380 α1,2-FUCOSYLATED AND β- N-ACETYLGALACTOMINYLATED PROSTATE SPECIFIC ANTIGEN AS AN EFFICIENT...
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cells as “human-only” control. Each probe was hybridized onto the whole human (U133A 2.0, Affymetrix) and mouse (430A 2.0, Af- fymetrix) genome array. The cDNA probe sequences were compared by bioinformatics analysis and cross-hybridizing probes were excluded from analysis by computational mask. RESULTS: Due to cross-hybridization between human and mouse 7.7% and 6.3% of genes, respectively were excluded. In the bone stromal compartment 77 genes were up-regulated 8-fold in presence of PC cells. Three genes encoding extracellular matrix (ECM) proteins over expressed by the stroma in response to cancer cells were evaluated as putative biomarkers of PC bone metastasis, namely periostin (POSTN), asporin (ASPN), hevin (SPARCL1). Real time RT PCR assays with mouse (stroma) specific probes validated the stromal expression of the 3 genes and their over expression in presence of osteoinductive PC cells. Immunohistochemistry performed on tumor xenografted mouse bone, primary PC, benign prostate hyperplasia (BPH) and bone metastasis samples confirmed the protein products to be exclusively localized in the stroma. mRNA expression levels of POSTN, ASPN and SPARCL1 in murine primary calvaria osteoblasts in co-cultures with C4-2B4 cells are significantly increased after 24h compared to expression levels in absence of C4-2B4. CONCLUSIONS: This experimental model allows to dissect and analyze stroma derived from cancer derived gene expression. Cancer cells induce expression of ECM proteins in bone marrow stroma. Genes up-regulated in the stroma in response to cancer cells may represent potential markers for bone micro-/macro-metastasis. Source of Funding: None 379 NADPH OXIDASE MEDIATED ROS-GENERATION IS REQUIRED FOR HYPOXIA ASSOCIATED ANGIOGENIC SIGNALING IN PROSTATE CANCER Binod Kumar, Paul Maroni, Sweaty Koul, Jeong Seok Hwa, Randall Meacham, Hari Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Reactive oxygen spe- cies (ROS) generation through extra-mitochondrial sources, like NADPH oxidase play critical roles in pathophisology of prostate cancer. We have demonstrated previously that NADPH oxidase-derived ROS are required for aggressive phenotype. Moreover, tissue hypoxia is known to initiate multiple events that allow cells to continue proliferat- ing, mainly by activating hypoxia inducible factor 1 (HIF-1), a transcrip- tion factor activating downstream targets responsible for angiogenesis and increased survival. The goal of this study was to determine the role for NADPH oxidase in hypoxia mediated signaling in prostate cancer. METHODS: All the cell lines (LNCaP, PC3) were purchased from ATCC, and maintained according to ATCC guidelines. ROS generation was determined by fluorimetry using specific fluorescent dye. Cell proliferation and growth were measured by MTT and crystal violet method, respectively. The expression of HIF-1a ´ and VEGF were determined by RT-PCR as well as by Western blot methods. Promoter assays were performed for hypoxia response element (HRE), a 5-RCGTG-3 consensus sequence where HIF-1a ´ heterodimers bind. Expression of vascular endothelial growth factor receptor (VEGFR) was determined by RT-PCR. RESULTS: Our results show that NADPH oxidase inhibitor (DPI) inhibited hypoxia induced cell growth and proliferation in prostate cancer cell line. Moreover, exposure of prostate cancer cells with hypoxia was associated with an increased VEGF and VEGF receptor expression, as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity. We also observed that increased generation of ROS was an early response to hypoxia, and addition of NADPH oxidase inhibitor to the cells, not only abolished hypoxia induced ROS genera- tion, but also inhibited the expression of VEGF, VEGFR and HIF1a ´. Treatments of DPI also inhibited hypoxia induced promoter activity of HRE in LNCaP cells. These results demonstrate a critical role for hypoxia-induced ROS in pathogenesis of prostate cancer. Thus, cross talk between the hypoxia and NADPH oxidase system might play a critical role in tumor modulation in case of prostate cancer. CONCLUSIONS: These finding demonstrate that the hypoxia induced signaling pathways in prostate cancer are redox-sensitive, and ROS derived from NADPH oxidase is essential for hypoxic survival of prostate cancer cells. Source of Funding: Academic Enrichment Grant from The University of Ciolorado School of Medicine and the Department of Surgery 380 1,2-FUCOSYLATED AND - N-ACETYLGALACTOMINYLATED PROSTATE SPECIFIC ANTIGEN AS AN EFFICIENT MARKER OF PROSTATE CANCER Takefumi Satoh*, Kanagawa, Japan; Keiko Fukushima, Katsuko Yamashita, Yokohama, Japan; Ichiro Ito, Ken-ichi Tabata, Kazumasa Matsumoto, Shiro Baba, Kanagawa, Japan INTRODUCTION AND OBJECTIVES: It is well known that the structures of carbohydrates on the cell surface change during carcino- genesis. The analysis of the carbohydrate structures is difficult because of low levels of glycoprotein markers in the serum, such as PSA; however, it has been proposed that glycoproteins containing cancer- specific glycans exist. Lectin column chromatography is a powerful tool to elucidate both core structures and peripheral structures of carbohy- drates linked to at least several hundred pg of glycoproteins that are detectable by enzyme-linked immunosorbent assay (ELISA). To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we investigated whether prostate cancer (PC)-specific N-glycans are linked to PSA. METHODS: Serum samples were obtained from 20 randomly selected patients who were newly diagnosed with PC at various clinical stages and 20 patients with benign prostatic hyperplasia (BPH). His- topathologic findings of the prostate needle biopsy specimens con- firmed the diagnosis for all PC patients and the findings of systemic 22-core transperineal ultrasound-guided template prostate biopsy specimens confirmed the diagnosis for all BPH patients. We analyzed the carbohydrate structures of PSA derived from seminal fluid, serum of BPH and PC patients, using 8 lectin-immobilized columns and with ELISA. RESULTS: Total serum PSA concentrations from BPH and PC patients are shown in figure A. The fraction of serum PSA from PC patients bound to Fuc 1-2Gal and -GalNAc binding Trichosanthes japonica agglutinin-II (TJA-II) column, while that from BPH patients did not exhibit this binding ability. The amount of TJA-II bound total PSA in the serum of PC patients was significantly more than that in the serum of BPH patients (p 0.05) (Fig. B). As determined by TJA-II-Sepha- rose column chromatography, the mean SD (%) of the ratio of TJA-II bound PSA to total PSA of PC and BPH patients were 8.3 5.6% and 1.0 0.55%, respectively, and the difference was statistically signifi- cant (Fig. C). CONCLUSIONS: This study is the first to find that PSA-con- taining cancer-specific glycans are efficient markers of PC and could be used to discriminate between PC and BPH. e150 THE JOURNAL OF UROLOGY Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
Transcript of 380 α1,2-FUCOSYLATED AND β- N-ACETYLGALACTOMINYLATED PROSTATE SPECIFIC ANTIGEN AS AN EFFICIENT...
cells as “human-only” control. Each probe was hybridized onto thewhole human (U133A 2.0, Affymetrix) and mouse (430A 2.0, Af-fymetrix) genome array. The cDNA probe sequences were comparedby bioinformatics analysis and cross-hybridizing probes were excludedfrom analysis by computational mask.
RESULTS: Due to cross-hybridization between human andmouse 7.7% and 6.3% of genes, respectively were excluded. In thebone stromal compartment 77 genes were up-regulated �8-fold inpresence of PC cells. Three genes encoding extracellular matrix (ECM)proteins over expressed by the stroma in response to cancer cells wereevaluated as putative biomarkers of PC bone metastasis, namelyperiostin (POSTN), asporin (ASPN), hevin (SPARCL1). Real time RTPCR assays with mouse (stroma) specific probes validated the stromalexpression of the 3 genes and their over expression in presence ofosteoinductive PC cells. Immunohistochemistry performed on tumorxenografted mouse bone, primary PC, benign prostate hyperplasia(BPH) and bone metastasis samples confirmed the protein products tobe exclusively localized in the stroma. mRNA expression levels ofPOSTN, ASPN and SPARCL1 in murine primary calvaria osteoblasts inco-cultures with C4-2B4 cells are significantly increased after 24hcompared to expression levels in absence of C4-2B4.
CONCLUSIONS: This experimental model allows to dissectand analyze stroma derived from cancer derived gene expression.Cancer cells induce expression of ECM proteins in bone marrowstroma. Genes up-regulated in the stroma in response to cancer cellsmay represent potential markers for bone micro-/macro-metastasis.
Source of Funding: None
379NADPH OXIDASE MEDIATED ROS-GENERATION IS REQUIREDFOR HYPOXIA ASSOCIATED ANGIOGENIC SIGNALING INPROSTATE CANCER
Binod Kumar, Paul Maroni, Sweaty Koul, Jeong Seok Hwa, RandallMeacham, Hari Koul*, Aurora, CO
INTRODUCTION AND OBJECTIVES: Reactive oxygen spe-cies (ROS) generation through extra-mitochondrial sources, likeNADPH oxidase play critical roles in pathophisology of prostate cancer.We have demonstrated previously that NADPH oxidase-derived ROSare required for aggressive phenotype. Moreover, tissue hypoxia isknown to initiate multiple events that allow cells to continue proliferat-ing, mainly by activating hypoxia inducible factor 1 (HIF-1), a transcrip-tion factor activating downstream targets responsible for angiogenesisand increased survival. The goal of this study was to determine the rolefor NADPH oxidase in hypoxia mediated signaling in prostate cancer.
METHODS: All the cell lines (LNCaP, PC3) were purchasedfrom ATCC, and maintained according to ATCC guidelines. ROSgeneration was determined by fluorimetry using specific fluorescentdye. Cell proliferation and growth were measured by MTT and crystalviolet method, respectively. The expression of HIF-1a and VEGF weredetermined by RT-PCR as well as by Western blot methods. Promoterassays were performed for hypoxia response element (HRE), a5-RCGTG-3 consensus sequence where HIF-1a heterodimers bind.Expression of vascular endothelial growth factor receptor (VEGFR)was determined by RT-PCR.
RESULTS: Our results show that NADPH oxidase inhibitor(DPI) inhibited hypoxia induced cell growth and proliferation in prostatecancer cell line. Moreover, exposure of prostate cancer cells withhypoxia was associated with an increased VEGF and VEGF receptorexpression, as well as increased hypoxia inducible factor (HIF-1alpha)levels and activity. We also observed that increased generation of ROSwas an early response to hypoxia, and addition of NADPH oxidaseinhibitor to the cells, not only abolished hypoxia induced ROS genera-tion, but also inhibited the expression of VEGF, VEGFR and HIF1a.Treatments of DPI also inhibited hypoxia induced promoter activity ofHRE in LNCaP cells. These results demonstrate a critical role forhypoxia-induced ROS in pathogenesis of prostate cancer. Thus, crosstalk between the hypoxia and NADPH oxidase system might play acritical role in tumor modulation in case of prostate cancer.
CONCLUSIONS: These finding demonstrate that the hypoxiainduced signaling pathways in prostate cancer are redox-sensitive, andROS derived from NADPH oxidase is essential for hypoxic survival ofprostate cancer cells.
Source of Funding: Academic Enrichment Grant from TheUniversity of Ciolorado School of Medicine and the Departmentof Surgery
380�1,2-FUCOSYLATED AND �- N-ACETYLGALACTOMINYLATEDPROSTATE SPECIFIC ANTIGEN AS AN EFFICIENT MARKER OFPROSTATE CANCER
Takefumi Satoh*, Kanagawa, Japan; Keiko Fukushima, KatsukoYamashita, Yokohama, Japan; Ichiro Ito, Ken-ichi Tabata, KazumasaMatsumoto, Shiro Baba, Kanagawa, Japan
INTRODUCTION AND OBJECTIVES: It is well known that thestructures of carbohydrates on the cell surface change during carcino-genesis. The analysis of the carbohydrate structures is difficult becauseof low levels of glycoprotein markers in the serum, such as PSA;however, it has been proposed that glycoproteins containing cancer-specific glycans exist. Lectin column chromatography is a powerful toolto elucidate both core structures and peripheral structures of carbohy-drates linked to at least several hundred pg of glycoproteins that aredetectable by enzyme-linked immunosorbent assay (ELISA). To applythe cancer-associated carbohydrate alteration to the improvement ofPSA assay, we investigated whether prostate cancer (PC)-specificN-glycans are linked to PSA.
METHODS: Serum samples were obtained from 20 randomlyselected patients who were newly diagnosed with PC at various clinicalstages and 20 patients with benign prostatic hyperplasia (BPH). His-topathologic findings of the prostate needle biopsy specimens con-firmed the diagnosis for all PC patients and the findings of systemic22-core transperineal ultrasound-guided template prostate biopsyspecimens confirmed the diagnosis for all BPH patients. We analyzedthe carbohydrate structures of PSA derived from seminal fluid, serum ofBPH and PC patients, using 8 lectin-immobilized columns and withELISA.
RESULTS: Total serum PSA concentrations from BPH and PCpatients are shown in figure A. The fraction of serum PSA from PCpatients bound to Fuc�1-2Gal and �-GalNAc binding Trichosanthesjaponica agglutinin-II (TJA-II) column, while that from BPH patients didnot exhibit this binding ability. The amount of TJA-II bound total PSA inthe serum of PC patients was significantly more than that in the serumof BPH patients (p � 0.05) (Fig. B). As determined by TJA-II-Sepha-rose column chromatography, the mean � SD (%) of the ratio of TJA-IIbound PSA to total PSA of PC and BPH patients were 8.3 � 5.6% and1.0 � 0.55%, respectively, and the difference was statistically signifi-cant (Fig. C).
CONCLUSIONS: This study is the first to find that PSA-con-taining cancer-specific glycans are efficient markers of PC and could beused to discriminate between PC and BPH.
e150 THE JOURNAL OF UROLOGY� Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
Source of Funding: None
381ABERRANT EXTRACELLULAR SIGNAL-REGULATED PROTEINKINASE 5 (ERK5) SIGNALLING PROMOTES CELLULARMOTILITY AND INVASION IN PROSTATE CARCINOGENESIS
Alison Ramsay*, Glasgow, United Kingdom; Stuart McCracken,Newcastle Upon Tyne, United Kingdom; Rosie Morland, JanisFleming, Laura Machesky, Xinzi Yu, Glasgow, United Kingdom;Dylan Edwards, Robert Nuttall, Norwich, United Kingdom; MoragSeywright, Glasgow, United Kingdom; Evan Keller, Ann Arbor, MI;Hing Leung, Glasgow, United Kingdom
INTRODUCTION AND OBJECTIVES: The MEK5/ERK5 path-way is implicated in a number of tumour types including prostatecancer. Its molecular mechanism for carcinogenesis remains to be fullycharacterised. We have further investigated the functional role of ERK5in prostate cancer and tested its relationship ERK1/2.
METHODS: Functional assays were performed in prostate can-cer cells (PC3 and PC3M cells) following transfection with ERK1, ERK2and ERK5 siRNA or treatment with the mitogen-activated protein ki-nase kinase (MEK) inhibitor (PD184352): proliferation by cell count,motility by culture chamber, migration/invasion by time-lapse micro-scropy. Matrix metalloproteinase (MMP) expression was studied bypromoter luciferase reporter assay and real time PCR. Invadopodiaassays were performed as described previously (J Cell Sci 2008, 121:369), and data were analysed using Image J software. Immunohisto-chemistry for ERK5 expression was also performed on metastaticprostate tissue (Oncogene 2008, 27: 2978).
RESULTS: Using siRNA and PD184352 to target ERK5 ex-pression and function respectively, both motility and invasion of PC3cells were significantly impaired (P�0.005 for all parameters). Q-PCRand promoter activity analysis revealed activation of MMP-2, -9, -12and TIMP2 expression, and suppression of MMP-16 by ERK5. Follow-ing ERK5 transfection, PC3 cells formed significantly more invadopodia(P�0.0048). Immunohistochemistry further confirmed upregulatedERK5 expression in both primary and metastatic prostate tumours.Among the informative metastatic lesions (n�11), strong ERK5 immu-noreactivity, cytoplasmic (55%) and nuclear (73%), was observed.Potential ‘cross-talk’ between ERK5 and ERK1/2 was explored. ERK1knockdown resulted in prolonged and enhanced activation of bothERK5 and ERK2. This was associated with a small but reproduciblemitogenic effect following ERK1 specific knockdown, supporting pub-lished data that ERK1 acts as a negative regulator and ERK2 as apositive regulator of cell proliferation.
CONCLUSIONS: Our results validate the importance of theERK5 signalling pathway as a potential target for therapy and highlighta novel functional and biochemical relationship between ERK1 andERK5 signalling.
Source of Funding: Royal College of Surgeons of Edinburgh/Royal College of Physicians and Surgeons of Glasgow
382BENZODIAZEPINES INHIBIT CELL PROLIFERATION ANDINCREASE APOPTOSIS IN PROSTATE CANCER BOTH IN VIVOAND IN VITRO BY BLOCKING TRANSLOCATOR PROTEIN
Ardavan Akhavan*, New York, NY; Arlee Fafalios, Anil Parwani,Robert Bies, Kevin McHugh, Beth Pflug, Pittsburgh, PA
INTRODUCTION AND OBJECTIVES: Translocator Protein(TSPO), formerly known as the peripheral benzodiazepine receptor,mediates translocation of cholesterol into the mitochondria, the rate-limiting step in steroidogenesis, and has been implicated in cell prolif-eration and apoptosis in epithelial tumors. We investigated the expres-sion of TSPO in prostate cancer and evaluated the impact of TSPOblockade with benzodiazepines both in vivo and in vitro.
METHODS: Tumor tissue microarrays for prostate cancer pro-gression, PSA failure and tumor metastases were stained and gradedfor TSPO. PPC-1, LN97, DU145 human prostate cancer cell lines, andHEK239 embryonic kidney cells transfected with a TSPO overexpress-ing vector were assessed for TSPO by immunoblot analysis. Cells weretreated with TSPO ligands, PK11195 or lorazepam and cell viability,apoptosis, and cell number were assessed. In vivo, tumor growth wasassessed in prostate cancer cell mouse xenografts treated daily withvehicle or lorazepam over 12 weeks. Tumors were then immunostainedand scored for markers of angiogenesis, apoptosis and cell prolifera-tion.
RESULTS: TSPO expression is progressively increased inprostatic intraepithelial neoplasia, primary prostate cancer, and meta-static disease over normal and BPH tissue, respectively. TSPO immu-nostaining is also higher with increasing Gleason sum as well as clinicalstage. All prostate cancer cell lines expressed TSPO. Treatment witheither PK11195 or lorazepam increased cell death and decreased cellviability and cell counts in all prostate cancer cell lines investigated.HEK239-TSPO overexpressing cells had significantly higher suscepti-bility to lorazepam over nontransfected HEK239 cells. In vivo, tumors ofxenograft mice treated for 12 weeks with lorazepam were approxi-mately 1⁄2 the size of the tumors of mice treated with vehicle. Immuno-histochemistry revealed lorazepam-treated tumors had increased apo-ptosis and decreased cell proliferation while no effect on angiogenesiswas evident.
CONCLUSIONS: TSPO is upregulated with prostate cancerprogression. TSPO blockade with benzodiazepines has significanteffects on cell proliferation and apoptosis and is an attractive potentialtherapeutic adjuvant for future clinical trials.
Source of Funding: National Center for Research ResourcesGrant 5 UL1 RR024153; Department of Defense GrantPC080062
383CHARACTERIZATION OF �4-INTEGRIN-MEDIATED CYTOTOXICEFFECT OF AEXU, A TYPE THREE SECRETION SYSTEMEFFECTOR, ON HUMAN PROSTATE CANCER PC3 CELLS
Masafumi Kumano*, Hideaki Miyake, Kobe, Japan; Takeshi Honda,Osaka, Japan; Masato Fujisawa, Kobe, Japan
INTRODUCTION AND OBJECTIVES: Type three secretionsystem (TTSS) of gram-negative bacteria is responsible for deliveringbacterial proteins, termed effectors, from the bacterial cytosol directlyinto the host cells. AexU, one of TTSS effectors, has been shown toplay an important role during A. hydrophila SSU strain pathogenesisthrough the ribosylation of ADP. Recently, several investigators have
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