined with Viscum album extract (VAE) in clinically relevant concentrations and proliferation and apoptosis were measured.ResultsVAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of our experimental settings. At higher concentrations VAE showed an additive inhibitory effect.ConclusionsOur in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE simultaneously.

The over-expression of Pim-2 promote the tumorigenesis of prostatic carcinoma through phosphorylating eIF4B

Cell experiments have found Pim-2 may take part in the tumorigenesis of prostatic carcinoma (PCA). More direct evidences are needed, and the detailed anti-apoptotic mechanism of Pim-2 in PCA cells is still unknown. Pim-2 expression levels were compared between benign prostatic hyperplasia (BPH) tissues and PCA tissues using real time PCR and Western blot, respectively. Then Pim-2 expression levels were detected in PCA cell lines DU-145 and LNCaP, as well as in nontumorous prostatic epithelial cell lines RWPE-1 and PNT1a, using real time PCR and Western blot, respectively. The co-expression of Pim-2 and eukaryotic initiation factor 4B (eIF4B) was examined by immunofluorescence cytochemistry using laser scanning confocal microscope. Finally, Pim-2 SiRNA was transfected into DU-145 cells and Pim-2 was transfected into RWPE-1 cells, and the level of Pim-2 and phosphorylated eukaryotic initiation factor 4B (p-eIF4B) were detected, as well as the apoptosis rate. The Pim-2 mRNA and protein level were significantly higher in PCA tissues than those in BPH tissues. The Pim-2 mRNA and protein level in DU-145 and LNCaP cells were significantly higher than those in RWPE-1 and PNT1a cells. Pim-2 and eIF4B could co-express in DU-145 cells. Pim-2 level determined the phosphorylation level of eIF4B and the apoptosis rate of prostatic cells. The higher Pim-2 expressed, the more eIF4B phosphorylated, then the less cell got apoptosis, and vice versa. Pim-2 was over-expressed in PCA cell lines and tissues. It may inhibit the apoptosis of PCA cells through phosphorylating eIF4B, thus promote the tumorigenesis of PCA.

Information Theoretic Entropy for Molecular Classification: Oxadiazolamines as Potential Therapeutic Agents

In this review we present algorithms for classification and taxonomy based on information entropy, followed by structure-activity relationship (SAR) models for the inhibition of human prostate carcinoma cell line DU-145 by 26 derivatives of N-aryl-N-(3-aryl-1,2,4-oxadiazol-5-yl)amines (NNAs). The NNAs are classified using two characteristic chemical properties based on different regions of the molecules. A table of periodic properties of inhibitors of DU-145 human prostate carcinoma cell line is obtained based on structural features from the amine moiety and from the oxadiazole ring. Inhibitors in the same group and period of the periodic table are predicted to have highly similar properties, and those located only in the same group will present moderate similarity. The results of a virtual screening campaign are presented.

Abstract 5231: Zoledronic acid encapsulated into liposomes and nanoparticles: Enhanced antitumor activity in 3D-prostate carcinoma spheroids

Abstract Introduction: Zoledronic acid is currently used for the treatment of bone metastases in hormone-resistant prostate cancer patients. The rapid plasma half-life and the high affinity for Osteoclasts, the primary targets of bisphosphonates, limit the use for extra-skeletal tissues. Moreover, it remains still unclear, whether cancer-associated fibroblasts (CAF) influence the toxicity of ZOL. The use of nanomedicine is one promising strategy to improve the availability of drugs within the tumor tissue. To investigate the benefit of such approach, we used two different nanovectors encapsulating ZOL on 3D-spheroids from prostate carcinoma cells and CAF in combination with standard toxicity assays. Material and Methods: Stealth liposomes (PGNP_ZOL) and a new generation of nanovector, named self-assembly nanoparticles (NP_ZOL), were prepared using standard protocols already described. The prostate carcinoma cell lines PC-3, DU-145 and LNCaP were cultivated in 96-well MTP's and as Spheroids on agarose coated 96-well MTP's. Heterologous spheroids were prepared by mixing tumor cells with different ratios of normal (NAF) or tumor-associated (CAF) fibroblasts. Cell cultures/spheroids were incubated with ZOL, NP_ZOL/PGNP_ZOL for up to 120 h. Changes in growth behavior and toxicity (apoptosis/necrosis) were quantified from spheroid-cultures by modifications of standard assays (CDD+, LDH+). The adaptability of these techniques for 3D- cultures was assessed using thapsygargin/tunicamycin as apoptosis-inducing agents. Results: Standard-viability assays revealed a significant enhancement of antitumor activity of NP_ZOL/PGNP_ZOL as compared to the free drug in cell cultures of DU-145 and LNCaP, whereas these effects were less pronounced in PC-3 cells. The antitumor efficacy of NP_ZOL/PGNP_ZOL was caused by a strong inhibition of tumor cell proliferation as revealed by the BrDU-assay; on the other hand, both CDD+ and LDH+ failed to detect both apoptosis and necrosis after drug treatment for up to 120 h. The average IC50 values in the tumor cells ranges from 10-20µM/L, but the fibroblasts exhibit complete growth inhibition at concentrations between 2-5µm/L ZOL. Conclusion: These results confirm the hypothesis that the use of nanomedicine enables the extension in therapeutic applications of drugs such as bisphosphonates. In this context, ZOL seems to exert benefits even onto the primary tumor cells. Homo-/heterologeous 3D-spheroids offer advanced models mimicking the microenvironment within a tumor tissue grows thus bridging the gap between cell culture and live tissue. Moreover, the rapid generation of single-tumor spheroids allows for a high-throughput cell function and toxicity analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5231. doi:1538-7445.AM2012-5231

Cell-to-Cell Signaling Influences the Fate of Prostate Cancer Stem Cells and Their Potential to Generate More Aggressive Tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44+CD24− phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.

PTEN Regulates PDGF Ligand Switch for β-PDGFR Signaling in Prostate Cancer

Platelet-derived growth factor (PDGF) family members are potent growth factors that regulate cell proliferation, migration, and transformation. Clinical studies have shown that both PDGF receptor β (β-PDGFR) and its ligand PDGF D are up-regulated in primary prostate cancers and bone metastases, whereas PDGF B, a classic ligand for β-PDGFR, is not frequently detected in clinical samples. In this study, we examined the role of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of PDGF expression levels using both a prostate-specific, conditional PTEN-knockout mouse model and mouse prostate epithelial cell lines established from these mice. We found an increase in PDGF D and β-PDGFR expression levels in PTEN-null tumor cells, accompanied by a decrease in PDGF B expression. Among Akt isoforms, increased Akt3 expression was most prominent in mouse PTEN-null cells, and phosphatidylinositol 3-kinase/Akt activity was essential for the maintenance of increased PDGF D and β-PDGFR expression. In vitro deletion of PTEN resulted in a PDGF ligand switch from PDGF B to PDGF D in normal mouse prostate epithelial cells, further demonstrating that PTEN regulates this ligand switch. Similar associations between PTEN status and PDGF isoforms were noted in human prostate cancer cell lines. Taken together, these results suggest a mechanism by which loss of PTEN may promote prostate cancer progression via PDGF D/β-PDGFR signal transduction.