- Email: [email protected]
Inhibition of prostatic epithelial invasion and proliferation in human bone marrow by zoledronic acid
137 Dads Sa-REDUCTASE ANDROGEN ACTION, CANCER CELL LINES
INHIBITOR,
DUTASTERIDE,
CELL GROWTH/VIABILITY
INHIBITS
IN PROSTATE
138 INHIBITION OF PROSTATIC EPITHELIAL PROLIFERATION IN HUMAN BONE MARROW ACID
INVASION AND BY ZOLEDRONIC
Montaeue R.J.‘. Hart C.A.‘. Brown M.‘. Ramam V.A C.‘, George N.J.R.‘, Clarke N.W.’
Lazier C.‘, Thomas L.‘, Douglas R.‘, Schmidt L.‘. Tindall D.’ ,Dalhousic University, Biochemistry and Molecular Biology, Halifax, Canada, :Mayo Clinic, Biochemistry and Molecular Biology, Rochester, United States of America, ‘Mayo Clinic, Urology, Rochester, United States of America INTRODUCTION ,& OBJECTIVES: See-Reductase (5aR) catalysis of teStosterone (T) to dthydrotestosterone (DHT) amplifies androgen action, Our obje&ve was to mvestigate,actions of dutasteride, a novel dual 5aR inhibitor, on prostate cancer (PCs) cell lmes (LNCaP, LAPC-4, PC-3). MATERIAL & METHODS: PSA (prostate specific antigen) levels were measured by radioimmunoassay. Cell growth and viability were assessed by specific assays including Annexin V staining and BrdU uptake. RESULTS: In LNCaP cells. dutasteride inhibited 5aRlactivity and T action. In addition, DHT-induced accumulation of PSA was inhibited with an IC50 of 0.5. JuM after incubation with 0. InM DILT for I8 hr or 6 days. LNCaP cells exhibited decreased viability after 48 hr incubation with ?IOpM dutasteride. The same effects were seen with LAPC-4 cells, which contain wild type AR, as opposed to the T877A mutant AR found in LNCaP cells. Both cell lines showed decreased proliferation after 48-72 hrs in IO uM dutasteride. PC-3 cells, which lack AR, had no decrease in viability/proliferation for up to 96 hrs with IOpM dutasteride but did at 50pM. Treatment of LNCaP cells with IOuM dutasteridc for I8 hrs resulted in increased cell death that was partly reduced by inclusion of the synthetic androgen, RI 881. CONCLUSIONS: Prostate cells containing mutant or wild-type AR are more sensitive to cytotoxic effects of dutasteride than cells lacking AR. R188 I partly protects against dutasteride’s impact on cell death. The dutasteride dose required for half-maximal inhibttion of DHT-Induced PSA accumulation is lower than the half-maximal cytotoxic dose. The molecular basis for these observations is under investigation.
‘South Manchester Universtty Hospital, Urology. Manchester. United Kingdom. ‘Paterson Institute. Kay Kendall. Manchester, United Kingdom. ‘Chrisite Hospital. Urology, Manchester, Umred Kingdom INTRODUCTION & OBJECTIVES: This study examines the effect of the Bisphosphonate, Zoledronic Acid (Zoledronate) on prostate epithelial proliferation and mvasion in vitro using epithelial / endothelial co-culture techniques and cytokme assay. MATERIAL & METHODS: Prostatic epithelial cells isolated from men undergoing TURP for benign and malignant disease and the PC-3 prostate cancer cell line were studied in co-culture using long term human bone marrow stroma harvested from men with bentgn disease. Prostatic eprthelial binding and colony growth in bone marrow stroma was measured using standardised quantitative techniques in the presence of escalating doses of control (EDTA), Clodronate. Pamidronate and Zoledronate. Prostatic cellular invasion through matrigel (synthetic basement membrane) and cultured cndothelial monolayers was measured etther rn invasion chambers m the presence absence of drugs using cytokeratin labelled quantitatton of migrating cells or by the tmeasurement of endothelial monolayer permeability using quantitative fluorescent dextran labelhng. Co-culture supematants were assayed for specific cytokine levels. Bone marrow cellular toxicity was assessed using a standard MIXassay. RESULTS: Zoledronate inhibited ~nvasron and prohferation of prostattc epithelial crllb by 70% and 45% respectively. at concentrattons of >40pM. Thus inhibition significantly exceeded that seen with Pamidronate, whtch inhibited invasron by 50% and proliferation by 17%. Clodronate had no discemable effect by comparison with control. None of the test drugs had a measurable effect on cell /matrix binding or on endothelial permeability. suggesting that the observed phenomenon was a consequence of direct cellular Inhibition. Cytokine assays of supematants showed reduced levels of VEGF, TNFo. TIMP-I and 2 and GM-CSF with increasing Zoledronate concentrations whilst levels of TGFIJ, MMP I, 7 and the MMP activator uPA increased. Bone marrow cellular suppression was seen with Zoledronate but only at doses exceeding those required for inhibition of proliferation and migration of epithelial cells (Zoledronate 50pM proliferation / migration, marrow inhibition SO-IOOpM). CONCLUSIONS: Zoledronate
significantly inhibits prostate epithelial proliferation and migration in bone marrow but ha? no demonstrable effect on cell / matrix binding or cndothelial permeability. fhe observed effect IS assoctated wrth srgmticant alteratrons rn the cytokine mrlieu. Bone marrow hacmopoictic proliferatton IS Inhibited hut thus effect 15 only seen at higher dose levels.
140
139 IN VITRO CYTOTOXIC EFFECTS OF A TYROSINE KINASE INHIBITOR IMATINIB IN HUMAN PROSTATE CANCER CELL LINES
UPM3 TEST - A NEW MOLECULAR ASSAY DETECTING PROSTATE CANCER IN URINE SAMPLES -A NEW FUTURE PERSPECTIVE
Kiibler H.. Van Randenborgh
TinLl M.. Djavan
Technische Universitat
H., Wutrler S.. Lehmer A.. llartung
R., Paul R
Munchen, Urology. Munich, Germany
ClnivrrGty
INTRODUCTION & OBJECTIVES: Hormonerefractory prostate cancer (HRPC) is a major cause of mortality and morbidity in mtn. Although rcccnt clinical trials of taxanc-based regimes have demonstrated significant clinical activity in HRPC no survival benefit has yet been shown. Therefore, there is a clear riced to explore new targets and agents in the search of more effective treatment. Imatinib (ST1571, Gleevec@) is a selective inhibitor of the plateletderived growth factor receptors (PDGF-R). Previous studies hav,e shown that PDGF-Rs arc cxprcssed in prostatic intraepithelial neoplasia and adenocarcinoma hut not in benign prostate hypcrplasia ot- normal prostatic epithelium. MATERIAL & METHODS: Human prostate cancer cell lines LNCaP, DU145 and PC-3 were purchased from the American Type Tissue Collection and were cultured in RPMI-medium. Imatinib was kindly provided by Novartts. Drug solutions of lmatinib at different concentrations were added to a 96-well tissue culture plate. Variable cell growth was determined by XTT reduction assay, RESULTS: The dose-response curves of lmatinib for LNCaP, DU-145 and PC3 were determined by XTT assay after continuously exposure to the drug for 4 days. The IC50.values of lmatinib for LNCaP were l7uM, for PC-3 12,5pM and for DU-145 21.2uM respectively. A significant inhibition ofprolifcrations could be achieved.
of Vienna,Urology.
that the administration of cancer cell lines. Although trials, the present findings clinical protocols involving
Imatimb there are provide Imatinib
M Vienna.
Austria
INTRODUCTION & OBJECTIVES: Serum markers currently available fat prostate cancer dctcction lack sensttivity and specially sufticient speciticity. Earlier results with urinary PSA were inconsistent, whereas recent preliminary reports with a newly developed molecular urine test (uPM3) suggested a significant improvement of the statistical performance of total PSA. The purpose of the prospective study was to evaluate the uPM3 test in men referred for early prostate cancer detection and a PSA form 2.5-10 ng/mL. MATERIAL & METHODS: A total of X2 pattents were included and uPM3 tcstmg performed prior to TRUS-guided prostate biopsy. After having carefully performed a dtgital-rectal examination the first voided urine (30 ml) was taken as sample for the assay. The uPM3 test consists of an isothermic nucleic acid based amplification assay (NASBA -technique). It detects the expression of PSAmRNA as a marker of prostate cells in the urine sample and PCA-3mRNA which selectively is expressed in the majority of prostate cancer patients. The two targets are detected in real-time using specific beacons as probes tn an EasyQ instrument. Final pathological results were compared and statistical analysis performed using the JMP V3.2.2, (SAS, Cary, USA). RESULTS: A total of X2 urine samples were obtained. Cancer detection rate was 3 I%. The overall uPM3 sensitivity and specificity wcrc 90.4% and 58% respectively. In patients with PSA levels between 2.5-4 ng/mL sensitivity was 98X and specificity
CONCLUSIONS: These findings suggest has cytotoxic effects against human prostate gaps between in vitro studies and clinical useful infonnation for the establtshment of in HRPC‘.
B., Marberger
77%.
CONCLUSIONS: The uPM3 test offers the advantage of an office based noninvasive test combining sensitivity and specificity values of over 90% and60%, respectively. More even, uPM3 significantly improved cancer dctcction in the low PS.4 range 2.5-4 ngirnl. European
Urology
Supplements
2 (2003)
No. 1, pp. 37
INHIBITOR,
DUTASTERIDE,
CELL GROWTH/VIABILITY
INHIBITS
IN PROSTATE
138 INHIBITION OF PROSTATIC EPITHELIAL PROLIFERATION IN HUMAN BONE MARROW ACID
INVASION AND BY ZOLEDRONIC
Montaeue R.J.‘. Hart C.A.‘. Brown M.‘. Ramam V.A C.‘, George N.J.R.‘, Clarke N.W.’
Lazier C.‘, Thomas L.‘, Douglas R.‘, Schmidt L.‘. Tindall D.’ ,Dalhousic University, Biochemistry and Molecular Biology, Halifax, Canada, :Mayo Clinic, Biochemistry and Molecular Biology, Rochester, United States of America, ‘Mayo Clinic, Urology, Rochester, United States of America INTRODUCTION ,& OBJECTIVES: See-Reductase (5aR) catalysis of teStosterone (T) to dthydrotestosterone (DHT) amplifies androgen action, Our obje&ve was to mvestigate,actions of dutasteride, a novel dual 5aR inhibitor, on prostate cancer (PCs) cell lmes (LNCaP, LAPC-4, PC-3). MATERIAL & METHODS: PSA (prostate specific antigen) levels were measured by radioimmunoassay. Cell growth and viability were assessed by specific assays including Annexin V staining and BrdU uptake. RESULTS: In LNCaP cells. dutasteride inhibited 5aRlactivity and T action. In addition, DHT-induced accumulation of PSA was inhibited with an IC50 of 0.5. JuM after incubation with 0. InM DILT for I8 hr or 6 days. LNCaP cells exhibited decreased viability after 48 hr incubation with ?IOpM dutasteride. The same effects were seen with LAPC-4 cells, which contain wild type AR, as opposed to the T877A mutant AR found in LNCaP cells. Both cell lines showed decreased proliferation after 48-72 hrs in IO uM dutasteride. PC-3 cells, which lack AR, had no decrease in viability/proliferation for up to 96 hrs with IOpM dutasteride but did at 50pM. Treatment of LNCaP cells with IOuM dutasteridc for I8 hrs resulted in increased cell death that was partly reduced by inclusion of the synthetic androgen, RI 881. CONCLUSIONS: Prostate cells containing mutant or wild-type AR are more sensitive to cytotoxic effects of dutasteride than cells lacking AR. R188 I partly protects against dutasteride’s impact on cell death. The dutasteride dose required for half-maximal inhibttion of DHT-Induced PSA accumulation is lower than the half-maximal cytotoxic dose. The molecular basis for these observations is under investigation.
‘South Manchester Universtty Hospital, Urology. Manchester. United Kingdom. ‘Paterson Institute. Kay Kendall. Manchester, United Kingdom. ‘Chrisite Hospital. Urology, Manchester, Umred Kingdom INTRODUCTION & OBJECTIVES: This study examines the effect of the Bisphosphonate, Zoledronic Acid (Zoledronate) on prostate epithelial proliferation and mvasion in vitro using epithelial / endothelial co-culture techniques and cytokme assay. MATERIAL & METHODS: Prostatic epithelial cells isolated from men undergoing TURP for benign and malignant disease and the PC-3 prostate cancer cell line were studied in co-culture using long term human bone marrow stroma harvested from men with bentgn disease. Prostatic eprthelial binding and colony growth in bone marrow stroma was measured using standardised quantitative techniques in the presence of escalating doses of control (EDTA), Clodronate. Pamidronate and Zoledronate. Prostatic cellular invasion through matrigel (synthetic basement membrane) and cultured cndothelial monolayers was measured etther rn invasion chambers m the presence absence of drugs using cytokeratin labelled quantitatton of migrating cells or by the tmeasurement of endothelial monolayer permeability using quantitative fluorescent dextran labelhng. Co-culture supematants were assayed for specific cytokine levels. Bone marrow cellular toxicity was assessed using a standard MIXassay. RESULTS: Zoledronate inhibited ~nvasron and prohferation of prostattc epithelial crllb by 70% and 45% respectively. at concentrattons of >40pM. Thus inhibition significantly exceeded that seen with Pamidronate, whtch inhibited invasron by 50% and proliferation by 17%. Clodronate had no discemable effect by comparison with control. None of the test drugs had a measurable effect on cell /matrix binding or on endothelial permeability. suggesting that the observed phenomenon was a consequence of direct cellular Inhibition. Cytokine assays of supematants showed reduced levels of VEGF, TNFo. TIMP-I and 2 and GM-CSF with increasing Zoledronate concentrations whilst levels of TGFIJ, MMP I, 7 and the MMP activator uPA increased. Bone marrow cellular suppression was seen with Zoledronate but only at doses exceeding those required for inhibition of proliferation and migration of epithelial cells (Zoledronate 50pM proliferation / migration, marrow inhibition SO-IOOpM). CONCLUSIONS: Zoledronate
significantly inhibits prostate epithelial proliferation and migration in bone marrow but ha? no demonstrable effect on cell / matrix binding or cndothelial permeability. fhe observed effect IS assoctated wrth srgmticant alteratrons rn the cytokine mrlieu. Bone marrow hacmopoictic proliferatton IS Inhibited hut thus effect 15 only seen at higher dose levels.
140
139 IN VITRO CYTOTOXIC EFFECTS OF A TYROSINE KINASE INHIBITOR IMATINIB IN HUMAN PROSTATE CANCER CELL LINES
UPM3 TEST - A NEW MOLECULAR ASSAY DETECTING PROSTATE CANCER IN URINE SAMPLES -A NEW FUTURE PERSPECTIVE
Kiibler H.. Van Randenborgh
TinLl M.. Djavan
Technische Universitat
H., Wutrler S.. Lehmer A.. llartung
R., Paul R
Munchen, Urology. Munich, Germany
ClnivrrGty
INTRODUCTION & OBJECTIVES: Hormonerefractory prostate cancer (HRPC) is a major cause of mortality and morbidity in mtn. Although rcccnt clinical trials of taxanc-based regimes have demonstrated significant clinical activity in HRPC no survival benefit has yet been shown. Therefore, there is a clear riced to explore new targets and agents in the search of more effective treatment. Imatinib (ST1571, Gleevec@) is a selective inhibitor of the plateletderived growth factor receptors (PDGF-R). Previous studies hav,e shown that PDGF-Rs arc cxprcssed in prostatic intraepithelial neoplasia and adenocarcinoma hut not in benign prostate hypcrplasia ot- normal prostatic epithelium. MATERIAL & METHODS: Human prostate cancer cell lines LNCaP, DU145 and PC-3 were purchased from the American Type Tissue Collection and were cultured in RPMI-medium. Imatinib was kindly provided by Novartts. Drug solutions of lmatinib at different concentrations were added to a 96-well tissue culture plate. Variable cell growth was determined by XTT reduction assay, RESULTS: The dose-response curves of lmatinib for LNCaP, DU-145 and PC3 were determined by XTT assay after continuously exposure to the drug for 4 days. The IC50.values of lmatinib for LNCaP were l7uM, for PC-3 12,5pM and for DU-145 21.2uM respectively. A significant inhibition ofprolifcrations could be achieved.
of Vienna,Urology.
that the administration of cancer cell lines. Although trials, the present findings clinical protocols involving
Imatimb there are provide Imatinib
M Vienna.
Austria
INTRODUCTION & OBJECTIVES: Serum markers currently available fat prostate cancer dctcction lack sensttivity and specially sufticient speciticity. Earlier results with urinary PSA were inconsistent, whereas recent preliminary reports with a newly developed molecular urine test (uPM3) suggested a significant improvement of the statistical performance of total PSA. The purpose of the prospective study was to evaluate the uPM3 test in men referred for early prostate cancer detection and a PSA form 2.5-10 ng/mL. MATERIAL & METHODS: A total of X2 pattents were included and uPM3 tcstmg performed prior to TRUS-guided prostate biopsy. After having carefully performed a dtgital-rectal examination the first voided urine (30 ml) was taken as sample for the assay. The uPM3 test consists of an isothermic nucleic acid based amplification assay (NASBA -technique). It detects the expression of PSAmRNA as a marker of prostate cells in the urine sample and PCA-3mRNA which selectively is expressed in the majority of prostate cancer patients. The two targets are detected in real-time using specific beacons as probes tn an EasyQ instrument. Final pathological results were compared and statistical analysis performed using the JMP V3.2.2, (SAS, Cary, USA). RESULTS: A total of X2 urine samples were obtained. Cancer detection rate was 3 I%. The overall uPM3 sensitivity and specificity wcrc 90.4% and 58% respectively. In patients with PSA levels between 2.5-4 ng/mL sensitivity was 98X and specificity
CONCLUSIONS: These findings suggest has cytotoxic effects against human prostate gaps between in vitro studies and clinical useful infonnation for the establtshment of in HRPC‘.
B., Marberger
77%.
CONCLUSIONS: The uPM3 test offers the advantage of an office based noninvasive test combining sensitivity and specificity values of over 90% and60%, respectively. More even, uPM3 significantly improved cancer dctcction in the low PS.4 range 2.5-4 ngirnl. European
Urology
Supplements
2 (2003)
No. 1, pp. 37