#627: Multitarget gene inhibition by synthetic nucleic acids in bladder cancer cells Burmeister Y, Kraemer K, Fuessel S, Kotzsch M #, Meye A*, Hakenberg OW, Wirth MP Department of Urology and # Institute of Pathology, Technical University of Dresden, Germany Department of Urology and # Institute of Pathology, Technical University of Dresden, Germany Introduction & Objectives A high proportion of bladder cancers (BCa) progress from superficial to invasive growth. Furthermore, highly malignant superficial BCa recur within a few years after resection. The currently used chemotherapy (CT) schedules seem to be inefficient. Moreover, BCa are frequently resistant to CT. Therefore, a tumor-specific and more efficient therapy with reduced adverse effects should be of great interest for the uro- oncological research. Specific down-regulation of genes associatedwith tumor onset or progression, and with resistance to CT is supposed to inhibit tumor cell growth and to sensitize tumor cells to CT. Suitable targets for gene silencing by antisense deoxynucleotides (AS- ODN) or small interfering RNAs (siRNA) are the human telomerase reverse transcriptase (hTERT), the inhibitor of apoptosis survivin (SVV) and the vascular endothelial growth factor (VEGF) since they are specifically expressed in the majority of BCa, associated with unlimited growth of tumors and resistance to CT. Here, we investigated possible enhancement effects of: i) the simultaneous down-regulation of hTERT, SVV and VEGF by AS-ODN ii) the simultaneous down-regulation of hTERT, SVV and VEGF by siRNA iii) a pretreatment with siRNA against VEGF prior to a CT with cisplatin (CDDP), gemcitabine (GEM) or mitomycin C (MMC) in different BCa cell lines. Materials & Methods selection of suitable, overexpressed targets for BCa treatment: -hTERT  telomere lengthening, cell immortalization, DNA stability, capping function of telomers -survivin  inhibitor of apoptosis protein, 4 th most common transcript in tumor cells, correlation with stage & grade -VEGF  major angiogenic factor, correlation with BCa progression selection of suitable BCa cell lines overexpressing the targets: EJ28, 5637, RT112 evaluation of selected AS-ODN against hTERT, SVV and VEGF and siRNA against hTERT and SVV in previous studies [Kraemer 2003 & 2006, Fuessel 2004, Ning 2004, Forster 2004]: -with regard to their potency to inhibit target expression (quantitative PCR & western blot or ELISA) -with regard to impair BCa cell growth as isolated treatment combination of AS-ODN or siRNA against 3 different targets: AS-ODN: 4h, ODN:lipofectin = 1:3 (w/w), serum-free medium (OptiMEM), 250nM+250nM or 500nM, control-ODN NS-K1 siRNA: 4h, ODN:DOTAP = 1:4 (w/w), serum-free medium (OptiMEM), 200nM+200nM or 400nM, control-siRNA NS-si determination of suitable CT concentrations, which provoke moderate inhibitory effects as single treatment combination of the most potent AS-ODN and siRNA with CT: -previous studies: AS-ODN against hTERT, SVV or VEGF [Kraemer 2004, Krause 2005, Fuessel 2006], siRNA against hTERT or SVV [Kraemer 2006, Fuessel 2006] -this study: siRNA against VEGF assessment of the inhibition of proliferation of BCa cells: -viability (WST-1 assay) Seeding Transfection (4h) CT 72h20h WST-1assay Cell counting Apoptosis detection 24h CDDP, GEM 2h MMC incubation for further 24, 48 or 72h WST-1assay Cell counting Apoptosis detection Cell cycle analysis 24h 2h MMC 24h 2h Results for VEGF-directed siRNA and siRNA-combinations efficient down-regulation of VEGF by 3 different VEGF-directed siRNA (siVEGF203, siVEGF437, siVEGF865) at mRNA and protein levels (Fig.2: results for siVEGF865) combined treatment with NS-si control and siRNA against hTERT (sihTERT3106), SVV (siSVV284) or VEGF (siVEGF865), respectively (each 200nM) combined treatment with each two of the siRNA against hTERT, SVV or VEGF, respectively (each 200nM)  significant enhancement of the effects of isolated treatments in EJ28 (Fig.3) chemosensitization by VEGF-directed siRNA (Fig.4): - 3 BCa cell lines (EJ28, 5637, RT112) - 3 VEGF-directed siRNA (siVEGF203, siVEGF437, siVEGF865) - 3 CT (CDDP, MMC, GEM)  enhanced inhibitory effects on tumor cell viability with highest potency of CDDP Conclusions The results demonstrate that the simultaneous inhibition of different tumor-related transcripts by target-specific inhibitors like siRNAs resulted in a more pronounced antiproliferative action than the attack of an individual transcript in human BCa cells. Furthermore, the pretreatment with expression inhibitors directed against single targets can efficiently sensitize BCa cell lines in vitro to a subsequent CT. The potential enhancement of CT by simultaneous downregulation of multiple targets should be evaluated in future studies. Definite conclusions concerning the impact of the VEGF- targeted constructs on tumor growth can be made only in BCa animal models, which should also give the possibility to evaluate effects on tumor neovascularization. Fig.1 Cellular viability of the BCa cell lines EJ28 (a) and 5637 (b) after transfection with AS-ODN against VEGF, SVV or hTERT in combination with the NS-K1 control or with each other (each 250 nM) 24 / 48 / 72 h after transfection. All values represent means of 5 parallel measurements and were normalized to the NS-K1 control (100%). a) EJ28 b) 5637 Fig.2 VEGF expression levels 24 / 48 / 72 h after transfection with siVEGF865 (200nM) in the BCa cell lines EJ28, 5637 and RT112 determined by qPCR and ELISA. The mRNA expression of VEGF was normalized to that of the reference gene PBGD. All expression values are shown in relation to the NS-si control. EJ RT112 Fig.3 Cellular viability of the BCa cell lines EJ28, 5637 and RT112 after transfection with siRNAs against VEGF, survivin or hTERT in combination with the NS-si control or with each other (each 200 nM) 72 h after transfection. All values represent means of quadruplicates + SD and were normalized to the NS-si control (100%). The combinations of target-directed siRNAs were compared to siRNA+NS-si by t-test ( * p<0.05, ** p<0.01, *** p<0.01). EJ RT112 Fig.4 Inhibition of viability after combined treatment of VEGF-directed siRNAs with chemotherapeutics (CT) 96 h after transfection in EJ28, 5637 and RT112 cells. NS-si control represents 100%. Two doses of CT per cell line were used (CT-1, CT-2). The data represent mean values + SD. Significant differences between VEGF-siRNA+CT and NS-si+CT ( * p<0.05, ** p<0.01, *** p<0.01) were indicated. CT-1/CT-2: CDDP: EJ28, 5637, RT /2.0 µg/ml; MMC: EJ28, /0.67 µg/ml, RT /1.67 µg/ml; GEM: EJ /2.5 ng/ml, /4.0 ng/ml, RT /4.0 ng/ml EJ28 RT112 + MMC-1 + MMC-2 + CDDP-1 + CDDP-2 + GEM-1 + GEM-2 Results for AS-ODN-combinations efficient inhibition of viability of the BCa cell lines EJ28 and 5637 by: isolated treatment with 500nM AS-ODN against hTERT (ASt2331), SVV (ASsvv286) and VEGF (ASvegf857) combined treatment with NS-K1 control and AS-ODN against hTERT, SVV or VEGF, respectively (each 250nM) combined treatment with each two of the AS-ODN against hTERT, SVV or VEGF, respectively (each 250nM)  but no significant enhancement of the effects of isolated treatments (Fig.1) References Forster Y. et al. Cancer Lett 2004;212: Fuessel S. et al. J Urol 2004;171: Fuessel S. et al. Cancer Lett 2006; 232: Kraemer K. et al. Clin Cancer Res 2003;9: Supported by the Jürgen Manchot Foundation (Duesseldorf, Germany) Kraemer K. et al. J Urol 2004;172: Kraemer K et al. Int J Cancer 2006; [in press]. 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