J Urol. 2013 Jun;189(6):2327-34.

The role of c-FLIP in cisplatin resistance of human bladder cancer cells.

Lee S, Yoon CY, Byun SS, Lee E, Lee SE.

Department of Urology, Seoul National University Bundang Hospital, Seongnam, Korea.



Purpose: 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.

Materials and Methods: 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.

Results: 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).

Conclusions: 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.

Copyright © 2013 American Urological Association Education and Research, Inc.

PMID: 23313194



Deregulation of apoptosis, which controls the balance between cell survival and death, is a well-known chemotherapeutic resistance mechanism that has been identified in many malignant diseases. c-FLIP, an important regulator of apoptosis, is a catalytically inactive pro-caspase-8/10 homolog associated with the signaling complex downstream of death receptors
and, thus, c-FLIP negatively interferes with apoptotic signaling.

Silencing c-FLIP expression with siRNA reportedly induced spontaneous apoptosis in some tumors, ie colorectal and lung cancer, and lymphoma. In MCF-7 breast cancer cells an apoptotic inhibitory complex composed of DR5, FADD, caspase-8 and c-FLIP was reported and the absence of c-FLIP from this complex induced DR5 and FADD mediated caspase-8 activation in DISC. The effect of c-FLIP on the apoptotic response to chemotherapy, ie 5-fluoro-uracil, oxaliplatin or irinotecan, for colorectal cancer was also evaluated. Although increased expression of anti-apoptotic c-FLIPS was noted in various cancers, a paucity of data exists in the literature on the role of c-FLIP in bladder cancer, especially in correlation with cisplatin resistance.

In the current study, we performed microarray analysis to explore differential gene expression between human bladder cancer cell lines with different sensitivity to cisplatin and found increased c-FLIP and related gene expression in cisplatin resistant human bladder cancer cell line T24R2 compared with cisplatin-sensitive T24 cells. T24R2 cells also showed significantly greater resistance to TRAIL, a potent ligand for the death receptor and a main target of c-FLIP, than T24 cells, suggesting the involvement of c-FLIP, one of the major regulators of death receptor signaling, in antitumor activity and also in the mechanism of resistance to cisplatin in human bladder cancer cells. Silencing c-FLIP expression with siRNA rendered cisplatin-resistant T24R2 cells more amenable to cisplatin treatment. These findings show that c-FLIP has an important role in modulating the antitumor effect of cisplatin in human bladder cancer cells, indicating that focusing on c-FLIPL may be a promising strategy for bladder cancer that relapses after first line cisplatin based chemotherapy.

In conclusion, the present study showed the important role of c-FLIP and related signaling pathways in the resistance of human bladder cancer cells to cisplatin, therefore suggesting possible targeting of c-FLIP-related pathway for the enhancement antitumor effect of cisplatin or the overcome of the cisplatin resistance in human bladder cancer.

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