ARCaP Cells Research Articles

Abstract 6319: Defense mechanism against oxidative damage in metastatic prostate cancer: Adipocyte-mediated modulation of mTOR and ferroptosis pathways

Abstract Prostate cancer (PCa) is the most common cancer amongst males and becomes incurable once it advances to the secondary site. The most common site of metastasis from PCa is the adipocyte-rich bone marrow. Adipocytes are metabolically active cells capable of secreting a variety of adipokines and free fatty acids, which can be utilized by tumor cells to enhance proliferation and cause resistance to chemotherapies. Our lab has shown that adipocytes promote PCa progression and therapy evasion through modulation of tumor metabolism and activation of pro-survival signaling; however, the molecular mechanisms behind tumor-promoting effects of fat cells are not understood. It is well known that high levels of iron within the tumor microenvironment help the growth of PCa cells. However, since iron overload can cause an increase in reactive oxygen species (ROS), which are harmful to cell structure, tumor cells have developed defense mechanisms to protect them from this oxidative damage. Intriguingly, our data show that the expression of ferritin, an iron storing protein, is reduced in PC3 and ARCaP(M) cells in co-culture with adipocytes. Since low ferritin levels have been associated with induction of ferroptosis, an iron and oxidative stress-regulated cell death process, we examined protein levels of ferroptosis markers GPX4 and NCOA4 in PCa cells. Surprisingly, our data show that interaction with adipocytes increases GPX4 and reduces NCOA4 expression in PCa cells suggesting an adipocyte-mediated defense mechanism against ferroptosis. The aim of the present study was to establish that adipocytes promote a defense mechanism against ferroptosis in PCa cells by the upregulation of GPX4 activity via the mTOR pathway. GPX4 is known to be an essential antioxidant enzyme that utilizes glutathione (GSH) to reduce lipid-peroxidation-induced ROS. We demonstrate that PCa cells exposed to adipocytes have reduced lipid-peroxidation induced ROS, which can be reversed with ferroptosis inducers. We also show that exposure of PC3 and ARCaP(M) cells to adipocytes leads to depletion of GSH, which may be indicative of higher GPX4 activity. It has been suggested that an increase in GPX4 can result from an interplay between ferroptosis and mTOR pathways, specifically mTORC1, which may mediate cysteine-induced GPX4 protein synthesis. Surprisingly, treatment of PCa cells co-cultured with adipocytes with mTORC1 inhibitor Everolimus (EVO) increases the protein and gene levels of GPX4. This coincides with reduced mRNA and protein expression of SLC7A11, transporter involved in the cysteine/glutamate antiporter system, which typically fuels GPX4 activity by bringing in the GSH precursor cysteine. Understanding the mechanisms of fat cell contribution to dysregulation of the mTOR pathway and escape from ferroptosis may have therapeutic implications for metastatic PCa. Citation Format: Alexis Wilson, Shane Mecca, Mackenzie Herroon, Laimar Garmo, Izabela Podgorski. Defense mechanism against oxidative damage in metastatic prostate cancer: Adipocyte-mediated modulation of mTOR and ferroptosis pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6319.

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaPM cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.

Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells

BackgroundSnail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE.MethodsARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments.ResultsSnail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells.ConclusionsThis study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.

Snail mediates invasion through uPA/uPAR and the MAPK signaling pathway in prostate cancer cells

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of vimentin, while epithelial-associated genes like E-cadherin are lost. This enables cells to be more metastatic. Factors that are able to induce EMT include growth factors such as transforming growth factor-β (TGF-β) and epidermal growth factor, and transcription factors such as Snail. Snail-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by the activity of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). LNCaP, 22Rv1 and ARCaP human prostate cancer (CaP) cells stably transfected with empty vector control (Neo) or constitutively active Snail exhibited increased cell invasion. Superarray analysis revealed an upregulation in uPA and uPAR RNA expression in Snail-transfected ARCaP cells compared with that of a Neo control. In addition, the protein expression levels of Snail, uPA and uPAR were measured by western blot analysis which showed that overexpression of Snail increased uPA and uPAR protein levels. The activity of uPA in conditioned media was measured using an ELISA which revealed that uPA activity was elevated in LNCaP, 22Rv1 and ARCaP cells overexpressing Snail. Additionally, transient silencing of uPAR in ARCaP cells overexpressing Snail using short interfering RNA resulted in abrogation of Snail-mediated invasion. Snail overexpression was associated with increased extracellular-signal-regulated kinase activity, and antagonism of this activity with mitogen-activated protein (MAPK) inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human CaP cells may occur via the regulation of uPA/uPAR and the MAPK signaling pathway.

Role of GLI-1 in epidermal growth factor-induced invasiveness of ARCaPE prostate cancer cells

Epidermal growth factor (EGF) signaling and Hedgehog (HH) signaling are both involved in prostate cancer (PCa) progression, yet the mechanisms through which these two pathways are synergistically linked require elucidation. In the present study, we aimed to ascertain how EGF and the HH signaling transcription factor GLI-1 are linked in prostate cancer invasiveness. ARCaP human prostate cancer cells, which included ARCaPE and ARCaPM cells, were used as a model in the present study. The expression of EGF receptor (EGFR) and the HH signaling transcriptional factor GLI-1 were detected in ARCaPE cells by immunofluorescence, and the ARCaPE cells were treated with human recombinant EGF protein (hrEGF) for 4 consecutive days invitro. Transwell invasion assays were performed in the ARCaPE cells following treatment with DMSO (vehicle control), hrEGF, GATN61 (GLI-1-specific inhibitor), hrEGF plus GANT61 and in the ARCaPM cells. The expression of phosphorylated extracellular signal regulated kinase (p-ERK), total ERK and GLI-1 was detected by western blotting in ARCaPE cells at different time-points following treatment with hrEGF. The expression of EGFR and GLI-1 was detected in ARCaPE cells, which exhibited a cobblestone-like morphology, while after treatment with hrEGF, the cell morphology was altered to a spindle-shaped mesenchymal cell morphology. Transwell invasion assays demonstrated that hrEGF dramatically enhanced the invasive capability of the ARCaPE cells (p<0.05). Additionally, western blot assay demonstrated that the expression levels of p-ERK and GLI-1 in ARCaPE cells increased in a time-dependent manner after treatment with hrEGF (p<0.05); however, the expression levels of total ERK in the cells remained relatively unchanged. It also demonstrated that the GLI-1 inhibitor GANT61 could reverse the enhanced invasive effect induced by EGF in ARCaPE cells (p<0.05). Our preliminary invitro study showed that EGF signaling may increase the invasive capability of ARCaPE human prostate cancer cells via upregulation of p-ERK and the HH signaling transcriptional factor GLI-1. Additionally, this enhanced cell invasive effect was reversed by a GLI-1-specific inhibitor invitro. Consequently, it indicates that both EGF and HH signaling are synergistically involved in the progression of human prostate cancer ARCaP cells, and GlI-1 may be one of the important effectors, which is activated by EGF downstream signaling, to promote the invasiveness of ARCaPE prostate cancer cells.

501 SONIC HEDGEHOG SIGNALING IS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN HUMAN PROSTATE CANCER ARCAP CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research (III)1 Apr 2013501 SONIC HEDGEHOG SIGNALING IS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN HUMAN PROSTATE CANCER ARCAP CELLS Guodong Zhu, Jiancheng Zhou, Haiyen E. Zhau, Linlin Zhang, Wenbin Song, Lei Li, Dalin He, and Leland W.K. Chung Guodong ZhuGuodong Zhu Xi'an, China, People's Republic of More articles by this author , Jiancheng ZhouJiancheng Zhou Xi'an, China, People's Republic of More articles by this author , Haiyen E. ZhauHaiyen E. Zhau Los Angeles, CA More articles by this author , Linlin ZhangLinlin Zhang Xi'an, China, People's Republic of More articles by this author , Wenbin SongWenbin Song Xi'an, China, People's Republic of More articles by this author , Lei LiLei Li Xi'an, China, People's Republic of More articles by this author , Dalin HeDalin He Xi'an, China, People's Republic of More articles by this author , and Leland W.K. ChungLeland W.K. Chung Los Angeles, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1895AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) progression from localized to disseminated bone and soft tissue metastases have been well characterized using ARCaPE to ARCaPM transition model. In this study, we further examined the role of sonic hedgehog (Shh) signaling activation upon the progression of ARCaP cells, with particular emphasis on their transition from epithelial to mesenchymal phenotype, as evidenced by cell morphology and gene expression profiles. METHODS The Shh expressed ARCaPE cell (ARCaPE-Shh) was established by lipofectamine transfection system. The expression of EMT markers (E-cadherin, CK18, CK19, N-cadherin and Vimentin) was examined in vector control ARCaPE (ARCaPE-Neo), ARCaPE-Shh, and ARCaPM by western blot. The migration and invasion of ARCaP cells, and the effect of cyclopamine (Cycl) on EMT were evaluated respectively by wound healing and trans-well assays. Zinc finger E-box Binding homeobox-1(ZEB-1), Slug, Snail, Gli-1, and Matrix Metallopeptidase 9 (MMP-9) protein expressions were detected by western blot. The biochemical characterizations of Shh signaling components in ARCaP EMT model were correlated with the growth of tumor xenografts in nude mice. Specifically, the expressions of E-cadherin, Vimentin, ZEB-1 and MMP-9 were analyzed in tumor xenografts by immunohistochemistry (IHC). RESULTS The ARCaPE-Shh, when compared to ARCaPE-Neo, exhibited spindle-like scattering morphology, and expressed higher levels of mesenchymal makers (Vimentin and N-cadherin) but lower levels of epithelial makers (E-cadherin, CK18 and CK19); these mesenchymal transition phenotypes coincided with increased cell migration and invasion capability of ARCaPE-Shh cells. The involvement of Shh signaling in ARCaP EMT was further supported by the observation that Cycl effectively inhibited the migratory and invasive capability of ARCaPE-Neo, ARCaPE-Shh and ARCaPM cells. Constitutive overexpression of GLI-1 increased MMP9 and ZEB-1 expression in ARCaPE-Shh cells and these increases were blocked by the simultaneous treatment with Cycl. Increased Shh signaling was also supported by in vivo studies. The average tumor xenograft size in ARCaPE-Shh group was 18±3 mm3 in comparison to ARCaPE-Neo group, 8±2mm3, and the average tumor formation latency for ARCaPE-Shh group was 38±5 days, shorter than ARCaPE-Neo group, 56±6 days. CONCLUSIONS Shh signaling may play an important role in initiation of EMT in PCa cells. Shh signaling inhibitor, Cycl, could be further developed as a therapeutic agent against PCa progression by targeting Shh-induced EMT. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e205-e206 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guodong Zhu Xi'an, China, People's Republic of More articles by this author Jiancheng Zhou Xi'an, China, People's Republic of More articles by this author Haiyen E. Zhau Los Angeles, CA More articles by this author Linlin Zhang Xi'an, China, People's Republic of More articles by this author Wenbin Song Xi'an, China, People's Republic of More articles by this author Lei Li Xi'an, China, People's Republic of More articles by this author Dalin He Xi'an, China, People's Republic of More articles by this author Leland W.K. Chung Los Angeles, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Anti-inflammatory Role of Endometase/Matrilysin-2 in Human Prostate Cancer Cells

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is an endopeptidase mostly produced by human carcinoma cells. While MMPs are thought to regulate the dynamics of extracellular matrix turnover, new evidence shows that these enzymes may play a critical regulatory role in inflammation. To investigate the role of MMP-26 in inflammation, three different variants of androgen repressed human prostate cancer (ARCaP) cells were investigated in the study: parental, MMP-26 sense cDNA-transfected, and MMP-26 antisense cDNA-transfected ARCaP cells. Protein lysates and RNA from control and genetically modified cells were analyzed by Western blotting and real-time reverse transcription polymerase chain reaction on arrays of genes critical to the inflammatory response. In comparison to parental controls, up-regulation of MMP-26 expression in MMP-26 sense cDNA-transfected cells resulted in a decrease in inflammatory genes expression. Conversely, inflammatory genes were up-regulated in MMP-26 antisense cDNA-transfected cells. Therefore, modulation of MMP-26 levels significantly affects the expression of inflammatory genes, suggesting an anti-inflammatory role of MMP-26. To determine a possible mechanism of action, further analysis, at both transcript and protein levels, revealed a dramatic down-regulation of interleukin-10 receptor B (IL10RB) in MMP-26 antisense cDNA-transfected cells. The low level of IL10RB was inversely correlated with matrix metalloproteinase-9 (MMP-9) expression. Collectively, our data suggest that the deficiency of MMP-26 may promote inflammation via inhibition of IL10RB-mediated signaling. These results propose a novel anti-inflammation function of MMP-26 and could provide novel molecular insight of therapeutic targeting.

The phytoalexin camalexin mediates cytotoxicity towards aggressive prostate cancer cells via reactive oxygen species

Camalexin is a phytoalexin that accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. Besides moderate antibacterial and antifungal activity, camalexin was reported to also exhibit antiproliferative and cancer chemopreventive effects in breast cancer and leukemia. We studied the cytotoxic effects of camalexin treatment on prostate cancer cell lines and whether this was mediated by reactive oxygen species (ROS) generation. As models, we utilized LNCaP and its aggressive subline, C4-2, as well as ARCaP cells stably transfected with empty vector (Neo) control or constitutively active Snail cDNA that represents an epithelial to mesenchymal transition (EMT) model and displays increased cell migration and tumorigenicity. We confirmed previous studies showing that C4-2 and ARCaP-Snail cells express more ROS than LNCaP and ARCaP-Neo, respectively. Camalexin increased ROS, decreased cell proliferation, and increased apoptosis more significantly in C4-2 and ARCaP-Snail cells as compared to LNCaP and ARCaP-Neo cells, respectively, while normal prostate epithelial cells (PrEC) were unaffected. Increased caspase-3/7 activity and increased cleaved PARP protein shown by Western blot analysis was suggestive of increased apoptosis. The ROS scavenger N-acetyl cysteine (NAC) antagonized the effects of camalexin, whereas the addition of exogenous hydrogen peroxide potentiated the effects of camalexin, showing that camalexin is mediating its effects through ROS. In conclusion, camalexin is more potent in aggressive prostate cancer cells that express high ROS levels, and this phytoalexin has a strong potential as a novel therapeutic agent for the treatment of especially metastatic prostate cancer.

Abstract 5332: Snail transcription factor contributes to prostate cancer tumor progression via reactive oxygen species and Rac1 activation

Abstract Epithelial-mesenchymal transition (EMT) represses cell adhesion associated genes, making cells more migratory. Snail is the master gene that regulates the EMT process by down regulating tight junction proteins, such as E-cadherin, and up-regulating mesenchymal genes, such as vimentin. Cell migration can also be regulated by the Rho family of GTPases such as Rac1, which can induce reactive oxygen species (ROS), specifically superoxide, via the NADPH oxidase (Nox) pathways. ROS, specifically hydrogen peroxide has been shown to mediate EMT induced by MMP-3. The objective of this study is to determine whether Snail Transcription factor contributes to tumor progression via ROS and Rac1 and to study the effect(s) of antioxidants on Snail signaling. We utilized androgen-dependent LNCaP and androgen-independent 22Rv1 and ARCaP cell lines overexpressing Snail to examine Rac1 activity in vitro. ROS (superoxide and hydrogen peroxide) levels were examined in vitro and in vivo. Western blot analysis of EMT markers, cell migration on collagen and Real Time PCR Array to assay oxidative stress enzymes was also performed. We found that LNCaP, 22Rv1 and ARCaP cells overexpressing Snail displayed increased Rac1 activity while LNCaP and ARCaP cells with Snail overexpression expressed higher levels of superoxide and hydrogen peroxide in vitro. Furthermore, ARCaP cells overexpressing Snail injected into nude mice showed higher tumorigenicity as compared to ARCaP Neo control cells and excised tumors displayed increased levels of superoxide and hydrogen peroxide ex vivo. Real Time PCR Array revealed significant increase in key enzymes involved in oxidative stress such as aldehyde oxidase I and peroxidasin. Treatment with the hydrogen peroxide scavenger, N-acetyl cysteine (NAC), could partially revert Snail-mediated EMT in ARCaP cells as evidenced by re-expression of E-cadherin after 3 days. The muscadine grape skin extract (MSKE) antioxidant decreased ROS levels and Rac1 activity and reverted Snail-mediated EMT as seen by decreased vimentin, re-induction of E-cadherin, and decreased cell migration. This is the first report of Snail regulation of ROS by regulating oxidative stress enzymes, leading to EMT and tumor progression. We are also showing novel data that Snail can regulate cell motility via Rac1 activation. Therefore, targeting Snail-mediated EMT with antioxidants offers a promising therapy to reduce prostate cancer progression in future. Research supported by NIH Grants: 1P20MD002285 and G12RR03062. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5332. doi:1538-7445.AM2012-5332

Abstract 2407: Snail 1 regulates invasion through uPA-uPAR and MMP-9 signaling in prostate cancer cells

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -α (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Since Snail1 can induce matrix metalloproteinases (MMP 2 and 9) which are also involved in cell invasion, we also examined MMP activity. Androgen-dependent LNCaP, and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. The levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. Gelatin zymography analysis was utilized to detect MMP-9 activity in the conditioned media. Currently, ARCaP-Snail cells are being transiently transfected with uPAR siRNA to assay for the effects of uPAR knockdown on uPA and MMP activity. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP cells. Also, MAPK inhibited MAPK expression in the cell and decreased uPA activity. In addition gelatin zymography showed higher MMP 9 activity in ARCaP-Snail overexpressing cells that was inhibited by MAPK inhibitor. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA and uPAR activities in addition to MMPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2407. doi:1538-7445.AM2012-2407

Abstract 542: Camalexin, the phytoalexin from Cruciferous Plants, decreases cell proliferation and induces apoptosis in prostate cancer cells

Abstract Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. Besides moderate antibacterial and antifungal activity, camalexin was reported to also exhibit antiproliferative and cancer chemopreventive effects. We studied the effects of camalexin treatment on viability of several prostate cancer (PCa) cell lines and whether cytostatic/cytotoxic effects of camalexin-treated prostate cancer cells were due to reactive oxygen species (ROS) generation. As models, we utilized LNCaP and its aggressive subline, C4-2, as well as ARCaP cells stably transfected with empty vector (Neo) control or constitutively active Snail cDNA that represents an EMT model. Previous studies have shown that C4-2 and ARCaP-Snail cells express more ROS than LNCaP and ARCaP-Neo, respectively. We hypothesized that camalexin would decrease cell proliferation and increase apoptosis in PCa cell lines and that the more aggressive cell lines expressing more ROS would be more sensitive to camalexin. Our results showed that camalexin treatments led to higher ROS as shown spectrofluorometrically by the oxidation of H2DCFDA, and this was decreased by co-treatment with NAC. Cell viability using MTS proliferation assay indicated that camalexin treatments decreased cell proliferation more significantly in C4-2 and ARCaP-Snail cells, as compared to LNCaP and ARCaP-Neo cells, respectively, while PrEC normal prostate epithelial cells were unaffected. Conversely, camalexin treatments increased apoptosis and mitocasp activity, which assesses caspase3/7 activity. This was associated with increased p21 and p27 anti-proliferative markers and cleaved PARP apoptotic marker as shown by western blot analysis. NAC reduced camalexin-induced ROS in ARCaP-Snail cells and abrogated camalexin-mediated cell proliferation decrease and apoptosis increase, showing that camalexin is mediating its effects through ROS. In conclusion, camalexin retarded prostate cancer cell proliferation and induced apoptosis via increased ROS generation. Interestingly, it was more potent in aggressive PCa cells expressing higher levels of ROS and did not affect normal cells. Hence, this phytoalexin has a strong potential as a novel therapeutic agent for the treatment of especially metastatic prostate cancer cells that express high levels of ROS. Supported by grants P20MD002285-01 &amp; G12RR003062-22. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 542. doi:1538-7445.AM2012-542

Characterization of the Phosphoproteome in Androgen-Repressed Human Prostate Cancer Cells by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.

Snail negatively regulates cell adhesion to extracellular matrix and integrin expression via the MAPK pathway in prostate cancer cells

Snail transcription factor induces epithelial-mesenchymal transition (EMT) in which the epithelial cells downregulate cell-cell adhesion genes such as E-Cadherin and upregulate mesenchymal genes such as vimentin, leading to increased invasion and migration. Very little is known about the role of Snail in cellular adhesion to the extracellular matrix. We hypothesized that Snail will lead to decreased cellular adhesion to fibronectin and collagen I matrix through integrin regulation, concomitant with increased cell migration. Androgen-independent C4-2 cells, an aggressive subline of androgen-dependent LNCaP cells, exhibited decreased cell adhesion and increased cell migration on fibronectin and collagen I as compared to LNCaP cells, which was reversed by Snail knock down in C4-2 cells. ARCaP and LNCaP prostate cancer cells stably transfected with Snail displayed decreased adhesion and increased cell migration on fibronectin and collagen I as compared to control Neo-transfected cells, which was reversed by Snail knockdown. Flow cytometry analysis revealed a decrease in a5, a2 and b1 integrin expression in ARCaP Snail-transfected cells that was reversed in Snail knock down cells. We also observed an increase in ERK phosphorylation in ARCaP Snail-transfected cells as compared to control ARCaP-Neo cells, and inhibition of the MAPK pathway with UO126 inhibitor in ARCaP Snail-transfected cells abrogated Snail-mediated decrease in cell adhesion and reinduced a5, a2 and b1 integrin expression. Collectively, these studies define a new role for Snail transcription factor in cell adhesion to the ECM, which may be mediated by MAPK signaling, and may be crucial for cell detachment and subsequent metastasis.

Abstract 3430: Snail1 regulates invasion through uPA-uPAR signaling in prostate cancer

Abstract PURPOSE: Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -β (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including migration and invasion. DESIGN METHODS: 22Rv1, LNCaP and ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail which led to increased cell migration and invasion. The levels of Snail, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Also, cells were treated with PI3K and MAPK inhibitors at set time points. RESULTS: The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from ARCaP, LNCaP and 22Rv1 cells transfected with Snail. In addition, MAPK inhibitor, UO126, inhibited ERK activity within 30 min up to 72 h, and decreased uPA activity within 24 h. CONCLUSION: Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA/uPAR and MAPK activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3430. doi:10.1158/1538-7445.AM2011-3430

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer.

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.

Snail-mediated regulation of reactive oxygen species in ARCaP human prostate cancer cells

Reactive oxygen species increases in various diseases including cancer and has been associated with induction of epithelial–mesenchymal transition (EMT), as evidenced by decrease in cell adhesion-associated molecules like E-cadherin, and increase in mesenchymal markers like vimentin. We investigated the molecular mechanisms by which Snail transcription factor, an inducer of EMT, promotes tumor aggressiveness utilizing ARCaP prostate cancer cell line. An EMT model created by Snail overexpression in ARCaP cells was associated with decreased E-cadherin and increased vimentin. Moreover, Snail-expressing cells displayed increased concentration of reactive oxygen species (ROS), specifically, superoxide and hydrogen peroxide, in vitro and in vivo. Real Time PCR profiling demonstrated increased expression of oxidative stress-responsive genes, such as aldehyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3days, characterized by cells returning back to the epithelial morphology and increased E-cadherin. In conclusion, this study shows for the first time that Snail transcription factor can regulate oxidative stress enzymes and increase ROS-mediated EMT regulated in part by ERK activation. Therefore, Snail may be an attractive molecule for therapeutic targeting to prevent tumor progression in human prostate cancer.

Phorbol ester phorbol-12-myristate-13-acetate induces epithelial to mesenchymal transition in human prostate cancer ARCaPE cells.

We have reported that human prostate cancer ARCaP(E) cells undertake epithelial to mesenchymal transition (EMT) when stimulated by certain soluble factors, and that EMT is regulated by surface receptor-elicited signaling pathways through protein phosphorylation. It is known that phorbol ester phorbol-12-myristate-13-acetate (PMA), a potent antagonist to both conventional and novel protein kinase C (PKC) isoenzymes, induces cancer cell scattering. To assess the effect of PMA on EMT, ARCaP(E) cells were treated with PMA and were assayed for EMT-related morphologic and behavioral changes. Specific inhibitors were used to investigate the PMA-induced EMT. PMA at 100 nM induced EMT in a time-dependent manner, resulting in a complete change from epithelial to mesenchymal stromal morphology. Concurrently, PMA inhibited expression of epithelial marker E-cadherin and increased the level of stromal marker protein vimentin, while the treated cells showed increased migratory and invasive capacities. Using specific inhibitors, we confirmed that the effect of PMA was mediated by PKC, while isoenzymes of the novel PKC subfamily were implicated as the main mediator. Finally, we determined that the EMT was dependent on newly synthesized proteins, because inhibitors for gene transcription and protein translation could both inhibit the initiation of EMT. Although PMA is well known for its effects on cell migration and tumor formation, this work is the first to define PMA as an EMT inducer in prostate cancer cells. Further investigation in this experimental model may reveal important regulatory mechanisms and additional molecular changes underlying EMT.

Differential expression of the α2 chain of the interleukin‐13 receptor in metastatic human prostate cancer ARCaPM cells.

The alpha2 chain of the interleukin-13 receptor (IL13Ralpha2) is a high-affinity receptor and a candidate target for cytotoxic killing of cancer cells. Availability of a human prostate cancer cell line with high level of IL13Ralpha2 expression will facilitate the development of therapeutic modalities. ARCaP(E) and ARCaP(M) human prostate cancer cell lines were subjected to comparative analyses of gene expression. Expression of the IL13Ralpha2 protein was confirmed by Western blotting and immunostaining. IL13Ralpha2 proteins in xenograft tumors and clinical human prostate cancer specimens were detected by specific antibodies. LNCaP prostate cancer cells stably transfected with IL13Ralpha2 were examined for accelerated growth in athymic mice. We found that IL13Ralpha2 proteins could be detected in both the ARCaP(E) and ARCaP(M) cells, but the expression level in ARCaP(M) was more than 17-fold higher than in ARCaP(E) cells. Importantly, the ARCaP lineage represented the only human prostate cancer cell line that expresses IL13Ralpha2 proteins at the level detectable by Western blotting. Expression of IL13Ralpha2 was accompanied by resistance to the anti-tumor activity of interleukin-13 (IL-13). ARCaP cells were found to be insensitive to growth inhibition upon IL-13 treatment, while overexpression of IL13Ralpha2 in LNCaP cells promoted intratibial tumor growth in athymic mice. Differential IL13Ralpha2 expression may account for the high tumorigenic and metastatic potential of ARCaP(M) cells. The unique expression of IL13Ralpha2 makes ARCaP lineage an attractive model for evaluating the targeting efficacy of therapeutic agents based on IL13Ralpha2 protein expression.

Progressive epithelial to mesenchymal transitions in ARCaP E prostate cancer cells during xenograft tumor formation and metastasis.

The mechanism of epithelial to mesenchymal transition (EMT) could be adopted by tumor cells for migration and invasion. We have reported that ARCaP(E) human prostate cancer cells undergo EMT-like changes during xenograft growth in athymic mice. In this report, we assessed the extent of EMT by tracking changes in cloned ARCaP(E) cells expressing red fluorescence protein during successive orthotopic prostate tumor formation. Cancer cells with stromal-like morphology were isolated and examined for EMT-like changes. EMT-like morphologic and expression changes were detected after one round of in vivo tumor formation. Importantly, when recovered tumor cells were used in second round xenograft tumor formation, a large fraction of ARCaP(E) cells showed drastic EMT-like changes, with markedly enlarged cell size and divergent cell shapes similar to those of mesenchymal stromal cells. The morphologic change was accompanied by increased growth and metastasis, as tumor incidence increased while red fluorescent tumor cells could be detected from circulating blood, bone marrow, peritoneal ascites, and lung of the tumor-bearing mice. Recovered clones from these samples had lost epithelial markers but many showed activated stromal marker vimentin expression. The EMT appeared permanent since the newly acquired morphology was sustained after continuous passages. Results from this study demonstrate that through interaction with the host tumor microenvironment, cancer cells acquire cellular plasticity. During xenograft tumor formation and metastasis, a single clone of cancer cells could yield a heterogeneous population, with a substantial number of tumor cells adopting mesenchymal stroma-like phenotypes.

Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells

Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.