1. siRNA-mediated inhibition of antiapoptotic genes in human bladder cancer cells siRNA-mediated inhibition of antiapoptotic genes in human bladder cancer cells Doreen Kunze, Daniela Wuttig, Susanne Fuessel, Axel Meye, Manfred P. Wirth Department of Urology, Technical University Dresden Department of Urology, Technical University Dresden http://urologie.uniklinikum-dresden.de, e-mail: doreen.kunze@uniklinikum-dresden.de This study was supported by a grant from the Dr. Robert Pfleger-Stiftung. Introduction: BCL2 and BCLX L two antiapoptotic members of the BCL2 family are mitochondrial-membrane proteins that act by preventing the release of mitochondrial cytochrome c into the cytoplasm. In tumor cells that express both BCL2 and BCLX L, the prediction which protein is more important for cell survival is difficult [1]. Furthermore, some tumor cells have been found to switch the expression from BCL2 to BCLX L [2]. Therefore, a combined inhibition of both targets seems to be promising. XIAP, an important member of the inhibitor of apoptosis family, directly binds to and inhibits both initiator caspase 9 and effector caspases 3 and 7. Therefore, XIAP can suppress apoptosis triggered by different stimuli as the mitochondrial- and the death receptor-mediated pathways. Overexpression of the three selected antiapoptotic genes in tumor cells is often associated with a resistance to chemotherapy (CT) [3,4,5]. Since these genes are frequently overexpressed in bladder cancer (BCa) their inhibition, e.g. using siRNAs, represents a promising possibility in the treatment of BCa and could sensitize BCa cells to a subsequent CT. targetsiRNA sequence (sense)siRNA sequence (antisense) BCL2-D1GGA UGA CUG AGU ACC UGA AdTdTUUC AGG UAC UCA GUC AUC CdCdA BCL2-D2GGC GCA CGC UGG GAG AAC AdTdTUGU UCU CCC AGC GUG CGC CdTdT BCLX L -D1CGG CGG CUG GGA UAC UUU UdTdTAAA AGU AUC CCA GCC GCC GdTdT BCLX L -D2GCA GCU UGG AUG GCC ACU UdTdTAAG UGG CCA UCC AAG CUG CdAdG XIAP-D1GUG GUA GUC CUG UUU CAG CdTdTGCU GAA ACA GGA CUA CCA CdTdT XIAP-D2GGA GAU ACC GUG CGG UGC UdTdTAGC ACC GCA CGG UAU CUC CdTdT ns-siRNAUUC UUC GAA CGU GUC ACG UdTdTACG UGA CAC GUU CGG AGA AdTdT siRNA cell count mRNA expression BCL2/TBP mRNA expression BCLX L /TBP mRNA expression XIAP/TBP apoptotic rate apoptotic rate after CT BCL2-D2- 42 %- 41 %- 15 %- 9 %+ 88 %+ 40 % BCLX L -D2- 31 %+ 11 %- 63 %- 5 %+ 94 %+ 33 % XIAP-D1- 45 %- 7 %- 4 %- 54 %- 13 % BCL2-D2 + BCLX L -D2- 44 %- 42 %- 31 %± 0 %+ 87 %+ 29 % untreated- 7 %+ 7 %- 17 %+ 1 %+ 12 %+ 3 % XIAP β-actin XIAP-D1 XIAP-D2 ns-siRNA untreated XIAP-D1 XIAP-D2 ns-siRNA untreated EJ28 T24 Material & Methods: For each target gene (XIAP, BCL2, BCLX L ) two siRNAs were designed (Tab. 1). EJ28 and T24 BCa cells were transfected for 4h lipid- mediated with 200nM of one siRNA or with selected combinations of two siRNAs (each 100nM) against one or different targets (Fig. 1a). Furthermore, siRNA treatment was combined with CT (Fig. 1b). The mRNA expression levels of the targets and the reference gene TBP were examined by quantitative PCR and normalized to the non-silencing siRNA (ns- siRNA) control. XIAP protein level was investigated by western immunoblotting (WB) using primary antibody: hILP - BD Bioscience and secondary antibody: anti-mouse HRP - Dako;  -actin served as a loading control. Clonogenic survival was assessed by colony formation assay, apoptosis by annexin V-staining and cell cycle distribution by propidium iodide staining. The influence of the siRNAs as well as the effects of a siRNA pretreatment followed by CT with mitomycin C (MMC; 0,9µg/ml) or cisplatin (CDDP; 2,1µg/ml) on cellular viability were examined by WST-1 assay. Results: Dependent on the cell line for all used siRNAs a reduction of the target mRNA expression down to 35-80% compared to ns-siRNA treated cells was measured 24h after transfection (data not shown). Even 96h after transfection there is a significant inhibition of the targeted mRNAs (Tab. 2). WB analyses revealed a distinct XIAP protein reduction in EJ28 and T24 cells (Fig. 2). The combined treatment with two siRNAs directed at the same target showed a comparable rate of target mRNA reduction compared to the monotherapies (Fig. 3). A combined transfection with the most effective siRNAs against BCL2 and BCLX L revealed the mRNA downregulation of both targets with an inhibition rate comparable to the monotherapies (Tab. 2). Treatment of EJ28 cells with siRNAs directed at BCL2 and BCLX L as monotherapy or in combination reduced cell count and increased apoptosis (Tab. 2) whereas cell viability (Fig. 4), cell cycle formation and clonogenic survival showed no alterations. Nevertheless, specific combinations of siRNA+MMC caused a significant reduction of the cellular viability compared to the ns ‑ siRNA+MMC control. Tab. 1: siRNA sequences Tab. 2: Cell count, mRNA expression relative to TBP and apoptotic rate of EJ28 cells 96h after transfection with siRNA. Measured values were normalized to ns-siRNA treated cells – the values in the table show the difference compared to ns-siRNA (=100%). Orange column: CDDP was added 20h after transfection and incubated for 24h. In this case cells treated with ns-siRNA + CDDP served as control. Fig. 1: Treatment scheme for siRNA transfection (a) and for combination of siRNA transfection with CT (b). Fig. 2: Western blot analysis of EJ28 and T24 cells 48h after transfection with siRNAs directed at XIAP.  -actin served as a loading control. Conclusions: BCL2, BCLX L and XIAP are promising antiapoptotic targets in the treatment of BCa. The used siRNAs inhibited the target mRNA expression significantly and long-lasting. Furthermore, cell count was reduced, apoptosis and the effects of a subsequent chemotherapy were increased. The combined inhibition of BCL2 and BCLX L caused comparable results to the monotherapies. As these genes are associated with treatment resistance and tumor progression and because of the possibility that the tumor cells switch the expression from one of these genes to the other the combined inhibition of BCL2 and BCLX L also in combination with chemotherapy represents a promising strategy in the treatment of chemoresistant BCa. seeding transfection 4h chemotherapy 2 or 24h analyses 24-72h 20h 46-70h 24-72h 20-92h (a) (b) Fig. 3: mRNA expression levels of the targeted gene 48h after transfection of EJ28 cells with 200nM of one siRNA or after combined treatment with two siRNAs (100nM each) directed at the same target. Expression levels were normalized to ns-siRNA treated cells. References: [1] Gleave ME, Monia BP. Antisense therapy for cancer. Nat Rev Cancer 2005;5(6):468-79. [2] Han Z et al. Isolation and characterization of an apoptosis-resistant variant of human leukemia HL-60 cells that has switched expression from Bcl-2 to Bcl-xL. Cancer Res 1996; 56(7):1621-8. [3] Schimmer AD et al. Targeting XIAP for the treatment of malignancy. Cell Death Differ 2006;13(2):179-88. [4] Hong JH et al. Antisense Bcl2 oligonucleotide in cisplatin-resistant bladder cancer cell lines. BJU Int 2002;90(1):113-117. [5] Lebedeva I et al. Chemosensitization of bladder carcinoma cells by bcl-xL antisense oligonucleotides. J Urol 2001;166(2):461-469. Fig. 4: Relative cell viability of EJ28 cells 96h after transfection with siRNA without and with subsequent CT with MMC or CDDP. Values are normalized to cells treated with ns ‑ siRNA (100%. left part) or to untreated cells (right part). Asterisks indicate a significant difference compared to ns-siRNA + CT (p<0.05). B2: BCL2-D2, BXL: BCLX L -D1, ns: ns-siRNA