385 EXPRESSION OF CYCLOOXYGENASE-2 IN HUMAN BLADDER TUMOR

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Expression of Cyclooxygenase-2 in Human Bladder Tumor Jang H.1, Seo J.W.1, Lee S.C.2, Lee H.l.3, Kim W.J.2

Daejeon Veterans Hospital, Urology, Daejeon, South Korea, 2College of Medicine, Chungbuk National University, Urology, Cheongju, South Korea, 3 College of Medicine, Kyunghee University, Urology, Seoul, South Korea 1



386

Effects of intravesical paclitaxel and antisense oligodeoxynucleotides on human transitional cell carcinoma in a new orthotopic rodent model

Bolenz C.1, Wenzel M.1, Gabriel U.1, Trojan L.1, Fernández M.1, Steidler A.1, Weiss C.2, Alken P.1, Michel M.S.1 University Hospital Mannheim, Department of Urology, Mannheim, Germany, 2University Hospital Mannheim, Department of Medical Statistics, Mannheim, Germany

1

Introduction & Objectives: Accumulating evidence indicates that cyclooxygenase-2 (COX-2) plays a critical role in tumorigenesis. To understand the role of COX-2 in tumorigenesis of the human bladder tumor, correlation between COX-2 expression and the clinico-pathologic variables, progression and recurrence has been examined.

Introduction & Objectives: Local recurrence and disease progression are still frequent events in superficial transitional cell carcinoma (TCC) of the urinary bladder despite intravesical chemotherapy. Promising results have been reported after combination of chemotherapeutic agents and antisense oligodeoxynucleotides (AS-ODNs) in TCC cell lines. Aim of our study was to evaluate potential antitumor effects of intravesically administered paclitaxel and bcl-xL AS-ODNs in a new orthotopic rodent model.

Material & Methods: Quantitative competitive polymerase chain reaction (QC-PCR) and real time PCR were performed to determine alteration in the expression levels of COX-2 mRNA derived from 249 human bladder tissues of transitional cell carcinoma (TCC), 92 tumor-matched surrounding normal mucosae and 34 normal bladder mucosae. Immunohistochemical stainging was applied to monitor the expression levels of COX-2 protein in 94 archival paraffin blocks from 43 bladder tumors and 50 normal bladder tissues.

Material & Methods: Human TCC (UMUC-3 cells) were induced in the study group consisting of 16 Foxnrnu athymic nude rats. Tumor diagnosis and follow-up examinations were performed by cystoscopy. Intravesical treatment was initiated after primary tumor diagnosis and consisted of one instillation weekly for two weeks. Instillation time was one hour. The urinary bladders were instilled with paclitaxel (group 1, n=5), bcl-xL AS-ODNs (group 2, n=4) or a combination of paclitaxel and bcl-xL-AS-ODNs (group 3, n=4). Untreated tumor-bearing animals served as controls (group 4, n=3). Tumor size was assessed in weekly intervals using a scaled wire (0.25 mm in diameter) which was introduced via the mini-endoscope’s working channel. Representative maximum tumor diameter served as a marker lesion.

Results: The expression levels of COX-2 mRNA were significantly higher in the bladder tumor tissues than in normal bladder mucosae (p < 0.001). However, no significant difference in the expression level was observed between tumor tissues and corresponding normal looking bladder mucosae surrounding tumor (p = 0.188). Furthermore, there was no correlation between the expression levels of COX-2 mRNA and the stage, grade, progression and recurrence of bladder tumors. Immunohistochemical analysis revealed the higher expression levels of COX-2 protein in the bladder tumor tissues than in normal bladder tissues (p < 0.001). Conclusions: COX-2 was over-expressed in human bladder tumor. COX-2 might play an important role on tumorigenesis of human bladder tumor rather than tumor progression and recurrence. Thus COX-2 might be useful tumor marker of bladder tumor.



387

Multitarget siRNA inhibition of antiapoptotic genes in bladder cancer cells Kunze D., Wuttig D., Stade J., Fuessel S., Hakenberg O.W., Meye A., Wirth M.P. Technical University of Dresden, Department of Urology, Dresden, Germany

Introduction: Overexpression of the three selected antiapoptotic genes BCL2, BCLXL and XIAP in tumor cells is often associated with a resistance to chemotherapy (CT). Since these genes are frequently upregulated in bladder cancer (BCa) their targeted inhibition, e.g. using siRNAs, represents a promising possibility in the treatment of BCa. Material & Methods: Protein and mRNA expression levels of the targets (XIAP, BCL2, BCLXL) were examined in tumor (tu) and tumorfree (tf) tissue samples obtained from radical cystectomy specimens. EJ28 and T24 BCa cells were transfected lipid-mediated with one siRNA (200nM) or with combinations of two/three siRNAs (each 100/67nM) against the different targets. The mRNA expression levels of the targets were examined by quantitative PCR. Treatments with target-directed siRNAs were normalized to nonsilencing-siRNA control. Cellular viability was analyzed by WST-1 assay, apoptosis by annexin V-propidium iodide staining. Also, cell cycle distribution and clonogenic survival were examined. Furthermore, the effects of a siRNA (mono- and multitarget) pretreatment followed by CT with different doses of mitomycin C, gemcitabine or cisplatin were examined.

Results: Intravesical treatment in group 1, 2 and 3 was well tolerated. Marker lesion size prior to treatment in group 1-3 and in group 4 was 0.504 cm (mean) and 0.217 cm, respectively. After two instillations mean marker lesion size was 0.256 cm in treated animals and 0.75 cm in untreated animals. Mean marker lesion size reduction in treated animals was 0.248 cm whereas marker lesion size in controls increased by a mean of 0.533 cm. No significant differences in marker lesion size reduction were found between group 1, 2 and 3. Two tumors completely disappeared after two instillations in group 3. Conclusions: Intravesical paclitaxel is well tolerated and should be considered for further experimental studies. Our preliminary results clearly demonstrate antineoplastic effects of intravesical paclitaxel as well as of the combined intravesical treatment with paclitaxel and bcl-xL AS-ODNs on TCC. Whether or not intravesical paclitaxel is superior to conventional agents such as mitomycin c or epirubicin and whether or not a combined treatment with bclxL AS-ODNs is superior to paclitaxel monotreatment is currently being evaluated in larger study groups.



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Overexpressed alternative survivin splice variants in human bladder cancer are not suitable targets for siRNA mediated in vitro inhibition Wuttig D., Kunze D., Füssel S., Stade J., Hakenberg O.W., Meye A., Wirth M.P. Technical University of Dresden, Department of Urology, Dresden, Germany

Introduction: Survivin‑wildtype (SVV‑wt) is an important inhibitor of apoptosis and a cell cycle regulator. Some of its alternative splice variants are also supposed to be critical for apoptosis (antiapoptotic SVV‑ΔEx3, proapoptotic SVV‑2B). Previous studies showed remarkable antiproliferative effects in various bladder cancer (BCa) cell lines caused by simultaneous inhibition of different SVV transcripts (including SVV‑wt) by small interfering RNAs (siRNAs) or antisense oligodeoxynucleotides. Since SVV plays a role in G2/M phase of the cell cycle in normal cells, it seems to be promising to reduce side effects of SVV‑inhibiting anticancer strategies by reducing only one specific SVV variant. Material & Methods: 18 tumor and 22 non‑malignant cryo‑preserved bladder tissue samples (including 17 autologous) were obtained from radical cystectomy specimens and used for expression analyses of the different SVV transcripts by quantitative PCR (qPCR). siRNAs (200 nM), each directed against one alternative SVV splice variant (SVV‑ΔEx3, SVV‑2B) or a combination of different SVV transcripts, were transfected lipid‑mediated in human BCa cells (EJ28). Expression of the different SVV forms was quantified by qPCR and western blot analyses, respectively. Furthermore, rate of apoptosis, cell cycle distribution (both by flowcytometry) and colony formation ability were examined. Chemosensitization effects to cisplatin, mitomycin c and gemcitabine were evaluated (WST‑1 viability test).

Results: Expression levels of XIAP, BCL2 and BCLXL were higher in tu than in tf tissues. Dependent on the cell line all siRNAs caused a reduction of the target mRNA expression down to 34-66% 24h after transfection. Even 72h later similar inhibition rates were measured. A combined treatment against BCL2+BCLXL or BCL2+BCLXL+XIAP revealed the mRNA downregulation of all targets which was comparable to the monotherapies. siRNA mono- and multitarget treatments reduced cell count and induced apoptosis. No significant changes in cellular viability and clonogenic survival were measured after single siRNA treatment. Nevertheless, specific combinations of siRNA+CT caused a significant reduction of the cellular viability compared to the ns‑siRNA+CT controls.

Results: Besides SVV‑wt also its alternative splice variants SVV‑ΔEx3 and ‑2B were significantly overexpressed in BCa tissues compared to non‑malignant bladder tissues. SVV‑wt was the quantitatively dominant transcript (74‑100% of total SVV). Except siRNA targeting SVV‑ΔEx3, the 4 tested siRNAs (targeting SVV‑ΔEx3, SVV‑2B, SVV‑wt + ‑ΔEx3 + ‑2B, SVV‑wt + ‑2B) caused a specific down‑regulation only of the targeted SVV transcript(s) in EJ28 cells. SVV‑wt inhibition was also observed at the protein level. All siRNAs increased apoptosis by 11‑46%, whereby strongest effects were caused by constructs directed against a combination of SVV-wt and one or both alternative SVV splice variants. Reduction of different SVV transcripts simultaneously also increased G2/M arrest by 28‑92% and decreased cell colony formation down to 68‑70%. Statistically significant chemosensitization effects were observed mainly by inhibiting different SVV transcripts.

Conclusions: Multitarget inhibition of BCL2, BCLXL and XIAP reduced mRNA expression of all target genes, increased apoptosis and sensitized BCa cells to CT. As expression levels of the three antiapoptotic genes differ between BCa patients their combined inhibition represents a more suitable therapeutic option than the reduction of only one antiapoptotic target.

Conclusions: Overexpression of SVV‑ΔEx3 and ‑2B in BCa tissues indicates accessibility of these transcripts for a siRNA‑mediated expression inhibition. In vitro studies showed, that specific inhibition of one alternative SVV splice variant did not cause remarkable antiproliferative effects. Therefore, the specific inhibition of an alternative SVV splice variant is not reasonable for anticancer treatment, at least in BCa therapy.

Eur Urol Suppl 2007;6(2):119