PC-3M Cells Research Articles

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

A Comprehensive Panel of Three-Dimensional Models for Studies of Prostate Cancer Growth, Invasion and Drug Responses

Prostate epithelial cells from both normal and cancer tissues, grown in three-dimensional (3D) culture as spheroids, represent promising in vitro models for the study of normal and cancer-relevant patterns of epithelial differentiation. We have developed the most comprehensive panel of miniaturized prostate cell culture models in 3D to date (n = 29), including many non-transformed and most currently available classic prostate cancer (PrCa) cell lines. The purpose of this study was to analyze morphogenetic properties of PrCa models in 3D, to compare phenotypes, gene expression and metabolism between 2D and 3D cultures, and to evaluate their relevance for pre-clinical drug discovery, disease modeling and basic research. Primary and non-transformed prostate epithelial cells, but also several PrCa lines, formed well-differentiated round spheroids. These showed strong cell-cell contacts, epithelial polarization, a hollow lumen and were covered by a complete basal lamina (BL). Most PrCa lines, however, formed large, poorly differentiated spheroids, or aggressively invading structures. In PC-3 and PC-3M cells, well-differentiated spheroids formed, which were then spontaneously transformed into highly invasive cells. These cell lines may have previously undergone an epithelial-to-mesenchymal transition (EMT), which is temporarily suppressed in favor of epithelial maturation by signals from the extracellular matrix (ECM). The induction of lipid and steroid metabolism, epigenetic reprogramming, and ECM remodeling represents a general adaptation to 3D culture, regardless of transformation and phenotype. In contrast, PI3-Kinase, AKT, STAT/interferon and integrin signaling pathways were particularly activated in invasive cells. Specific small molecule inhibitors targeted against PI3-Kinase blocked invasive cell growth more effectively in 3D than in 2D monolayer culture, or the growth of normal cells. Our panel of cell models, spanning a wide spectrum of phenotypic plasticity, supports the investigation of different modes of cell migration and tumor morphologies, and will be useful for predictive testing of anti-cancer and anti-metastatic compounds.

Synthesis and antitumor properties of selenocoxib‐1 against rat prostate adenocarcinoma cells

Hormone refractory prostate cancer poses a huge problem and standard of care chemotherapy has not been very successful. We used a novel strategy to combine properties of 2 well-studied class of compounds (selenium and COX-2 inhibitor) and examined the resulting effectiveness against prostate cancer. Bearing in mind that sulfonamide moiety and pyrazole ring is important for the proapoptotic activity of Celecoxib, we synthesized a selenium derivative, Selenocoxib-1, by modifying Celecoxib at position 3 of the pyrazole ring. The PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund-Wistar (LW) rat were used to examine the efficacy of Selenocoxib-1 in vitro. In addition, human metastatic prostate cancer cells, PC-3M, were tested for antitumor effect of Selenocoxib-1 in vitro. The IC(50) in PAIII and PC-3M cells for Selenocoxib-1 was about 5 microM, while for Celecoxib it was more than 20 microM. Selenocoxib-1 induced apoptosis in a dose-dependent manner in the PAIII cells. COX-2 expression in PAIII cells was downregulated by Celecoxib and Selenocoxib-1 at 20 and 5 microM, respectively; the COX-2 activity was, however, not affected by Selenocoxib-1. Following treatment with Selenocoxib-1, PAIII cells resulted in dose-dependent decrease in HIF-1alpha, p-AKT and Bcl-2 levels. A reduction in weights was observed in subcutaneous tumors produced by PAIII cells pretreated with Selenocoxib-1 as compared to Celecoxib in LW rats. Further, following 1 week Selenocoxib-1 treatment of PAIII tumors resulted in significant reduction of tumor weights. This study demonstrates that Selenocoxib-1 is more effective against prostate cancer than Celecoxib.

Abstract LB-85: TGF-ß regulates its own expression and down-regulates its own receptors in prostate cancer (CaP) cells through a positive feedback pathway mediated by DNA methyltransferase

Abstract Introduction: Over expression and loss insensitivity to TGF-ß are two features which are coincident with CaP progression. In present study, we investigated whether a positive feedback pathway exists in which TGF-ß activates DNA methyltransferase (DNMT), which induces promoter hypermethylation. This hypermethylation may down-regulate TGF-ß receptors and in turn stimulate increased expression of TGF-ß. Materials and Methods: Human CaP cells (including the less aggressive lines, PC-3 and PC-3M-Pr04; and more aggressive lines, PC-3M and PC-3M-LN4) were rendered insensitive to TGF-ß using a retroviral vector containing a dominant negative TGF-ß type II receptor gene (TßRIIDN). ELISA were performed to quantify the secreted TGF-ß 1 (pg/105 cells/48 hours) in the pooled conditioned medium from CaP cells expressing T RIIDN and mock transfected. Semi-quantification western blot analyses were performed to evaluate the expression of DNA methyltransferase (DNMT), TßRI and TßRII. A [3H]-Thymidine Incorporation Assay was performed to determine cell proliferation rates in relation to TGF-ß. Results: Compared to PC-3 and PC-3M-Pr04, PC-3M and PC-3M-LN4 secreted almost 2 folds higher baseline levels of TGF-ß1 and DNMTs (1, 3α and 3ß). In contrast, TßRI and TßRII expression was significantly reduced in these same cell lines. In all cell lines, TßRIIDN expression was associated with significantly higher (∼1-2×) TGF-ß secretion, a greater than 3-fold decrease in the expression of DNMT1, DNMT3a, and DNMT 3b, and a correspondingly increased expression of both TßRI and TßRII (naïve intracellular domain). Although there was no significant difference in the original proliferation rate among PC3 variants, PC-3 and PC-3M-Pr04 cells were more sensitive to growth inhibition (>30%) by TGF-ß1 compared to PC-3M-LN4 and PC-3M cells (<7.5%). The growth inhibitory effects of TGF-ß1 disappeared when these cells were rendered insensitive to TGF-ß by infection with TßRIIDN. Conclusion: Higher expression of TGF-ß and lower expression of its own receptors are associated with more aggressive CaP phenotypes. Overexpression of tumor derived TGF-ß can activate DNMTs which will then mediate promoter methylation and down regulation of its receptors, subsequently, blockade of TGF-ß signaling may activate a feedback system which can stimulate the secretion of more TGF-ß. This novel mechanism has yet been reported, and may provide a new insight on TGF-ß regulation in prostate cancer. This study was supported in part by grants from the American Cancer Society, Illinois (#08-22), Department of Defense (W81XWH-09-1-0311), Portes Center/Institute of Medicine of Chicago (QZ), American Cancer Society Institutional Research Grant (ACS-IRG 93-037-12), American Urological Association Foundation (AUA Foundation, QZ), National Cancer Institute (P50CA090386), Illinois Department of Public Health (Contract #:83284034), a grant from the Genzyme Corporation, and a gift from Mr. Fred L. Turner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-85.

The apoptosis of human prostate cancel cell line (PC-3M) at different levels of Omi/HtrA2 when treated with TRAIL

Objective To study the apoptosis of human prostate cancel cell line (PC-3M) at dif-ferent levels of Omi/HtrA2 when treated with TNF-related apoptosis-inducing ligand (TRAIL). Methods siRNA expressing vector was constructed. The expression of omi/HtrA2 in PC-3M cells was detected by u-sing reverse transcription-polymerase chain reaction (RT-PCR) techniques. According to the software of Invivogen company, Omi/HtrA2 specific siRNA sequence was designed, synthesized in vitro and cloned in-to the expression vector psiRNA-hH1neo. The constructed psiRNA-Omi/HtrA2 was transiently transfected into PC-3M cells and the inhibitory effect of psiRNA-Omi/HtrA2 on the expression of Omi/HtrA2 in PC-3M cells was detected using RT-PCR and Western blotting. The apoptesis index (AI) of PC-3M cells was determined by in situ end labeling techniques under different TRAIL concentrations (50, 100,200 and 500 μg/L). Results There was high level of the Omi/HtrA2 expression in PC 3M cells. Enzyme digestion a-nalysis and DNA sequencing confirmed the Omi/HtrA2 specific siRNA expression vector was constructed successfully. The expression of Omi/HtrA2 was down-regulated in PC-3M cells after the transfection of psiRNA-Omi/HtrA2. The AI of PC-3M cells in transfection group was 7. 23, 14. 87, 22. 65 and 31.78 re-spectively, and that in un-transfection group was 15.28, 24. 17, 36. 33 and 47. 76, respectively (P < 0. 05). Conclusion Omi/HtrA2 plays an important role in the apoptosis of PC-3M cells. TRAIL can in-duce the apoptosis of PC-3M cells concentration-dependently, and its effect was correlated with the expres-sion level of omi/HtrA2. Key words: siRNA; Prostate carcinoma; TNF-related apoptosis-inducing ligand; Serine protease

390 ONCOLYTIC ADENOVIRUS TARGETING TRANSFORMING GROWTH FACTOR BETA SIGNALING: A NOVEL GENE THERAPY APPROACH FOR THE TREATMENT OF PROSTATE CANCER METASTASES

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2010390 ONCOLYTIC ADENOVIRUS TARGETING TRANSFORMING GROWTH FACTOR BETA SIGNALING: A NOVEL GENE THERAPY APPROACH FOR THE TREATMENT OF PROSTATE CANCER METASTASES Zebin Hu, Janhavi Gupta, Zhenwei Zhang, Charles Brendler, and Prem Seth Zebin HuZebin Hu More articles by this author , Janhavi GuptaJanhavi Gupta More articles by this author , Zhenwei ZhangZhenwei Zhang More articles by this author , Charles BrendlerCharles Brendler More articles by this author , and Prem SethPrem Seth More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.458AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The goal of this project is to develop a gene therapy approach to treat prostate cancer metastases. Recent literature suggests that high levels of transforming growth factor-ß (TGFß) can enhance tumor invasion and metastasis of prostate cancer. We have developed an oncolytic adenovirus (Ad.sTßRFc) that destroys tumor cells and simultaneously expresses soluble TGFßRII fused with Fc domain of human IgG (sTGFßRIIFc), a protein that can inhibit TGFß signaling, together inhibiting prostate cancer metastases. METHODS In Vitro Assays. Prostate tumor cells (PC3-M, DU-145) were infected with Ad.sTßRFc and sTGFßRIIFc expression examined by Western blots and ELISA, and viral replication examined by plaque forming assays. To study the effect of sTGFßRIIFc on TGFß1 signaling, SMAD-2 and SMAD-3 phosphorylation by Western blots and IL-11 production by ELISA were examined. Metastasis Studies. PC3-M cells were injected into the left heart ventricles of nude mice. After four days, Ad.sTßRFc was injected intravenously. On day 45, animals were subjected to radiographic analysis; tumors were identified by histology; blood samples were analyzed for sTGFßRIIFc, and tumors were examined for viral replication. RESULTS In Vitro Assays. Exposure of PC3-M and DU-145 cells to Ad.sTßRFc produced 1.4 μg/ml and 0.9 μg/ml sTGFßRIIFc respectively in the extracellular media of the two cell lines, and resulted in at-least 9,000-fold increase in the viral titers. Ad.sTßRFc-mediated production of sTGFßRIIFc inhibited TGFß-dependent SMAD-2 and SMAD-3 phosphorylation, IL-11 production and osteoclast formation. Metastasis Studies. In the group that received buffer alone, 7 of 9 animals (78%) had large lesions either in bone or soft tissues. However, in the group that received the Ad.sTßRFc, only 2 of 9 animals (22%) developed tumors by day 45, indicating significant inhibition of metastases. The Ad.sTßRFc treated group had high levels (> 300 μg/ml) of sTGFßRIIFc in the blood, as well as viral replication in the tumors, suggesting that both viral replication and sTGFßRIIFc production are critical for the anti-tumor response of Ad.sTßRFc. CONCLUSIONS Data presented here shows that it is feasible to create an oncolytic adenovirus that can induce tumor cell destruction and simultaneously inhibit TGFß signaling. More importantly, the systemic administration of Ad.sTßRFc can inhibit metastases, suggesting that Ad.sTßRFc can be developed as an anti-tumor agent for the treatment of prostate cancer metastases. Evanston, IL© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e154-e155 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zebin Hu More articles by this author Janhavi Gupta More articles by this author Zhenwei Zhang More articles by this author Charles Brendler More articles by this author Prem Seth More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

381 ABERRANT EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE 5 (ERK5) SIGNALLING PROMOTES CELLULAR MOTILITY AND INVASION IN PROSTATE CARCINOGENESIS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2010381 ABERRANT EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE 5 (ERK5) SIGNALLING PROMOTES CELLULAR MOTILITY AND INVASION IN PROSTATE CARCINOGENESIS Alison Ramsay, Stuart McCracken, Rosie Morland, Janis Fleming, Laura Machesky, Xinzi Yu, Dylan Edwards, Robert Nuttall, Morag Seywright, Evan Keller, and Hing Leung Alison RamsayAlison Ramsay Glasgow, United Kingdom More articles by this author , Stuart McCrackenStuart McCracken Newcastle Upon Tyne, United Kingdom More articles by this author , Rosie MorlandRosie Morland Glasgow, United Kingdom More articles by this author , Janis FlemingJanis Fleming Glasgow, United Kingdom More articles by this author , Laura MacheskyLaura Machesky Glasgow, United Kingdom More articles by this author , Xinzi YuXinzi Yu Glasgow, United Kingdom More articles by this author , Dylan EdwardsDylan Edwards Norwich, United Kingdom More articles by this author , Robert NuttallRobert Nuttall Norwich, United Kingdom More articles by this author , Morag SeywrightMorag Seywright Glasgow, United Kingdom More articles by this author , Evan KellerEvan Keller Ann Arbor, MI More articles by this author , and Hing LeungHing Leung Glasgow, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.449AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The MEK5/ERK5 pathway is implicated in a number of tumour types including prostate cancer. Its molecular mechanism for carcinogenesis remains to be fully characterised. We have further investigated the functional role of ERK5 in prostate cancer and tested its relationship ERK1/2. METHODS Functional assays were performed in prostate cancer cells (PC3 and PC3M cells) following transfection with ERK1, ERK2 and ERK5 siRNA or treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor (PD184352): proliferation by cell count, motility by culture chamber, migration/invasion by time-lapse microscropy. Matrix metalloproteinase (MMP) expression was studied by promoter luciferase reporter assay and real time PCR. Invadopodia assays were performed as described previously (J Cell Sci 2008, 121: 369), and data were analysed using Image J software. Immunohistochemistry for ERK5 expression was also performed on metastatic prostate tissue (Oncogene 2008, 27: 2978). RESULTS Using siRNA and PD184352 to target ERK5 expression and function respectively, both motility and invasion of PC3 cells were significantly impaired (P<0.005 for all parameters). Q-PCR and promoter activity analysis revealed activation of MMP-2, -9, -12 and TIMP2 expression, and suppression of MMP-16 by ERK5. Following ERK5 transfection, PC3 cells formed significantly more invadopodia (P=0.0048). Immunohistochemistry further confirmed upregulated ERK5 expression in both primary and metastatic prostate tumours. Among the informative metastatic lesions (n=11), strong ERK5 immunoreactivity, cytoplasmic (55%) and nuclear (73%), was observed. Potential ‘cross-talk' between ERK5 and ERK1/2 was explored. ERK1 knockdown resulted in prolonged and enhanced activation of both ERK5 and ERK2. This was associated with a small but reproducible mitogenic effect following ERK1 specific knockdown, supporting published data that ERK1 acts as a negative regulator and ERK2 as a positive regulator of cell proliferation. CONCLUSIONS Our results validate the importance of the ERK5 signalling pathway as a potential target for therapy and highlight a novel functional and biochemical relationship between ERK1 and ERK5 signalling. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e151 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Ramsay Glasgow, United Kingdom More articles by this author Stuart McCracken Newcastle Upon Tyne, United Kingdom More articles by this author Rosie Morland Glasgow, United Kingdom More articles by this author Janis Fleming Glasgow, United Kingdom More articles by this author Laura Machesky Glasgow, United Kingdom More articles by this author Xinzi Yu Glasgow, United Kingdom More articles by this author Dylan Edwards Norwich, United Kingdom More articles by this author Robert Nuttall Norwich, United Kingdom More articles by this author Morag Seywright Glasgow, United Kingdom More articles by this author Evan Keller Ann Arbor, MI More articles by this author Hing Leung Glasgow, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract A21: Cancer stem cell in multi-drug-resistant prostate cancer and green tea polyphenols restore drug sensitivity by reversal of epithelial mesenchymal transition

Abstract Background: Prostate cancer (PC) is the most common cause of cancer death in men. In advanced PC, many patients suffer chemo-resistance therefore; establishment of a drug-resistant cell model to study the mechanism of action of drug resistance and increase in drug sensitivity is urgently needed. Recently, cancer stem cells (CSCs), and epithelial mesenchymal transition (EMT) have been suggested to play important roles in drug resistance in various cancer types. Green tea polyphenols, particularly the major component epigallocatechin-3-gallate (EGCG), have been shown to have chemopreventive properties in the malignant setting and recent studies have suggested that green tea catechins may enhance the chemo-sensitivity of drug-resistant cancer cells, such as breast cancer, although the mechanism of action is still unclear. Aims: (1) To establish a multi-drug-resistant prostate cancer cell line enriched for CSCs and (2) To investigate whether EGCG can restore drug sensitivity by limiting CSC expansion and/or by reversal of EMT. Experimental procedures: Chemo-resistant (CR) PC3M cells were developed by growth in increasing concentrations of a chemotherapeutic agent. Drug resistance was confirmed using increasing concentrations of doxorubicin (Dox), Taxotere (Tax), 5-fluorouracil (5FU) and cisplatin in fluorometric cell viability assays on resistant and wild-type (WT) cells. Immunofluorescent (IF) staining and flow cytometry were used to identify CSCs. Gene expression was assessed using quantitative real time-PCR. Cell morphology was observed using Nikon invert microscopy. Results: A chemo-resistant variant of WT-PC3M was derived by long-term culture in increasing concentrations of the chemotherapeutic, 5FU. As a result, the IC50 for WT PC3M to 5FU was 30μM as compared to &amp;gt;500μM for CR PC3M. In addition, the CR cells were also shown to be resistant to other chemotherapeutics including Dox and Tax, but not cisplatin. Enrichment for cells with CSC properties was determined in WT and CR cells. In comparison to WT PC3M, gene expression of both CD133 and ATP-binding cassette subfamily G2 (ABCG2), which have been reported as CSC markers in various cancer including prostate cancer, were significantly up-regulated by 10 and 2-folds, respectively in drug resistant cells (p&amp;lt;0.01). Protein expression of both CSC markers was confirmed to be elevated in CR PC3M by IF staining. Data from flow cytometry showed that there were 10–15% double-positive CD133/ABCG2 cells within CR cells whereas the percentage is &amp;lt;2% in WT PC3M cells. In addition, CD133 expression was confirmed to be down-regulated by EGCG. When EGCG was combined with 5FU, the IC50 of 5FU in CR PC3M decreased from 500μM to 100μM in CR PC3M, but it did not show a significant effect in WT PC3M, suggesting that EGCG had restored drug sensitivity in CR cells. It was noted that chemo-resistant PC3M had a more mesenchymal appearance when compared to WT PC3M. The expression of key EMT transcriptional regulator genes such as twist, snail, slug and zeb-1, were up-regulated whereas the epithelial marker gene E-cadherin was down-regulated in CR PC3M. After 48hours treatment with EGCG, all four EMT regulatory genes were significantly down-regulated by up to 3-folds whereas expression of E-cadherin was elevated. Conclusion: Green tea polyphenols restore drug sensitivity in chemo-resistant prostate cancer by reversal of EMT. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A21

Apoptosis induction by E2F decoy DNA of the prostate cancer cell line

To observe effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M the binding specificity of the E2F decoy DNA to the PC-3M nuclear protein was detected by electrophoretic mobility shift assay (EMSA). E2F decoy DNA, ARE decoy DNA and Control decoy DNA were respectively transfected into PC-3M cells with lipofectamine.Their influence on cell proliferative activity was detected by MTT assay.The cell apoptotic rate was determined by flow cytometric(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The change of mRNA expression of C-myc and CyclinD1 were detected by RT-PCR.The change of mRNA expression of C-myc and CyclinD1 were detected by Western-blot. EMSA demonstrated specific binding of the E2F decoy to E2F transcription factor.The PC-3M cell growth was inhibited after transfection. The apoptotic rate was 26.35% and DNA ladder could be observed after transfection.The expression of C-myc and CyclinD1 were inhibited. All these results indicated that E2F decoy DNA induced apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibited cell proliferation via inhibiting expression of C-myc and CyclinD1.

Iridoids and Lignans from Valeriana jatamansi

Thirteen new iridoids including seven iridolactones, jatamanins A-M (1-13), and a new lignan, (+)-9'-isovaleroxylariciresinol (14), together with seven known iridoids and 13 lignans were obtained from whole plants of Valeriana jatamansi. Structures of the new compounds were determined by spectroscopic and crystallographic methods, and the absolute configuration of compound 1 was assigned by application of the modified Mosher method. Jatamanins H (8) and I (9) are iridolactones with an unusual C-8-C-11 oxygen bridge, forming a cage-like structure. (+)-9'-Isovaleroxylariciresinol (14) showed significant in vitro cytotoxicity against PC-3M and HCT-8 cell lines, with IC(50) values of 8.1 and 5.3 microM, respectively.

Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro

To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro. Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells. A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1E8. Western blot revealed that PAG1 protein was downregulated in PC-3M-1E8 cell line which was in agreement with the gene expression at mRNA level. The proliferation ability and clonogenicity of PAG1 overexpression cells by stable transfection were markedly decreased in comparing with that of the control cells (P < 0.05). Colonies formed in stably PAG1-transfected cells, the vector-transfected clones and parental cells were 26.7 ± 5.2, 47.2 ± 3.2 and 52.3 ± 3.4 respectively (P < 0.05). Flow cytometry analysis showed that the stable PAG1-transfected cells at G₀-G₁ phase were significantly more than that of the control cells (P < 0.05). However, no difference of the apoptosis rate was found between PAG1-transfected cells and control cells (P > 0.05). The number of cells passing through the matrigel and multipore membrane was also decreased in the stable PAG1-transfected cells (35.1 ± 4.9) compared with those of the vector-transfected clones (127.6 ± 6.6) and parental cells (135.0 ± 5.0, P < 0.05). Using cDNA microarray technique and differential gene expression analysis of sublines of the parental cancer cell lines enable of revealing the metastasis-related genes, among which PAG1 represents one of those under-expressed genes in the high metastatic subline PC-3M-1E8. Transfection expression of PAG1 suppresses cell proliferation, anchorage-independent growth and invasive ability of PC-3M-1E8 cells in vitro. Conclusively, PAG1 may play an important role in inhibiting the proliferation, invasion and metastasis of the cancer cells.

Stable alterations of CD44 isoform expression in prostate cancer cells decrease invasion and growth and alter ligand binding and chemosensitivity

BackgroundDysregulated CD44 expression characterizes most human cancers, including prostate cancer (PCa). PCa loses expression of CD44 standard (CD44s) that is present in benign epithelium, and overexpresses the novel splice variant isoform, CD44v7-10.MethodsUsing retroviral gene delivery to PC-3M PCa cells, we expressed luciferase-only, enforced CD44s re-expression as a fusion protein with luciferase at its C-terminus or as a protein separate from luciferase, or knocked down CD44v7-10 by RNAi. Invasion, migration, proliferation, soft agar colony formation, adhesion, Docetaxel sensitivity, and xenograft growth assays were carried out. Expression responses of merlin, a CD44 binding partner, and growth-permissive phospho-merlin, were assessed by western blot.ResultsCompared to luciferase-only PC-3M cells, all three treatments reduced invasion and migration. Growth and soft agar colony formation were reduced only by re-expression of CD44s as a separate or fusion protein but not CD44v7-10 RNAi. Hyaluronan and osteopontin binding were greatly strengthened by CD44s expression as a separate protein, but not a fusion protein. CD44v7-10 RNAi in PC-3M cells caused marked sensitization to Docetaxel; the two CD44s re-expression approaches caused minimal sensitization. In limited numbers of mouse subcutaneous xenografts, all three alterations produced only nonsignificant trends toward slower growth compared with luciferase-only controls. The expression of CD44s as a separate protein, but not a fusion protein, caused emergence of a strongly-expressed, hypophosphorylated species of phospho-merlin.ConclusionStable re-expression of CD44s reduces PCa growth and invasion in vitro, and possibly in vivo, suggesting CD44 alterations have potential as gene therapy. When the C-terminus of CD44s is fused to another protein, most phenotypic effects are lessened, particularly hyaluronan adhesion. Finally, CD44v7-10, although it was not functionally significant for growth, may be a target for chemosensitization.

Involvement of Fatty Acid Binding Protein 5 and PPARβ/δin Prostate Cancer Cell Growth

Fatty acid binding protein 5 (FABP5) delivers ligands from the cytosol directly to the nuclear receptor PPARβ/δ and thus facilitates the ligation and enhances the transcriptional activity of the receptor. We show here that expression levels of both FABP5 and PPARβ/δ are correlated with the tumorigenic potential of prostate cancer cell lines. We show further that FABP5 comprises a direct target gene for PPARβ/δ and thus the binding protein and its cognate receptor are engaged in a positive feedback loop. The observations demonstrate that, similarly to effects observed in mammary carcinomas, activation of the FABP5/PPARβ/δ pathway induces PPARβ/δ target genes involved in cell survival and growth and enhances cell proliferation and anchorage-independent growth in prostate cancer cells. Furthermore, the data show that downregulation of either FABP5 or PPARβ/δ inhibits the growth of the highly malignant prostate cancer PC3M cells. These studies suggest that the FABP5/PPARβ/δ pathway may play a general role in facilitating tumor progression and that inhibition of the pathway may comprise a novel strategy in treatment of cancer.

Nuclear localization of insulin-like growth factor binding protein-6

To study the nuclear localization of insulin-like growth factor binding protein-6(IGFBP-6) in PC-3M cells. The two fragments of the nuclear localization sequence (NLS)-deleted IGFBP-6 and the NLS-mutated IGFBP-6 were obtained by overlapping PCR, and then the fragment was inserted into a pEGFP-C1 vector. PC-3M cells were transfected with the expression constructs containing wild-type IGFBP-6 or the two mutants (pEGFP-C1-BP6 Delta NLS and pEGFP-C1-BP6-Mut), and the different distribution of the three EGFP-fusion proteins was observed by confocal laser microscope. The statistical analysis of the ratio of the nuclear fluorescence to the cytoplasmic fluorescence (Fn/c) was performed. Results Confocal microscopic images of transfected cells showed that the green fluorescence of EGFP-IGFBP-6 was concentrated mostly in the nuclei, whereas the control cells expressing EGFP showed green fluorescence distributed uniformly. The results of Fn/c from EGFP and EGFP-IGFBP-6 were significant different (P<0.05). The NLS-deleted IGFBP-6 completely eliminated nuclear accumulation of the green fluorescent signal; in contrast, nuclear accumulation was only slightly reduced for the NLS-mutated IGFBP-6; compared with wild-type IGFBP-6, both mutants were significantly different (P<0.05). Conclusions IGFBP-6 can be translocated to the nucleus in PC-3M cell that is mediated by a putative NLS sequence. Our study provides new evidence for further studies on the insulin-like growth factor-independent activity of IGFBP-6.

Atelocollagen-delivered siRNA targeting the FABP5 gene as an experimental therapy for prostate cancer in mouse xenografts

The gene FABP5 encodes cutaneous fatty acid binding protein (C-FABP) that is up-regulated in prostate cancer where it acts as a putative oncogene. To test the hypothesis that siRNA to FABP5 delivered to the external environment of a prostate cancer would reduce the level of C-FABP in vivo, experiments were established whereby siRNA to FABP5 suspended in atelocollagen was injected around tumour masses produced by PC-3M cells in Balb/c nude mice and compared with the effect of non-specific scrambled siRNA in atelocollagen. At autopsy, the average size of tumours from the groups treated with 10 and 15 microM siRNA in atelocollagen was significantly (p=0.02) reduced by more than 3-fold, when compared to the controls. In contrast, when compared to the tumours produced by the group treated with scrambled siRNA, treatment with 10 microM FABP5 siRNA in buffer and 1 or 5 microM siRNA in atelocollagen did not produce significant differences. Although the dosage of 15 microM siRNA produced a greater reduction in tumour sizes when compared with 10 microM, this difference was not significant (p=0.9). Immunohistochemistry and Western blotting revealed that the levels of C-FABP expression in tumours from mice treated with 10 and 15 microM dosages were lower than those from the other groups. These data demonstrate that FABP5 siRNA delivered by atelocollagen to the external environment surrounding a tumour mass can effectively inhibit prostate cancer cell growth in nude mice when administered in a dose-dependent manner at concentrations of >10 microM.

Endoglin phosphorylation by ALK2 contributes to the regulation of prostate cancer cell migration

Endoglin, a transmembrane glycoprotein that acts as a transforming growth factor-beta (TGF-beta) coreceptor, is downregulated in PC3-M metastatic prostate cancer cells. When restored, endoglin expression in PC3-M cells inhibits cell migration in vitro and attenuates the tumorigenicity of PC3-M cells in SCID mice, though the mechanism of endoglin regulation of migration in prostate cancer cells is not known. The current study indicates that endoglin is phosphorylated on cytosolic domain threonine residues by the TGF-beta type I receptors ALK2 and ALK5 in prostate cancer cells. Importantly, in the presence of constitutively active ALK2, endoglin did not inhibit cell migration, suggesting that endoglin phosphorylation regulated PC3-M cell migration. Therefore, our results suggest that endoglin phosphorylation is a mechanism with relevant functional consequences in prostate cancer cells. These data demonstrate for the first time that TGF-beta receptor-mediated phosphorylation of endoglin is a Smad-independent mechanism involved in the regulation of prostate cancer cell migration.

The Chemopreventive Bioflavonoid Apigenin Inhibits Prostate Cancer Cell Motility through the Focal Adhesion Kinase/Src Signaling Mechanism

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratch-wound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of "exaggerated filopodia," which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness.

Inhibition of Tumor Cell Motility by the Interferon-inducible GTPase MxA

To identify pathways controlling prostate cancer metastasis we performed differential display analysis of the human prostate carcinoma cell line PC-3 and its highly metastatic derivative PC-3M. This revealed that a 78-kDa interferon-inducible GTPase, MxA, was expressed in PC-3 but not in PC-3M cells. The gene encoding MxA, MX1, is located in the region of chromosome 21 deleted as a consequence of fusion of TMPRSS2 and ERG, which has been associated with aggressive, invasive prostate cancer. Stable exogenous MxA expression inhibited in vitro motility and invasiveness of PC-3M cells. In vivo exogenous MxA expression decreased the number of hepatic metastases following intrasplenic injection. Exogenous MxA also reduced motility and invasiveness of highly metastatic LOX melanoma cells. A mutation in MxA that inactivated its GTPase reversed inhibition of motility and invasion in both tumor cell lines. Co-immunoprecipitation studies demonstrated that MxA associated with tubulin, but the GTPase-inactivating mutation blocked this association. Because MxA is a highly inducible gene, an MxA-targeted drug discovery screen was initiated by placing the MxA promoter upstream of a luciferase reporter. Examination of the NCI diversity set of small molecules revealed three hits that activated the promoter. In PC-3M cells, these drugs induced MxA protein and inhibited motility. These data demonstrate that MxA inhibits tumor cell motility and invasion, and that MxA expression can be induced by small molecules, potentially offering a new approach to the prevention and treatment of metastasis.

Identification of a small molecule class to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer, and activated CT-CTR autocrine axis plays a pivotal role in tumorigenicity and metastatic potential of multiple prostate cancer cell lines. Recent studies suggest that CT promotes prostate cancer metastasis by reducing cell-cell adhesion through the disassembly of tight and adherens junctions and activation of beta-catenin signaling. We attempted to identify a class of molecules that enhances cell-cell adhesion of prostate cells and reverses the disruptive actions of CT on tight and adherens junctions. Screening several compounds led to the emergence of phenyl-methylene hydantoin (PMH) as a lead candidate that can augment cell-cell adhesion and abolish disruptive actions of CT on junctional complexes. PMH reduced invasiveness of PC-3M cells and abolished proinvasive actions of CT. Importantly, PMH did not display significant cytotoxicity on PC-3M cells at the tested doses. I.p. administered PMH and its S-ethyl derivative remarkably decreased orthotopic tumor growth and inhibited the formation of tumor micrometastases in distant organs of nude mice. PMH treatment also reduced the growth of spontaneous tumors in LPB-Tag mice to a significant extent without any obvious cytotoxic effects. By virtue of its ability to stabilize cell junctions, PMH could reverse the effect of CT on junctional disruption and metastasis, which strengthens the possibility of using PMH as a potential drug candidate for CT-positive androgen-independent prostate cancers.

Osteopontin Mediates Dense Culture‐Induced Proliferation and Adhesion of Prostate Tumour Cells: Role of Protein Kinase C, p38 Mitogen‐Activated Protein Kinase and Calcium

Cells growing in high density were observed to undergo a variety of responses due to cell-cell contact, pericellular hypoxia, etc. In order to investigate the influence of cell density on cell proliferation and adhesion and to elucidate possible mechanisms, we tested the growth ability of human prostate tumour (PC-3M) cells in dense culture and the influences of density on cell adhesion. Our results demonstrate that increasing cell density exerted stress on PC-3M cells, which decreased cell proliferation in dense cultures, but tended to facilitate tumour metastasis since cell adhesion ability was elevated and the cells showed an increased growth rate after being moved to a favourable growth environment. We conclude that higher cell density-mediated pericellular hypoxia was an important factor inducing expression of the intrinsic hypoxia marker osteopontin, another mechanism contributing to cell adhesion enhancement in PC-3M cells. In addition, cell density enhanced adhesion ability due to the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C. Intracellular calcium also played positive roles at least partially through activating p38 MAPK.