The Role of c-FLIP in Cisplatin Resistance of Human Bladder Cancer Cells

Abstract

We investigated the mechanisms underlying cisplatin resistance in human bladder cancer cells to provide novel molecular targets for the treatment of cisplatin resistant bladder cancer. The differential gene expression of cisplatin sensitive (T24) and resistant (T24R2) human bladder cancer cell lines was analyzed and validated by microarray and Western blot analysis. Changes in cisplatin sensitivity by c-FLIP knockdown and related mechanisms in T24R2 cells were assessed using the Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, Maryland) and Western blot. siRNA oligonucleotides that specifically target c-FLIP were prepared and siRNA transfection was done. Microarray analysis revealed that the expression of 1,086 and 322 genes showed more than twofold and fourfold changes in the T24R2 and T24 cell lines, respectively. Especially genes involved in the c-FLIP related death receptor apoptosis pathway, including caspase 2 and 9, NF-kB, BID, c-FLIP, XIAP, and cIAP1 and 2, showed differential expression in the 2 cell lines. Western blot demonstrated complete cisplatin mediated suppression of c-FLIP expression in T24 cells but no change in c-FLIP expression was observed in T24R2 cells after cisplatin treatment in the same dose range. Suppression of c-FLIP expression in T24R2 cells by siRNA transfection rendered these cells significantly more sensitive to cisplatin treatment than untransfected T24R2 cells (p <0.05). Results reveal that c-FLIP has an important role in the cisplatin resistance of human bladder cancer cells and c-FLIP modulation may at least partially reverse cisplatin resistance in bladder cancer cells.