Human Prostate Cancer Cell Lines LNCaP Research Articles

GPX2 predicts recurrence-free survival and triggers the Wnt/β-catenin/EMT pathway in prostate cancer.

ObjectiveThis study aimed to establish a prognostic model related to prostate cancer (PCa) recurrence-free survival (RFS) and identify biomarkers.MethodsThe RFS prognostic model and key genes associated with PCa were established using Least Absolute Shrinkage and Selection Operator (LASSO) and Cox regression from the Cancer Genome Atlas (TCGA)-PRAD and the Gene Expression Omnibus (GEO) GSE46602 datasets. The weighted gene co-expression network (WGCNA) was used to analyze the obtained key modules and genes, and gene set enrichment analysis (GSEA) was performed. The phenotype and mechanism were verified in vitro.ResultsA total of 18 genes were obtained by LASSO regression, and an RFS model was established and verified (TCGA, AUC: 0.774; GSE70768, AUC: 0.759). Three key genes were obtained using multivariate Cox regression. WGCNA analysis obtained the blue module closely related to the Gleason score (cor = –0.22, P = 3.3e − 05) and the unique gene glutathione peroxidase 2 (GPX2). Immunohistochemical analysis showed that the expression of GPX2 was significantly higher in patients with PCa than in patients with benign prostatic hyperplasia (P < 0.05), but there was no significant correlation with the Gleason score (GSE46602 and GSE6919 verified), which was also verified in the GSE46602 and GSE6919 datasets. The GSEA results showed that GPX2 expression was mainly related to the epithelial–mesenchymal transition (EMT) and Wnt pathways. Additionally, GPX2 expression significantly correlated with eight kinds of immune cells. In human PCa cell lines LNCaP and 22RV1, si-GPX2 inhibited proliferation and invasion, and induced apoptosis when compared with si-NC. The protein expression of Wnt3a, glycogen synthase kinase 3β (GSK3β), phosphorylated (p)-GSK3β, β-catenin, p-β-catenin, c-myc, cyclin D1, and vimentin decreased; the expression of E-cadherin increased; and the results for over-GPX2 were opposite to those for over-NC. The protein expression of GPX2 decreased, and β-catenin was unchanged in the si-GPX2+ SKL2001 group compared with the si-NC group.ConclusionWe successfully constructed the PCa RFS prognostic model, obtained RFS-related biomarker GPX2, and found that GPX2 regulated PCa progression and triggered Wnt/β-catenin/EMT pathway molecular changes.

A Novel Splice Variant of the Inhibitor of Growth 3 Lacks the Plant Homeodomain and Regulates Epithelial-Mesenchymal Transition in Prostate Cancer Cells.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial–mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

The Effect of Quercetin Nanosuspension on Prostate Cancer Cell Line LNCaP via Hedgehog Signaling Pathway.

Prostate cancer (PCa) is the second leading cause of cancer death in American population. In this manner, novel therapeutic approaches for identification of therapeutic targets for PCa has significant clinical implications. Quercetin is a potent cancer therapeutic agent and dietary antioxidant present in fruit and vegetables. To investigate the underlying mechanism by which the PCa was regulated, nanoparticles of quercetin were administrated to cells. For in vitro experiments, human PCa cell line LNCaP were involved. Cell viability assay and quantitative RT-PCR (qRT-PCR) for hedgehog signaling pathway genes were used to determine the key signaling pathway regulated for PCa progression. The cell viability gradually decreased with increased concentration of quercetin nanoparticles. At 48 h, 40 mM concentration of quercetin treatment showed near 50% of viable cells. Quercetin nanoparticles upregulates Su(Fu) mRNA expressions and downregulates gli mRNA expressions in the LNCaP cells. The results showed that the hedgehog signaling targeted inhibition may have important implications of PCa therapeutics. Additionally, the outcomes provided new mechanistic basis for further examination of quercetin nanoparticles to discover potential treatment strategies and new targets for PCa inhibition.

Abstract 3983: Direct contact of heterogeneous fibroblasts with cancer cells regulates the invasive phenotype of prostate cancer cells

Abstract In the tumor microenvironment of prostate cancer (PCa), cancer cells frequently invade the surrounding stroma in the way of single cell migration (SCM) or collective cell migration (CCM). This phenomenon can be responsible for the heterogeneous formation of structural atypia such as Gleason pattern. Generally, PCa cells undergo a phenotypic change which is called the epithelial-to-mesenchymal transition (EMT), e.g., downregulation of epithelial cell-cell adhesion molecules such as E-cadherin and gain of mesenchymal morphology and motility. The surrounding stroma is mainly composed of fibroblasts and extracellular matrix (ECM). We hypothesize that direct contact-mediated interactions at the interface between PCa cells and fibroblasts/ECM can trigger numerous changes in PCa cells including morphology and motility. In this study, we investigated the roles of fibroblasts in the three-dimensional (3D) morphogenesis of PCa cells on a viscous substrate. To examine the effects of fibroblasts on the 3D morphogenesis of PCa cells on Matrigel, we mixed human PCa cell line LNCaP cells with commercially available normal human prostate stromal cells (PrSC) or nine primary cultures of prostate fibroblasts (pcPrFs and NPFs) isolated from patients with PCa and bladder cancer. The 3D morphogenesis of LNCaP cells mixed with fibroblasts on Matrigel was confirmed by E-cadherin immunohistochemical staining. All ten fibroblasts alone were aggregated on Matrigel, whereas LNCaP cells alone were not. LNCaP cells mixed with six fibroblasts (PrSC, pcPrF-M5, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a deep crater-like hole on Matrigel. In the 3D structure, the upper and lower tiers were composed of LNCaP cells and fibroblasts, respectively. Of note, direct contact of four fibroblasts (NPF-M13, pcPrF-M7, pcPrF-M10, and pcPrF-M26) with LNCaP cells decreased the aggregation of fibroblasts on Matrigel. Five of six fibroblasts (PrSC, pcPrF-M5, pcPrF-M23, pcPrF-M28, and pcPrF-M31) induced SCM of LNCaP cells, while only one fibroblasts (pcPrF-M24) induced CCM. In the scratch assay, the cell motility of CCM-inducing fibroblasts was slower than that of SCM-inducing fibroblasts. Expression pattern of primary cilia in SCM-inducing fibroblasts was changed by mixing with LNCaP cells, whereas that in CCM-inducing fibroblasts was not. PCa is quite interesting because of the multi-focal and heterologous progression of primary tumors. The heterogeneous stromal compartment in PCa tissues contains multiple populations of fibroblasts that are associated with morphology and motility of PCa cells. Here our results showed that direct contact of heterogeneous fibroblasts with PCa cells on Matrigel regulated the invasive phenotype of PCa cells such as SCM and CCM. Impairing the malignant phenotype of fibroblasts in PCa tissues, e.g., differentiation therapy targeting tumor stroma, may abrogate PCa cell invasion. Citation Format: Kenichiro Ishii, Yasuhisa Nakagawa, Taku Shirai, Masako Ichishi, Masaya Fujiwara, Daisuke Kato, Chise Matsuda, Yoshifumi Hirokawa, Yoshiki Sugimura, Masaki Inagaki, Masatoshi Watanabe. Direct contact of heterogeneous fibroblasts with cancer cells regulates the invasive phenotype of prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3983.

Abstract 1650: Non-invasive imaging tool to predict BRCA2 silencing in the context of prostate cancer

Abstract Background: We recently demonstrated that biallelic as well as monoallelic loss of the DNA damage response (DDR) gene BRCA2 in prostate cancer (PC) cell lines and mCRPC organoids leads to an aggressive form of PC and early androgen deprivation therapy (ADT) resistance (Chakraborty et al.). Clinical studies have established that DDR deficiency is generally associated with a poor prognosis. Very recently, immunohistochemical analysis of mCRPC patient samples by Paschalis et al. revealed that defects in DDR genes (in particular BRCA2 and ATM) are associated with increased prostate-specific membrane antigen (PSMA; folate hydrolase, FOLH1) expression on the cell membrane. PSMA expression can be measured non-invasively in pre-clinical models and human subjects using one of the several positron emission tomography (PET) imaging agents such as [68Ga]-PSMA11 or [124I]-MSK-PSMA11 that are being evaluated in pre-clinical and/or clinical setting. Therefore, we hypothesized that upregulation of PSMA expression can be a marker for BRCA2 loss and this increased expression can be quantified using PET agents both in vitro and in vivo. Experimental design: We investigated the effect of BRCA2 deletion on PSMA expression in the castration sensitive human PC cell line LNCaP at the transcriptional and translational level and quantified the changes using saturation binding assays with [124I]-MSK-PSMA11. Using CRISPR-Cas9 and RNAi-based methods, we silenced BRCA2 in the castration sensitive cell line LNCaP and evaluated its effect on PSMA at the transcriptional and translational level. We carried out saturation binding assay using [124I]-MSK-PSMA11 to measure changes in cell surface PSMA receptor density. Results: BRCA2 knockout was achieved successfully using CRISPR-Cas9 based methods. Immunoblotting analysis revealed that BRCA2 loss resulted in a significant increase in PSMA levels when compared to control LNCaP cell line. Immunohistochemical analysis confirmed this obervation. Cell binding assays demonstrated that BRCA2 null LNCaP cell lines have about 5-6 fold higher uptake of the PET tracer [124I]-MSK-PSMA11. We will be conducting in vivo studies to demonstrate that BRCA2 deletion leads to a significant increase in PSMA signal in mice xenograft models. Conclusions: Our results indicate that BRCA2 silencing leads to significant upregulation of PSMA expression in PC cell lines, which can be imaged using a PSMA targeted PET tracer. These studies were partly supported by DOD-PCRP-Grant # W81XWH-19-1-0536 and PCF Young Investigator Award to Goutam Chakraborty.

Sea cucumber extract TBL-12 inhibits the proliferation, migration, and invasion of human prostate cancer cells through the p38 mitogen-activated protein kinase and intrinsic caspase apoptosis pathway.

Sea cucumber is a kind of nutritious echinoderm that has multiple biological activities, including antioxidant, antibacterial, and antitumor activities. However, there is no extensive study on the antitumor effect of sea cucumber extract on prostate cancer (PCa). TBL-12 is a new sea cucumber extract. In this study, we investigated the in vivo anti-PCa effect of TBL-12 and its in vitro effects on the proliferation, apoptosis, migration, and invasion of the human PCa cell lines LNCaP, 22RV1, PC-3, and DU145, and evaluated its possible mechanisms. Cell proliferation was analyzed by cell counting kit-8 and colony formation assays. Scratch migration assay and transwell invasiveness assay were used to observe TBL-12 effect on the migration and invasion of PCa cells. Matrix metalloproteinase 2 (MMP-2) and MMP-9 expression and enzymatic activity was determined by Western blot analysis, quantitative reverse-transcription polymerase chain reaction, and gelatin zymography. Apoptosis level was detected by flow cytometry analysis. Western blot analysis was used to analyze p38 mitogen-activated protein kinase (MAPK) and apoptosis pathways. Angiogenic array analysis was used to explore autocrine and paracrine growth factors in PCa cell lines. Xenograft tumor model was built to observe the in vivo anticancer effect. TBL-12 could significantly inhibit tumor growth in xenograft PCa mice in vivo, and dramatically inhibit the proliferation, colony formation, migration, and invasiveness of PCa cells in vitro (P < 0.05 and P < 0.001). The expression and enzyme activity of MMP-2 and MMP-9 were significantly suppressed by TBL-12 ( P < 0.01), and decreased phosphorylation level of p38 in PCa cells was detected ( P < 0.001). Furthermore, TBL-12 could reinforce the MMP-2/MMP-9 inhibitory effect of SB203580, a specific inhibitor of the p38 MAPK pathway ( P < 0.05). Besides, TBL-12 could induce the apoptosis of PCa cells by activating caspase-9, caspase-7, and poly(ADP-ribose) polymerase and suppressing survivin, and inhibit the secretion of angiogenin, angiopoietin-2, and vascular endothelial growth factor in PCa cells. Sea cucumber extract TBL-12 could suppress the proliferation and metastasis of human PCa cells by inhibiting MMP-2 and MMP-9 via blocking the p38 MAPK pathway, inducing apoptosis through intrinsic caspase apoptosis pathway and inhibiting the secretion of angiogenic factors. Our findings may be of importance and significance for the research and clinical applications of sea cucumber extract in PCa treatment.

Abstract 5220: Chronic exposure to a novel AR-NTD inhibitor induces resistance via a selective metabolism pathway

Abstract Background: The androgen receptor (AR) is recognized as playing a crucial role in prostate cancer (PCa) maintenance and progression; therefore its inhibition has been the cornerstone of modern therapy for men who fail primary treatment. Current treatments are initially effective, however resistance ultimately develops and the disease progresses to a lethal form termed castration-resistant prostate cancer (CRPC). Our lab has discovered a new class of molecules (EPI) which inhibit the AR by binding to the N-terminal domain (NTD). The NTD interacts with transcriptional machinery, and its presence is vital for a transcriptionally functional receptor. We have previously shown that EPI-002 specifically inhibits both full-length AR and constitutively active AR splice variants. The efficacy of EPI-506, the prodrug of EPI-002, is currently being tested in a Phase I/II clinical trial for CRPC. Here we propose novel resistance mechanisms arising from sustained AR-NTD inhibition, with the goal of preemptively developing backup compounds. Methods: The androgen sensitive human PCa cell line LNCaP was used in all experiments. A resistant cell line (LNCaP-EPIR) was generated by passaging parental LNCaP cells weekly in media supplemented with EPI-002 beginning in September 2012. In vitro and in vivo studies using LNCaP and LNCaP-EPIR cells were employed to confirm biological resistance. A human affymetrix microarray identified possible resistance mechanisms and was validated using qRT-PCR, western blot and functional studies. LNCaP and LNCaP-EPIR cells were challenged with EPI-045, an EPI-analog predicted to remain effective in the context of EPI-002 resistance. Results: LNCaP-EPIR cells treated with 25 µM EPI-002 displayed similar growth rates to vehicle treatment, both in vitro and in vivo. Conversely, parental LNCaP cells showed significant growth inhibition in response to EPI-002. LNCaP-EPIR cells retained sensitivity to anti-androgens and AR knock-down by targeted siRNA, implying functional AR remains essential for growth. qRT-PCR data demonstrated that EPI-002 had reduced ability to block AR mediated gene transcription. Interrogation of microarray data revealed candidate genes (UGT2B family) which were specifically upregulated in the resistant line, and may function to metabolize EPI-002. Supporting this hypothesis EPI-045 which is predicted to be resistant to UGT2B metabolism, was able to significantly inhibit LNCaP-EPIR proliferation and AR transcriptional activity. Conclusions: Taken together these data suggest an EPI-specific mechanism of resistance, whereby EPI-002 is preferentially metabolized and removed from the cell. LNCaP-EPIR cells remain dependent upon AR signalling, and are sensitive to anti-androgens used clinically as well as a novel EPI-analog. This work highlights the potential for combination or sequential therapy in the context of drug-resistant CRPC. Citation Format: Jonathon K. Obst, Jun Wang, David Williams, Amy H. Tien, Nasrin R. Mawji, Yu Chi Yang, Raymond J. Andersen, Marianne D. Sadar. Chronic exposure to a novel AR-NTD inhibitor induces resistance via a selective metabolism pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2017-5220

Dietary Early Glycation Products Promote Prostate Tumorigenesis through Modulating Macrophages

Abstract Glycation products are ubiquitous in food. Moreover, the well-controlled glycation, that results in the production of early glycation products (EGPs), has been proposed as a strategy to improve the physicochemical properties of the food proteins. However, the health effects of EGPs are rarely known. Since there is evidence showing that Western diet (glycation prone) is associated with higher mortality of prostate patients than Prudent diet, we investigated the role of EGPs in prostate tumorigenesis. EGPs were produced from a whey protein isolate-glucose model system, with non-reacted samples (NR) and advanced glycation end-products (AGEs) as controls. They were used to gavage male C57BL6 mice, and the mice were then subcutaneously injected with TRAMP-C2 cells (a mouse prostate cancer (PCa) cell line) in the flank area. The tumor length, width, and the proliferation index were measured, and the splenic leukocytes were studied. In addition, the mouse TRAMP-C2 cells and two human PCa cell lines LNCaP and PC-3 were cultured with NR, EGPs and AGEs, and LNCaP and PC-3 were further cultured with the conditioned medium generated from human macrophages. The PCa cell proliferation, and the cytokine/chemokine production and iNOS expression by macrophages were determined. The results showed that EGPs promoted prostate tumor growth most in vivo, and PCa cell proliferation only when the presence of macrophages in vitro. In addition, EGPs activated the production of most cytokines/chemokines examined, with IL-10 and MIP-1β unregulated most, and decreased the iNOS expression in macrophage. In conclusion, EGPs promoted PCa possibly by modulating macrophages to become prostate tumor-associated M2, which produced high levels of IL-10 and MIP-1β.

Amino-terminal enhancer of split gene AES encodes a tumor and metastasis suppressor of prostate cancer.

A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration‐resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR‐defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium‐specific inactivation of Aes in Pten flox/flox mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways.

Prostate Cancer Associated Lipid Signatures in Serum Studied by ESI-Tandem Mass Spectrometryas Potential New Biomarkers.

Prostate cancer (PCa) is one amongst the most common cancersin western men. Incidence rate ofPCa is on the rise worldwide. The present study deals with theserum lipidome profiling of patients diagnosed with PCa to identify potential new biomarkers. We employed ESI-MS/MS and GC-MS for identification of significantly altered lipids in cancer patient’s serum compared to controls. Lipidomic data revealed 24 lipids are significantly altered in cancer patinet’s serum (n = 18) compared to normal (n = 18) with no history of PCa. By using hierarchical clustering and principal component analysis (PCA) we could clearly separate cancer patients from control group. Correlation and partition analysis along with Formal Concept Analysis (FCA) have identified that PC (39:6) and FA (22:3) could classify samples with higher certainty. Both the lipids, PC (39:6) and FA (22:3) could influence the cataloging of patients with 100% sensitivity (all 18 control samples are classified correctly) and 77.7% specificity (of 18 tumor samples 4 samples are misclassified) with p-value of 1.612×10−6 in Fischer’s exact test. Further, we performed GC-MS to denote fatty acids altered in PCa patients and found that alpha-linolenic acid (ALA) levels are altered in PCa. We also performed an in vitro proliferation assay to determine the effect of ALA in survival of classical human PCa cell lines LNCaP and PC3. We hereby report that the altered lipids PC (39:6) and FA (22:3) offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis.

Arsenic trioxide inhibits viability and induces apoptosis through reactivating the Wnt inhibitor secreted frizzled related protein-1 in prostate cancer cells.

BackgroundGrowing evidence suggests that arsenic trioxide (As2O3) induces apoptosis and inhibits tumor cell growth in prostate cancer (PCa), although details of the mechanism are still inconclusive. We investigated the antitumor effect of As2O3 in human PCa cell lines LNCaP and PC3 and the underlying mechanisms by focusing on the Wnt signaling pathway.MethodsThe effect of As2O3 on the viability and apoptosis of PCa cells was investigated by cholecystokinin-8 and flow cytometry. The expression of the related proteins in the Wnt signaling pathway and the downstream target genes of the Wnt signaling pathway was examined by Western blot and quantitative real-time PCR assay. The methylation status of the SFRP1 gene promoter was assessed by bisulfite sequencing.ResultsAs2O3 inhibited the viability of PCa cells and induced apoptosis of PCa cells in a dose-dependent manner. The protein level of phosphoglycogen synthase kinase-3β was upregulated, whereas the protein level of β-catenin and the mRNA levels of c-MYC, MMP-7, and COX-2 were downregulated in a dose-dependent manner in PCa cells treated with As2O3. In addition, As2O3 upregulated the protein and mRNA levels of secreted frizzled related protein-1, and increased the demethylation of the SFRP1 gene promoter.ConclusionOur results suggest that As2O3 may inhibit cell viability and induce apoptosis through reactivating the Wnt inhibitor secreted frizzled related protein-1 in both androgen-dependent and -independent human PCa.

Overexpression of p53 activated by small activating RNA suppresses the growth of human prostate cancer cells.

Previous research has reported that a particular double-stranded RNA, named dsP53-285, has the capacity to induce expression of the tumor suppressor gene TP53 in chimpanzee cells by targeting its promoter. Usually, it is the wild-type p53 protein, rather than mutants, which exhibits potent cancer-inhibiting effects. In addition, nonhuman primates, such as chimpanzees, share almost identical genome sequences with humans. This prompted us to speculate whether dsP53-285 can trigger wild-type p53 protein expression in human prostate cancer (PCa) cells and consequently suppress cell growth. The human PCa cell lines LNCaP and DU145 were transfected with dsP53-285 for 72 hours. Compared with the dsControl and mock transfection groups, expression of both p53 messenger RNA and p53 protein was significantly enhanced after dsP53-285 transfection, and this enhancement was followed by upregulation of p21, which indirectly indicated that dsP53-285 induced wild-type p53 expression. Moreover, overexpression of wild-type p53 mediated by dsP53-285 downregulated the expression of Cyclin D1 and cyclin-dependent kinase 4/6, thereby inducing PCa cell cycle arrest in G0/G1 phase and then inhibiting cell proliferation and clonogenicity. More importantly, dsP53-285 suppressed PCa cells mainly by modulating wild-type p53 expression. In conclusion, our study provides evidence that dsP53-285 can significantly stimulate wild-type p53 expression in the human PCa cell lines LNCaP and DU145 and can exert potent antitumor effects.

A role for the dehydrogenase DHRS7 (SDR34C1) in prostate cancer.

Several microarray studies of prostate cancer (PCa) samples have suggested altered expression of the “orphan” enzyme short-chain dehydrogenase/reductase DHRS7 (retSDR4, SDR34C1). However, the role of DHRS7 in PCa is largely unknown and the impact of DHRS7 modulation on cancer cell properties has not yet been studied. Here, we investigated DHRS7 expression in normal human prostate and PCa tissue samples at different tumor grade using tissue microarray and immunovisualization. Moreover, we characterized the effects of siRNA-mediated DHRS7 knockdown on the properties of three distinct human prostate cell lines. We found that DHRS7 protein expression decreases alongside tumor grade, as judged by the Gleason level, in PCa tissue samples. The siRNA-mediated knockdown of DHRS7 expression in the human PCa cell lines LNCaP, BPH1, and PC3 significantly increased cell proliferation in LNCaP cells as well as cell migration in all of the investigated cell lines. Furthermore, cell adhesion was decreased upon DHRS7 knockdown in all three cell lines. To begin to understand the mechanisms underlying the effects of DHRS7 depletion, we performed a microarray study with samples from LNCaP cells treated with DHRS7-specific siRNA. Several genes involved in cell proliferation and adhesion pathways were found to be altered in DHRS7-depleted LNCaP cells. Additionally, genes of the BRCA1/2 pathway and the epithelial to mesenchymal transition regulator E-cadherin were altered following DHRS7 knockdown. Based on these results, further research is needed to evaluate the potential role of DHRS7 as a tumor suppressor and whether its loss-of-function promotes PCa progression and metastasis.

Regulation of androgen receptor expression by Z‐isochaihulactone mediated by the JNK signaling pathway and might be related to cytotoxicity in prostate cancer

The androgen receptor (AR) is a main therapeutic target for treatment of prostate cancer (PCa). The natural compound isochaihulactone (K8), which has a chiral center ring and two racemic forms (E-K8 and Z-K8), has anti-tumor effects on multiple cancer types both in vitro and in vivo. Here, we determined which form of K8 contains significant tumor cytotoxicity and examined how this form regulates AR expression in PCa cells and xenografts. We chose the androgen-dependent human PCa cell line LNCaP and the androgen-independent cell lines DU145 and PC-3 to study the anti-tumor potency and AR regulation mediated by Z-K8. We measured cell viability and used flow cytometry, RT-PCR, and Western blotting. Growth inhibition in vivo was evaluated with an LNCaP xenograft animal model. In LNCaP cells, Z-K8 significantly repressed cell proliferation, induced apoptosis, repressed AR mRNA and protein expression in a time-dependent manner, and induced JNK phosphorylation. Furthermore, treatment with a JNK inhibitor significantly abolished Z-K8-induced AR downregulation. Z-K8 did not significantly inhibit reporter gene expression of constructs containing the AR promoter when it contained a mutated Sp1 binding site. Z-K8 also showed anti-tumor effects in the xenograft animal model. Z-K8 not only induced LNCaP apoptosis but also reduced AR expression. These results indicate that Z-K8 may be a potential anti-tumor drug for PCa therapy.

Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.

229 HOW DOES ETS FAMILY CONTRIBUTE TO PROSTATE CANCER PROGRESSION?

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010229 HOW DOES ETS FAMILY CONTRIBUTE TO PROSTATE CANCER PROGRESSION? Takeo Kosaka, Akira Miyajima, Shuji Mikami, Eiji Kikuchi, Suguru Shirotake, Takahiro Maeda, and Mototsugu Oya Takeo KosakaTakeo Kosaka More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , Shuji MikamiShuji Mikami More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Suguru ShirotakeSuguru Shirotake More articles by this author , Takahiro MaedaTakahiro Maeda More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.287AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recurrent gene fusions between TMPRSS2 and the ETS transcription factors have been identified in prostate cancer (PCa) development. However, the function of fusion products has not been well characterized. We previously showed long-term androgen ablation induced Ets-1 up-regulation in prostate cancer (Kosaka T, Prostate 2009). In this study, we examined the expression and the function of Ets-1, and determined its prognostic significance and correlation with hormone-independency. METHODS Total 139 human PCa specimens: 119 specimens (group AD) from patients who underwent radical prostatectomy for localized PCa and 20 specimens (group HR) from patients who died of hormone-refractory prostate cancer (HRPC) were enrolled in this study. Immunohistochemistry (IHC) was used to investigate Ets-1 and CD34:MVD. The median follow up was 70.5 months. Progression free survival was calculated by the Kaplan-Meier method. Multivariate analysis was performed using Cox regression model. Three human PCa cell lines LNCaP, C4-2, and C4-2AT6 (Established at our laboratory under androgen-ablated condition medium, Prostate 2009) were analyzed in vitro and in vivo. Ets-1 expression was transiently down-regulated using siRNA directed against Ets-1 (si-Ets-1). RESULTS The expression of Ets-1 in the nucleus and MVD in group HR were significantly higher than group AD (p<0.001). There was s significant positive correlation between Ets-1 and MVD (Spearman′s correlation, r=0.752, p<0.001). There were statistically significant correlation between Ets-1 expression and MVD (p<0.001), and extracapsular extension of cancer (ECE) (p<0.001). During a median 70.5 months follow-up period, PSA failure was confirmed in 30 patients. There were significant differences in PSA failure-free survival (log-rank test, p<0.001), ECE (p<0.001) with regard to Ets-1 expression, MVD, ECE, and LVI. The 5-year PSA failure-free survival was 43.0% for patients with higher Ets-1 expression compared to 88.8% for patients with lower Ets-1 expression. Multivariate Cox regression analysis indicated that higher Snail expression (p=0.002) and LVI (p=0.008) were independent prognostic factors. C4-2AT6 expressed Ets-1 in the nucleus more strongly than C4-2 and LNCaP cells in vitro and in vivo, accompanied by higher VEGF production. Knockdown of Ets-1 by si-Ets-1 resulted in a matched reduction in VEGF production in C4-2AT6 and C4-2. CONCLUSIONS These results indicated that increased expression of Ets-1 in the nucleus, which found to be prognostic indicator, were associated with angiogenesis and hormone-independency. Tokyo, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e89-e90 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeo Kosaka More articles by this author Akira Miyajima More articles by this author Shuji Mikami More articles by this author Eiji Kikuchi More articles by this author Suguru Shirotake More articles by this author Takahiro Maeda More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Activation of the Y1 Receptor by Neuropeptide Y Regulates the Growth of Prostate Cancer Cells

This study deals with the role of neuropeptide Y (NPY) in the regulation of cell proliferation. NPY is expressed in the normal and tumoral prostate, but no data on its possible role in prostate cancer (PCa) progression are available. Therefore, we evaluated the direct effect of NPY on the growth of the human PCa cell lines LNCaP (androgen dependent) and DU145 and PC3 (androgen independent). All PCa cell lines expressed Y1-R gene and protein. NPY treatment reduced the proliferation of LNCaP and DU145 cells and increased that of PC3 cells. The Y1-R antagonist BIBP3226 abolished such effects, suggesting a mandatory role of Y1-R in this process. LNCaP cells showed elevated constitutive levels of phosphorylated ERK1/2, which were not affected by NPY. In DU145 cells, NPY stimulated a long-lasting ERK1/2 activation, whereas, in PC3 cells, this effect was rapid and transient and required activation of protein kinase C. Moreover, in both cell lines, pretreatment with BIBP3226 prevented the NPY-induced ERK1/2 phosphorylation, further supporting Y1-R involvement. NPY treatment reduced forskolin-stimulated cAMP accumulation only in PC3 cells and did not change intracellular calcium concentration in any PCa cell line. These data indicate that NPY may directly regulate PCa cell growth via Y1-R. The direction of this effect appears to be related to the time kinetics of MAPK activation, i.e. long-lasting vs. transient, and to the clone-specific involvement of other intracellular signals. These findings suggest that NPY-related mechanisms might play a relevant role in the progression of PCa, at both androgen dependent and independent stages.

Evaluation of β-adrenergic receptor subtypes in the human prostate cancer cell line-LNCaP

The present study was undertaken to determine the effects of catecholamines, agonists, and antagonists of β-adrenergic receptors (AR) in the LNCaP cell line. Changes in cellular cyclic adenosine-3′,5′-monophosphate (cAMP) levels were quantified by the use of a 6 cAMP response element (CRE)-luciferase reporter gene assay. LNCaP cells were transiently transfected with this gene construct, incubated in 96-well microtiter plates for 24 hr, and then treated with β-AR agonists and/or antagonists for 4 hr. The rank order of potency for catecholamines and known β-AR agonists was terbutaline (3.31 nM)> isoproterenol (8.31 nM)≥ fenoterol (15 nM)= epinephrine (16.2 nM)> norepinephrine (77.5 nM)> BRL-37344 [( R ∗, R ∗)-(±)4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy acetic acid, sodium salt] (1000 nM)> dobutamine (1770 nM)> CGP 12177 (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2 H-benzimidazole-2-one hydrochloride) (inactive). The non-selective β 1-/-β 2-AR antagonists; propranolol and CGP 12177, at 10 −7 M, inhibited luciferase activity induced by these agonists by 80–96%. Propranolol blocked isoproterenol-induced luciferase responses in a competitive manner ( K B =1.4 nM). In addition, isoproterenol-activated luciferase expression was blocked more potently by ICI 118,551 [(±)-1-[2,3-(dihydro-7-methyl-1 H-inden-4-yl)oxy]-3-[(1-methylethy) amino]-2-butanol], a β 2-AR antagonist than by ICI 89,406 [(±)- N-[2-[3-(2-cyanophenoxy-)]-2-hydroxypropylamino]ethyl- N-phenylurea], a β 1-AR antagonist, giving K B values of 1.07 and 161 nM, respectively. These results suggest that the β 2-AR is the major subtype mediating catecholamine-induced cAMP changes in LNCaP cells.

Role of PI3K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state.

The mechanisms by which prostate cancer (PCa) cells progress to a hormone refractory state are poorly understood. The progression process under androgen ablation conditions involves the survival of at least a portion of malignant cells and their eventual proliferation in an androgen-independent manner. The goal of this study was to investigate the role of PI3K signaling in such a progression. Using an in vitro model of androgen ablation, we show that after removal of androgen support, the human PCa cell line LNCaP initially arrested in G(1) and trans-differentiated into neuroendocrine-like cells that eventually resumed androgen-independent proliferation. Both acute and chronic androgen ablation resulted in an increase in basal levels of PI3K and Akt activity, which were sustained throughout the progression process. Under these conditions, inhibition of PI3K, pharmacologically or with ectopic expression of PTEN, arrested cell proliferation and blocked progression to the androgen-independent state. In contrast, LNCaP cells in the presence of androgens were marginally sensitive to PI3K inhibition. During the chronic stage of androgen deprivation, androgen-independent proliferation correlated with diminished p27(kip1) protein levels, whereas PI3K and Akt activity remained elevated. At this stage, PI3K inhibition rapidly triggered accumulation of p27(kip1), cell cycle arrest, and cell death. PI3K modulated p27(kip1) levels at least in part by regulating its rate of degradation. Taken together, these data show that androgen ablation alone can increase PI3K-Akt activation, which supports survival after acute androgen ablation and proliferation during chronic androgen deprivation. Successful progression to the androgen-independent state in the LNCaP cell line model requires intact PI3K signaling.

Molecular determinants of cell death induction following adenovirus-mediated gene transfer of wild-type p53 in prostate cancer cells.

Adenoviral vectors expressing wild-type p53 (Ad-p53) induce apoptosis in different types of cancer cells. The therapeutic utility of Ad-p53 is now being evaluated in prostate-cancer patients. Bcl-2 is frequently expressed by prostate-cancer cells and has previously been shown to inhibit p53-mediated cell death following genotoxic stress. We studied the impact of bcl-2 on Ad-p53-induced cell death in human prostate-cancer cells. Human prostate-cancer cell lines LNCaP (p53 wt) and PC3 (p53 mut) were stably transfected with bcl-2. After p53 transduction, cell viability, apoptosis induction and modulation of specific apoptosis-regulatory proteins were assessed. LNCaP vector control and bcl-2-expressing cells underwent similar decreases in viability associated with apoptosis induction following Ad-p53 infection. Increased bcl-2 expression provided significant protection to PC3 cells transduced with Ad-p53. These findings are correlated with modulations in bax, bcl-2, bcl-x(L) and p21 protein levels. These data suggest that Ad-p53 may be useful in the treatment of some prostate cancers.