Lymph Node Carcinoma Of The Prostate Research Articles

Abstract 5682: Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors

Abstract INTRODUCTION: Prostate Cancer (PCa) is the most common non-cutaneous cancer and the second leading cause of cancer death among men in the United States. Most of the PCa cases are diagnosed in men between the ages of 65-74. Due to the recent push for frequent prostate screenings, these cancers are being detected early and treated much faster, leading to a 5-year survival of a localized or regional PCa to >99%. However, the 5-year survival of PCa with distant metastasis is significantly reduced to 32%, especially with involvement of osteoblastic bone lesions, lung lesions, and brain lesions. Therefore, there is a clinical need to identify prognostic markers and new targets for developing therapies and effectively treating advanced PCa. Previous studies have shown that levels of MDMX and MDM2, oncogenic inhibitors of p53 tumor suppressor protein, are significantly elevated in PCa cells. Inhibition of MDM2 and MDMX using specific inhibitors has been shown to elevate the levels of p21, p27, and p53 in LNCaP (Lymph Node Carcinoma of the Prostate) cells leading to cell death. Recent studies have demonstrated that micro RNAs (miRNAs) play an important role in supporting the tumor suppressor function of p53 through regulating the balance between p53 and MDM2. In this regard, several miRNAs were found to be significantly altered to modulate the level and intracellular mechanisms of p53 and MDM2 in various cancers. OBJECTIVES: To determine the relative levels of miRNA31, miRNA-34a and miRNA-128 in LNCaP cells that were treated with NSC-207895 (MDMX inhibitor), SJ-172550 (MDMX inhibitor), and RG-7388 (MDM2 inhibitor). METHODS: LNCaP cells were treated with NSC-207895 (20 uM), SJ-172550 (20 uM) or RG-7388 (2 uM) for 24 hrs. After completing the treatments, the miRNAs were extracted from the LNCaP cells using the miRNeasy Tissue/Cells Advanced Kit from Qiagen. We analyzed the levels of miRNA-31, miRNA-34a, and miRNA-128 in the treated samples using qRT-PCR analysis method and compared the levels to the control. During the qRT-PCR analysis the cDNA was amplified using Meridian Biosciences qPCR Master Mix and specific primers for miRNAs from Origene were used according to the manufacturer's protocol. RESULTS: Our experimental results indicate that inhibition of MDMX using NSC-207895 and SJ-172550 can elevate the levels of p21, p27, and p53 leading to cell death. Determination of cell death markers in the past revealed induction of necroptosis following drug treatments. Our current results indicate measurable changes in the levels of miRNA-31 and miRNA-34a following inhibition of MDMX using SJ-172550. We are in the process of determining the possible link between the changes in the levels of miRNAs and cell death. ACKNOWLEDGEMENT: This research was supported by the Royal Dames of Cancer Research, Ft. Lauderdale, Florida. Citation Format: Rojin Fatirkhorani, Ahmed Saleh Alsarrani, Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5682.

Antioxidant, AChE inhibitory, and anticancer effects of Verbascum thapsus extract.

Verbascum thapsus (VT) is a medicinal plant that is used in folk medicine to treat a variety of ailments. For this study, the biological functions of VT methanol extract were determined in vitro. The plant's methanol extract was created through the maceration process. The phytochemical composition of plant extracts was investigated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The antioxidant capacity of the extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazil) and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) tests and its cytotoxicity was assessed using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)) assay on the Caco-2 (human colorectal adenocarcinoma cells), LNCaP (Lymph Node Carcinoma of the Prostate), and HEK293 cell lines (Human embryonic kidney 293 cells) used to model colon, prostate, and non-cancerous cells. VT extract showed low DPPH and ABTS radical scavenging activities compared to standard antioxidants at 30 mg/ml concentration. In addition, it was determined that VT extract inhibited acetylcholinesterase enzyme.

In Vitro Cytotoxicity Efficiency of Extracellular L-Glutaminase Produce by Mesophilic Bacteria, Kurthia Gibsonii on Lncap, MDA-MB 231 and Hepg-2 Cell Lines

Mesophilic bacteria from soil habitat have been reported to produce extracellular L-glutaminase. The present study was carried out to anticancer screening of L-glutamiase producing bacteria (Kurthia gibsonii) from soil sample of cattle feeding site from Satara parisar, Chhatrapati Sambhajinagar. Results showed, among three soil samples of cattle feeding farms, Kurthia gibsonii was isolated. From that exhibited the highest L-glutaminase activity. Moreover, the in vitro cytotoxic activity of L-glutaminase against the (Lymph Node Carcinoma of the Prostate) LNCaP, (an epithelial, human breast cancer cell line) MDA-MB 231 and hepatocellular (HepG-2) carcinoma cell lines at different concentration (0.47, 0.94, 1.88, 3.75, 7.50, 15.00, 30.00 and 60.00 μg/ml) by the MTT assay and compared with the standard Doxrubcin. The antitumor effect against human liver carcinoma cell line revealed that L-glutaminase produced by Kurthia gibsonii showed potent cytotoxic activity of tested cell line in a dose-dependent manner with an LC50 value of 4.1 μg/ml.

Chemical composition and antiproliferative activity on prostate and cervical cancer cell lines of Lantana camara Linn. essential oil

Lantana camara (LC) is an aromatic plant used in traditional medicine in Burkina Faso in association with other plants for the management of many microbial and inflammatory pathologies. The objective of this study was to determine the chemical composition, the antioxidant and antitumor activities on prostate and cervical cancer cells in culture of the essential oil (EO) of this plant. The chemical composition of Lantana camara EO was determined by Gas Chromatography with Flame-Ionization Detection (GC-FID) and Gas Chromatography–Mass spectrometry (GC-MS) analysis and the antiproliferative activity was evaluated through the 3[4.5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) test assay. A total of 48 compounds have been identified in the essential oil of Lantana camara of which cabinene, beta-caryophyllene, eucalyptol and alpha-humulene are found to be the majority. However, 4 compounds, unknown in the literature, present in this essential oil have not been identified. Lantana camara essential oil inhibits both the proliferation of HeLa cells from cervical cancer and LNCaP (Lymph Node Carcinoma of the Prostate) cells from prostate cancer. Beyond constituting the scientific basis for the use of Lantana camara in traditional medicine through its antiproliferative effect. These results require further study by determining the nature of its unidentified compounds and its mechanism of molecular action on these two cancer cell lines in vitro.

The effect of encomir-93 mimic transfection on the expression of miR-93 and PSA and androgen receptor in prostate cancer LNcap cell line.

Prostate cancer (PCa) is one of the most common cancers in men with high mortality rate which is a major concern for men's health. However, the molecular mechanisms remain poorly understood. miR-93 is an important oncogene which may have important function inprostate cancer.So, this study aimed to predict that encomir-93 mimic transfection on the expression of miR-93 and PSA and AR in prostate cancer LNcap cell line. Lymph node carcinoma of the prostate (LNCaP) was cultured and then miR-93 mimics wasdesigned, synthesized and the transfected to LNCaP. Theexpression level of prostate-specific antigen (PSA) andandrogen receptor (AR) was determined via Real-time PCR after treated with 15pmol of miR-93 mimics. miR-93 mimic transfection led to significant increase in PSA and AR expression in comparison with control group (p≤0.05). The miR-93 and its target genes has important role in PCa progression via enhancement in PSA and AR expression. Further research on the function of the miR-93 and its target genes in tumorgenesis and progression PCa could be helpful for the treatment of prostate cancer.

Protective effects of Yerba mate (IIex paraguariensis) on prostate cancer development

ObjectivesProstate cancer (PCa) is the most common adenocarcinoma in men >50 y of age. It has a long latency period, which provides time for preventive strategies like incorporating healthy eating habits. Yerba mate (YM) intake has been associated with numerous health benefits. Since YM is one of the most popular infusions in Argentina, the of this study was to examine the influence of YM on PCa development. MethodsWe carried out an in vivo model of PCa through subcutaneous inoculation of transgenic adenocarcinoma of the mouse prostate-C1 cells in C57BL/6 mice. Subsequently, the animals were divided into two groups: mate (25 mg/mL of YM in drinking water, n = 15), and control (only drinking water, n = 15). We also developed an in vitro model to study the direct effects of YM on three human PCa cell lines: lymph node carcinoma of the prostate (LNCaP), PC-3, and DU-145. ResultsOur in vivo model showed that YM intake slightly reduced body weight, increased the latency of tumor appearance (P <0.01), and diminished the tumor volume (P <0.05) compared with the control group. In agreement, the expression of proliferating cell nuclear antigen, and nuclear estrogen receptor α were lower in the tumors of the mate animals (P <0.05). In vitro, YM decreased the viability, proliferation, and adhesion of the three tumor cell lines (P < 0.001) and retarded the migration of LNCaP (P <0.05) and DU-145 (P <0.005), without modifying the migration of PC-3 cells. ConclusionsYM showed anticancer effects in vitro and in vivo and were more effective on the androgen-sensitive cell line (LNCaP).

Study on essential oil, antioxidant activity, anti-human prostate cancer effects, and induction of apoptosis by Equisetum arvense

Abstract In this study, we have reported the chemical composition of Equisetum arvense essential oil and the anti-cancer activity of the plant against the prostate cancer cell line. The essential oil was obtained using the hydro-distillation assay. The chemical composition was identified using the gas chromatographic methods including gas chromatography/flame ionization detector and gas chromatography/mass spectrometry. The antioxidant activity of the essential oil and extract was evaluated using classical methods. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was run to evaluate the cytotoxic effect of the essential oil and extract on the prostate cancer cell line of lymph node carcinoma of the prostate. The induction apoptosis of the extract was analyzed by a flow cytometer. Thymol acetate (14.7%), trans-carveol (12.5%), thymol (11.8%), and δ-elemene (9.4%) were identified as the main compounds for the essential oil. The extract scavenged the free radical of 2,2-diphenyl-1-picrylhydrazyl with a half maximal inhibitory concentration (IC50) of 15.2 ± 1.4 μg/mL for the plant extract. In the MTT assay, the IC50 of the extract and essential oil were 25.2 ± 0.3 and 218.9 ± 10.7 μg/mL after 72 h. The highest apoptosis was 31.6% for the plant extract. The obtained results of the present study revealed that E. arvense can be introduced as a potent agent to prevent the growth of prostate tumors.

Refined Chelator Spacer Moieties Ameliorate the Pharmacokinetics of PSMA-617.

Prostate-specific membrane antigen (PSMA) binding tracers are promising agents for the targeting of prostate tumors. To further optimize the clinically established radiopharmaceutical PSMA-617, novel PSMA ligands for prostate cancer endoradiotherapy were developed. A series of PSMA binding tracers that comprise a benzyl group at the chelator moiety were obtained by solid-phase synthesis. The compounds were labeled with 68Ga or 177Lu. Competitive cell-binding assays and internalization assays were performed using the cell line C4-2, a subline of the PSMA positive cell line LNCaP (human lymph node carcinoma of the prostate). Positron emission tomography (PET) imaging and biodistribution studies were conducted in a C4-2 tumor bearing BALB/c nu/nu mouse model. All 68Ga-labeled ligands were stable in human serum over 2 h; 177Lu-CA030 was stable over 72 h. The PSMA ligands revealed inhibition potencies [Ki] (equilibrium inhibition constants) between 4.8 and 33.8 nM. The percentage of internalization of the injected activity/106 cells of 68Ga-CA028, 68Ga-CA029, and 68Ga-CA030 was 41.2 ± 2.7, 44.3 ± 3.9, and 53.8 ± 5.4, respectively; for the comparator 68Ga-PSMA-617, 15.5 ± 3.1 was determined. Small animal PET imaging of the compounds showed a high tumor-to-background contrast. Organ distribution studies revealed high specific uptake in the tumor, that is, approximately 34.4 ± 9.8% of injected dose per gram (%ID/g) at 1 h post injection for 68Ga-CA028. At 1 h p.i., 68Ga-CA028 and 68Ga-CA030 demonstrated lower kidney uptake than 68Ga-PSMA-617, but at later time points, kidney time–activity curves converge. In line with the preclinical data, first diagnostic PET imaging using 68Ga-CA028 and 68Ga-CA030 revealed high-contrast detection of bone and lymph node lesions in patients with metastatic prostate cancer. The novel PSMA ligands, in particular CA028 and CA030, are promising agents for targeting PSMA-positive tumor lesions as shown in the preclinical evaluation and in a first patient, respectively. Thus, clinical translation of 68Ga-CA028 and 68Ga/177Lu-CA030 for diagnostics and endoradiotherapy of prostate cancer in larger cohorts of patients is warranted.

A novel biorecognition receptor Citropin-A modified impedimetric biosensor for detection of LNCaP prostate cancer cells

In this study, Citropin-A (Cit-A) as a biorecognition receptor was used for the first time to develop electrochemical impedance spectroscopy (EIS) based biosensor for the detection of Lymph Node Carcinoma of the Prostate (LNCaP) cancer cells. The biosensor was engineered by modification of a gold electrode (AuE) with cysteamine (Cys), Poliamidoamin (PAMAM (G4)) dendrimers, avidin, and biotinylated Cit-A, respectively. The detection time of the LNCaP cells was determined as 300 s by chronoimpedance (CI). Chronoimpedance also provided an exact detection time to avoid non-specific adsorptions. The biosensor showed good linearity between 1500 cells/L and 12000 cells/L, limit of detection (LOD), and limit of quantification (LOQ) values were 518 cells/L and 1570 cells/L, respectively.

Inflammation induced by lipopolysaccharide advanced androgen receptor expression and epithelial-mesenchymal transition progress in prostatitis and prostate cancer.

BackgroundTo explore the mechanism of prostatic inflammation on prostate cancer (PCa) by comparing the changes of prostate epithelial cells and PCa cells in an inflammatory environment.MethodsFirst, immunohistochemistry (IHC) was used to compare the level of expression of TNF-α, IL-1β, IL-6, and TGF-β between benign prostatic hyperplasia (BPH), prostatitis, and PCa. Then primary prostate epithelial cells were sampled from patients who were suspected of PCa and had histological prostatitis (HP) confirmed by pathological biopsy. Lipopolysaccharide (LPS) or BAY11-7082 were used to investigate the change of androgen receptor (AR) and AR-mediated transcription, epithelial-mesenchymal transition (EMT) in primary prostate epithelial cells, and lymph node carcinoma of the prostate (LNCap) cells.ResultsTNF-α, IL-1β, IL-6, and TGF-β were significantly increased in HP and PCa compared with those in BPH patients. The proliferation of primary prostate epithelial cells and LNCap cells got the inflection point at LPS 10 µg/mL. In an inflammatory environment with 10 µg/mL LPS, both primary prostate epithelial cell and LNCap cell viability increased, and AR, AR-mediated transcription, and EMT processes were significantly increased. Inhibitors of NF-κB with 10 nM BAY11-7082 decreased AR, AR-mediated transcription, and EMT processes.ConclusionsNF-κB regulates AR expression and EMT in prostatitis and PCa, and NF-κB inhibitors may have potential therapeutic value.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Standardization of the [68Ga]Ga-PSMA-11 Radiolabeling Protocol in an Automatic Synthesis Module: Assessments for PET Imaging of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a glycoprotein present in the prostate, that is overexpressed in prostate cancer (PCa). Recently, PSMA-directed radiopharmaceuticals have been developed, allowing the pinpointing of tumors with the Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) imaging techniques. The aim of the present work was to standardize and validate an automatic synthesis module-based radiolabeling protocol for [68Ga]Ga-PSMA-11, as well as to produce a radiopharmaceutical for PET imaging of PCa malignancies. [68Ga]Ga-PSMA-11 was evaluated to determine the radiochemical purity (RCP), stability in saline solution and serum, lipophilicity, affinity to serum proteins, binding and internalization to lymph node carcinoma of the prostate (LNCaP) cells, and ex vivo biodistribution in mice. The radiopharmaceutical was produced with an RCP of 99.06 ± 0.10%, which was assessed with reversed-phase high-performance liquid chromatography (RP-HPLC). The product was stable in saline solution for up to 4 h (RCP > 98%) and in serum for up to 1 h (RCP > 95%). The lipophilicity was determined as −3.80 ± 0.15, while the serum protein binding (SPB) was <17%. The percentages of binding to LNCaP cells were 4.07 ± 0.51% (30 min) and 4.56 ± 0.46% (60 min), while 19.22 ± 2.73% (30 min) and 16.85 ± 1.34% (60 min) of bound material was internalized. High accumulation of [68Ga]Ga-PSMA-11 was observed in the kidneys, spleen, and tumor, with a tumor-to-contralateral-muscle ratio of >8.5 and a tumor-to-blood ratio of >3.5. In conclusion, an automatic synthesis module-based radiolabeling protocol for [68Ga]Ga-PSMA-11 was standardized and the product was evaluated, thus verifying its characteristics for PET imaging of PCa tumors in a clinical environment.

Val16A SOD2 Polymorphism Promotes Epithelial–Mesenchymal Transition Antagonized by Muscadine Grape Skin Extract in Prostate Cancer Cells

Epithelial–mesenchymal transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration, and can be induced by reactive oxygen species (ROS). Val16A (Ala) SOD2 polymorphism has been associated with increased prostate cancer (PCa) risk. We hypothesized that SOD2 Ala single nucleotide polymorphism (SNP) may promote EMT. We analyzed SOD2 expression and genotype in various prostate cell lines. Stable overexpression of Ala-SOD2 or Val-SOD2 allele was performed in Lymph Node Carcinoma of the Prostate (LNCaP) cells followed by analysis of intracellular ROS and EMT marker protein expression. Treatments were performed with muscadine grape skin extract (MSKE) antioxidant, with or without addition of H2O2 to provide further oxidative stress. Furthermore, MTS cell proliferation, cell migration, and apoptosis assays were completed. The results showed that SOD2 expression did not correlate with tumor aggressiveness nor SOD2 genotype. We demonstrated that the Ala-SOD2 allele was associated with marked induction of EMT indicated by higher Snail and vimentin, lower E-cadherin, and increased cell migration, when compared to Val-SOD2 allele or Neo control cells. Ala-SOD2 SNP cells exhibited increased levels of total ROS and superoxide and were more sensitive to co-treatment with H2O2 and MSKE, which led to reduced cell growth and increased apoptosis. Additionally, MSKE inhibited Ala-SOD2 SNP-mediated EMT. Our data indicates that treatment with a combination of H2O2-generative drugs, such as certain chemotherapeutics and antioxidants such as MSKE that targets superoxide, hold promising therapeutic potential to halt PCa progression in the future.

Ectopically expressed olfactory receptors OR51E1 and OR51E2 suppress proliferation and promote cell death in a prostate cancer cell line

Olfactory receptors (ORs), the largest family of G protein–coupled receptors, are expressed in the nasal epithelium where they mediate the sense of smell. However, ORs are also found in other non-nasal tissues, but the role of these ectopic ORs in cell signaling, proliferation, and survival is not well understood. Here, using an inducible expression system in the lymph node carcinoma of the prostate (LNCaP) cell line, we investigated two ectopic ORs, OR51E1 and OR51E2, which have been shown to be upregulated in prostate cancer. We found that, consistent with previous studies, OR51E1 stimulated adenylyl cyclase in response to treatment by short-chain to medium-chain organic acids (C3–C9) but not by acetate. OR51E2 responded to acetate and propionate but not to the longer chain organic acids. Stimulation of LNCaP cells with butyrate inhibited their growth, and the knockdown of the endogenous OR51E1 negated this cytostatic effect. Most significantly, overexpression of OR51E1 or OR51E2 suppressed LNCaP cell proliferation. Overexpression of another ectopic OR OR2AT4, β2-adrenergic receptor, or treatment of cells with forskolin did not suppress cell proliferation, indicating that a rise in cAMP is not sufficient to induce cytostasis. Overexpression of OR51E1 caused an upregulation of cytostatic and cell death markers including p27, p21, and p53, strongly increased annexin V staining, and stimulated extracellular signal–regulated protein kinases 1 and 2. Overexpression and/or activation of OR51E1 did not affect human embryonic kidney 293 cell proliferation, indicating that cytotoxicity of OR51E1/OR51E2 is specific for LNCaP cells. Together, our results further our understanding of prostate cancer etiology and suggest that ectopic ORs may be useful therapeutic targets.

Epigallocatechin-3-gallate Enhances the Efficacy of MicroRNA-34a Mimic and MiR-93 Inhibitor Co-transfection in Prostate Cancer Cell Line

The potential role of microRNAs (miRNA) as therapeutic molecules has moved them from basic research to the field of cancer therapy. High expression of miR-93 and low expression of miR-34a have previously been indicated in prostate cancer (PC), which is the second leading cause of cancer-related death in men. Androgen receptor (AR) and prostate-specific antigen (PSA) play key roles in the initiation and progression of this cancer. Therefore, this study aimed to investigate the effects of the transfection and co-transfection of miR-34a mimic and miR-93 inhibitor with or without epigallocatechin-3-gallate (EGCG) on prostate cancer cell line and also to evaluate their effects on the expression of AR, PSA. Human lymph node carcinoma of the prostate (LNCaP) cells were treated with miR-34a mimic or/and miR-93 inhibitor with or without EGCG. Gene or protein expressions were assessed by real-time PCR or western blotting of lysates. The transfection with miR-34a mimics significantly reduced the mRNA expression of AR (p=0.0016), and PSA (p=0.038) compared to the control. Also, the miR-93 inhibitor led to a decrease in the mRNA expression of AR (p=0.0057) and PSA (p&gt;0.05) compared to the control group. Furthermore, the co-transfection, along with EGCG, caused more decrease in both the AR (p&lt;0.001) and the PSA (p=0.003) expression compared with the co-transfection without EGCG. Our study indicates that the reduced expression of AR and PSA in PC cells followed by treatment with miR-34a mimic and miR-93 inhibitor and their combination with EGCG as a natural substance may be a promising therapeutic way for controlling the growth of these malignant cells.

Androgen Deprivation Induces Transcriptional Reprogramming in Prostate Cancer Cells to Develop Stem Cell-Like Characteristics.

Enzalutamide, an antiandrogen, is approved for therapy of castration resistant prostate cancer. Clinical applications have shown that approximately 30% of patients acquire resistance after a short period of treatment. However, the molecular mechanisms underlying this resistance is not completely understood. To identify transcriptomic signatures associated with acquisition of drug resistance we profiled gene expression of paired enzalutamide sensitive and resistant human prostate cancer LNCaP (lymph node carcinoma of the prostate) and C4-2B cells. Overlapping genes differentially regulated in the enzalutamide resistant cells were ranked by Ingenuity Pathway Analysis and their functional validation was performed using ingenuity knowledge database followed by confirmation to correlate transcript with protein expression. Analysis revealed that genes associated with cancer stem cells, such as POU5F1 (OCT4), SOX2, NANOG, BMI1, BMP2, CD44, SOX9, and ALDH1 were markedly upregulated in enzalutamide resistant cells. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with RUNX2, hedgehog, integrin signaling, and molecules associated with elastic fibers. Further examination of a patient cohort undergoing ADT and its comparison with no-ADT group demonstrated high expression of POU5F1 (OCT4), ALDH1, and SOX2 in ADT specimens, suggesting that they may be clinically relevant therapeutic targets. Altogether, our approach exhibits the potential of integrative transcriptomic analyses to identify critical genes and pathways of antiandrogen resistance as a promising approach for designing novel therapeutic strategies to circumvent drug resistance.

Roles of Interleukin-1 Receptor Antagonist in Prostate Cancer Progression.

Background: Inflammation is known to promote tumor formation and progression; however, we found a natural anti-inflammatory factor, interleukin (IL)-1 receptor antagonist (IL1RN), in a mouse transgenic adenocarcinoma of the mouse prostate (TRAMP)-C1-derived tumor microenvironment (TME). We sought to characterize the functions of the IL1RN-secreting cells in the TME. Methods: We compared tumors collected from two syngeneic mouse models and isolated tumor-infiltrating leukocytes (TILs) with different cluster of differentiation 11b (CD11b) statuses. We examined the proliferation functions of the TILs and the IL1RN using several approaches, including a colony-formation assay and DNA synthesis levels. Results: We demonstrated that CD11b-deficient TILs (TILs/CD11b−) secreted the IL1RN and promoted proliferation by analyzing conditioned media. In addition to mouse TRAMP-C1, proliferation functions of the IL1RN were confirmed in several human castration-resistant prostate cancer (CRPC) cell lines and one normal epithelial cell line. The androgen-sensitive lymph node carcinoma of the prostate (LNCaP) cell line showed cytotoxic responses to IL1β treatment and androgen-dependent regulation of IL-1 receptor type 1 (IL1R1), while the C4-2 CRPC cell line did not. IL1RN rescued LNCaP cells from the cytotoxic effects of IL1β/IL1R1 signaling. Conclusions: Our results support TILs/CD11b− cells being able to protect androgen-dependent cells from inflammatory damage and promote the malignant progression of prostate cancers partly through the IL1RN in the TME.

Analysis of novel enzalutamide-resistant cells: upregulation of testis-specific Y-encoded protein gene promotes the expression of androgen receptor splicing variant 7.

BackgroundEnzalutamide, a second-generation antiandrogen, is an approved medicine for the treatment of metastatic castration-resistant prostate cancer (CRPC); however, the mechanisms behind the resistance are not completely understood. In the present study, we established enzalutamide-resistant cells derived from lymph node carcinoma of the prostate (LNCaP) cells and characterized their androgen receptor (AR) status and changes in the gene expression with an aim to elucidate these mechanisms.MethodsSAS MDV No. 3–14 enzalutamide-resistant cells were established from LNCaP xenograft castrated male mice under continuous administration of enzalutamide. Then, the AR status and expression of AR target genes were evaluated by western blotting or real-time polymerase chain reaction analysis. The role of AR in the proliferation was also analyzed using the AR siRNA approach. The gene expression profiling in SAS MDV No. 3–14 cells was evaluated by microarray analysis. The role of testis-specific Y-encoded protein (TSPY), one of the upregulated genes, in the expression of AR and AR target genes and cell growth was also verified using siRNA.ResultsSAS MDV No. 3–14 cells expressed AR-v7, leading to the increased expression of AR target genes. Gene silencing of AR showed that both AR-FL and AR-v7 function as proliferative drivers in SAS MDV No. 3–14 cells. Microarray analysis revealed that TSPY is upregulated genes in these cells. TSPY siRNA inhibited cell proliferation, decreased the expression of AR-v7 and AR-v7 targeted genes.ConclusionsThis study demonstrated that SAS MDV No. 3–14 cells increase the expression of AR-v7 by upregulating TSPY, leading to acquired resistance to enzalutamide.

CD147 promotes epithelial‑mesenchymal transition of prostate cancer cells via the Wnt/β‑catenin pathway

The majority of deaths among patients with prostate cancer (PCa) occur following metastasis; therefore, there is a critical need for effective treatment of metastatic PCa. Epithelial-mesenchymal transition (EMT) is vital in the early stage of cancer cell metastasis and CD147 has been reported to be associated with various types of cancer. The goal of this study was to investigate the role of CD147 in the EMT of PCa cells via short hairpin (sh)RNA-mediated knockdown of CD147 in lymph node carcinoma of the prostate (LNCaP) cells. Reverse transcription-quantitative PCR and western blotting were performed to examine gene and protein expression. Cell migration and invasion were detected using a Transwell assay. Cell Counting Kit-8 assay was performed to investigate cell viability. The knockdown of CD147 in LNCaP cells (LNCaP/shCD147 cells) resulted in an increase in the expression of E-cadherin (an epithelial marker), and a decrease in the expression of N-cadherin and vimentin (mesenchymal markers). Importantly, the downregulation of CD147 in LNCaP cells inhibited the expression levels of nuclear β-catenin and Snail, and phosphorylation of glycogen synthase kinase (GSK)-3β on Ser 9, and increased the expression of phosphorylated (p)-β-catenin (Ser33/37/Thr41). Treatment with lithium chloride (LiCl), a Wnt/β-catenin pathway agonist or a GSK-3β inhibitor, attenuated CD147 downregulation-induced p-β-catenin (Ser33/37/Thr41) expression, which resulted in the upregulation of β-catenin in the nucleus. LiCl treatment prompted β-catenin-mediated expression of target proteins such as Snail and vimentin in LNCaP/shCD147 cells, and prevented E-cadherin expression, a molecule downstream to Snail. In conclusion, these findings revealed an important role of CD147 in the regulation of the invasive and metastatic potential of PCa cells. CD147, via modulation of the Wnt/β-catenin pathway, may be implicated in the regulation of EMT of PCa cells and could be a potential therapeutic target for PCa.

The Effect of Arbutin on The Expression of Tumor Suppressor P53, BAX/BCL-2 Ratio and Oxidative Stress Induced by Tert-Butyl Hydroperoxide in Fibroblast and LNcap Cell Lines.

Objective Arbutin (p-hydroxyphenyl-β-D-glucopyranoside) possesses beneficial functions including antioxidant, anti- inflammatory, and anti-tumoral activities. Due to the important role of oxidative stress and apoptosis in the successful treatment of cancer, understanding mechanisms that lead to apoptosis in cancer cells, is essential. The purpose of the current study was to evaluate the effect of arbutin on tert-butyl hydroperoxide (t-BHP)-induced oxidative stress and the related mechanisms in fibroblast and Lymph Node Carcinoma of the Prostate (LNCaP) cells. Materials and Methods In this experimental study, the LNCaP and fibroblast cell lines were pre-treated with arbutin (50, 250 and 1000 μM). After 24 hours, t-BHP (30 and 35 μM) was added to the cells. Viability was measured (at 24 and 48 hours) using MTT assay. The antioxidant effect of arbutin was measured by FRAP assay. The mRNA expression of P53 and BAX/BCL-2 ratio were measured using quantitative polymerase chain reaction (PCR). The percentage of apoptotic or necrotic cells was determined using a double staining annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit. Results Arbutin pre-treatment increased the total antioxidative power and cell viability in the MTT assay and reduced BAX/BCL-2 ratio, P53 mRNA expression and necrosis in fibroblasts exposed to the oxidative agent (P<0.001). In addition, our results showed that arbutin can decrease cell viability, induce apoptosis and increase BAX/BCL-2 ratio in LNCaP cells at some specific concentrations (P<0.001). ConclusionArbutin as a potential functional β-D-glucopyranoside has strong ability to selectively protect fibroblasts against t-BHP-induced cell damage and induce apoptosis in LNCaP cells.