Chemosensitization of gemcitabine-resistant human bladder cancer cell line both in vitro and in vivo using antisense oligonucleotide targeting the anti-apoptotic gene, clusterin

To characterize changes in clusterin (sCLU-2) expression in bladder cancer cells after continuous treatment with gemcitabine and to determine whether knockdown of sCLU-2 can re-introduce sensitivity of gemcitabine-resistant cells to treatment with gemcitabine. A human bladder cancer cell line, UM-UC-3, was continuously exposed to increasing doses of gemcitabine in vitro, and a gemcitabine-resistant cell line UM-UC-3R was developed. The role of sCLU-2 in chemoresistant phenotype acquired in both in vitro and in vivo was then analysed using antisense oligonucleotide targeting the sCLU-2 gene (OGX-011). Treatment of parental UM-UC-3 cells (UM-UC-3P) with gemcitabine induced transient up-regulation of sCLU-2 protein. There was a sustained increase in sCLU-2 expression levels in UM-UC-3R compared with UM-UC-3P cells (6.4-fold). Treatment of UM-UC-3R cells with OGX-011 resulted in a dose-dependent and sequence- specific inhibition in sCLU-2 expression. Furthermore, OGX-011 chemo-sensitized UM-UC-3R cells to gemcitabine in vitro with a reduction in the concentration that reduces the effect by 50% (IC50) from 100 nm to 10 nm. Tumour volume and the incidence of metastasis in nude mice injected with UM-UC-3R cells was significantly greater than those of nude mice injected with UM-UC-3P cells; however, systemic administration of OGX-011 plus a low dose of gemcitabine significantly suppressed tumour volume and the incidence of metastasis in both groups. These findings suggest that sCLU-2 plays a significant role in the acquisition of chemoresistant phenotype in bladder cancer cells and the knockdown of sCLU-2 using OGX-011 combined with a chemotherapeutic agent could be an attractive approach for advanced bladder cancer through the enhancement of chemosensitivity.