Androgen-dependent Cell Line Research Articles

Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer.

Estrogens are considered to potentially play some roles in the development and progression of prostate cancer through estrogen receptor beta (ERβ). However, additional factors which could influence the clinical outcome of the patients through modulating these steroid signalings have also been proposed. Among these, increased expression of serotonin receptor especially that of 5-hydroxytryptamine receptor Type 4 (5-HTR4) has been recently proposed to be involved in autocrine/paracrine mechanisms of castration-resistant prostate cancer, but the presence and clinical significance of 5-HTR4 in hormone-naive prostate cancer (HNPC) and its interaction with hormonal signaling pathways have remained virtually unknown. We evaluated the status of 5-HTR4 in 112 human HNPC cases (acinar adenocarcinoma) using immunohistochemistry and correlated the findings with clinicopathological features of individual patients and the status of androgen receptor (AR) and ERβ. To further elucidate its underlying mechanisms, androgen-dependent human prostate carcinoma cell line, LNCaP, expressing 5-HTR4, was treated by 5-HTR4 agonist. 5-HTR4 immunoreactivity was detected in 34% of prostate cancer cases examined (38/112) and was significantly correlated with the status of ERβ but not with that of AR and other clinicopathological factors of the patients. Results of in vitro studies demonstrated that 24 h incubation with 5-HTR4 agonist (10 nM) increased the expression level of ERβ messenger RNA compared to controls. 5-HTR4 agonist (100 nM) significantly inhibited LNCaP carcinoma cell migration (P < 0.05). Results of our present study indicated that 5-HTR4 signaling upregulated ERβ expression in HNPCs and could impact on biological processes in HNPC.

Investigating the role of caveolin-2 in prostate cancer cell line

Prostate cancer is a worldwide problem. While the role of caveolin-1 has been extensively studied, little is known about the role of caveolin-2 (CAV2) in prostate cancer. Up-regulation of CAV2 in androgen independent PC3 cells compared to normal prostate cell line and androgen dependent prostate cancer cell lines has been observed. Recent studies suggest that up-regulation of CAV2 plays an important role in androgen independent prostate cancer. This study investigates whether CAV2 is important in mediating the aggressive phenotypes seen in androgen independent prostate cancer cells. The androgen independent prostate cancer cell line, PC3 was used that has been shown to express CAV2, and CAV2 knock down was performed using siRNA system. Changes to cell number, migration and invasion were assessed after knocking down CAV2. Our results showed that down-regulating CAV2 resulted in reduced cell numbers, migration and invasion in PC3 cells. This preliminary study suggests that CAV2 may act to promote malignant behavior in an androgen independent prostate cancer cell line. Further studies are required to fully elucidate the role of CAV2 in androgen independent prostate cancer.

Identification of Mutations, Expression Alterations and Fusion Transcripts by Next Generation RNAseq in Castration-Resistant Prostate Cancer Cell lines with Possible Clinical Relevance

Androgen Deprivation Therapy (ADT) would benefit prostate cancer patients initially but cancer cells can eventually develop castration resistance. In this study, we compared androgen-dependent and androgenindependent cell lines to find potential genes associated with acquired resistance to ADT. Using RNAseq, we found 4397 mutations distributed in 2579 genes, out of which, 1574 mutated genes could also be found in prostate cancer tumor samples collected in Cosmic database (http://cancer.sanger.ac.uk/cosmic). We also discovered 157 and 549 genes which were down and up-regulated respectively in both PC3 and DU145 compared to LNCaP. Network analysis resulted in 3 dominant connection notes: GCR/MCR (NR3C1) and PKA-cat kinase (PRKACB) and PKC family (PRKD1). By ChimeraScan analysis, 48, 117 and 60 chimeric transcripts were discovered in DU145, LNCaP and PC3 respectively. Among them, six predicted fusions expressed specifically in androgen-independent cell lines (DU145 and PC3). Some of these gene mutations and transcription alterations have been reported in tumor samples from prostate cancer patients and may have certain associations with acquired resistance to anti-hormone therapy in prostate cancer. A proportion of mutations are enriched in genes involved in immune response pathways, suggesting new targets to develop effective treatments to overcome castration resistance.

Bifunctional conjugates with potent inhibitory activity towards cyclooxygenase and histone deacetylase

We herein disclose a series of compounds with potent inhibitory activities towards histone deacetylases (HDAC) and cyclooxygenases (COX). These compounds potently inhibited the growth of cancer cell lines consistent with their anti-COX and anti-HDAC activities. While compound 2b showed comparable level of COX-2 selectivity as celecoxib, compound 11b outperformed indomethacin in terms of selectivity towards COX-2 relative to COX-1. An important observation with our lead compounds (2b, 8, 11b, and 17b) is their enhanced cytotoxicity towards androgen dependent prostate cancer cell line (LNCaP) relative to androgen independent prostate cancer cell line (DU-145). Interestingly, compounds 2b and 17b arrested the cell cycle progression of LNCaP in the S-phase, while compound 8 showed a G0/G1 arrest, similar to SAHA. Relative to SAHA, these compounds displayed tumor-selective cytotoxicity as they have low anti-proliferative activity towards healthy cells (VERO); an attribute that makes them attractive candidates for drug development.

UGT2B17 Expedites Progression of Castration-Resistant Prostate Cancers by Promoting Ligand-Independent AR Signaling

Castration-resistant prostate cancer (CRPC) is characterized by a shift in androgen receptor (AR) signaling from androgen-dependent to androgen (ligand)-independent. UDP-glucuronosyltransferase 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into inactive forms. Although enhanced UGT2B17 expression by antiandrogens has been reported in androgen-dependent prostate cancer, its roles in regulating AR signaling transformation and CRPC progression remain unknown. In this study, we show that higher UGT2B17 protein expression in prostate tumors is associated with higher Gleason score, metastasis, and CRPC progression. UGT2B17 expression and activity were higher in androgen-independent compared to androgen-dependent cell lines. UGT2B17 stimulated cancer cell proliferation, invasion, and xenograft progression to CRPC after prolonged androgen deprivation. Gene microarray analysis indicated that UGT2B17 suppressed androgen-dependent AR transcriptional activity and enhanced of ligand-independent transcriptional activity at genes associated with cell mitosis. These UGT2B17 actions were mainly mediated by activation of the c-Src kinase. In CRPC tumors, UGT2B17 expression was associated positively with c-Src activation. These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand-independent AR signaling to activate cell mitosis in cancer cells. Cancer Res; 76(22); 6701-11. ©2016 AACR.

The anti-proliferative and anti-androgenic activity of different pomegranate accessions

This study monitored the anti-proliferative activity of pomegranate (Punica granatum L.) peel homogenates against breast (MCF7 and MDA-MB-453) and prostate (LNCaP and PC-3) cancer cell lines using 29 different accessions to study the natural diversity in these accessions. Using MTT method, it was revealed that high anti-proliferative activity was observed against MCF7, MDA-MB-453 and LNCaP (androgen-dependent) cell lines for most accessions, while the androgen-independent prostate cancer cell line, PC-3, exhibited relatively high resistance. Peel extracts displayed the highest LNCaP inhibition activity compared to aril juice and seed homogenates. Eight accessions exhibiting the highest anti-proliferative activity were studied further using the LNCaP and PC-3 cancer cells. IC50 and clonogenicity inhibition were determined. Several accessions were found to inhibit androgen receptor activity but not NF-κB transcription function. This study illustrated the importance of exploiting natural pomegranate variation to identify accessions exhibiting potential selective activity for the treatment of specific types of cancer.

11-Ketotestosterone and 11-Ketodihydrotestosterone in Castration Resistant Prostate Cancer: Potent Androgens Which Can No Longer Be Ignored.

Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and is implicated in the development and progression of castration resistant prostate cancer (CRPC). Under castrate conditions, DHT is produced from the metabolism of the adrenal androgen precursors, DHEA and androstenedione. Recent studies have shown that the adrenal steroid 11β-hydroxyandrostenedione (11OHA4) serves as the precursor to the androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). In this study we comprehensively assess the androgenic activity of 11KT and 11KDHT. This is the first study, to our knowledge, to show that 11KT and 11KDHT, like T and DHT, are potent and efficacious agonists of the human androgen receptor (AR) and induced both the expression of representative AR-regulated genes as well as cellular proliferation in the androgen dependent prostate cancer cell lines, LNCaP and VCaP. Proteomic analysis revealed that 11KDHT regulated the expression of more AR-regulated proteins than DHT in VCaP cells, while in vitro conversion assays showed that 11KT and 11KDHT are metabolized at a significantly lower rate in both LNCaP and VCaP cells when compared to T and DHT, respectively. Our findings show that 11KT and 11KDHT are bona fide androgens capable of inducing androgen-dependant gene expression and cell growth, and that these steroids have the potential to remain active longer than T and DHT due to the decreased rate at which they are metabolised. Collectively, our data demonstrates that 11KT and 11KDHT likely play a vital, but overlooked, role in the development and progression of CRPC.

Abstract 1360: Genistein-AR antagonist conjugate potently induce growth arrest in both androgen dependent and independent prostate cancer cell lines

Abstract The ability of prostate cancer to transition from androgen dependent- to androgen independent state (castration resistant), during androgen deprivation therapy, requires that new chemotherapeutic agents be introduced that will retain activity in the two prostate cancer types. Though described as androgen independent, castration resistant prostate cancer (CRPC) still relies heavily on androgen receptor (AR) signaling for survival. Enzalutamide, an AR antagonist, is probably that most effective drug used in androgen deprivation therapy for CRPC and AR dependent prostate cancer. Despite its clinical success, there is the fear of resistant to enzalutamide due to mutation in the ligand binding domain of AR. This poses a challenge that requires developing new agents devoid of resistance, in the event of mutation. One of the approaches being proposed is to develop new agents that can selectively induce AR degradation, an approach that was successfully used to develop the selective estrogen receptor degrader, fulvestrant. In our study, we incorporate genistein, a soy isoflavone known to perturb multiple biochemical pathways relevant for cancer survival, into an AR binding template to give AG-1-33. AG-1-33 was evaluated for growth inhibitory activity in LNCaP (AR dependent) and DU-145 (AR independent) prostate cancer cell lines. This compound retained its AR antagonist activity and also showed low micromolar anti-proliferation activity in the two prostate cancer cell lines. We also evaluated the effect of AG-1-33 on AR expression, as well as its ability to act as a selective AR degrader in LNCaP. Citation Format: Idris O. Raji, Alex George, Omer Kucuk, Adegboyega K. Oyelere. Genistein-AR antagonist conjugate potently induce growth arrest in both androgen dependent and independent prostate cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1360.

Design and evaluation of novel oxadiazole derivatives as potential prostate cancer agents

Various 1,3,4-oxadiazole derivatives have been synthesized and their antiproliferative properties have been studied. The in vitro screening was performed against androgen dependent (LNCaP) and androgen independent (PC-3) prostate cancer cell lines. Most of the compounds showed promising activity. Among them, compounds 2d (IC50=0.22 and 1.3μM) and 2a (IC50=8.34 and 2,5μM) have shown significant activities on PC-3 and LNCaP cell lines respectively. To investigate the mechanism of cell death we performed cell apoptosis staining and cell cycle arrest assay on more sensitive PC-3 cell lines on 2d. The results demonstrated that 2d induced apoptosis and shifted the cells to the sub G0/G1 and S phase. Our study evidently identified the potency of compound 2d as potential anti-prostate cancer agent.

A Signaling Network Controlling Androgenic Repression of c-Fos Protein in Prostate Adenocarcinoma Cells

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.

Abstract A03: Procyanidin B2 3,3”-di-O-gallate from grape seed causes metabolic alterations in prostate cancer cells: 1H-, 13C- and 31P-NMRS-based metabolomics study

Abstract Herein, we assessed the efficacy of procyanidins from grape seed against a panel of human prostate cancer (PCa) cell lines which ranged from ‘classical’ cell lines to the new variants that differed in their androgen responsiveness, castration resistance, and metastatic potential. The classical cell lines chosen were PC3, DU145 and LNCaP. Of these cell lines: PC3 and DU145 do not need androgens for growth, i.e, these are androgen independent (AI); they also lack androgen receptors (AR). The LNCaP cell line on the other hand, demonstrates androgen sensitivity (AS) and also requires androgens for its growth; while it harbors a mutated AR. Of the new variants of PCa cell lines available, we further chose a castration resistant variant of LNCaP, C4-2B, derived from the xenografts of castration resistant LNCaP subline-C4 in castrated mice. C4-2B thus represents castration resistant PCa (CRPC) cell line; while it does not require androgen for growth it demonstrates androgen sensitivity due to the presence of AR and is thus categorized as CRPC (AI/ AS) variant. Another PCa cell line, 22Rv1, which was chosen, does not require androgens for growth but is androgen responsive and like C4-2B is categorized as CRPC (AI/ AS).Trypan Blue dye exclusion assays utilizing total procyanidins from grape seed or its active constituent procyanidin B2 3,3”-di-O-gallate (B2G2) were performed to determine procyanidin effects, as a function of time, on the viability of the selected human PCa cell lines. Results indicated that the androgen dependent LNCaP cell line and its castration resistant variant, C4-2B, were more sensitive to procyanidins treatment at lower doses; importantly, another castration resistant cell line, 22Rv1, also showed significant decrease in cell viability at lower doses. While at these lower doses significant cell death was observed even at time points as early as 12 h in these cell lines; the classical AI cell lines PC3 and DU145 were most resistant to procyanidin treatment at lower doses and showed sensitivity (growth inhibition and cell death) only at higher doses after 72 h of procyanidins exposure. On similar lines, cancer stem cells (CD44+alpha2beta1high cells) isolated from PC3 and DU145 cells were also more resistant to treatment with procyanidins in prostasphere formation assays compared to other PCa cell lines. To understand the differential efficacy of procyanidins or its active constituent B2G2 towards different PCa cell lines, we carried out a metabolomics study utilizing quantitative high-resolution nuclear magnetic resonance spectroscopy (1H-, 13C- and 31P-NMR) to assess the metabolic profile and energy state of the B2G2 treated PCa cells. We examined global metabolic profile, including glucose metabolism, energy state, and lipid metabolism in these cells after B2G2 treatment. The time-course of B2G2 effects on glucose uptake and lactate release was also studied in the media of B2G2-treated PCa cells from 4-72 hrs of treatment. Results indicated that there was differential effect of B2G2 on mitochondrial glucose metabolism in these cells. While glucose uptake was markedly reduced as a function of time, there was also a significant decrease in extracellular lactate export with increased B2G2 exposure-time. Importantly, B2G2 preserved citrate concentration in the PCa cells as indicated by increase in citrate levels after treatments; citrate generation is indicative of normal secretion functions of the prostate epithelial cells. The fact that the citrate was not further utilized for cholesterol synthesis was also confirmed by decrease in cholesterol levels in these cells, indicating decreased cholesterogenesis by B2G2. Taken together these studies show that the procyanidins from grape seed differentially induce metabolic alterations in various PCa cell lines; these modulatory effects can be correlated with the effects on cell growth, proliferation and death, as well as the androgen dependency/ or lack of it. Citation Format: Komal Raina, Dileep Kumar, Rajesh Agarwal, Chapla Agarwal. Procyanidin B2 3,3”-di-O-gallate from grape seed causes metabolic alterations in prostate cancer cells: 1H-, 13C- and 31P-NMRS-based metabolomics study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A03.

TM4SF3 and AR: A Nuclear Complex that Stabilizes Both Proteins

Transmembrane 4 superfamily 3 (TM4SF3) was identified as a novel androgen-regulated gene in prostate cancer (PCa) cells. Our data demonstrate that TM4SF3 exhibits androgen-induced repression of the mRNA but up-regulation of the protein. The androgen positive effect on the TM4SF3 protein is of significant interest in view of the procancer functions of both androgens and tetraspanin proteins. Androgen positively regulates TM4SF3 protein stability by inhibiting its proteasome-dependent degradation. This androgen stabilization of TM4SF3 is involved in promoting PCa cell invasion and migration of both androgen-dependent and androgen-independent PCa cells. Although confirming androgen up-regulation of the TM4SF3 protein, we observed that TM4SF3 is localized not only to the membrane, but also, surprisingly, the nuclei of PCa cells. This novel nuclear localization of TM4SF3 depends on androgen-induced nuclear localization of androgen receptor (AR) in both androgen-dependent and androgen-independent PCa cell lines. TM4SF3 interacts with AR both in PCa cell types and in vitro, strongly suggesting a direct interaction. This direct interaction is required for the stabilization of not only TM4SF3, but also remarkably AR, because down-regulation of TM4SF3 resulted in reduced AR protein levels. As expected of an important AR regulator, TM4SF3 regulates androgen-dependent gene expression in and proliferation of PCa cells. Importantly, a direct correlation between AR and TM4SF3 protein levels and nuclear colocalization were also observed in prostate tumors, strongly suggesting that the mutual stabilization resulting from the AR-TM4SF3 interaction is found in tumors and that this interaction is important in PCa biology.

Abstract 2065: TMEFF2 is a novel androgen receptor co-activator in prostate cancer cells

Abstract The goal of this study is to delineate the role of a novel androgen receptor (AR) coactivator in advanced prostate cancer (PCa). While great advances have been made in the ability to detect PCa, the disease remains the second leading cause of cancer related deaths in men. The AR is a steroid nuclear receptor that serves a central function in both normal prostate cell homeostasis and in PCa; in fact, the vast majority of PCa tumors require androgens for growth. This dependency has led to the development and use of androgen deprivation therapies (ADTs) for the treatment of PCa. ADT is initially a successful form of treatment for most PCa patients; however, the majority of patients eventually relapse with castration-resistant prostate cancer (CRPC), which is currently incurable. Because sustained AR activity under androgen depleted conditions is a major contributor to the development of CRPC, identifying molecular mechanisms that contribute to AR activity in CRPC is paramount for the development of successful therapies. Our lab has been studying the role of the transmembrane protein with epidermal-like growth factor domain and two follistatin domains 2 (TMEFF2) in PCa. While TMEFF2 gene expression is repressed by hypermethylation in multiple cancers, TMEFF2 is overexpressed in PCa and CRPC, suggesting that TMEFF2 may have a unique function in PCa. Importantly, TMEFF2 expression is activated by the androgen receptor (AR) at the level of transcription and translation, which may contribute to its overexpression in PCa. Our data presented here indicate that TMEFF2 functions in PCa cells as a coactivator of the AR. Using shRNA to stably knockdown TMEFF2 expression in androgen dependent PCa and CRPC cell lines, we demonstrate that TMEFF2 activates the basal and androgen-induced expression of multiple androgen responsive genes, including prostate-specific antigen (PSA), at the mRNA and protein level. Importantly, the knock-down of TMEFF2 does not alter AR protein levels, indicating that TMEFF2 influences AR activity. Additionally, by coimmunoprecipitation analysis, we demonstrate that TMEFF2 interacts with the AR, and western blot analyses with cellular fractionation samples indicate that TMEFF2 has the ability to translocate to the nucleus of PCa cells. Based on these results, we conclude that TMEFF2 is a novel AR coactivator in PCa. Future studies will focus on understanding the nature and biological effects of the interaction between TMEFF2 and the AR, and delineating the molecular mechanism through which TMEFF2 activates AR-mediated gene expression. Because sustained AR activity in androgen depleted conditions is a major contributor to CRPC relapse and PCa mortality, the TMEFF2-mediated AR activation mechanism may be clinically useful as a therapeutic target in CRPC. Citation Format: Joshua Corbin, Thomas Green, Maria J. Ruiz-Echevarria. TMEFF2 is a novel androgen receptor co-activator in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2065. doi:10.1158/1538-7445.AM2015-2065

Abstract LB-017: Minnelide reduces castration-resistant and enzalutamide-resistant prostate cancer via downregulation of androgen receptor-mediated signaling

Abstract Prostate cancer is the second leading cause of cancer death in men in western countries. Advanced prostate cancer is often resistant to hormonal treatment and systemic chemotherapy has limited efficacy. Androgen receptor (AR), a ligand dependent transcription factor plays pivotal role in the development and progression of prostate cancer. While majority of prostate cancers are initially androgen dependent and respond to androgen ablation therapy, most patients eventually recur with more aggressive castration-resistant prostate cancer (CRPC) where AR signaling is reactivated even in the absence of androgen stimulation. Therefore developing novel chemotherapeutic agents for castrate resistant prostate cancer (CRPC) treatment is critical to improve survival in men with CRPC. Triptolide, a diterpene triepoxide isolated from a chinese herb, is extremely effective against several cancers like pancreatic cancer, colorectal cancer and liver cancer both in vivo and in vitro. The water-soluble pro-drug of triptolide, Minnelide, downregulates HSP70 via inhibition of the activity of transcription factor Sp1. Since both Sp1 and HSP70 have been reported to be critical in functionality of AR, we assessed therapeutic potential of Minnelide on androgen dependent, CRPC in vitro and in vivo. Triptolide treatment resulted in dose- and time-dependent cell death in an androgen dependent cell line LNCaP, CRPC cell line C4-2 and enzalutamide resistant CRPC tumor cell line 22RV1. Triptolide treatment decreased expression of AR full length, AR splice variants and its downstream targets (PSA, NKX3.1) at the mRNA and protein levels. Further, reporter assay with AR responsive elements showed that triptolide decreased transcriptional activity of AR. Expression levels of Sp1 and HSP70 were also reduced following treatment with triptolide these cell lines. To test the efficacy of Minnelide in vivo, male athymic nude mice were castrated 7 days prior to implantation of enzalutamide resistant CRPC (22RV1) cells subcutaneously. The animals received daily intraperitoneal injection of Minnelide and tumor volume was measured weekly until tumor size reached 2cm3.Mice receiving daily injection of Minnelide had significantly smaller tumors than controls as early as two week of treatment (p = 0.008). Triptolide therapy inhibited enzalutamide resistant CRPC growth both in vitro and in vivo. Further, our studies for the first time showed that triptolide induces prostate tumor cell death by reducing expression of both full length AR and AR splice variants in a similar manner. Citation Format: Sumit Isharwal, Shrey Modi, Usman Barlass, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee, Badrinath Konety. Minnelide reduces castration-resistant and enzalutamide-resistant prostate cancer via downregulation of androgen receptor-mediated signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-017. doi:10.1158/1538-7445.AM2015-LB-017

14C]Fluciclovine (alias anti-[14C]FACBC) uptake and ASCT2 expression in castration-resistant prostate cancer cells

Introductiontrans-1-Amino-3-[18F]fluorocyclobutanecarboxylic acid ([18F]fluciclovine, also known as anti-[18F]FACBC), is a tracer for positron emission tomography (PET) imaging for detection of tumors such as prostate cancer (PCa). Our previous study showed that ASCT2 (Na+-dependent amino acid transporter (AAT)) mediates fluciclovine uptake in androgen-dependent PCa cells; its expression is influenced by androgen, a key hormone in the progression of primary PCa and castration-resistant prostate cancer (CRPC). In this study, we investigated the uptake mechanisms and feasibility of [18F]fluciclovine for CRPC in the androgen-dependent PCa cell line LNCaP and LNCaP-derivatives LNCaP-SF and LN-REC4. MethodsLNCaP-SF was established after long-term cultivation of LNCaP in steroid-free conditions, and LN-Pre and LN-REC4 were established from LNCaP inoculated in intact and castrated severe combined immunodeficient mice, respectively. Uptake and competitive inhibition experiments were performed with trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid ([14C]fluciclovine) to characterize the involvement of AATs in androgen-dependent PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cell lines. AAT expression was analyzed by Western blotting, and [14C]fluciclovine uptake in androgen-dependent PCa and CRPC-like cell lines were investigated in the presence or absence of dihydrotestosterone (DHT). ResultsThe contribution of Na+-dependent AATs to [14C]fluciclovine uptake in all cell lines was 88−98%, and [14C]fluciclovine uptake was strongly inhibited by l-glutamine and l-serine, the substrates for Na+-dependent alanine-serine-cysteine (system ASC) AATs, in the presence of Na+. DHT enhanced ASCT2 expression in LNCaP, LN-Pre, and LN-REC4, but not in LNCaP-SF, and the responses of ASCT2 expression to DHT correlated with [14C]fluciclovine uptake. ConclusionsSystem ASC, especially ASCT2, could play a major role in [14C]fluciclovine uptake into CRPC-like and androgen-dependent PCa cells, suggesting [18F]fluciclovine-PET is applicable to the detection of CRPC as well as androgen-dependent PCa. Advance in knowledge[18F]fluciclovine-PET may be applied for the detection of CRPC. Implication for patient care[18F]fluciclovine-PET may permit early intervention for CRPC treatment.

Antioxidant, antiangiogenic and antiproliferative activities of root methanol extract of Calliandra portoricensis in human prostate cancer cells

ObjectiveProstate cancer (PCa) is a major health concern. Calliandra portoricensis (CP) is traditionally known for its analgesic, anti-ulcerogenic and anticonvulsant properties. However, its antiproliferative properties for PCa still need to be investigated. MethodsAntioxidant activities of CP were determined by 1,1-diphenyl-2-picryhydrazyl (DPPH) and hydroxyl (OH−) radicals-scavenging methods. PC-3 and LNCaP (androgen-refractory and androgen-dependent PCa-derived cell lines) were cultured and treated with CP (10, 50 and 100 μg/mL). Effects of CP on cells were determined by cytotoxicity assay (lactate dehydrogenase, LDH) and viability assay (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate, XTT). DNA fragmentation was detected by cell death detection enzyme-linked immunosorbent assay plus kit. CP was tested as an inhibitor of angiogenesis using chicken chorioallantoic membrane (CAM) assay. ResultsCP showed significant scavenging of DPPH and OH- radicals. CP significantly (P<0.05) inhibited lipid peroxidation in a dose-dependent manner. Precisely, CP (10, 50 and 100 μg/mL) inhibited PC-3 and LNCaP growth by 7%, 74% and 92%, and 27%, 73%, and 85% respectively at 48 h. CP had low toxicity in vitro at its half inhibitory concentration dose. Detection of cell death induced by CP at 50 μg/mL showed higher enrichment factors in LNCaP (7.38±0.95) than PC-3 (3.48±0.55). Also, treatment with CP (50 μg/mL) significantly reduced network of vessels in CAM, suggesting its antiangiogenic potential. ConclusionCalliandra portoricensis elicited antioxidant, antiangiogenic and antiproliferative effects in PCa cells.

Micro-ribonucleic acid 29b inhibits cell proliferation and invasion and enhances cell apoptosis and chemotherapy effects of cisplatin via targeting of DNMT3b and AKT3 in prostate cancer.

BackgroundMicro-ribonucleic acids (miRNAs) are crucial regulators in malignant tumors. miRNA-29b (miR-29b) has been identified as a tumor suppressor in prostate cancer (PCa). However, very few studies have investigated the effects of miR-29b in PCa, especially the mechanism and its association with chemotherapy. Our study aimed to explore the role and mechanism of miR-29b in PCa.Materials and methodsThe expression levels of miR-29b were detected in ten clinical PCa specimens and four different PCa cell lines through quantitative real-time polymerase chain reaction. After miR-29b mimics and inhibitors were successfully transfected into LNCaP, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was then used to investigate cell proliferation and cisplatin sensitivity of PCa cells. Cell cycle, cell apoptosis, and cell invasion were detected via flow cytometry, annexin V–fluorescein isothiocyanate labeling, and transwell assay, respectively. Based on bioinformatic methods, Western blot analysis, and dual-luciferase reporter assay, novel target genes of miR-29b were identified.ResultsmiR-29b was downregulated in PCa tissues compared with matched adjacent nontumor tissues. In the androgen-independent PCa cell line (LNCaP-AI), the expression of miR-29b was much lower than the androgen-dependent PCa cell line (LNCaP). Subsequent studies showed that forced expression of miR-29b inhibited cell proliferation and cell invasion and induced cell apoptosis in PCa. Upregulation of miR-29b also enhanced the chemosensitivity of PCa cells to cisplatin. Moreover, we identified DNMT3b and AKT3 as novel target genes of miR-29b in PCa.ConclusionTaken together, the results showed that miR-29b plays a tumor-suppressive role in PCa. It inhibits cell biological behavior and enhances the chemotherapy effects of cisplatin through its involvement in epigenetic regulation and PI3K/AKT pathway.

Diagnostic value of SFRP1 as a favorable predictive and prognostic biomarker in patients with prostate cancer.

Growing genetic and molecular biological evidence suggests that the disruption of balance between Secreted Frizzled-Related Protein-1 (SFRP1) and β-catenin plays an important role in the initiation and development of multiple cancers. The aim of this study was to examine whether the expression of SFRP1 and β-catenin is associated with the clinical-pathologic features of patients with prostate cancer (PCa), and to evaluate their potential roles as predictive and prognostic biomarkers. In this study, a total of 61 patients with PCa and 10 patients with benign prostatic hyperplasia were included, and we showed that the expression of SFRP1 and β-catenin was correlated with the Gleason score, survival rate and response for endocrine therapy of PCa. The survival rates of PCa patients with low SFRP1 expression (P = 0.016) or high β-catenin expression (P = 0.004) were significantly poorer. A negative correlation (r = -0.275, P = 0.032) between SFRP1 and β-catenin was observed by Chi-square test. Multivariate analysis suggested that SFRP1 (hazard ratio, 0.429; 95% confidence intervals, 0.227–0.812; P = 0.009) may serve as an independent predictive and prognostic factor for PCa. We also showed that the protein and mRNA levels of SFRP1 in androgen-dependent PCa cell line LNCaP were significantly higher than those in androgen-independent PCa cell lines DU145 and PC3. However, the protein level of β-catenin in LNCaP cells was significantly lower than that in DU145 and PC3 cells, and no significant difference of β-catenin mRNA level was observed in LNCaP, DU145 and PC3 cells. Bisulfite sequencing PCR assay revealed significantly lower methylation level of SFRP1 promoter in LNCaP cells than that in DU145 and PC3 cells. Taken together, these findings suggest that SFRP1, which expression inversely correlates with that of β-catenin, is a favorable predictive and prognostic biomarker.

Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells.

New therapies are required for castrate-resistant prostate cancer (CRPC), and growth-arrest specific 5 (GAS5) lncRNA, which riborepresses androgen receptor action, may offer novel opportunities in this regard. This lncRNA promotes the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate-resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies. Since GAS5 is a member of the 5' terminal oligopyrimidine gene family, we have examined mTOR inhibition as a strategy to increase GAS5 expression. Furthermore, we have determined if GAS5 itself mediates the action of mTOR inhibitors, as demonstrated for other chemotherapeutic agents in prostate cancer cells. The effects of mTOR inhibitors on GAS5 lncRNA levels and cell growth were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNAs and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. First generation mTORC1, combined mTORC1/mTORC2 and dual PI3K/mTOR inhibitors all increased cellular GAS5 levels and inhibited culture growth in androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU 145) cell lines. The latter exhibited low endogenous GAS5 expression, and GAS5 silencing in LNCaP and 22Rv1 cells decreased the sensitivity to mTOR inhibitors, whereas transfection of GAS5 lncRNA sensitized PC-3 and DU 145 cells to these agents. mTOR inhibition enhances GAS5 transcript levels in certain prostate cancer cell lines. This selectivity is likely to be related to endogenous GAS5 expression levels, since GAS5 lncRNA is itself required for mTOR inhibitor action in prostate cancer cells.

Transfected Poly(I:C) Activates Different dsRNA Receptors, Leading to Apoptosis or Immunoadjuvant Response in Androgen-independent Prostate Cancer Cells

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression.