956: Systematic in Vitro Evaluation of Survivin-Directed Antisense Oligodeoxynucleotides in Bladder Cancer Cells

Abstract

Proc Amer Assoc Cancer Res, Volume 45, 2004 2952 Objectives: The rather poor responses to conventional treatment of bladder cancer (BCa) require novel, specific therapy approaches. The down-regulation of BCa-associated genes may represent a new option to specifically inhibit BCa cell growth and to induce cell death. Survivin, an inhibitor of apoptosis upregulated in the vast majority of malignancies including BCa provides an attractive target for molecular therapies such as treatment with specific antisense oligodeoxynucleotides (AS ODN). Material and Methods: We used a mRNA secondary structure prediction to design survivin-directed AS ODN. After lipid-mediated transfection with 30 selected anti-survivin AS ODN inhibitory effects on cell growth properties (viability, clonogenic survival, induction of apoptosis, cell cycle alterations) were measured. Survivin expression was analyzed by a quantitative real-time PCR assay and ELISA techniques, respectively. Results: Three out of 30 tested constructs remarkably impaired growth characteristics of four BCa cell lines. Detailed analysis of the cell line EJ28 treated with the constructs SVV261, SVV264 and SVV286 revealed an impressive reduction of viability (down to 35%) and long-term proliferation (down to 14%) which were caused by a cell cycle arrest and a dramatic increase of apoptosis (from 19.5% to 51.3% maximum). This strong inhibition of tumor cell growth was associated with the down-regulation of survivin expression of up to 60-80%. Interestingly, all three evolved AS ODN are directed against the putative single stranded survivin mRNA motif between 274-285 nt identified by secondary structure prediction. The reported accessibility of this motif to other nucleic acid based inhibitors such as ribozymes and small interfering RNAs emphasizes the rationale of a systematic selection of mRNA target sites. Conclusions: The survivin-directed AS ODN shown to inhibit remarkably the proliferation of BCa cells in the present study, may provide suitable adjuvant therapeutic agents for the specific local treatment of BCa.