EFFECTIVITY AND AVAILABILITY OF AN INTRAVESICAL ANTISENSE APPROACH IN A BLADDER CANCER MODEL

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DICOUMAROL POTENTIATES CISPLATIN-INDUCED APOPTOSIS MEDIATED BY C-JUN N-TERMINAL KINASE IN P53 WILD-TYPE UROGENITAL CANCER CELL LINES

EFFECTIVITY AND AVAILABILITY OF AN INTRAVESICAL ANTISENSE APPROACH IN A BLADDER CANCER MODEL

Watanabe J., Nishiyama H., Matsui Y., Kawanishi H., Ito M., Kamoto T., Ogawa O.

1

Blietz C.E.1, Kausch I.1, Kynast B.2, Doehn C.1, Jocham D.1 University of Schleswig-Holstein, Campus Lübeck, Urology, Lübeck, Germany, 2Faustus Forschungs GmbH, Urology, Leipzig, Germany

Kyoto University, Department of Urology, Kyoto, Japan INTRODUCTION & OBJECTIVES: Dicoumarol (3-3’-methylene-bis [4-hydroxycoumarin]), an inhibitor of NADPH: quinone oxidoreductase 1 (NQO1), has been widely used as anti-coagulant at clinical setting. On the other hand, recent studies suggest that this reagent possess the ability to exert anti-neoplastic effect through the oxidative stress-associated pathway. However, its effectiveness and detailed mechanisms in urogenital cancer remains still to be elucidated. In the present study, we investigated the cytotoxic effects of dicoumarol in combination with cisplatin (CDDP), using a panel of urogenital cancer cell lines, and underlying molecular mechanisms were analysed. MATERIAL & METHODS: Four bladder (RT112, 253J, J82 and UMUC3) and two prostate (LNCap and PC3) cancer cell lines were employed in this study. Activation status of each molecular events were monitored by Western blotting or immunocyotochemistry. For the evaluations of cell viability or degree of apoptosis, MTT assay and flow cytometry were employed. RESULTS: Single treatment with 0.1mM dicoumarol suppressed cell proliferation but did not induce apoptosis at 24h in all cell lines examined. On the other hand, pretreatment with dicoumarol enhanced cytotoxicity of CDDP in three cell lines with wild-type of p53 (RT112, 253J and LNCap), but not in three other cell lines with mutant p53 or in RT112 stable transfectants with a dominant-negative mutant of p53. In RT112 and LNCap, CDDP induced p53 and p21 expression, while pretreatment of dicoumarol suppressed induction of p21 but not Bax; remarkable decrease of p21/Bax ratio was observed. The specific suppression of p21 resulted in sequential activation of c-Jun N-terminal kinase (JNK) and increase of activated form of Bax in a time-dependent manner. Furthermore, inhibition of JNK, using SP600125, completely suppressed Bax activation, activity of caspases and poly (ADP-ribose) polymerase (PARP) cleavage, leading to suppression of enhancement of CDDP-mediated apoptosis by dicoumarol. Also Bax-inhibitory peptide could partially blocked this enhancing effect. CONCLUSIONS: Our study revealed that dicoumarol could enhance CDDP-induced apoptosis in urogenital cancer cells with wild-type p53 specifically. Enzymatic inhibition of NQO-1 resulted in the strong suppression of p21, through the alleviation of CDDP-induced p53 accumulation. Sequential activation of JNK pathway caused by this p21 suppression, and JNK-mediated Bax activation were critical molecular events for the robust apoptosis achieved by combination use of dicoumarol and CDDP.

INTRODUCTION & OBJECTIVES: Antisense Oligonucleotides (AS-ON) against different target genes are currently evaluated in numerous oncological trials. High local effectivity of these compounds after topical treatment has been frequently described. The urinary bladder is an ideal organ for local instillation of therapeutical agents. However, no studies have yet examined conclusively the preclinical or clinical availability of AS-ON after intravesical treatment. We have analysed the effectivity, availability and toxicity of AS-ON in murine animal models. ON were targeted against the Ki-67 antigen which has previously shown to be a highly effective antitumoural target. MATERIAL & METHODS: This study investigated toxicology and pharmacokinetics of systemically and intravesically applicated AS-ON against the Ki-67 antigen. NMRI mice received 215mg/kg body weight (b.w.), 464 mg/kg bw and 1000 mg/kg bw of Ki-67-AS-ON intravenously (in groups of 5). The animals were daily observed for clinical signs of toxicity. Euthanization and necropsy was performed after 14 days. To obtain preliminary information on the penetration and the kinetics of the test substance after administration into the urinary bladder 3H labelled AS-ON were given in 12 female rat’s intravesically. In order to keep the test substance in the bladder the urethra was ligated at its orifice and reopened after two hours. Euthanization was performed after exposure time. The bladders were investigated histopathologically and microautoradiographs were obtained. Additionally, the uptake of FITC labelled oligonucleotides and downregulation of target gene was examined in MB-49 bladdertumour-bearing C57/BL6 mice by fluorescence microscopy, RT-PCR and Western Blot. RESULTS: After a single intravenous application of the AS-ON against the Ki-67 antigen at a dose of 215 mg/kg bw no clinical signs of toxicity were seen. After administration of 464 mg/kg bw slightly reduced motility in all animals, slight ataxia in 8 of 10 animals and slight dyspnoea in 7 of 10 animals was noted. No premature mortality was seen. Slightly too severely reduced motility, ataxia and dyspnoea in all animals was noted after application of 1000 mg/kg bw. One mouse died within 24 hours after administration and one mouse was found dead on test day 3. The body weight was not influenced, no macroscopic changes were noted at necropsy. FITC-uptake studies confirmed the high local availability. Furthermore, molecular studies of orthotopic tumour samples demonstrated significant target gene and target protein down-regulation. CONCLUSIONS: In this study we investigated the intravesical uptake and effectivity of antisense oligonuleotides. The high local and low systemic concentrations indicate the urinary bladder as an ideal organ for local cancer treatment. Significant down-regulation of the target gene in these experiments underlines the usefulness of this approach. Results from a subsequent phase I study in bladder tumour patients are awaited.

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FLUORESCENT-IN-SITU HYBRIDISATION (FISH) FOR DETECTION OF RECURRENT TRANSITIONAL CELL CARCINOMA OF THE BLADDER

IS HIGH-RESOLUTION ARRAY-BASED COMPARATIVE GENOMIC HYBRIDISATION ABLE TO PICK UP GERMLINE GENOMIC ABERRATIONS IN HEREDITARY BLADDER CANCER?

Marin M.1, Mengual L.1, Algaba F.2, Arce Y.2, Burset M.1, Ribal M.J.3, Ars E.1, Izquierdo L.3, Villavicencio H.4, Alcaraz A.3

Kiemeney L.A.1, Kuiper R.P.2, Pfundt R.2, Van Reijmersdal S.2, Schoenberg M.P.3, Aben K.A.4, Niermeijer M.F.2, Witjes J.A.5, Schoenmakers E.F.P.M.2

Fundacio Puigvert, Molecular Biology Laboratory, Barcelona, Spain, 2Fundacio Puigvert, Pathology, Barcelona, Spain, 3Hospital Clinic, Urology, Barcelona, Spain, 4Fundacio Puigvert, Urology, Barcelona, Spain

1

INTRODUCTION & OBJECTIVES: Cystoscopy and cytology are currently the gold standard to detect and monitor superficial urothelial carcinoma. Despite the poor sensitivity of cytology and the invasivity of cystoscopy, these methods are being used at regular intervals to monitor patients for recurrence or progression of previously treated tumours. The development of a non-invasive technique, with the same sensitivity and specificity as that of the cystoscopy and cytology, would allow a new approach for clinical monitoring of the recurrent bladder tumours. Objective: To evaluate the use of UroVysion FISH assay for a non-invasive detection of recurrent superficial bladder cancer and to compare the performance of the technique with that of current gold standard. MATERIAL & METHODS: Ninety-three voided urines from patients affected with TCC of the bladder, 12 from patients with history of tumour but negative result in their last cystoscopy, and 14 from healthy volunteers were collected. All samples were analysed by FISH and cytology. Results from both techniques were correlated with the corresponding tumoural stage and grade. RESULTS: Overall specificity was 100% (14 out of 14) for cytology and 92.9% (13 out of 14) for FISH (p=NS). Overall sensitivity favoured FISH 76.3% versus 47.3% for cytology (p=0.00005). Results by stage and grade are summarized in the table. of remark, 68.8% of the 16 patients with tumour that were only suspicious by cytology resulted positive by FISH. Table. Sensitivity of FISH and cytology by stages and grades Stage/grade (n)

FISH

Total (93)

76.3%

CYTOLOGY 47.3%

p: 0.00005

pTis (4)

75%

50%

p: 0.4652

pTa (50)

64%

22%

p: 0.00002

pT1 (20)

85%

65%

p: 0.144

pT2 (19)

100%

94.7%

p: 0.311

G1 (13)

46.2%

7.7%

p: 0.027

G2 (39)

62.8%

30.8%

p: 0.0152

G3 (41)

95.1%

78%

p: 0.02332

CONCLUSIONS: UroVysion FISH assay has higher sensitivity than cytology in detecting recurrent urothelial carcinomas. It is remarkable its ability to detect recurrent low grade tumours and maybe of help in suspicious cytology.

Eur Urol Suppl 2006;5(2):76

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Radboud University Nijmegen Medical Centre, Urology, Epidemiology & Biostatistics, Comprehensive Cancer Centre IKO, Nijmegen, The Netherlands, 2Radboud University Nijmegen Medical Centre, Human Genetics, Nijmegen, The Netherlands, 3Johns Hopkins Hospital, Urology, Baltimore, United States, 4Radboud University Nijmegen Medical Centre, Epidemiology & Biostatistics, Comprehensive Cancer Centre IKO, Nijmegen, The Netherlands, 5 Radboud University Nijmegen Medical Centre, Urology, Nijmegen, The Netherlands INTRODUCTION & OBJECTIVES: Linkage studies for the identification of high penetrance bladder cancer genes are impossible, as extended bladder cancer families are rare. Traditional karyotyping or conventional CGH may reveal constitutional chromosomal anomalies that point to the location of susceptibility genes but have limited resolution. The recent development of array-based CGH consisting of 32,447 overlapping BAC clones has increased this resolution to ~100 kb. MATERIAL & METHODS: From a large study on familial bladder cancer, we identified 10 families who fulfilled (arbitrary) criteria for hereditary bladder cancer. After informed consent, one index case from each family donated a blood sample. Genomic DNA was isolated from PBLs, labelled and hybridized against a sex-mismatched reference pool. Spot identification and two-colour fluorescence intensity measurements were obtained, normalized and analysed. Data normalization and copy number detection were performed according to De Vries et al, Am J Hum Genet 2005; 77: 606-616. All identified genomic copy-number alterations were compared to both public and private databases of known disease-unrelated large-scale copynumber variations (LCVs) (http://projects.tcag.ca/variation/). RESULTS: In all ten patients, we detected one or more genomic copy-number alterations that varied in size from 0.25-5.8 Mb. All 41 alterations, however, have previously been categorized as 21 different disease-unrelated LCVs. CONCLUSIONS: Our findings confirm the high resolution of arrayCGH. Unfortunately, we did not find any candidate region for a gene that may predispose to the development of bladder cancer but germline point mutations in tumour-suppressor or DNA repair genes were below our detection limit. Nevertheless, high-resolution arrayCGH studies may point the way to bladder cancer susceptibility genes.