Human Prostate Tumor Cell Lines Research Articles

Sensitivity of magnetic resonance elastography to extracellular matrix and cell motility in human prostate cancer cell line-derived xenograft models

Prostate cancer (PCa) is a significant health problem in the male population of the Western world. Magnetic resonance elastography (MRE), an emerging medical imaging technique sensitive to mechanical properties of biological tissues, detects PCa based on abnormally high stiffness and viscosity values. Yet, the origin of these changes in tissue properties and how they correlate with histopathological markers and tumor aggressiveness are largely unknown, hindering the use of tumor biomechanical properties for establishing a noninvasive PCa staging system.To infer the contributions of extracellular matrix (ECM) components and cell motility, we investigated fresh tissue specimens from two PCa xenograft mouse models, PC3 and LNCaP, using magnetic resonance elastography (MRE), diffusion-weighted imaging (DWI), quantitative histology, and nuclear shape analysis. Increased tumor stiffness and impaired water diffusion were observed to be associated with collagen and elastin accumulation and decreased cell motility. Overall, LNCaP, while more representative of clinical PCa than PC3, accumulated fewer ECM components, induced less restriction of water diffusion, and exhibited increased cell motility, resulting in overall softer and less viscous properties. Taken together, our results suggest that prostate tumor stiffness increases with ECM accumulation and cell adhesion - characteristics that influence critical biological processes of cancer development.MRE paired with DWI provides a powerful set of imaging markers that can potentially predict prostate tumor development from benign masses to aggressive malignancies in patients. Statement of significanceXenograft models of human prostate tumor cell lines, allowing correlation of microstructure-sensitive biophysical imaging parameters with quantitative histological methods, can be investigated to identify hallmarks of cancer.

Discovery of a Dual Tubulin and Neuropilin-1 (NRP1) Inhibitor with Potent In Vivo Anti-Tumor Activity via Pharmacophore-based Docking Screening, Structure Optimization, and Biological Evaluation.

Dual inhibition of tubulin and neuropilin-1 (NRP1) may become an effective method for cancer treatment by simultaneously killing tumor cells and inhibiting tumor angiogenesis. Herein, we identified dual tubulin/NRP1-targeting inhibitor TN-2, which exhibited good inhibitory activity against both tubulin polymerization (IC50 = 0.71 ± 0.03 μM) and NRP1 (IC50 = 0.85 ± 0.04 μM). Importantly, it significantly inhibited the viability of several human prostate tumor cell lines. Further mechanism studies indicated that TN-2 could inhibit tubulin polymerization and cause G2/M arrest, thereby inducing cell apoptosis. It could also suppress cell tube formation, migration, and invasion. Moreover, TN-2 showed obvious antitumor effects on the PC-3 cell-derived xenograft model with negligible side effects and good pharmacokinetic profiles. These data demonstrate that TN-2 could be a promising dual-target chemotherapeutic agent for the treatment of prostate cancer.

Protein Kinase C Epsilon Overexpression in Prostate Adenocarcinoma is Associated with Oncogenesis

Background: PKCε, an isozyme of serine-threonine kinase, has been implicated in epithelial cancer metastasis and progression. This study investigates the impact of the oncogenic PKCε, overexpressed abnormally in human Prostate tumor samples and cell lines, to understand its efficacy. Methods: The microarray dataset, GSE86257, was processed for normalization. The identification of upregulated and downregulated genes was based on FDR >1 and p <0.05 values. Cytoscape analysis and functional enrichment of significant genes were done. The identified genes were validated on the TCGA dataset and survival analysis was performed by Kaplan-Meier analysis. Results: A total of 1524 DEGs were identified with 728 upregulated genes and 818 downregulated genes. The two significant modules with MCODE score:9.0 and Venn analysis provided cyclin-dependent kinase inhibitor protein (CDK1), Cyclin B1 (CCNB1), Phospholipase C Gamma 1 (PLCG1), Cyclin Dependent Kinase 9 (CDK9), Phosphoinositide-3-Kinase Regulatory Subunit 3 (PIK3R3), H4 Clustered Histone 6 (H4C6), Phospholipase C Gamma 2 (PLCG2) as most interacting genes. TCGA data analysis and Prognostic analysis revealed CCNBI, CDK9, and PLCG1 associated with poor prognosis. Conclusion: PKCε regulates genes that are responsible for cancer progression. Therefore, targeting PKCε in Prostate cancer may serve as an important regulatory effect and may improve the prognosis of the disease.

Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18

NK cell-based therapies have shown promise for hematological cancer forms, but their use against solid tumors is hampered by their poor ability to infiltrate the tumor. NK cells release extracellular vesicles (EVs) containing cytolytic proteins, indicating that NK-cell derived EVs may have therapeutic potential. In this study, we compared the tumor-targeting potential of EVs derived from either primary NK cells or the NK cell lines NK-92 and KHYG-1 cultured in IL-15 alone or in combination with IL-12 and IL-18. Primary NK cells were also stimulated through the activating receptor CD16. Tumor cell apoptosis was measured using a panel of human colon, melanoma, glioblastoma, prostate, breast, and ovarian tumor cell line spheroids. NK cells or NK-92 cells stimulated with IL-12, IL-15, and IL-18 generated EVs with higher efficiency than EVs from resting cells, although similar amounts of EVs were produced under both conditions. Proteomic analysis indicated similar distribution of cytolytic proteins in EVs from primary NK cells and NK-92, but lower levels in KHYG-1 EVs that translated into poor capacity for KHYG-1 EVs at targeting tumor cell lines. Further, we show that CD16-stimulated NK cells release low amounts of EVs devoid of cytolytic proteins. Importantly, EVs from cytokine-stimulated NK cells penetrate into the spheroid core, and tumor spheroid susceptibility to NK-cell derived EVs was linked to differential expression of the NKG2D ligands MICA/B, which was blocked with an anti-NKG2D antibody. We conclude that EVs from activated primary NK cells or NK-92 cells has the best potential to infiltrate and target solid tumors.

Novel σ1 antagonists designed for tumor therapy: Structure - activity relationships of aminoethyl substituted cyclohexanes.

Depending on the substitution pattern and stereochemistry, 1,3-dioxanes 1 with an aminoethyl moiety in 4-position represent potent σ1 receptor antagonists. In order to increase the stability, a cyclohexane ring first replaced the acetalic 1, 3-dioxane ring of 1. A large set of aminoethyl substituted cyclohexane derivatives was prepared in a six-step synthesis. All enantiomers and diastereomers were separated by chiral HPLC at the stage of the primary alcohol 7, and their absolute configuration was determined by CD spectroscopy. Neither the relative nor the absolute configuration had a large impact on the σ1 affinity. The highest σ1 affinity was found for cis-configured benzylamines (1R,3S)-11 (Ki=0.61nM) and (1S,3R)-11 (Ki=1.3nM). Molecular dynamics simulations showed that binding of (1R,3S)-11at the σ1 receptor is stabilized by the typical polar interaction of the protonated amino moiety with the carboxy group of E172 which is optimally oriented by an H-bond interaction with Y103. The lipophilic interaction of I124 with the N-substituent also contributes to the high σ1 affinity of the benzylamines. The antagonistic activity was determined in a Ca2+ influx assay in retinal ganglion cells. The enantiomeric cis-configured benzylamines (1R,3S)-11 and (1S,3R)-11 were able to inhibit the growth of DU145cells, a highly aggressive human prostate tumor cell line. Moreover, cis-11 could also inhibit the growth of further human tumor cells expressing σ1 receptors. The experimentally determined logD7.4 value of 3.13 for (1R,3S)-11 is in a promising range regarding membrane penetration. After incubation with mouse liver microsomes and NADPH for 90min, 43% of the parent (1R,3S)-11 remained unchanged, indicating intermediate metabolic stability. Altogether, nine metabolites including one glutathione adduct were detected by means of LC-MS analysis.

Characterization of the in vitro cytotoxic effects of brachydins isolated from Fridericia platyphylla in a prostate cancer cell line

ABSTRACT Brachydins (Br) A, B, and C are flavonoids extracted from Fridericia platyphylla (Cham.) L.G. Lohmann roots (synonym Arrabidaea brachypoda), whose extract previously exhibited cytotoxic and antitumor activity. In vitro cell culture of human prostate tumor cell line (PC-3) was used to determine cell viability as evidenced by MTT, neutral red, and LDH release using nine concentrations (0.24 to 30.72 µM) of each brachydin. A triple-fluorescent staining assay assessed the mechanism resulting in cell death. Genomic instability and protein expression were evaluated using comet assay and western blot analysis, respectively. The pro-oxidant status was analyzed using the5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) probe. The IC50 values for brachydins BrA, BrB, and BrC were 23.41, 4.28, and 4.44 µM, respectively, and all compounds induced apoptosis and necrosis. BrB and BrC increased p21 levels indicating a possible G1 cell cycle arrest. BrA (6 µM) and BrB (3.84 µM) decreased phospho-AKT (AKT serine/threonine kinase) expression, which also influenced cell cycle and proliferation. BrA, BrB, and BrC elevated cleaved PARP (poly (ADP-ribose) polymerase), a protein related to DNA repair and induction of apoptotic processes. Therefore, this study determined the IC50 values of brachydins in the PC-3 cell line as well as the influence on cell proliferation and cell death processes, such as apoptosis and necrosis, indicating the proteins involved in these processes. Abbreviations ANOVA: Analysis of Variance; BrA: Brachydin A; BrB: Brachydin B; BrC: Brachydin C; CGEN: Genetic Heritage Management Council; CID: Compound identification number; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; CO2: Carbon dioxide; DMSO: Dimethyl sulfoxide; DNA: Deoxyribonucleic acid; DTT: Dithiothreitol; DXR: Doxorubicin; ECL: Chemiluminescence; EDTA: Ethylenediaminetetraacetic acid; FBS: Fetal bovine serum; H2O2: Hydrogen peroxide; HRMS: High-Resolution Mass Spectrometry; IC50: Half maximal inhibitory concentration; LDH: Lactate dehydrogenase; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Na3VO4: Sodium Orthovanadate; NaOH: Sodium hydroxide; NCBI: National Center for Biotechnology Information; NMR: Nuclear Magnetic Resonance; PBS: Phosphate buffer saline; PCR: Polymerase chain reaction; PSMF: Phenylmethylsulfonyl fluoride; RPMI: Roswell Park Memorial Institute Medium; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis; STR: Short tandem repeat; TBS-T: Tris-buffered saline and Polysorbate 20; UPHLC: Ultra-Performance Liquid Chromatography

Abstract B068: Methionine aminopeptidase II (MetAP2) activated in situ self-assembly of small-molecule probes for imaging prostate cancer

Abstract Prostate cancer is the most common noncutaneous malignancy among men in the United States and a leading cause of cancer death worldwide. The major clinical challenges in prostate cancer diagnosis include accurate staging, localizing tumors within the prostate, and developing imaging prognostic biomarkers. PET is routinely used in the clinical management of many cancers dues to its high sensitivity (10-15 M), spatial resolution (2-4 mm3), rapid whole-body scan times (<20 min), noninvasiveness, and its ability to provide both anatomic and molecular information when combined with computed tomography (PET/CT). Several PET tracers (e.g., 18F-FDG, 18F-NaF, 11C-choline, 11C-acetate, 18F-fluciclovine, etc.) have been studied for early stage prostate cancer, bone metastases, and recurrent disease; nevertheless, all of these tracers rely on cell uptake and their applications are often limited by the low metabolic activity and a low growth rate of prostate cancer. In this project, we aim to develop a novel enzymatic activity-based PET tracer for in vivo imaging of prostate cancer by targeting methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that catalyzes the cotranslational removal of the N-terminus initiator methionine residue from nascent proteins. We hypothesize that MetAP2 can serve as a novel diagnostic biomarker of prostate cancer. A MetAP2 activatable molecular imaging probe that self-aggregates in situ can be utilized to image and define this disease. To test our hypothesis, we first measured the expression level of MetAP2 in primary benign human prostate epithelial cells (BS403) and prostate tumor cell lines by Western blot. BS403 cells express significantly lower levels of MetAP2 than PC3 (by 4-fold), DU145 (3-fold), and 22Rv1 (3-fold). The expression of MetAP2 in DU145 tumor xenografts is 12-fold higher and for 22Rv1 14-fold higher, compared to BS403 cells. Immunohistologic analysis of tissues derived from DU145 and 22Rv1 xenografts confirms strong staining of MetAP2. We performed immunohistochemistry on a prostate cancer tissue microarray containing 225 independent patient samples represented in quadruplicate. Positive staining for MetAP2 was found in nearly all of the cancer samples. Encouraged by these data, we designed and synthesized a nonradioactive MetAP2 sensitive nano-aggregation probe called M-SNAP. Incubation of M-SNAP with recombinant MetAP2 enzyme confirmed the formation of the cyclized and aggregated product M-SNAP-cycl by HPLC, TEM (transmission electron microscopy), and DLS (dynamic light scattering). Treatment of PC3 and DU145 cells with or without M-SNAP and the MetAP2 specific inhibitor TNP470 demonstrated MetAP2 dependent intracellular probe aggregation/signal retention using post fluorescent labeling of the cyclized and aggregated products. In future work, we will prepare and study an MetAP2 activatable radioisotope-labeled probe in vitro for sensing and imaging and will evaluate the PET probe in different human prostate xenograft models. MetAP2 sensing probes could provide a highly sensitive and noninvasive PET imaging approach for prostate cancer detection that could be useful in prostate cancer detection, staging, and monitoring of therapeutic response. Citation Format: Jinghang Xie, Meghan Rice, Yunfeng Cheng, Guosheng Song, Christian Kunder, James D. Brooks, Tanya Stoyanova, Jianghong Rao. Methionine aminopeptidase II (MetAP2) activated in situ self-assembly of small-molecule probes for imaging prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B068.

Zika Virus Selectively Kills Aggressive Human Embryonal CNS Tumor Cells In Vitro and In Vivo.

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKVBR) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKVBR was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKVBR in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated Wnt signaling were more susceptible to the oncolytic effects of ZIKVBR Furthermore, modulation of Wnt signaling pathway significantly affected ZIKVBR-induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKVBR could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects.Significance: Brazilian Zika virus strain kills aggressive metastatic forms of human CNS tumors and could be a potential oncolytic agent for cancer therapy. Cancer Res; 78(12); 3363-74. ©2018 AACR.

Quantitative [Fe]MRI determination of the dynamics of PSMA-targeted SPIONs discriminates among prostate tumor xenografts based on their PSMA expression.

There is a need for a quantitative MRI method for iron concentration magnetic resonance imaging suitable for measuring the delivery of targeted superparamagnetic iron oxide nanoparticles (SPIONs) to tumors. To apply our newly developed [Fe]MRI method to the quantitative imaging in both space and time of the iron dynamics of anti-prostate specific membrane antigen (PSMA) conjugated SPIONs within human prostate tumor xenografts in nude mice. Longitudinal. 45 Harlan Sprague Dawley athymic nude mice bearing xenografts from PSMA-positive LNCaP, C4-2 and PSMA-negative DU145 tumors from human prostate tumor cell lines. 1.0 Tesla/ T1 and T2 weighted spin echo. Image intensity and contrast measurements. Student's t-test. The SPION diffusion coefficient within tumors was D = 44.8 ± 2.4 × 10-6 mm2 /s. The iron taken up by PSMA-positive LNCaP and C4-2 tumors was proportional to the tail-vein injected dose from 60 nmol to 1.6 μmol; injection of 1 μmol of iron in anti-PSMA conjugated SPIONs resulted in a tumor [Fe] of 76 μM. Even at the highest iron dose of 1.6 μmol, the PSMA-negative DU145 tumors took up no significant iron from the anti-PSMA conjugated SPIONs. A similar lack of nonspecific uptake was observed when the antibodies against PSMA were omitted from the injected SPION preparation. The fraction of the initial iron dose that was taken up by PSMA-positive tumors was 2.32 ± 0.75% (n = 10); uptake by the PSMA-negative DU145 tumors and for SPIONs without anti-PSMA antibodies was 0.16 ± 0.34% (n = 7) giving a ratio of [Fe] in PSMA + versus PSMA- tumors greater than 15:1 (P = 0.01). Quantitative [Fe]MRI of anti-PSMA conjugated SPIONs discriminated between PSMA-positive LNCaP and C4-2 and PSMA-negative DU145 human prostate tumor xenografts in vivo. 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017. J. MAGN. RESON. IMAGING 2018;48:469-481.

Inhibition of cell proliferation through an ATP-responsive co-delivery system of doxorubicin and Bcl-2 siRNA.

Herein, DNA duplex was constructed through the hybridization of adenosine triphosphate (ATP)-responsive aptamer and its cDNA in which GC-rich motif could be used to load doxorubicin (DOX), and then, cationic polymer PEI25K was used as a carrier to simultaneously condense DOX-Duplex and Bcl-2 siRNA to prepare the ternary nanocomplex polyethylenimine (PEI)/DOX-Duplex/siRNA. The ATP concentration gradient between the cytosol and extracellular environment could achieve the stable loading of DOX in duplex and the rapid drug release in an ATP-responsive manner. Using human prostate tumor cell line PC-3 as a model, an obvious induction of cell proliferation could be detected with a cell viability of 53.3%, which was stronger than single cargo delivery, indicating the synergistic effect between these two components. The enhanced anti-proliferative effect of ternary nanocomplex could be attributed to the improved induction of cell apoptosis in a mitochondria-mediated pathway and cell-cycle arrest at the G2 phase. Overall, the ATP-responsive nanocarrier for co-delivering DOX and Bcl-2 siRNA has been demonstrated to be a smart delivery system with favorable anti-proliferative effect, especially for solving the multidrug resistance of tumors.

Secreted Frizzled-related protein 4 (sFRP4) chemo-sensitizes cancer stem cells derived from human breast, prostate, and ovary tumor cell lines

This study investigated molecular signals essential to sustain cancer stem cells (CSCs) and assessed their activity in the presence of secreted frizzled-related protein 4 (sFRP4) alone or in combination with chemotherapeutic drugs. SFRP4 is a known Wnt antagonist, and is also pro-apoptotic and anti-angiogenic. Additionally, sFRP4 has been demonstrated to confer chemo-sensitization and improve chemotherapeutic efficacy. CSCs were isolated from breast, prostate, and ovary tumor cell lines, and characterized using tumor-specific markers such as CD44+/CD24−/CD133+. The post-transcription data from CSCs that have undergone combinatorial treatment with sFRP4 and chemotherapeutic drugs suggest downregulation of stemness genes and upregulation of pro-apoptotic markers. The post-translational modification of CSCs demonstrated a chemo-sensitization effect of sFRP4 when used in combination with tumor-specific drugs. SFRP4 in combination with doxorubicin/cisplatin reduced the proliferative capacity of the CSC population in vitro. Wnt/β-catenin signaling is important for proliferation and self-renewal of CSCs in association with human tumorigenesis. The silencing of this signaling pathway by the application of sFRP4 suggests potential for improved in vivo chemo-responses.

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

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

Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription

Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the “AR-interactome.” Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope (streptavidin-binding peptide-tagged wild-type androgen receptor; SBP-AR). A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex in vitro. Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers.

Health-benefits of date fruits produced in Saudi Arabia based on in vitro antioxidant, anti-inflammatory and human tumor cell proliferation inhibitory assays

Date fruits are reported to exhibit health-beneficial effects in addition to its nutritional value. Fruits collected from commercial date palm trees were sequentially extracted with water and methanol. All varieties of date fruits contained sugars, phenolics, triterpenoids, triglycerides, fatty acids and steroids, where sugars were the predominant components. Water and methanolic extracts of date fruits were assayed for antioxidant, antiinflammatory and human tumor cell proliferation inhibitory activities. In MTT antioxidant assay, methanolic extracts at 250μg/mL exhibited moderate activity with absorbance values between 0.14 and 0.41. Water and methanolic extracts at 100μg/mL inhibited lipid peroxidation (LPO) by 50–67% and 58–82%, respectively. In anti-inflammatory assay using cyclooxygenase enzymes (COX-1 and -2), water and methanolic extracts at 100μg/mL showed COX-1 enzyme inhibition by 26–36% and 33–41%, and COX-2 by 45–48% and 48–52%, respectively. At 100μg/mL concentrations, methanolic extracts of all date fruits showed marginal cell proliferation inhibitory activity against human gastric, prostate, colon, breast and lung tumor cell lines. The bioassay results suggest that varietal difference is not a significant factor among the 29 date fruits studied when compared for health-beneficial effects resulting from non-nutritional components present in it.

Abstract 1566: Notch-1 regulation of the PTEN - mTOR axis in prostate

Abstract Prostate cancer affects one in three men over the age of 60. Loss of expression or function of PTEN is the most commonly observed molecular defect in human prostate cancer. Thirty to 70% of clinical cases exhibit loss of this critical tumor suppressor. PTEN is an essential negative regulator of PI3K/Akt signaling. Together with the mTOR pathway, the PTEN/PI3K/Akt cascade forms a network for cellular responses to growth factors and nutrients. Studies in mice have demonstrated that small changes in PTEN dose can influence cancer development. Thus, understanding the regulation of PTEN expression and function is key to understanding tumor progression. We have previously reported that signaling through the Notch-1 receptor pathway results in increased transcriptional expression of the tumor suppressor PTEN. We further reported that Notch-1 signaling was lost in the tumor foci of clinical prostate cancer cases as compared to the surrounding benign tissue. Herein, we report that Notch-1 signaling alters the pool of active, unphosphorylated PTEN in the human metastatic prostate tumor cell line DU145. Retroviral transduction was used to generate DU145 cells that express constitutively active Notch-1 (DU/ICN1). In cells with constitutively active Notch-1 (DU/ICN1), the pool of unphosphorylated PTEN was increased roughly 2.5 fold compared to the vector only control. Constitutive Notch-1 signaling also resulted in decreased mTOR signaling as determined by a decrease in phosphorlyated 4E-BP1 and phosphorylated S6 ribosomal protein. Decreased expression of Raptor and Rictor and decreased phosphorylation of mTOR were also observed in the presence of constitutively active Notch-1. To test if Notch-1 signaling influences tumor engraftment and growth in a syngeneic model, the tumorigenic C2 cell line, derived from TRAMP mice, was transduced with constitutively active Notch-1. C2/ICN1 and parental C2 cells were engrafted into C57/BL6 mice. All recipients of the parental C2 cell line developed tumors within 46 days, whereas none that received cells expressing constitutively active Notch-1 (C2/ICN1) had developed tumors. Only after 61 days did one of the four mice that received C2/ICN1 cells develop tumors. We next tested if loss of Notch-1 expression promotes characteristics associated with tumorigenicity by using lentiviral transduction to knock down endogenous Notch-1 expression in DU145 cells (DU/shN1). DU145 cells with loss of Notch-1 (DU/shN1) exhibited decreased expression of PTEN protein and a decreased ability to migrate in transwell experiments as compared with control cells expressing endogenous Notch-1. Collectively, these data indicate a role for Notch-1 receptor signaling in modulating the activity of PI3K/Akt and mTOR axis through regulation of the PTEN tumor suppressor, and suggest a mechanistic basis for Notch-1 tumor suppressive activity in prostate cells. Citation Format: Jennifer M. Nutter, C William Angus, Fred E. Bertrand. Notch-1 regulation of the PTEN - mTOR axis in prostate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2014-1566

SPARCL1 suppresses metastasis in prostate cancer

PurposeMetastasis, the main cause of death from cancer, remains poorly understood at the molecular level. Experimental designBased on a pattern of reduced expression in human prostate cancer tissues and tumor cell lines, a candidate suppressor gene (SPARCL1) was identified. We used in vitro approaches to determine whether overexpression of SPARCL1 affects cell growth, migration, and invasiveness. We then employed xenograft mouse models to analyze the impact of SPARCL1 on prostate cancer cell growth and metastasis in vivo. ResultsSPARCL1 expression did not inhibit tumor cell proliferation in vitro. By contrast, SPARCL1 did suppress tumor cell migration and invasiveness in vitro and tumor metastatic growth in vivo, conferring improved survival in xenograft mouse models. ConclusionsWe present the first in vivo data suggesting that SPARCL1 suppresses metastasis of prostate cancer.

6r, a novel oxadiazole analogue of ethacrynic acid, exhibits antitumor activity both in vitro and in vivo by induction of cell apoptosis and S-phase arrest

This study investigated the in vitro and in vivo antitumor effects of 5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl) phenoxymethyl]-3-methyl-1,2,4- oxadiazole (6r), a novel ethacrynic acid (EA) derivative. The in vitro effect of 6r on cell proliferation of human colon, leukemia, prostate, lung, breast, ovarian and cervical tumor cell lines was assessed using MTT assay and the in vivo effect was determined with an SW620 xenografts nude mice model. The effect of 6r on expressions of GST P1-1 and apoptosis-related proteins were measured by western blotting and the effect on cell apoptosis was analysed by Hoechst 33258 nuclear staining as well as by cell surface staining of annexin V/propidium iodide. The effect on cell cycle was assessed by flow cytometry. Results showed that 6r inhibit proliferation of a range of human cancer cells in vitro and growth of SW620 tumor xenografts in vivo. The anti-proliferative effect of 6r is associated with cell apoptosis as a result of increased ratio of cellular Bax/bcl-2 expression and subsequent cytochrome-c and caspase-3 activation. Unlike EA, 6r did not show any influence on cellular GST P1-1 expression and its anti-proliferative action was associated with cell cycle arrest in G1/S-phase. In conclusion, 6r has the potential to be developed as a chemotherapeutic agent by induction of cell apoptosis but not regulating GST P1-1.

Abstract 2340: CPS49 and Flavopiridol: a new selective drug combination for advanced prostate cancer

Abstract Prostate cancer (PCa) still ranks as the second most commonly diagnosed cancer and metastatic castration-resistant prostate cancer (CRPC) is a leading cause of cancer death in men around the world. The common treatment for patients with CRPC is chemotherapy based on docetaxel. While there are currently seven agents approved for CRPC and four regimens have shown some survival benefit, those survival prolongations have been modest and unfortunately all patients will eventually progress. Thus, there is a need for new agents and regimens for this disease. 2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin dependent kinases and also provokes lethality against leukemic cells. We previously found that CPS49 and flavopiridol combination induced selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. The goal of this study was to investigate the selectivity and efficacy of CPS49-flavopiridol combination in prostate cancer preclinical models. Our results showed that flavopiridol enhanced CPS49 cytotoxicity in all human prostate tumor cell lines analyzed (PC3, C4-2, LNCaP and 22RV1); however non tumor cell lines (293HEK and MCF10) were resistant to the tested doses. Furthermore, it was previously reported, high doses of flavopiridol (10 μM) or CPS49 (12 μM) were needed to inhibit tumor growth in PC3 xenograft mice compared with vehicle treated mice. Here, we demonstrated that combining these two agents, antitumor activity was synergistically enhanced with low doses. Injecting subcutaneously PC3 cells in nu/nu mice, we found that CPS49-flavopiridol administration reduced tumor volume approximately 83% after 2 weeks of co-treatment and 54% after 1 week of low dose flavopiridol pretreatment and 2 weeks of drug combination. In addition, we performed RT-qPCR array containing 26 genes from PC3 cells exposed to CPS49 and flavopiridol combination. We determined that this treatment shut down the expression of several genes involved in cell cycle, DNA damage and tumor progression. Histological analysis of xenograft PC3 tumor samples showed extensive areas of necrosis induced by the treatment. Furthermore, we assessed the efficacy of CPS49 in combination with paclitaxel (docetaxel analog). All the prostate tumor cell lines tested were highly sensitive to this combination. However, this combination did not reduce the tumor volume in PC3 xenografts. These results indicate that the CPS49 and flavopiridol is a promising new alternative for the treatment of CRPC. 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 2340. doi:1538-7445.AM2012-2340

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.

SH3BP1, an Exocyst-Associated RhoGAP, Inactivates Rac1 at the Front to Drive Cell Motility

The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.