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829 The epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (‘iressa’, ZD-1839) and the protein prenyltransferase inhibitor M365408 as potential therapeutics in bladder cancer
828
829
BCL-XL ANTISENSE OLIGONUCLEOTIDES AS AN ENHANCER OF THE CYTOTOXIC EFFECTS OF CISPLATIN, MITOMYCIN C AND PACLITAXEL IN THE TREATMENT OF BLADDER CANCER
THE EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE KINASE INHIBITOR GEFITINIB (‘IRESSA’, ZD-1839) AND THE PROTEIN PRENYLTRANSFERASE INHIBITOR M365408 AS POTENTIAL THERAPEUTICS IN BLADDER CANCER
Schaaf A., Sagi S., Trojan
Domineuez-Escrie
University
Hospital
L., Alken
Mannheim,
P., Michel
Department
M.S of Urology,
Mannheim,
J.L.‘, Kelly J.2, Davies B.R.’
‘University of Newcastle, Northern Institute Cancer Research School of Surgical and Reproductive Sciences, Newcastle Upon Tyne, United Kingdom, ‘Cambridge University- Addenbrookes Hospital, Department of Surgical Oncology, Cambridge, United Kingdom
Germany
INTRODUCTION & OBJECTIVES: Toxicity and the progressive resistance of conventional chemotherapeutic drugs are the main barriers of cancer chemotherapy in our times. The in-depth understanding of tumour cell biology and antiapoptotic proteins such as Bcl-x, offers many new possibilities to overcome these problems. By the help of antisense technology the expression of antiapoptotic proteins can be modified and therefore an increase in the efficacy of chemotherapeutic drugs can be suggested. The aim of our study was therefore to examine the effects of the combined application of cisplatin, mitomycin C or paclitaxel in combination with Bcl-x, antisense oligonucleotides on four different human bladder cancer cell lines to determine possible synergistic effects in cytotoxicity. MATERIAL & METHODS: Human bladder cancer cell lines (UM-UC 3, RT 112, T24/83 and HT 1197) were treated with Bcl- xL antisense oligonucleotides, cisplatin, mitomycin C, paclitaxel individually and a combination of the Bcl- xL antisense oligonucleotides with each cytostatic agent. After incubation for 48 hours under standard conditions, cell survival was determined using a Neubauer haemocytometer or standard MTT assay. RESULTS: The combination of Bcl-x, antisense oligonucleotides with the three different cytostatic agents cisplatin, mitomycin C and paclitaxel resulted in significant lower cell survival rates of the used human bladder carcinoma cell lines RT 112, UM-UC 3, T 24/83 and HT 1197 compared to the individual treatments with each cytostatic agent. CONCLUSIONS: For the combined treatment with Bcl-T, antisense oligonucleotides plus cisplatin, mitomycin C and paclitaxel a synerglstlc effect can be strongly suggested. Therefore further investigations and in-vivo trials have to be done to determine the possible benefits for clinical applications.
INTRODUCTION & OBJECTIVES: The epidermal growth factor receptor (EGFR) is an independent predictor of stage progression and mortality in transltlonal cell carcinoma of the bladder ITCCYThe Ras gene oroduct is a membrane-localized G orotein functionine as a molecular switch iinkini receptor&d Aon-receptor tymsin kinase activatioh to downstream &toplasnuc and nuclear events. Activation of H-ras by mutation occurs in 6-40% of TCCs. EGFR slgnalling mvolves ICISdependent/independent mechanisms, resulting in pleiotrophic effects on cancer cells including induction of proliferation, motility, invasion and enhanced cell survival. EGFR-tyrosine kinase Inhibitors (TKIs) and protein prenyltransferase inhibitors (PTIs) are new “small molecules” with potential impact in bladder cancer. The aim of this stidy was to evaluate the effects of the EGFR-TKI gefitinib (‘Iressa’, ZD-1839) and the PTI M35640S on three human bladder cancer cell lines. MATERIAL & METHODS: Human bladder cancer cell lines expressing EGFR: T24 (harbouring an activating mutation of Ras), RT112 and 2535 Bv. Viable cell number and DNA synthesis was determined by MTT and 3H-Thymidine incorporation assays respectively. Relative EGFR expression and the effects of geftimb on EGF-induced EGFR phosphorylation was determined by Western Blotting. Signal mtensity was measured by Scion Software. The effect of gefitinib on tunour growth in vwo, was studied following S.C.inJection of RTI 12 and 2535 Bv cells into nude mice. Gefitinib and M365408 were kindly donated by AsrraZeneca UK Ltd. RESULTS: In Vitro. Both gefitinib and M365408 (1 to 10pM) caused a dose-dependent sigmficant reduction of viable cell number in all lines. Reduction by M365408 was greater in T24 (64%, p
831
830 EVIDENCE UROTHELIAL
FOR FREQUENT CARCINOMAS
Rouuret M.‘, Amira N.‘, Haab F.‘, Cussenot 0.’ ‘Hopital Pathology,
Tenon, Urology, Paris, France
Sibony
MICROSATELLITE OF THE UPPER M.‘,
Paris,
Fromont
France,
INSTABILITY URINARY TRACT
G.2, Doublet
21nstitut
J.d.‘,
Mutualiste
Gattegno
IN
B.‘,
Montsouris,
INTRODUCTION & OBJECTIVES: Transitional cell carcinoma of upper urinary tract (TCC-UUT) may develop with a high frequency in patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) syndrome. Tumours occurring in HNPCC syndrome display genomic lesions in DNA mismatch repair genes and are detectable as microsatellite instability (MSI). Because little is known about genetic lesions in TCC-UUT as compared to TCC of the bladder, we determined the genetic profiles (MSI) in a series of 71 TCC-UUT using five informative microsatellite markers. MATERIAL & METHODS: 71 paraffin embedded samples from 69 patients with clinically diagnosed TCC-UUT (renal pelvis and /or ureter) were tested for MS1 with dinucleotide markers D9Sl71 (9~21) and D5S346 (5q22), and mononucleotide repeats BAT25 (4q12), BAT26 (2pl6), BAT40 (1~13.1). RESULTS: MS1 was detected at one or more microsatellite loci in 46% of the tumours. In TCC-UUT, markers BAT40, BAT25, BAT26, D9S171 and D5S346 showed instability at mono and di-nucleotides in 19/71 (27%) tumours, of which lo/71 (14%) were MSI-High. CONCLUSIONS: Our findings show that, as for colorectal cancer, the pattern of MS1 varies with location in urinary tract. Furthermore, our study demonstrates a high level of MS1 in TCC-UUT. although it is a rare event in bladder cancers (1%). The establishment of distinct genetic profiles between upper and lower urinary tract tumours, could provide an additional tool to improve diagnosis, disease monitoring and prediction of prognosis. European
Urology
Supplements
3 (2004)
No. 2, pp. 210
NO DIRECT PRO-APOPTOTIC DNA (CPG-ODN) ON MB-49 CELLS IN VITRO
EFFECT OF IMMUNOSTIMULATIVE TRANSITIONAL CELL CARCINOMA
Heeele A.l, Dalpke A.*, Von Knobloch
R.‘, Heeg K.2, Hofinann
‘Philipps University, Urology, Marburg Microbiology, Marburg /l., Germany
/l., Germany,
R.‘, Olbert P.’
2Philipps University,
Hygiene
and
INTRODUCTION & OBJECTIVES: A Th-1 biased immune response is postulated in the mechanism of action of BCG as well as of immunostimulative DNA sequences (CpG-ODN). Antineoplastic properties of CpG-ODN and mycobacterial cell wall-DNA complex could be demonstrated in viva for different tumour entities. Investigating the mode of action of CpG-ODN in TCC, aim of our studies was to evaluate a potential direct pro-apoptotic effect on MB-49 TCC cells in vitro. MATERIAL & METHODS: MB-49 cell culture is maintained at our laboratory under standardized conditions (DMEM+Glutamax+lO%FCS+l%Pen/Strep, 37”, 5% CO,). 1~10~ cells were incubated for 24 hours. Thereafter the following agents were added: 1. Immunostimulative 1668CpG (Phosphorothioate (PTO)-backbone); 2. 1668GpC as direct GC control (PTO); 3. Immunostimulative Phosphodiester ODN (PO, to exclude immunostimulative PTO-effect); 4. Lipopolysaccharide (LPS); 5. PBS as unstimulated control. After 4 hours of stimulation, quantitative FACS-analysis was performed after Annexin V/Propidium-Jodine staining (Annexin-V-Fluos-Staining-Kit, Roche, Germany). Additionally, we performed fluorescence-microscopic examination of the stimulated and stained cells. RESULTS: Compared to unstimulated controls, neither CpG, GpC, PO nor LPS was able to increase the rate of apoptotic cells in FACS analysis (see table). This was confirmed by immunofluorescence microscopy.
Although pro-apoptotic effects of immunostimulatory DNA could be demonstrated in viva, we could not confirm these results in a TCC cell culture model. We conclude that the pro-apoptotic and potential antineoplastic effect of CpG-ODN is mediated immunologically. A direct interaction between CpG-ODN and tumour cell could not be demonstrated.
CONCLUSIONS:
829
BCL-XL ANTISENSE OLIGONUCLEOTIDES AS AN ENHANCER OF THE CYTOTOXIC EFFECTS OF CISPLATIN, MITOMYCIN C AND PACLITAXEL IN THE TREATMENT OF BLADDER CANCER
THE EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE KINASE INHIBITOR GEFITINIB (‘IRESSA’, ZD-1839) AND THE PROTEIN PRENYLTRANSFERASE INHIBITOR M365408 AS POTENTIAL THERAPEUTICS IN BLADDER CANCER
Schaaf A., Sagi S., Trojan
Domineuez-Escrie
University
Hospital
L., Alken
Mannheim,
P., Michel
Department
M.S of Urology,
Mannheim,
J.L.‘, Kelly J.2, Davies B.R.’
‘University of Newcastle, Northern Institute Cancer Research School of Surgical and Reproductive Sciences, Newcastle Upon Tyne, United Kingdom, ‘Cambridge University- Addenbrookes Hospital, Department of Surgical Oncology, Cambridge, United Kingdom
Germany
INTRODUCTION & OBJECTIVES: Toxicity and the progressive resistance of conventional chemotherapeutic drugs are the main barriers of cancer chemotherapy in our times. The in-depth understanding of tumour cell biology and antiapoptotic proteins such as Bcl-x, offers many new possibilities to overcome these problems. By the help of antisense technology the expression of antiapoptotic proteins can be modified and therefore an increase in the efficacy of chemotherapeutic drugs can be suggested. The aim of our study was therefore to examine the effects of the combined application of cisplatin, mitomycin C or paclitaxel in combination with Bcl-x, antisense oligonucleotides on four different human bladder cancer cell lines to determine possible synergistic effects in cytotoxicity. MATERIAL & METHODS: Human bladder cancer cell lines (UM-UC 3, RT 112, T24/83 and HT 1197) were treated with Bcl- xL antisense oligonucleotides, cisplatin, mitomycin C, paclitaxel individually and a combination of the Bcl- xL antisense oligonucleotides with each cytostatic agent. After incubation for 48 hours under standard conditions, cell survival was determined using a Neubauer haemocytometer or standard MTT assay. RESULTS: The combination of Bcl-x, antisense oligonucleotides with the three different cytostatic agents cisplatin, mitomycin C and paclitaxel resulted in significant lower cell survival rates of the used human bladder carcinoma cell lines RT 112, UM-UC 3, T 24/83 and HT 1197 compared to the individual treatments with each cytostatic agent. CONCLUSIONS: For the combined treatment with Bcl-T, antisense oligonucleotides plus cisplatin, mitomycin C and paclitaxel a synerglstlc effect can be strongly suggested. Therefore further investigations and in-vivo trials have to be done to determine the possible benefits for clinical applications.
INTRODUCTION & OBJECTIVES: The epidermal growth factor receptor (EGFR) is an independent predictor of stage progression and mortality in transltlonal cell carcinoma of the bladder ITCCYThe Ras gene oroduct is a membrane-localized G orotein functionine as a molecular switch iinkini receptor&d Aon-receptor tymsin kinase activatioh to downstream &toplasnuc and nuclear events. Activation of H-ras by mutation occurs in 6-40% of TCCs. EGFR slgnalling mvolves ICISdependent/independent mechanisms, resulting in pleiotrophic effects on cancer cells including induction of proliferation, motility, invasion and enhanced cell survival. EGFR-tyrosine kinase Inhibitors (TKIs) and protein prenyltransferase inhibitors (PTIs) are new “small molecules” with potential impact in bladder cancer. The aim of this stidy was to evaluate the effects of the EGFR-TKI gefitinib (‘Iressa’, ZD-1839) and the PTI M35640S on three human bladder cancer cell lines. MATERIAL & METHODS: Human bladder cancer cell lines expressing EGFR: T24 (harbouring an activating mutation of Ras), RT112 and 2535 Bv. Viable cell number and DNA synthesis was determined by MTT and 3H-Thymidine incorporation assays respectively. Relative EGFR expression and the effects of geftimb on EGF-induced EGFR phosphorylation was determined by Western Blotting. Signal mtensity was measured by Scion Software. The effect of gefitinib on tunour growth in vwo, was studied following S.C.inJection of RTI 12 and 2535 Bv cells into nude mice. Gefitinib and M365408 were kindly donated by AsrraZeneca UK Ltd. RESULTS: In Vitro. Both gefitinib and M365408 (1 to 10pM) caused a dose-dependent sigmficant reduction of viable cell number in all lines. Reduction by M365408 was greater in T24 (64%, p
831
830 EVIDENCE UROTHELIAL
FOR FREQUENT CARCINOMAS
Rouuret M.‘, Amira N.‘, Haab F.‘, Cussenot 0.’ ‘Hopital Pathology,
Tenon, Urology, Paris, France
Sibony
MICROSATELLITE OF THE UPPER M.‘,
Paris,
Fromont
France,
INSTABILITY URINARY TRACT
G.2, Doublet
21nstitut
J.d.‘,
Mutualiste
Gattegno
IN
B.‘,
Montsouris,
INTRODUCTION & OBJECTIVES: Transitional cell carcinoma of upper urinary tract (TCC-UUT) may develop with a high frequency in patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) syndrome. Tumours occurring in HNPCC syndrome display genomic lesions in DNA mismatch repair genes and are detectable as microsatellite instability (MSI). Because little is known about genetic lesions in TCC-UUT as compared to TCC of the bladder, we determined the genetic profiles (MSI) in a series of 71 TCC-UUT using five informative microsatellite markers. MATERIAL & METHODS: 71 paraffin embedded samples from 69 patients with clinically diagnosed TCC-UUT (renal pelvis and /or ureter) were tested for MS1 with dinucleotide markers D9Sl71 (9~21) and D5S346 (5q22), and mononucleotide repeats BAT25 (4q12), BAT26 (2pl6), BAT40 (1~13.1). RESULTS: MS1 was detected at one or more microsatellite loci in 46% of the tumours. In TCC-UUT, markers BAT40, BAT25, BAT26, D9S171 and D5S346 showed instability at mono and di-nucleotides in 19/71 (27%) tumours, of which lo/71 (14%) were MSI-High. CONCLUSIONS: Our findings show that, as for colorectal cancer, the pattern of MS1 varies with location in urinary tract. Furthermore, our study demonstrates a high level of MS1 in TCC-UUT. although it is a rare event in bladder cancers (1%). The establishment of distinct genetic profiles between upper and lower urinary tract tumours, could provide an additional tool to improve diagnosis, disease monitoring and prediction of prognosis. European
Urology
Supplements
3 (2004)
No. 2, pp. 210
NO DIRECT PRO-APOPTOTIC DNA (CPG-ODN) ON MB-49 CELLS IN VITRO
EFFECT OF IMMUNOSTIMULATIVE TRANSITIONAL CELL CARCINOMA
Heeele A.l, Dalpke A.*, Von Knobloch
R.‘, Heeg K.2, Hofinann
‘Philipps University, Urology, Marburg Microbiology, Marburg /l., Germany
/l., Germany,
R.‘, Olbert P.’
2Philipps University,
Hygiene
and
INTRODUCTION & OBJECTIVES: A Th-1 biased immune response is postulated in the mechanism of action of BCG as well as of immunostimulative DNA sequences (CpG-ODN). Antineoplastic properties of CpG-ODN and mycobacterial cell wall-DNA complex could be demonstrated in viva for different tumour entities. Investigating the mode of action of CpG-ODN in TCC, aim of our studies was to evaluate a potential direct pro-apoptotic effect on MB-49 TCC cells in vitro. MATERIAL & METHODS: MB-49 cell culture is maintained at our laboratory under standardized conditions (DMEM+Glutamax+lO%FCS+l%Pen/Strep, 37”, 5% CO,). 1~10~ cells were incubated for 24 hours. Thereafter the following agents were added: 1. Immunostimulative 1668CpG (Phosphorothioate (PTO)-backbone); 2. 1668GpC as direct GC control (PTO); 3. Immunostimulative Phosphodiester ODN (PO, to exclude immunostimulative PTO-effect); 4. Lipopolysaccharide (LPS); 5. PBS as unstimulated control. After 4 hours of stimulation, quantitative FACS-analysis was performed after Annexin V/Propidium-Jodine staining (Annexin-V-Fluos-Staining-Kit, Roche, Germany). Additionally, we performed fluorescence-microscopic examination of the stimulated and stained cells. RESULTS: Compared to unstimulated controls, neither CpG, GpC, PO nor LPS was able to increase the rate of apoptotic cells in FACS analysis (see table). This was confirmed by immunofluorescence microscopy.
Although pro-apoptotic effects of immunostimulatory DNA could be demonstrated in viva, we could not confirm these results in a TCC cell culture model. We conclude that the pro-apoptotic and potential antineoplastic effect of CpG-ODN is mediated immunologically. A direct interaction between CpG-ODN and tumour cell could not be demonstrated.
CONCLUSIONS: