PC-3 Invasion Research Articles

Abstract 5563: Targeting the PH domain of TIAM1 to inhibit prostate cancer metastasis.

Abstract TIAM1 (T-lymphoma invasion and metastasis-inducing protein-1) is a highly conserved guanine nucleotide exchange factor and contains an Dbl Homology (DH)/C-terminal Pleckstrin Homology (PH) domain. The crystal structure of DH-PH of TIAM1 in complex with Rac-1 has been reported. TIAM1 has been found to be over-expressed cancers such as breast, colon and prostate cancers. An increase in TIAM1 expression has been shown to be associated with increased metastatic potential of breast cancer cell lines and is also correlated with poor prognosis of patients with prostate cancer. TIAM1 is thus a novel PH domain-containing drug target directly related to cancer progression, metastasis and patient survival. We have identified and characterized novel small molecule inhibitors targeting the phosphoinositide lipid binding PH domain of TIAM1 in order to selectively inhibit cell migration, invasion and survival. An In Silico screen of our internal and Maybridge library using the crystal structure of TIAM1 has identified 11 compounds that bind to the PH domain. In vitro assays revealed that compounds TPH-3 and TPH-15 significantly reduced the amount of active Rac1 in PC-3 prostate cancer cells (EC50 of 2.73±0.13 and 2.38±0.98 μM) by binding to PH-TIAM1 with high affinity (KD in the micromolar range) using surface plasmon resonance (SPR) spectrometry. Both compounds displaced PtdIns-3,4,5-P3 in a SPR competitive binding assay. TPH-3 and TPH-15 inhibited cell proliferation with EC50 of 19.8±3.2 and 28.7±1.7 μM in prostate cancer cells LnCaP and EC50 of 18.6±1.8 and 33.3±6.7 μM in PC-3. Wound healing assays and lamellipodia formation were both inhibited by the compounds. TPH-15 inhibited ≈70% of PC-3 invasion using a matrigel invasion assay. Finally, TPH-15 exhibits anti-tumor properties in a PC-3 mouse xenograft study (%T/C≈38.8) with good pharmacokinetic properties (T1/2≈5 hours). Cardio-injection of prostate PC-3 cells in SCID mice was used to test the effects of TPH-15 in the prevention of bone metastasis. Mice were treated with 50mg/kg ip of TPH-15 once a day for 5 days. TPH-15-treated mice developed significantly less pelvic bone lesions than the untreated group and TPH-15 treated mice lived longer, on average, than the untreated group. Overall, we have identified novel compounds which exhibit the ability to reduce prostate cancer bone metastasis by binding to the PH domain of TIAM1, an important GEF in the process of metastasis. Citation Format: Emmanuelle J. Meuillet, Sylvestor A. Moses, Jana Jandova, Eugene A. Mash, Shuxing Zhang. Targeting the PH domain of TIAM1 to inhibit prostate cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5563. doi:10.1158/1538-7445.AM2013-5563

Deregulated expression of urokinase and its inhibitor type 1 in prostate cancer cells: Role of epigenetic mechanisms

Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2′-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2±1.7%) in comparison to adjacent non-tumor tissue (6.0±0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expressions were changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.

MicroRNA-182 and MicroRNA-200a Control G-protein Subunit α-13 (GNA13) Expression and Cell Invasion Synergistically in Prostate Cancer Cells

G protein-coupled receptors (GPCRs) and their ligands have been implicated in progression and metastasis of several cancers. GPCRs signal through heterotrimeric G proteins, and among the different types of G proteins, GNA12/13 have been most closely linked to tumor progression. In this study, we explored the role of GNA13 in prostate cancer cell invasion and the mechanism of up-regulation of GNA13 in these cells. An initial screen for GNA13 protein expression showed that GNA13 is highly expressed in the most aggressive cancer cell lines. Knockdown of GNA13 in highly invasive PC3 cells revealed that these cells depend on GNA13 expression for their invasion, migration, and Rho activation. As mRNA levels in these cells did not correlate with protein levels, we assessed the potential involvement of micro-RNAs (miRNAs) in post-transcriptional control of GNA13 expression. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-182 and miR-141/200a showed an inverse correlation to the protein expression in LnCAP and PC3 cells. Ectopic expression of miR-182 and miR-141/200a in PC3 cells significantly reduced protein levels, GNA13-3'-UTR reporter activity and in vitro invasion of these cells. This effect was blocked by restoration of GNA13 expression in these cells. Importantly, inhibition of miR-182 and miR-141/200a in LnCAP cells using specific miRNA inhibitors elevated the expression of GNA13 and enhanced invasion of these cells. These data provide strong evidence that GNA13 is an important mediator of prostate cancer cell invasion, and that miR-182 and miR-200 family members regulate its expression post-transcriptionally.

A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells

Methylglyoxal is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Methylglyoxal cytotoxicity appears to occur through cell-cycle arrest but, more often, through induction of apoptosis. In this study we examined whether, and through which molecular mechanism, methylglyoxal affects the growth of poorly aggressive LNCaP and invasive PC3 human prostate cancer cells, where its role has not been exhaustively investigated yet. We demonstrated that methylglyoxal is cytotoxic on LNCaP and PC3 and that such cytotoxicity occurs not via cell proliferation but apoptosis control. Moreover, we demonstrated that methylglyoxal cytotoxicity, potentiated by the silencing of its major scavenging enzyme Glyoxalase I, occurred via different apoptotic responses in LNCaP and PC3 cells that also showed a different susceptibility to this metabolite. Finally, we showed that the observed methylglyoxal apoptogenic role involved different molecular pathways, specifically mediated by methylglyoxal or methylglyoxal-derived argpyrimidine intracellular accumulation and NF-kB signaling-pathway. In particular, in LNCaP cells, methylglyoxal, through the accumulation of argpyrimidine, desensitized the key cell survival NF-kB signaling pathway, which was consistent with the modulation of NF-kB-regulated genes, triggering a mitochondrial apoptotic pathway. The results suggest that this physiological compound merits investigation as a potential chemo-preventive/-therapeutic agent, in differently aggressive prostate cancers.

Copper Modulates Zinc Metalloproteinase-Dependent Ectodomain Shedding of Key Signaling and Adhesion Proteins and Promotes the Invasion of Prostate Cancer Epithelial Cells

A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at the molecular level. In the current study, we investigated whether copper might regulate the ectodomain shedding of two key cell surface proteins implicated in the invasion and metastasis of prostate cancer, the Notch ligand Jagged1 and the adhesion molecule E-cadherin, and whether the metal was able to influence the invasion of the prostate cancer epithelial cell line PC3. Physiological copper concentrations stimulated the ZMP-mediated proteolysis of Jagged1 and E-cadherin in cell culture models, whereas other divalent metals had no effect. Copper-mediated Jagged1 proteolysis was also observed following the pretreatment of cells with cycloheximide and in a cell-free membrane system, indicating a posttranslational mechanism of sheddase activation. Finally, the concentrations of copper that stimulated ZMP-mediated protein shedding also enhanced PC3 invasion; an effect that could be negated using a sheddase inhibitor or copper chelators. Collectively, these data implicate copper as an important factor in promoting prostate cancer cell invasion and indicate that the selective posttranslational activation of ZMP-mediated protein shedding might play a role in this process.

TGFβ1 induces apoptosis in invasive prostate cancer and bladder cancer cells via Akt-independent, p38 MAPK and JNK/SAPK-mediated activation of caspases

Recent findings indicate that advanced stage cancers shun the tumor suppressive actions of TGFβ and inexplicably utilize the cytokine as a tumor promoter. We investigated the effect of TGFβ1 on the survival and proliferation of invasive prostate (PC3) and bladder (T24) cancer cells. Our study indicated that TGFβ1 decreased cell viability and induced apoptosis in invasive human PC3 and T24 cells via activation of p38 MAPK-JNK-Caspase9/8/3 pathway. Surprisingly, no change in the phosphorylation of pro-survival Akt kinase was observed. We postulate that TGFβ1 pathway may be utilized for specifically targeting urological cancers without inflicting side effects on normal tissues.

Role of glyoxalase I in the proliferation and apoptosis control of human LNCaP and PC3 prostate cancer cells

Glyoxalase I (GLOI) detoxifies reactive dicarbonyls, as methylglyoxal (MG) that, directly or through the formation of MG-derived adducts, is a growth inhibitor and apoptosis inducer. GLOI has been considered a general marker of cell proliferation, but a direct link between the two has yet to be demonstrated. The aim of the present work was to clarify whether GLOI was involved in the proliferation control of LNCaP and PC3 human prostate cancer cells or might play a different role in the growth regulation of these cells. RNA interference was used to study the role of GLOI in cell proliferation or apoptosis. Cell proliferation was evaluated by [3H]thymidine incorporation assay and flow cytometry, that was also used to analyze apoptosis. Real-time TaqMan polymerase chain reaction and spectrophotometric analyses were used to study transcript levels or specific activity, respectively. Proteins levels were analyzed by Western blot. MG was measured by high-performance liquid chromatography. We found that GLOI is not implicated in the proliferation control of LNCaP and PC3 cells but plays a role in the apoptosis of invasive prostate cancer PC3 cells, through a mechanism involving a specific MG-adduct and NF-kB signaling pathway. Our data represent the first systematic demonstration that GLOI cannot be considered a general marker of cell proliferation and that acts as a pro-survival factor in invasive PC3 cells by elusing apoptosis. GLOI may be involved in prostate cancer progression, via the control of key molecules in the mitochondrial apoptotic mechanism, through NF-kB signaling pathway.

Abstract 4322: Iron increases the invasiveness of prostate cancer cells in vitro: Mechanisms and inhibition by the antioxidant ebselen

Abstract The effect of iron on PC-3 prostate cancer cell invasiveness was investigated using Neuroprobe filter membrane transmigration assays. PC-3 cells were pre-incubated with ferric ammonium citrate (FAC) for 6 hours. Excess iron was removed before the assay. The PC-3 cells were placed on an extracellular membrane preparation (Matrigel®) in the upper wells of the chamber and allowed to migrate onto the underside of a porous membrane that allowed cell transmigration. The number of cells migrating to the underside of the membrane was determined and used as a marker of cell invasion. Pre-treatment with 100 µM FAC caused a 4-fold increase in PC-3 cell invasion when measured at 24 hours (P< 0.05). The FAC concentrations that induced PC-3 invasion were 100 times smaller than concentrations which interfered with cell viability or proliferation rate. Western blot analysis suggested that the effect of FAC exposure on PC-3 cell invasion was mediated by p42/44 MAP kinase activity. Furthermore, the antioxidant ebselen, a hydrogen peroxide inhibitor, decreased FAC-induced PC-3 cell invasion. We also examined the mRNA expression of invasion-related genes using a cDNA array and found a positive correlation between the exposures to FAC and the invasion phenotype of prostate cancer cells. The data demonstrated that the upregulation of genes, such as fractalkine and plasminogen, after FAC treatment may be associated with iron-induced invasion of prostate cancer. Our data suggest that iron overload may be detrimental to patients with prostate cancer and that the effect of iron on invasion may be inhibited by ebselen. 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 4322. doi:1538-7445.AM2012-4322

321 HDAC BLOCKADE COUNTERACTS RESISTANCE DEVELOPMENT CAUSED BY CHRONIC MTOR INHIBITION

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2012321 HDAC BLOCKADE COUNTERACTS RESISTANCE DEVELOPMENT CAUSED BY CHRONIC MTOR INHIBITION Jasmina Makarevic, Igor Tsaur, Eva Weich, Axel Haferkamp, and Roman Blaheta Jasmina MakarevicJasmina Makarevic Frankfurt, Germany More articles by this author , Igor TsaurIgor Tsaur Frankfurt, Germany More articles by this author , Eva WeichEva Weich Frankfurt, Germany More articles by this author , Axel HaferkampAxel Haferkamp Frankfurt, Germany More articles by this author , and Roman BlahetaRoman Blaheta Frankfurt, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.381AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR-targeting is an attractive approach to prostate cancer therapy. However, the benefit of long-term application has not been evaluated in detail. Growth and invasive behavior of prostate cancer cells under chronic exposure to the mTOR-inhibitor temsirolimus was, therefore, investigated. Further investigation was designed to determine whether additionally targeting histone deacetylase (HDAC) by an HDAC-inhibitor (valproic acid, VPA) might counteract undesired modification of the tumor caused by chronic use of temsirolimus. METHODS Prostate cancer cells were either treated with temsirolimus over 12 months, starting at 1 nM and increasing stepwise to 5 μM (PC3TEM), or only treated with medium (PC3). 3 days before experiment begin, temsirolimus containing culture medium was replaced by a temsirolimus free medium and tumor cells analyzed thereafter, or PC3TEM (versus PC3) then re-incubated with low-dosed (10 nM) temsirolimus to evaluate drug responsiveness. Growth, proliferation, adhesion to matrix proteins or endothelial cells and invasion of PC3 versus PC3TEM were investigated. Expression of cell cycle regulating proteins (cdk1, cdk2, cdk4, cyclin A,B,D3,E, p21, p27), mTOR related intracellular signaling (Akt, mTOR, rictor, raptor, p70S6k) and the expression profile of alpha and beta integrin subtypes were also evaluated. Additionally, HDAC was blocked in PC3TEM cells and the consequences on invasive growth investigated. RESULTS PC3 TEM accumulated in the G2/M-phase, accompanied by cdk1, cdk2, cyclin B elevation and reduction of p21 and p27. Application of 10 nM temsirolimus to PC3TEM did not diminish cell growth and proliferation, whereas proliferative activity in PC3 was significantly diminished by 10 nM temsirolimus. PC3TEM cell adhesion and invasion was enhanced by 10 nM temsirolimus, compared to temsirolimus treated PC3 control cells. The target proteins Akt, mTOR, rictor (but not raptor) and p70S6k were all elevated (total and activated) in PC3TEM compared to PC3 cells. Distinct modifications were also seen with respect to alpha and beta integrin expression. Additional application of VPA blocked growth and adhesion of PC3TEM cells, reverted integrin modulation and also deactivated proteins of the mTOR signaling pathway. CONCLUSIONS Chronic use of the mTOR inhibitor temsirolimus causes prostate cancer cell resistance. HDAC-inhibition counteracts this process, possibly by cross-communicating with the mTOR signaling axis. Specific targeting of HDAC may, therefore, enhance the benefit of an mTOR-inhibitor based regimen. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e130 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jasmina Makarevic Frankfurt, Germany More articles by this author Igor Tsaur Frankfurt, Germany More articles by this author Eva Weich Frankfurt, Germany More articles by this author Axel Haferkamp Frankfurt, Germany More articles by this author Roman Blaheta Frankfurt, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Altered CXCR3 isoform expression regulates prostate cancer cell migration and invasion

BackgroundCarcinoma cells must circumvent the normally suppressive signals to disseminate. While often considered 'stop' signals for adherent cells, CXCR3-binding chemokines have recently been correlated positively with cancer progression though the molecular basis remains unclear.ResultsHere, we examined the expression and function of two CXCR3 variants in human prostate cancer biopsies and cell lines. Globally, both CXCR3 mRNA and protein were elevated in localized and metastatic human cancer biopsies compared to normal. Additionally, CXCR3A mRNA level was upregulated while CXCR3B mRNA was downregulated in these prostate cancer specimens. In contrast to normal prostate epithelial cells (RWPE-1), CXCR3A was up to half the receptor in the invasive and metastatic DU-145 and PC-3 prostate cancer cells, but not in the localized LNCaP cells. Instead of inhibiting cell migration as in RWPE-1 cells, the CXCR3 ligands CXCL4/PF4 and CXCL10/IP10 promoted cell motility and invasiveness in both DU-145 and PC-3 cells via PLCβ3 and μ-calpain activation. CXCR3-mediated diminution of cell motility in RWPE-1 cells is likely a result of cAMP upregulation and m-calpain inhibition via CXCR3B signal transduction. Interestingly, overexpression of CXCR3B in DU-145 cells decreased cell movement and invasion.ConclusionThese data suggest that the aberrant expression of CXCR3A and down-regulation of CXCR3B may switch a progression "stop" to a "go" signal to promote prostate tumor metastasis via stimulating cell migration and invasion.

Circulation times of prostate cancer and hepatocellular carcinoma cells by in vivo flow cytometry

In metastasis, the cancer cells that travel through the body are capable of establishing new tumors in locations remote from the site of the original disease. To metastasize, a cancer cell must break away from its tumor and invade either the circulatory or lymphatic system, which will carry it to a new location, and establish itself in the new site. Once in the blood stream, the cancer cells now have access to every portion of the body. Here, we have used the "in vivo flow cytometer" to study if there is any relationship between metastatic potential and depletion kinetics of circulating tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labelled cells in vivo. We have improved the counting algorithm and measured the depletion kinetics of cancer cells with different metastatic potential. Interestingly, more invasive PC-3 prostate cancer cells are depleted faster from the circulation than LNCaP cells. In addition, we have measured the depletion kinetics of two related human hepatocellular carcinoma (liver cancer) cell lines, high-metastatic HCCLM3 cells, and low-metastatic HepG2 cells. More than 60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

CrkI and p130Cas complex regulates the migration and invasion of prostate cancer cells

Prostate cancer metastasis is often associated with poor prognosis. The molecular coupling of the adaptor protein Crk to the docking protein p130(Cas) serves as a switch that regulates cell migration in several invasive cancer cells and Ack appears to act upstream of CrkII to modulate the cell motility. However, the precise role of Ack, Crk and p130(Cas) complex in prostate cancer migration remains unknown. In this study we examined the expression of Crk and p130(Cas) in prostate cancer cell lines, and found that CrkI and p130(Cas) protein level was higher in highly invasive PC-3M and PC-3 cell lines than in moderately invasive DU-145 cells. Upon shRNA mediated knockdown of CrkI and p130(Cas) in PC-3M cells, cell migration and invasion were significantly inhibited as analyzed by wound healing assay and transwell invasion assay. Furthermore, co-immunoprecipitation assay showed that p130(Cas) interacted with CrkI in PC-3M cells and the stability of p130(Cas) and CrkI depended on each other. AckI interacted with both CrkI and p130(Cas) and the interaction of AckI with CrkI seemed to be independent of p130(Cas) . Taken together, our results demonstrate the high expression of CrkI and p130(Cas) in invasive prostate cancer cells and the important role of CrkI/p130(Cas) complex in the migration and invasion of prostate cancer cells. These data suggest that CrkI/p130(Cas) could be exploited as potential molecular therapeutic target for prostate cancer metastasis.

Wild-Type p53 Attenuates Cancer Cell Motility by Inducing Growth Differentiation Factor-15 Expression

A major function of the p53 tumor suppressor is the regulation of the cell cycle and apoptosis. In addition to its well-documented functions in malignant cancer cells, p53 can also regulate cell migration and invasion, which contribute to metastasis. Growth differentiation factor-15 (GDF-15), a member of the TGF-β superfamily, has been shown to be a downstream target of p53 and is associated with diverse human diseases and cancer progression. In this study, we examined the potential role of GDF-15 in p53-regulated cancer cell motility. We show that overexpression of wild-type p53 in two highly invasive p53-null human cancer cell lines, SKOV3 and PC3, attenuated cell migration and the movement through Matrigel. Using wild-type p53 and DNA-binding-deficient p53 mutants, we found that the transcriptional activity of p53 is required in the induction of GDF-15 expression. Cell movement through uncoated and Matrigel-coated transwell decreased in response to treatment with recombinant GDF-15, whereas the cell proliferation was not affected by GDF-15 treatment. Moreover, the induction of GDF-15 expression and secretion by p53 and the reduction in cell movement through Matrigel were diminished by treatment with GDF-15 small interfering RNA. This study demonstrates a mechanism by which p53 attenuates cancer cell motility through GDF-15 expression. In addition, our results indicate that GDF-15 mediates the functions of p53 by autocrine/paracrine action.

1610 STATINS REDUCE THE RISK OF PROSTATE CANCER PROGRESSION: AN IN VITRO STUDY IN TO THE MECHANISM OF METASTASIS

INTRODUCTION AND OBJECTIVES: Exposure of cancer cells to statins result in the down-regulation of the mevalonate pathway by HMG-CoA reductase inhibition, inducing inhibition of a range of cellular functions such as membrane integrity, cell signalling, protein synthesis and cell cycle progression. Disruption of these processes may result in changes in the regulation of tumour initiation, growth and metastasis. Clinical studies have reported that statins do not effect the incidence of clinically significant prostate cancer but usage reduces the risk of progression and mortality (0.26; 95% CI 0.08–0.83 0.64; 95% CI 0.44–0.93). It is currently unclear how statins reduce the clinical progression of prostate cancer. Here we present in vitro data showing that statins act directly on the prostate epithelial cells, reducing their ability to invade through and form metastatic colonies in the bone marrow stroma (BMS). METHODS: BMS was isolated with ethical approval from consenting patients undergoing surgery for non-malignant disease. All statins were used at non-toxic doses as determined by colony forming assays (atorvastatin, mevastatin, simvastatin (1 M), rosuvastatin (5 M) & pravastatin (100 M)). Binding of statin treated PC-3GFP cells to BMS was assessed using a FLUOstar OPTIMA spectrometer. Invasion of statin treated PC-3 cells towards untreated BMS or 7 day statin pre-treated BMS was assessed using the FluoroBlok tumour co-culture cell invasion system. Colony formation was assessed by seeding statin treated PC-3 cells on to confluent BMS. Statins were refreshed daily for 8 days before fixing and staining with anti pancytokeratin, counting and measuring epithelial colonies. RESULTS: Statins do not affect the ability of PC-3 cells to bind to BMS. Pravastatin had no effect on PC-3 invasion or colony formation in BMS. Treating PC-3 cells with atorvastatin, mevastatin, simvastatin and rosuvastatin significantly reduced their ability to invade towards BMS as compared with untreated controls on average 66.68% (range 53.93%–77.04% p 0.05). These statins also significantly reduced the number (76.2 vs 118; p 0.05) and size (0.2mm vs 0.29mm; p 0.05) of colonies formed in BMS co-culture, with colonies displaying a more compact morphology indicative of a reduction in the ability to migrate through the BMS. CONCLUSIONS: Statins reduce the risk of prostate cancer progression by inhibiting the ability of malignant prostate epithelial cells to invade towards and through human BMS, reducing the formation and the spread of metastatic prostate colonies.

Expression and shedding of endothelial protein C receptor in prostate cancer cells

BackgroundIncreasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.ResultsEPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H2O2, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells.ConclusionsThe study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.

Role of monocyte‐lineage cells in prostate cancer cell invasion and tissue factor expression

Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

Role of CXCL16/CXCR6 axis in the metastasis of human prostate cancer

To explore the roles of chemokine CXCL16 and its receptor CXCR6 in the directional invasion of human prostate cancer (PCa). The expression of CXCL16/CXCR6 in PCa samples and osseous tissues was determined by immunohistochemistry. The expression of CXCR6 in PC3 and LNCap cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Then the effects of CXCL16 upon the migration and invasion of human PC3 and LNCap cells were examined by Matrigel invasion assay. The expression of CXCR6 protein was detected in all clinical PCa samples. But no CXCL16 protein was detected. Positive CXCL16 expression was observed in human osseous tissues. Both PC3 and LNCap cells expressed CXCR6 mRNA (0.38+/-0.054 vs 0.41+/-0.019 respectively) and protein. In addition, CXCL16 could promote the in vitro migration and invasion of PC3 and LNCap cell lines (invading cells 211.50+/-5.60 vs 89.25+/-3.31 respectively). Such a promoting effect of CXCL16 could not be blocked influenced by antiCXCL12 or antiCXCR4. CXCL16/CXCR6 axis may be another independent chemokine factor playing a significant role in the metastasis of prostate cancer.

Influence of omega-6 PUFA arachidonic acid and bone marrow adipocytes on metastatic spread from prostate cancer

Background:Prostate cancer (CaP) preferentially metastasises to the bone, and we have previously shown that the poly-unsaturated fatty acid (PUFA) arachidonic acid (AA) is a potent stimulator of CaP invasion. Here we present that AA promotes CaP invasion by inducing bone marrow adipocyte formation.Methods:Boyden invasion-chamber assays assessed the ability of dietary oils, their PUFA components, and specific PUFA-loaded adipocytes to induce PC-3 invasion. Lipid transfer and metabolism was followed using deuterated AA and Fourier Transform Infrared spectroscopy (FTIR).Results:Poly-unsaturated fatty acid constituents, but not their corresponding dietary oils, induced PC-3 invasion. PUFAs induce bone marrow adipocyte (BM-Ad) differentiation with AA inducing higher levels of BM-Ad differentiation, as compared with other PUFAs (3998±514.4 vs 932±265.8; P=0.00002), which stimulated greater PC-3 invasion than free AA (22 408.5±607.4 vs 16 236±313.9; P=0.01111) or adipocytes generated in the presence of other PUFAs. In bone marrow co-culture PC-3 and BM-Ad interactions result in direct uptake and metabolism of AA by PC-3 cells, destruction of the adipocyte and subsequent formation of a bone metastasis.Conclusion:The data supports the hypothesis that AA not only promotes CaP invasion, it also prepares the ‘soil’, making it more supportive for implantation and propagation of the migrating metastatic cell.

Suppression of Wnt/β-catenin signaling inhibits prostate cancer cell proliferation

Although mounting evidence has demonstrated an important role of Wnt/β-catenin signaling in the development and progression of cancer, the therapeutic potential of small molecules that target this pathway for prostate cancer remains largely unknown. We reported herein that the highly invasive androgen-independent PC-3 and DU145 human prostate cancer cells exhibited higher levels of Wnt/β-catenin signaling than the androgen-dependent LNCaP prostate cancer cells and non-cancerous PZ-HPV-7 and PWR-1E prostate cells, and that exogenous Wnt3A treatment exaggerated the difference of the Wnt/β-catenin signaling levels among these prostate cells. Furthermore, we demonstrated that the non-steroidal anti-inflammatory drug, sulindac sulfide, the cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, and the nitric oxide-donating aspirin derivative, NO-ASA, blocked Wnt/β-catenin signaling in PC-3 and DU145 cells. These effects occurred at concentrations comparable to those required to inhibit cell proliferation, indicating that the inhibitory effect of these drugs on prostate cancer cell proliferation may involve the suppression of Wnt/β-catenin signaling. Finally, we showed that a novel small molecule inhibitor of Wnt/β-catenin signaling, PKF118- 310, inhibited Wnt/β-catenin signaling and proliferation in prostate cancer cells within the same concentration range. Together, these results suggest that small molecules that inhibit Wnt/β-catenin signaling have therapeutic potential for the prevention or treatment of prostate cancer.

TCEB1 promotes invasion of prostate cancer cells

Amplification of the long arm of chromosome 8 is one of the most recurrent findings in prostate cancer and it is associated with poor prognosis. Several minimal regions of amplification suggest multiple target genes which are yet to be identified. We have previously shown that TCEB1, EIF3S3, KIAA0196 and RAD21 are amplified and overexpressed in prostate cancer and they are located in the 8q area. In this study, we examined the functional effects of these genes to prostate cancer cell phenotype. We overexpressed and inhibited the genes by lentivirus mediated overexpression and RNA interference, respectively. shRNA mediated TCEB1 silencing decreased significantly cellular invasion of PC-3 and DU145 cells through Matrigel. TCEB1 silencing reduced the anchorage-independent growth of PC-3 cells. Similar effects were not seen with any other genes. When overexpressed in NIH 3T3 cells, TCEB1 and EIF3S3 increased the growth rate of the cells. Transcriptional profiling of TCEB1 silenced PC-3 cells revealed decrease of genes involved in invasion and metastasis. Finally, we also confirmed here the overexpression of TCEB1 in hormone-refractory prostate tumors. This study indicates that TCEB1 promotes invasion of prostate cancer cells, is involved in development of hormone-refractory prostate cancer and is thereby a strong candidate to be one of the target genes for the 8q gain.