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DO SEROTONIN AND MELATONIN HAVE A ROLE IN THE GROWTH REGULATION OF PROSTATE CANCER CELL LINES?
5
MASS SPECTROMETRIC IDENTIFICATION OF HUMAN PROSTATE CANCER-DERIVED PROTEINS IN SERUM OF XENOGRAFT-BEARING MICE Van Den Bemd G.J.1 , Krijgsveld J.2 , Luider T.3 , Demmers J.4 , Jenster G.1 1 Erasmus MC, Urology, Rotterdam, The Netherlands; 2 University Utrecht, Biomolecular Mass Spectrometry, Utrecht, The Netherlands; 3 Erasmus MC, Neurology, Rotterdam, The Netherlands; 4 Erasmus MC, Biochemistry, Rotterdam, The Netherlands
INTRODUCTION & OBJECTIVES: Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells, but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human, will by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. MATERIAL & METHODS: PC346 and PC339 human prostate cancer xenografts were grown subcutaneous in nude mice. Serum from these male mice were collected and abundant proteins removed. Depleted serum was separated by one-dimensional gel electrophoresis and the gel segmented in 3 mm gel slices. Each slice was trypsin digested and fractionated by nano-liquid chromatography and the mass and sequence of the peptides determined using LTQ-FT mass spectrometry. RESULTS: We identified approximately 1000 serum proteins, 300 of which were recognized as part of the human proteome while 200 had one or more peptides with a human-specific sequence. Interestingly, 9 glycolysis enzymes and 7 proteasome subunits were identified as human-specific proteins. Proteins related to glycolysis and proteasome have been detected in plasma and their expression is linked to tumorigenesis. Other known markers for cancer metastasis identified as xenograftderived proteins are NME1 and NME2. To prove independently that these proteins are xenograft-derived, western blot analyses were performed using antibodies directed against GAPDH, NME1, NME2, and proteasomes, showing their unique presence in xenograft-bearing mice. CONCLUSIONS: Using our novel method, we have shown for the first time that it is possible to identify tumor-derived proteins in a complex protein sample (serum) in an in vivo xenograft system. Although the usefulness of these proteins in the diagnosis and prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of novel prostate cancer biomarkers.
6
DO SEROTONIN AND MELATONIN HAVE A ROLE IN THE GROWTH REGULATION OF PROSTATE CANCER CELL LINES? Pirozhok I.1 , Meye A.2 , Hakenberg O.W.2 , F¨ussel S.2 , Wirth M.P.2 1 Central Emergency Hospital, Department of Urology, Chernivtsy, Ukraine; 2 “Carl Gustav Carus” Technical University of Dresden, Fetscherst 74, D-01307, Department of Urology, Dresden, Germany
INTRODUCTION & OBJECTIVES: The aims of the study were the investigation of the effects of serotonin (5-HT) and melatonin (MLT) on the regulation of malignant growth in prostate cancer cell lines and the activity of serotonin receptors (5HTR1a/1b) in prostate cancer (PCa) cell lines. MATERIAL & METHODS: In four human PCa cell lines (LNCaP, 22RV1, PC3, DU145) and two reference cell lines (BPH-1, human fibroblasts) 5-HTR1a and -1b relativemRNA expression was assessed by quantitative PCR (QPCR). Serotonin and Melatonin receptor agonists and antagonists (8-OH-DPAT (5HTR1a agonist), CGS-12066A maleate salt (5HTR1b agonist); NAN-190 hydrobromide (5HTR1a antagonist); Methiothepin mesylate salt (5HTR1b antagonist)) were used in stimulation and inhibition experiments in which apoptosis and changes in cell cycle phases were measured by flowcytometry. Cell proliferation WST-1 was used as the cellular viability test. RESULTS: The 5HTR mRNA expression in all PCa cell lines was higher for 5HTR1b than for 5HTR1a receptors and depended on cellular density. 5-HT at 10−4 M showed a significant growth stimulatory effect in all PCa cell lines after 24h of incubation. The 5-HTR1a and -1b agonists/antagonists did not significantly affect cellular viability. Melatonin showed an inhibitory activity in only one PCa cell line (PC3) and at a concentration of 10−3 –10−2 M after 72h of incubation. Similarly, the 5HTR1a antagonist NAN-190 induced apoptotic changes in PC3 cells only at high concentrations of 10−4 M, while methiothepin (5HTR1b antagonist) induced necrosis at concentrations (10−4 M) in all cell lines. Cell cycle alterations were seen in PC3 and DU 145 lines (G0 –G1 phase arrest) under the influence of the 5HTR1a antagonist (at 10−4 M). CONCLUSIONS: Our results only show effects of serotonin receptor antagonists and agonists as well as melatonin on cell viability and apoptosis of prostate cancer cell lines at high (supraphysiologic) concentrations. In contrast to recently published reports, our results thus do not support a regulatory role of serotonin or melatonin receptor activation or inhibition in prostate cancer growth. This research was supported by EUSP- EAU grant (S-02-2004) to I.P.
Eur Urol Suppl 2006;5:788
7
A NEW REAL-TIME RT-PCR. METHOD FOR THE DETECTION OF PSA POSITIVE CELLS IN BLOOD OF PROSTATE CANCER PATIENTS Barbero G.1 , Destefanis P.2 , Procida S.1 , Fiori C.2 , Ceruti C.2 , Bosio A.2 , Bisconti A.2 , Demaria C.2 , Ulliers D.1 , Mandili G.1 , Turrini F.1 , Fontana D.2 1 Universit`a Degli Studi di Torino, Dipartimento di Genetica, Biologia e Biochimica, Torino, Italy; 2 Ospedale San Giovanni Battista Molinette, Divisione Universitaria di Urologia 2, Torino, Italy
INTRODUCTION & OBJECTIVES: Since the introduction of PSA, test for the detection of prostate cancer (PCa) has rapidly become the mainstay of PCa diagnosis and follow-up. Molecular techniques have been widely used in research and clinical practice applied to PCa diagnosis and staging. Different results have been obtained. A new Real time PCR test, optimized in our Institution for the research of micrometastasis in colorectal and breast cancer patients, suggested us to reassess the role of real time PCR for the detection of PSA expressing tumor cells in the blood of patients with PCa. The new method has been applied to a large population of PCa patients to clarify the role of this test for diagnosis, staging and prognosis of PCa patients. MATERIAL & METHODS: Between 2003 and 2005, one or more venous blood samples were obtained from a population of PCa patients. All patients had given informed written consent. 5 mL of blood were collected and analyzed by a Real Time PCR method utilizing two novel features: a primer overlapping two adjacent exon in order to inhibit non specific amplification both in healthy donors and cancer patients and a non-end-point first round amplification step to increase sensitivity. The following parameters were considered for statistical analysis: age, PSA serum level at diagnosis, biopsy Gleason Score, clinical stage. RESULTS: Preliminary results are available for 54 patients (mean age 67,82±5,97 years) with newly diagnosed PCa, that undergone the test before any kind of treatment. Mean PSA value at diagnosis was 14,04±18,75 ng/ml. Median Gleason Score value was 6 with a range from 4 to 9. As far as NCCN recurrence risk is concerned, 21 patients were classified as low risk, 14 as intermediate risk, 15 as high risk and 4 as very high risk. Six patients had a positive result at Real time PCR test: the mean value was 3,47±3,26 PSA positive cells. Patients with positive test had a PSA value higher than patients with negative test but the difference didn’t prove to be statistically significant (p = 0,055). No difference was observed between real time PCR positive patients and negative ones regarding Gleason Score. All healthy donors analyzed (50) resulted negative. CONCLUSIONS: PSA positive blood circulating cell detection in patients affected by PCa using real-time RT-PCR could offer an extraordinary tool for diagnosis, staging and prognostic evaluation of this disease, but at this moment can only be applied to laboratory research due to the high rate of contradictory results provided by literature.
8
QUANTITATIVE REAL-TIME PCR OF SELECTED PROSTATE- AND PROSTATE CANCER-RELATED TRANSCRIPTS ON NATIVE NEEDLE BIOPSIES: ESTABLISHMENT OF THE TECHNOLOGY AND FIRST COMPARATIVE RESULTS Unversucht S.1 , Meye A.1 , Haase M.2 , F¨ussel S.1 , Wirth M.P.1 1 Technical University of Dresden, Department of Urology, Dresden, Germany; 2 Technical University of Dresden, Oncoray, Centre of Innovation Competence “Radiation Research in Oncology”, Dresden, Germany
INTRODUCTION & OBJECTIVES: At present diagnostic verification of prostate cancer (PCa) is limited to determination of serum PSA level and digital-rectal examinations normally followed by prostate needle core biopsies. In contrast prostateassociated or/and PCa-specific transcripts like DD3/PCA3 may present as sensitive and specific markers already in urine analysis for accurately molecular genetic determination of this disease and/or for the decision of new treatment strategies for prostate cancer patients. MATERIAL & METHODS: Immediately after radical prostatectomy multiple needle core biopsies were collected from fresh prostate specimens. Using cryomicrotome thin sections of biopsies for RNA isolations as well as for histological examination were generated. Quantitative PCR (QPCR) assays with site-specific hybridization probes were performed for nine different prostate-associated genes (like DD3/PCA3, EZH2, TRP-M8 or AMACR) as well as for the housekeeping gene TATA-box binding protein (TBP). Furthermore, the H&E-stained cryo-sections of needle core biopsies were histologically examined. RESULTS: The quality criterion for isolated RNA samples was defined as minimum amount of at least 50 molecules of the reference gene (TBP) per QPCR. In PCa-derived tissue specimens the amounts of relative transcript expression of the nine markers were comparable to those obtained from whole frozen PCa tissue specimens. After histological examinations on 6 cryo-sections which were uniformly distributed over the whole biopsy core by a trained pathologist, percentages of tumor/ stromal cells as well as Gleason Scores of collected prostate specimens were determined. A positive correlation of the relative expression levels and the percentage of tumor content within the biopsy were evaluated, such as for DD3. Moreover, the relative expression levels and the obtained Tu:Tf ratios were similar to those found in our previous studies for whole tissue samples originated from RPE explants. CONCLUSIONS: The presented analyses provided reproducible findings. The RNA derived from one half of a prostate needle core biopsy was sufficient to carry out QPCR for at least 10 different genes. The methodology combines the analysis of biopsy histology and the transcript quantification of different potential diagnostic and prognostic markers.
MASS SPECTROMETRIC IDENTIFICATION OF HUMAN PROSTATE CANCER-DERIVED PROTEINS IN SERUM OF XENOGRAFT-BEARING MICE Van Den Bemd G.J.1 , Krijgsveld J.2 , Luider T.3 , Demmers J.4 , Jenster G.1 1 Erasmus MC, Urology, Rotterdam, The Netherlands; 2 University Utrecht, Biomolecular Mass Spectrometry, Utrecht, The Netherlands; 3 Erasmus MC, Neurology, Rotterdam, The Netherlands; 4 Erasmus MC, Biochemistry, Rotterdam, The Netherlands
INTRODUCTION & OBJECTIVES: Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells, but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human, will by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. MATERIAL & METHODS: PC346 and PC339 human prostate cancer xenografts were grown subcutaneous in nude mice. Serum from these male mice were collected and abundant proteins removed. Depleted serum was separated by one-dimensional gel electrophoresis and the gel segmented in 3 mm gel slices. Each slice was trypsin digested and fractionated by nano-liquid chromatography and the mass and sequence of the peptides determined using LTQ-FT mass spectrometry. RESULTS: We identified approximately 1000 serum proteins, 300 of which were recognized as part of the human proteome while 200 had one or more peptides with a human-specific sequence. Interestingly, 9 glycolysis enzymes and 7 proteasome subunits were identified as human-specific proteins. Proteins related to glycolysis and proteasome have been detected in plasma and their expression is linked to tumorigenesis. Other known markers for cancer metastasis identified as xenograftderived proteins are NME1 and NME2. To prove independently that these proteins are xenograft-derived, western blot analyses were performed using antibodies directed against GAPDH, NME1, NME2, and proteasomes, showing their unique presence in xenograft-bearing mice. CONCLUSIONS: Using our novel method, we have shown for the first time that it is possible to identify tumor-derived proteins in a complex protein sample (serum) in an in vivo xenograft system. Although the usefulness of these proteins in the diagnosis and prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of novel prostate cancer biomarkers.
6
DO SEROTONIN AND MELATONIN HAVE A ROLE IN THE GROWTH REGULATION OF PROSTATE CANCER CELL LINES? Pirozhok I.1 , Meye A.2 , Hakenberg O.W.2 , F¨ussel S.2 , Wirth M.P.2 1 Central Emergency Hospital, Department of Urology, Chernivtsy, Ukraine; 2 “Carl Gustav Carus” Technical University of Dresden, Fetscherst 74, D-01307, Department of Urology, Dresden, Germany
INTRODUCTION & OBJECTIVES: The aims of the study were the investigation of the effects of serotonin (5-HT) and melatonin (MLT) on the regulation of malignant growth in prostate cancer cell lines and the activity of serotonin receptors (5HTR1a/1b) in prostate cancer (PCa) cell lines. MATERIAL & METHODS: In four human PCa cell lines (LNCaP, 22RV1, PC3, DU145) and two reference cell lines (BPH-1, human fibroblasts) 5-HTR1a and -1b relativemRNA expression was assessed by quantitative PCR (QPCR). Serotonin and Melatonin receptor agonists and antagonists (8-OH-DPAT (5HTR1a agonist), CGS-12066A maleate salt (5HTR1b agonist); NAN-190 hydrobromide (5HTR1a antagonist); Methiothepin mesylate salt (5HTR1b antagonist)) were used in stimulation and inhibition experiments in which apoptosis and changes in cell cycle phases were measured by flowcytometry. Cell proliferation WST-1 was used as the cellular viability test. RESULTS: The 5HTR mRNA expression in all PCa cell lines was higher for 5HTR1b than for 5HTR1a receptors and depended on cellular density. 5-HT at 10−4 M showed a significant growth stimulatory effect in all PCa cell lines after 24h of incubation. The 5-HTR1a and -1b agonists/antagonists did not significantly affect cellular viability. Melatonin showed an inhibitory activity in only one PCa cell line (PC3) and at a concentration of 10−3 –10−2 M after 72h of incubation. Similarly, the 5HTR1a antagonist NAN-190 induced apoptotic changes in PC3 cells only at high concentrations of 10−4 M, while methiothepin (5HTR1b antagonist) induced necrosis at concentrations (10−4 M) in all cell lines. Cell cycle alterations were seen in PC3 and DU 145 lines (G0 –G1 phase arrest) under the influence of the 5HTR1a antagonist (at 10−4 M). CONCLUSIONS: Our results only show effects of serotonin receptor antagonists and agonists as well as melatonin on cell viability and apoptosis of prostate cancer cell lines at high (supraphysiologic) concentrations. In contrast to recently published reports, our results thus do not support a regulatory role of serotonin or melatonin receptor activation or inhibition in prostate cancer growth. This research was supported by EUSP- EAU grant (S-02-2004) to I.P.
Eur Urol Suppl 2006;5:788
7
A NEW REAL-TIME RT-PCR. METHOD FOR THE DETECTION OF PSA POSITIVE CELLS IN BLOOD OF PROSTATE CANCER PATIENTS Barbero G.1 , Destefanis P.2 , Procida S.1 , Fiori C.2 , Ceruti C.2 , Bosio A.2 , Bisconti A.2 , Demaria C.2 , Ulliers D.1 , Mandili G.1 , Turrini F.1 , Fontana D.2 1 Universit`a Degli Studi di Torino, Dipartimento di Genetica, Biologia e Biochimica, Torino, Italy; 2 Ospedale San Giovanni Battista Molinette, Divisione Universitaria di Urologia 2, Torino, Italy
INTRODUCTION & OBJECTIVES: Since the introduction of PSA, test for the detection of prostate cancer (PCa) has rapidly become the mainstay of PCa diagnosis and follow-up. Molecular techniques have been widely used in research and clinical practice applied to PCa diagnosis and staging. Different results have been obtained. A new Real time PCR test, optimized in our Institution for the research of micrometastasis in colorectal and breast cancer patients, suggested us to reassess the role of real time PCR for the detection of PSA expressing tumor cells in the blood of patients with PCa. The new method has been applied to a large population of PCa patients to clarify the role of this test for diagnosis, staging and prognosis of PCa patients. MATERIAL & METHODS: Between 2003 and 2005, one or more venous blood samples were obtained from a population of PCa patients. All patients had given informed written consent. 5 mL of blood were collected and analyzed by a Real Time PCR method utilizing two novel features: a primer overlapping two adjacent exon in order to inhibit non specific amplification both in healthy donors and cancer patients and a non-end-point first round amplification step to increase sensitivity. The following parameters were considered for statistical analysis: age, PSA serum level at diagnosis, biopsy Gleason Score, clinical stage. RESULTS: Preliminary results are available for 54 patients (mean age 67,82±5,97 years) with newly diagnosed PCa, that undergone the test before any kind of treatment. Mean PSA value at diagnosis was 14,04±18,75 ng/ml. Median Gleason Score value was 6 with a range from 4 to 9. As far as NCCN recurrence risk is concerned, 21 patients were classified as low risk, 14 as intermediate risk, 15 as high risk and 4 as very high risk. Six patients had a positive result at Real time PCR test: the mean value was 3,47±3,26 PSA positive cells. Patients with positive test had a PSA value higher than patients with negative test but the difference didn’t prove to be statistically significant (p = 0,055). No difference was observed between real time PCR positive patients and negative ones regarding Gleason Score. All healthy donors analyzed (50) resulted negative. CONCLUSIONS: PSA positive blood circulating cell detection in patients affected by PCa using real-time RT-PCR could offer an extraordinary tool for diagnosis, staging and prognostic evaluation of this disease, but at this moment can only be applied to laboratory research due to the high rate of contradictory results provided by literature.
8
QUANTITATIVE REAL-TIME PCR OF SELECTED PROSTATE- AND PROSTATE CANCER-RELATED TRANSCRIPTS ON NATIVE NEEDLE BIOPSIES: ESTABLISHMENT OF THE TECHNOLOGY AND FIRST COMPARATIVE RESULTS Unversucht S.1 , Meye A.1 , Haase M.2 , F¨ussel S.1 , Wirth M.P.1 1 Technical University of Dresden, Department of Urology, Dresden, Germany; 2 Technical University of Dresden, Oncoray, Centre of Innovation Competence “Radiation Research in Oncology”, Dresden, Germany
INTRODUCTION & OBJECTIVES: At present diagnostic verification of prostate cancer (PCa) is limited to determination of serum PSA level and digital-rectal examinations normally followed by prostate needle core biopsies. In contrast prostateassociated or/and PCa-specific transcripts like DD3/PCA3 may present as sensitive and specific markers already in urine analysis for accurately molecular genetic determination of this disease and/or for the decision of new treatment strategies for prostate cancer patients. MATERIAL & METHODS: Immediately after radical prostatectomy multiple needle core biopsies were collected from fresh prostate specimens. Using cryomicrotome thin sections of biopsies for RNA isolations as well as for histological examination were generated. Quantitative PCR (QPCR) assays with site-specific hybridization probes were performed for nine different prostate-associated genes (like DD3/PCA3, EZH2, TRP-M8 or AMACR) as well as for the housekeeping gene TATA-box binding protein (TBP). Furthermore, the H&E-stained cryo-sections of needle core biopsies were histologically examined. RESULTS: The quality criterion for isolated RNA samples was defined as minimum amount of at least 50 molecules of the reference gene (TBP) per QPCR. In PCa-derived tissue specimens the amounts of relative transcript expression of the nine markers were comparable to those obtained from whole frozen PCa tissue specimens. After histological examinations on 6 cryo-sections which were uniformly distributed over the whole biopsy core by a trained pathologist, percentages of tumor/ stromal cells as well as Gleason Scores of collected prostate specimens were determined. A positive correlation of the relative expression levels and the percentage of tumor content within the biopsy were evaluated, such as for DD3. Moreover, the relative expression levels and the obtained Tu:Tf ratios were similar to those found in our previous studies for whole tissue samples originated from RPE explants. CONCLUSIONS: The presented analyses provided reproducible findings. The RNA derived from one half of a prostate needle core biopsy was sufficient to carry out QPCR for at least 10 different genes. The methodology combines the analysis of biopsy histology and the transcript quantification of different potential diagnostic and prognostic markers.