Human Prostate Epithelial Cell Line Research Articles

Triptonide acts as a novel antiprostate cancer agent mainly through inhibition of mTOR signaling pathway.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drugs in PCa therapy. Triptonide has been reported to have a strong inhibition activity in cancers through screening of Chinese herbal medicine. This study aims to investigate the effects of triptonide on anti-PCa activity and its mechanisms. Three human advanced PCa cell lines PC3, DU145, and LNCap, and a human normal prostate epithelial cell line RWPE were treated with a range (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 nM) of triptonide concentrations for 72 hours respectively. Then, cell viability was assessed by cell counting kit-8. PCa cells were treated with different doses (0-20 nM) of triptonide for 72 hours. Cell cycle and apoptosis were assessed by flow cytometry assays. Nude mice bearing human PCa xenografts were intraperitoneally injected daily with either triptonide (10 mg/kg/d) or phosphate-buffered saline as a control for 35 days. RNA-sequencing (RNA-seq) was performed by an Illumina Hiseq Sequencing platform and confirmed by a real-time polymerase chain reaction. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and ingenuity pathway analysis were used to analyze RNA-seq results. Triptonide effectively inhibits the proliferation of human PCa cells PC3, DU145, and LNCap in vitro with their IC50 values as 11.961, 10.259, and 12.012 nM, respectively. Triptonide (10 mg/kg) potently inhibits the growth of PCa cell xenografts in vivo at an inhibition rate of over 97.95%. Treatment with triptonide (5 nM) significantly promotes cell apoptosis and retaining cell-cycle arrest in the G2/M phase. RNA-seq data revealed that total of 936 genes were upregulated or downregulated in triptonide treated. Moreover, the phosphorylation of mechanistic target of rapamycin (mTOR) and the downstream protein p70S6K were both inhibited, most obviously in PCa cells. Our findings suggest that triptonide can efficaciously suppress PCa growth in vitro and in vivo via inhibiting the phosphorylation of mTOR and the activities of related downstream signaling pathways.

CID-6033590 inhibits p38MAPK pathway and induces S-phase cell cycle arrest and apoptosis in DU145 and PC-3 cells

Metastatic prostate cancer, with no effective treatment, is among the leading causes of cancer-associated deaths in men. Overexpression of p38αMAPK has been observed in neuroendocrine prostate cancer patients and in both DU145 and PC-3 cell lines and represents a good drug target. Sulfonamide derivatives have shown biological activities against many human diseases, including cancer. CID-6033590, a sulfonylhydrazide compound, screened from PubChem database by molecular docking with p38αMAPK, was evaluated for anti-cancerous activities. CID-6033590 induced toxicity in both DU145 and PC-3 cells in a concentration and time-dependent manner with an IC50 value of 60 μM and 66 μM, respectively. Sub-cytotoxic concentrations of the compound significantly induced S-phase cell cycle arrest, inhibited cyclinA/CDK2 complex and blocked cell proliferation. Further, CID-6033590 downregulated phosphorylation of p38MAPK (P-p38) as well as its downstream targets, Activating transcription factor 2 (ATF-2) and Heat shock protein 27 (Hsp27). The compound increased ROS and decreased mitochondrial membrane potential (Δψm), downregulated Bcl-2 and survivin and cleaved poly ADP ribose polymerase (PARP) and caspase-3, indicating the induction of apoptosis. The evaluaion of the compound on noncancerous, human prostatic epithelial cell line RWPE-1, and healthy murine tissues yielded no significant toxicity. Taken together, we suggest CID-6033590 as a potential candidate for prostate cancer therapy.

Corrigendum: Berberine Improves Benign Prostatic Hyperplasia via Suppression of 5 Alpha Reductase and Extracellular Signal-Regulated Kinase in Vivo and in Vitro.

[This corrects the article DOI: 10.3389/fphar.2018.00773.].

Overexpression of lncRNA GAS5 suppresses prostatic epithelial cell proliferation by regulating COX-2 in chronic non-bacterial prostatitis

ABSTRACT Chronic non-bacterial prostatitis (CNP) is a common urologic disease that is linked to the development of prostate cancer. Long non-coding RNA (lncRNA) GAS5 has been identified to mediate cell proliferation in prostate cancer, although its role in CNP is still unclear. Human prostate epithelial cell line RWPE-1 was induced by lipopolysaccharide (LPS) to mimic CNP model in vitro. Real-time PCR was performed to determine the expression of GAS5 and COX-2, while western blotting was used to evaluate the protein expression of COX-2. The interaction between GAS5 and COX-2 was determined using RNA pull-down and RNA immunoprecipitation (RIP). Cell proliferation was determined using MTT assay. The expression of GAS5 was decreased, while COX-2 was increased in prostatitis tissues and in LPS-induced RWPE-1 cells. The overexpression of GAS5 suppressed the protein level of COX-2, and inhibited cell proliferation of LPS-induced RWPE-1 cells and HPECs, which was rescued by the co-transfection with pcDNA-GAS5 and pcDNA-COX-2. GAS5 was confirmed to promote the ubiquitination of COX-2, and the in vivo GAS5-overexpressed CNP rat model decreased the motor scores, the volume of prostate tissues, the average number of inflammatory cells, prostatic proliferation, and COX-2 expression. Our findings revealed that overexpression of GAS5 inhibited cell proliferation via negatively regulating the expression of COX-2, thus alleviating the progression of CNP.

The role of NLRP3 inflammasome in regulating the progression of prostate cancer

Chronic inflammation is a hallmark of cancers and plays a critical role in modulating tumor progression and metastasis. Inflammasomes are a group of cytosolic protein complexes, composed of an NOD‐like protein (NLR), the adaptor apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and caspase‐1, which are responsible for the cleavage of pro‐IL‐1β and pro‐IL‐18 proteins into mature and biologically active IL‐1β and IL‐18, respectively. IL‐1β levels in tumor tissues were previously connected to poor prognosis for cancer patients. However, it remains unclear the function of inflammasomes in tumor growth and metastasis. The aim of this study is to examine whether the activation of NLRP3 inflammasome regulates the progression of prostate cancer. First, we screened the ability of NLRP3 inflammasome activation in prostate cells due to cancer heterogeneity. Prostate cell lines were primed with LPS, followed by treated with ATP or nigericin to induce NLRP3 inflammasome activation. Our results showed that the secretion of IL‐1β was significantly increased in PC3 (human prostate cancer cell line), BPH‐1 (human benign prostate hyperplasia epithelial cell line) and RWPE‐1 (human prostate epithelial cell line) cells. Second, we tested the effect of hypoxic condition in regulating the activation of NLRP3 inflammasome. Our results showed that hypoxia also induced the secretion of IL‐1β in PC3, BPH‐1 and RWPE‐1 cells. Third, we found that NLRP3 inflammasome activation involved in regulating tumor growth and metastasis. Our results indicated that the activation of NLRP3 inflammation plays an important role in modulating the progression of prostate cancer. Therefore, targeting NLRP3 inflammasome could be a strategy for prostate cancer therapy.Support or Funding InformationThis study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (Grant No. MOST 107‐2320‐B‐037‐019).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Transcription Factor ERG Regulates Super-Enhancers Associated With an Endothelial-Specific Gene Expression Program.

The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG's homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers. The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.

MiR-515-5p acts as a tumor suppressor via targeting TRIP13 in prostate cancer

MiR-515-5p has been suggested to function as tumor suppressor in various human cancers. However, the role of miR-515-5p in prostate cancer was still unclear. In this study, we observed miR-515-5p expression was reduced in prostate cancer tissues and prostate cancer cell lines compared with paired adjacent normal prostatic tissues and normal human prostate epithelial cell lines, respectively. Furthermore, we found miR-515-5p was negatively correlated with TRIP13 mRNA and protein expression in prostate cancer tissue samples, and miR-515-5p directly targeted TRIP13 3'-UTR and negatively regulated TRIP13 mRNA and protein expression. Moreover, miR-515-5p acted as a tumor suppressor to regulate cell proliferation, migration and invasion via targeting TRIP13 in prostate cancer. Besides, we observed that miR-515-5p expression in prostate cancer patients with advanced T stage subgroup or high Gleason score was significantly lower than its expression in prostate cancer patients with early T stage or low Gleason score. Survival analysis suggested that prostate cancer cases with high miR-515-5p expression had better prognosis than prostate cancer cases with low miR-515-5p expression. In conclusion, our study highlights the clinical and biological role of miR-515-5p as tumor suppressive microRNA in prostate cancer.

Botanical Formulation HX109 Ameliorates TP-Induced Benign Prostate Hyperplasia in Rat Model and Inhibits Androgen Receptor Signaling by Upregulating Ca2+/CaMKKβ and ATF3 in LNCaP Cells.

Benign prostatic hyperplasia (BPH) is a common disease in the elderly male population throughout the world. Among other factors, androgen dysregulation has been known to play major roles in its pathogenesis. HX109 is a botanical formulation prepared from a mixture of Taraxacum officinale, Cuscuta australis, and Nelumbo nucifera, which have traditionally been used—usually along with other plants—to treat urinary diseases. An ethanol extract was prepared from a mixture of these three plants, and its quality was controlled through cell-based bioassays and by quantification of several marker compounds by high-performance liquid chromatography (HPLC). In the testosterone propionate (TP)-induced prostate hyperplasia rat model, oral administration of HX109 ameliorated prostate enlargement and histological changes induced by TP. In LNCaP cells, a human prostate epithelial cell line, HX109 repressed AR-mediated cell proliferation and the induction of androgen receptor (AR) target genes at the transcriptional level without affecting the translocation or expression of AR. Such effects of HX109 on AR signaling were mediated through the control of activating transcriptional factor 3 (ATF3) expression, phosphorylation of calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ), and increases in intracellular calcium, as evidenced by data from experiments involving ATF3-specific siRNA, CaMKKβ inhibitor, and calcium chelator, respectively. Taken together, our data suggest that HX109 might be used as a starting point for developing therapeutic agents for the treatment of BPH.

AMD3100 inhibits epithelial-mesenchymal transition, cell invasion, and metastasis in the liver and the lung through blocking the SDF-1α/CXCR4 signaling pathway in prostate cancer.

Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have been found to be tightly correlated with the progression of prostate cancer (PC). In this study, we investigated the effects of an SDF-1α/CXCR4 inhibitor, AMD3100, on cell progression and metastasis potential of human PC cells. Human PC cell lines (LNCaP, PC3, and DU145) were cultured to detect SDF-1α/CXCR4, which showed higher SDF-1α and CXCR4 expression than the normal human prostate epithelial cell line, RWPE-1. AMD3100 was confirmed to be an inhibitor of SDF-1α, and to detect the effect of SDF-1α/CXCR4 inhibition on PC, PC cells were treated with AMD3100 or/and CXCR4 siRNA. The results suggested that inhibition of the SDF-1α/CXCR4 pathway could promote the E-cadherin level but inhibit the levels of invasion and migration of vimentin, N-cadherin and α5β1 integrin. Finally, tumor formation in nude mice was conducted, and the cell experiment results were verfied. These data show that AMD3100 suppresses epithelial-mesenchymal transition and migration of PC cells by inhibiting the SDF-1α/CXCR4 signaling pathway, which provides a clinical target in the treatment of PC.

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.

Berberine Improves Benign Prostatic Hyperplasia via Suppression of 5 Alpha Reductase and Extracellular Signal-Regulated Kinase in Vivo and in Vitro.

Benign prostate hyperplasia (BPH) is a common disease in elderly men, characterized by proliferated prostate and urinary tract symptoms. The hormonal cascade starting by the action of 5-alpha-reductase (5AR) is known to be one of the pathways responsible for the pathogenesis of BPH. Present investigation evaluated the capacity of berberine (BBR), a nature-derived compound abundant in Coptis japonica, in testosterone-induced BPH rats. Experimental BPH was induced by inguinal injection with testosterone propionate (TP) for 4 weeks. BBR or finasteride, a 5AR inhibitor as positive control, was treated for 4 weeks during BPH. BPH induced by TP evoked weight gaining and histological changes of prostate and BBR treatment improved all the detrimental effects not only weight reduction and histological changes but also suppression of prostate-specific antigen (PSA), which is elevated during BPH. Additionally, BBR suppressed TP-associated increase of 5AR, androgen receptor (AR) and steroid coactivator-1 (SRC-1), the key factors in the pathogenesis of BPH. To evaluate the underlying molecular mechanisms responsible for beneficial effects of BBR, we investigated whether these effects were associated with the mitogen-activated protein kinase pathway. BPH induced by TP showed increased phosphorylation of extracellular signal-regulated kinase (ERK), whereas this was suppressed by BBR treatment. On the other hand, c-jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase was not changed in BPH rats. In in vitro study using RWPE-1 cells, a human prostate epithelial cell line. TP increased cell proliferation and BPH-related key factors such as PSA, AR, and 5AR in RWPE-1 cells, and those factors were significantly decreased in the presence of BBR. Furthermore, these proliferative effects in RWPE-1cells were attenuated by treatment with U0126, an ERK inhibitor, confirming BBR can relieve overgrowth of prostate via ERK-dependent signaling. The cotreatment of U0126 and BBR did not affect the change of 5AR nor proliferation compared with U0126 alone, suggesting that the effect of BBR was dependent on the action of ERK. In conclusion, this study shows that BBR can be used as a therapeutic agent for BPH by controlling hyperplasia of prostate through suppression of ERK mechanism.

URG11 Regulates Prostate Cancer Cell Proliferation, Migration, and Invasion.

Upregulated gene 11 (URG11), a new gene upregulated by hepatitis B virus X protein, is involved in the development and progression of several tumors, including liver, stomach, lung, and colon cancers. However, the role of URG11 in prostate cancer remains yet to be elucidated. By determined expression in human prostate cancer tissues, URG11 was found significantly upregulated and positively correlated with the severity of prostate cancer, compared with that in benign prostatic hyperplasia tissues. Further, the mRNA and protein levels of URG11 were significantly upregulated in human prostate cancer cell lines (DU145, PC3, and LNCaP), compared with human prostate epithelial cell line (RWPE-1). Moreover, by the application of siRNA against URG11, the proliferation, migration, and invasion of prostate cancer cells were markedly inhibited. Genetic knockdown of URG11 also induced cell cycle arrest at G1/S phase, induced apoptosis, and decreased the expression level of β-catenin in prostate cancer cells. Overexpression of URG11 promoted the expression of β-catenin, the growth, the migration, and invasion ability of prostate cancer cells. Taken together, this study reveals that URG11 is critical for the proliferation, migration, and invasion in prostate cancer cells, providing the evidence of URG11 to be a novel potential therapeutic target of prostate cancer.

Monoamine Oxidase Deficiency Causes Prostate Atrophy and Reduces Prostate Progenitor Cell Activity.

Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5+ or p63+ basal cells, accompanied by lower Sca-1 expression in p63+ basal cells, but intact differentiated CK8+ luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133+ /CD44+ /CD24- stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance. Stem Cells 2018;36:1249-1258.

Tumor-associated myeloid cells promote tumorigenesis of non-tumorigenic human and murine prostatic epithelial cell lines

The etiology of prostate cancer is poorly understood, but it is a multi-step process that has been linked to environmental factors that induce inflammation within the gland. Glands of prostate cancer patients frequently contain multiple zones of disease at various stages of progression. The factors that drive disease progression from an indolent benign stage to aggressive disease are not well-defined. Prostate inflammation and carcinoma are associated with high levels of myeloid cell infiltration; these cells are linked to disease progression in other cancers, but their role in prostate cancer is unclear. To determine whether myeloid cells contribute to prostate cancer progression, the ability of prostate tumor-associated CD11b+ cells (TAMC) to drive prostate epithelial cell tumorigenesis was tested. Co-culture of CD11b+ TAMC with non-tumorigenic genetically primed prostate epithelial cells resulted in stable transformation and induction of tumorigenesis. RNA sequencing identified the IL-1α pathway as a potential molecular mechanism responsible for tumor promotion by TAMC. Inhibition of IL-1α delayed growth of TAMC-induced tumors. Further analysis showed that IL-1α inhibition led to decreased angiogenesis within tumors, suggesting that IL-1α promotes prostate tumor progression, potentially through augmentation of angiogenesis.

Highly Selective Photodynamic Therapy with a Short Drug-Light Interval Using a Cytotoxic Photosensitizer Porphyrus Envelope for Drug-Resistant Prostate Cancer Cells

Background: Photodynamic therapy (PDT) is a less invasive cancer treatment using photochemical reactions induced by light irradiation to a photosensitizer (PS). Highly selective PDT with fast accumulation of the PS in target site might be a promising treatment option for drug-resistant prostate cancer facing high incidence rate of elderly men who have no effective treatment options and require a minimally invasive treatment. Hemagglutinating virus of Japan envelope (HVJ-E) allows selective and fast drug delivery to the drug-resistant prostate cancer cells via rapid cell membrane fusion. PS named porphyrus envelope (PE) has been developed by insertion of lipidated protoporphyrin IX (PpIX lipid) into HVJ-E. In this study, we investigated the optimal conditions for PE preparation and laser irradiation for highly selective PDT using PE with a short drug-light interval. Materials and Methods: Human hormon refractory prostate cancer cell line PC-3 and human normal prostate epithelial cell line PNT2 were cultured. PpIX lipid uptake and cytotoxicity of PDT in the cells incubated with PE for 10 min were evaluated by measuring fluorescence intensity and by using a cell counting reagent 24 h after PDT, respectively. Results: PpIX lipid uptake and cytotoxicity of PDT were increased with PpIX lipid concentration. Cytotoxicity of PDT using PE was more than 9 times as strong as that with PpIX lipid and PpIX induced by 5-aminolevulinic acid. Much stronger cytotoxicity was induced in PC-3 cells than PNT2 cells with the ratio of cell death rate for cancer to normal cells up to 4.64 ± 0.09. Conclusions: Fast PS delivery with HVJ-E allows highly selective PDT with a short drug-light interval. Therefore, PDT using PE has a potential to shorten treatment period and reduce side effects of PDT.

Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway.

Notch1 is a type-1 transmembrane receptor which has been demonstrated to be involved in proliferation in various organisms. A number of studies have proposed that Notch signaling may be aberrantly activated, thus contributing to development, invasion and metastasis in a variety of human cancers. In the present study, the function and mechanism of Notch1 in human prostate cancer (PCa) LNCaP cells in vitro was investigated. Notch1 and cleaved-Notch1 expression were evaluated in human PCa cell lines, including LNCaP, PC-3 and DU 145, and the human prostate epithelial RWPE-1 cell line. LNCaP cells were transfected with Notch1-targeting short hairpin RNAs (shRNAs) and the level of proliferation, the ability to invade and the expression of genes associated with cancer cell invasion were subsequently investigated. Notch1 was highly expressed in LNCaP, PC-3 and DU 145 cells compared with RWPE-1 cells, while cleaved-Notch1 was expressed in LNCaP, PC-3 and DU 145 cells, and only to a minimal extent in RWPE-1 cells. Knockdown of Notch1 by shRNA in LNCaP cells markedly decreased cell invasion through Matrigel and inhibited cell proliferation 48 h following transfection. Reverse transcription-quantitative polymerase chain reaction analysis indicated that Notch1-knockdown resulted in a significant reduction of metastasis-associated 1 (MTA1) and increase of KiSS-1 metastasis-suppressor (KISS-1), mitogen-activated protein kinase 4 (MKK4) and cluster of differentiation 82 (KAI1). The present data demonstrated that expression of Notch1 was significantly associated with the invasion of prostate cancer. Knockdown of Notch1 decreased the invasive ability of LNCaP cells, which may be caused by downregulating MTA1 and upregulating KISS-1, MKK4 and KAI1. These findings indicated that targeting Notch1 may provide a novel method of suppressing or treating metastasis in prostate cancer.

Vanillic acid attenuates testosterone-induced benign prostatic hyperplasia in rats and inhibits proliferation of prostatic epithelial cells.

Benign prostatic hyperplasia (BPH) is a common disease in the male population, especially in elderly men. Vanillic acid (VA), a dihydroxybenzoic derivative used as a flavoring agent, is reported to have an anti-inflammatory effect. However, there are no reports of its effects on BPH to date. BPH was induced with a pre-4-week treatment of daily subcutaneous injections of testosterone propionate (TP), and the normal control group received injections of ethanol with corn oil instead. Six weeks of further injections were done with (a) ethanol with corn oil, (b) TP only, (c) TP + finasteride, and (d) TP + VA. Finasteride was used as a positive control group. VA had protective effects on the TP-induced BPH. In the VA treatment group, the prostate weight was reduced, and the histological changes including the epithelial thickness and lumen area were restored like in the normal control group. Furthermore, in the VA treatment group, two proliferation related factors, high molecular weight cytokeratin 34βE12 and α smooth muscle actin, were significantly down-regulated compared to the TP-induced BPH group. The expressions of dihydrotestosterone and 5α-reductase, the most crucial factors in BPH development, were suppressed by VA treatment. Expressions of the androgen receptor, estrogen receptor α and steroid receptor coactivator 1 were also significantly inhibited by VA compared to the TP-induced BPH group. In addition, we established an in vitro model for BPH by treating a normal human prostatic epithelial cell line RWPE-1 with TP. VA successfully inhibited proliferation and BPH-related factors in a concentration-dependent manner in this newly established model. These results suggest a new and potential pharmaceutical therapy of VA in the treatment of BPH.

Abstract 344: Chondroitin sulfatases regulate Wnt signaling through effects on Shp2, phospho-Erk1,2, c-Myc, and histone methylation of DKK3 in prostate cancer and prostate stem cells

Abstract The chondroitin sulfatases N-acetylgalactosamine 4-sulfatase (arylsulfatase B; ARSB) and N-acetylgalactosamine 6-sulfatase (galactose 6-sulfatase, GALNS) remove the 4-sulfate and 6-sulfate groups of chondroitin sulfates. ARSB acts to remove 4-sulfate groups of chondroitin 4-sulfate (C4S) or dermatan sulfate, and GALNS acts to remove 6-sulfate groups of chondroitin 6-sulfate (C6S) or chondroitin 4,6-disulfate, as well as keratan sulfate. In human prostate cancer tissues, ARSB activity was markedly reduced and GALNS activity increased. By immunohistochemistry of malignant prostate tissue captured by laser microdissection and by assay of human prostate stromal and epithelial cell lines, ARSB was predominant in stromal cells of the extracellular matrix, whereas GALNS predominated in prostate epithelial cells. When ARSB was reduced by silencing in human prostate stem cells or GALNS was increased by overexpression in the stem cells, SHP2 binding to chondroitin 4-sulfate increased, leading to inhibition of phosphatase action and sustained phosphorylation of Erk1,2. Erk activation enabled increase in c-Myc binding to nuclear DNA and increased expression of DNMT 1a, 3a, and 3b. DNMT activation increased following ARSB silencing or GALNS overexpression, and was blocked by ERK inhibition. Increased DNMT activity led to the increased promoter methylation of DKK3, and the subsequent inhibition of nuclear β-catenin nuclear translocation and Wnt signaling. Decline in Wnt signaling was manifested by reduced TCF-LEF nuclear binding, and decline in the mRNA expression of c-Myc and GATA. Since c-Myc activation can lead to increased expression of cell cycle regulators, the changes in chondroitin sulfation mediated by sulfatases can lead to profound effects in cell proliferation. Hence, the modification of sulfatase activity, manifested as increased chondroitin 4-sulfate, provides the platform for an extensive extracellular-intracellular signaling network that can regulate phosphorylation, DNA promoter methylation, and proliferation, and affect development and malignant transformation. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Chondroitin sulfatases regulate Wnt signaling through effects on Shp2, phospho-Erk1,2, c-Myc, and histone methylation of DKK3 in prostate cancer and prostate stem cells [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 344. doi:10.1158/1538-7445.AM2017-344

Inhibition of autophagy aggravated 4-nitrophenol-induced oxidative stress and apoptosis in NHPrE1 human normal prostate epithelial progenitor cells

4-Nitrophenol (PNP), a well-established human carcinogen, has been proven to have detrimental effects on reproductive system of male rats in previous studies. The molecular mechanisms involved PNP-induced damage remain to be established. Autophagy can exert protective effects on various cytotoxic factors that induce injury. In the present study, we aim to investigate whether autophagy is induced by PNP and the function of autophagy in PNP-induced injury in NHPrE1, a normal human prostate epithelial progenitor cell line. Our results indicate that PNP induced oxidative stress as evidenced by increased MDA levels and decreased activity of SOD and GSH-Px. PNP also increased apoptosis of NHPrE1 cells as evidenced by western blot and Hoechst 33258 staining and activated autophagy in NHPrE1 cells detected by RT-PCR and western blot. Inhibition of autophagy by 3-MA further increased PNP-induced oxidative stress and apoptosis of NHPrE1 cells. We also found that PNP-induced apoptosis was suppressed by N-acetylcysteine, suggesting oxidative stress may play an important role in PNP cytotoxicity. Furthermore, phosphorylation of mTOR protein was inhibited by PNP, indicating that PNP might induce autophagy in NHPrE1 cells via inhibiting mTOR pathway. In conclusion, these results suggest that activation of autophagy should play a protective role in PNP-induced oxidative stress and apoptosis of NHPrE1 cells, which might be mediated through mTOR pathway.

Zinc Induces Lipopolysaccharide-Mediated Upregulation of Human β-defensin-2 in Human Prostate Epithelial Cells

Zinc is considered to be one of the most essential trace minerals for the immune system. High levels of prostatic zinc are associated with prostatic antimicrobial activities. Zinc-deficient individuals experience increased susceptibility to a variety of pathogens. Zinc appears to play a crucial role in maintaining prostate health. This study aimed to clarify the role of zinc in human prostate epithelial cell defense against bacterial infection. To explore the effect of zinc on lipopolysaccharide (LPS)-mediated induction of human β-defensin-2 (HBD-2), the normal human prostate epithelial cell lines (RWPE-1) were co-treated with zinc/LPS and HBD-2 mRNA expression was quantitated by the reverse transcription polymerase chain reaction (RT-PCR). We also conducted a Western blot analysis to determine whether zinc stimulates p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To investigate the involvement of the p38MAPK and ERK1/2 signaling pathways in zinc-mediated upregulation of HBD-2, quantitative real-time PCR and immunocytochemical staining were then used to quantify HBD-2 mRNA expression and protein production, respectively, which was treated with either U0126 (ERK1/2 inhibitor) or SB203580 (p38MAPK inhibitor) prior to each analysis of HBD-2.