High-risk superficial urothelial carcinoma of the bladder (UCB) is commonly treated with intravesical bacillus Calmette-Guerin (BCG), but with significant side effects. We recently showed that the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibited high therapeutic potential against UCB cells and with only limited toxic effects in normal cells. However, many cancer cells are refractory to TRAIL during monotherapy. Therefore, our experimental aim was to develop combinatorial approaches with other pro- apoptotic agents to reactivate apoptosis in resistant phenotypes. We demonstrate that UCB cells varied in their response to TRAIL, and the effect was caspase-dependent (reduced or abrogated by pre-incubation of cells with caspase-inhibitor peptides). In contrast wortmannin, a PI3K/Akt inhibitor, enhanced the TRAIL effect. Furthermore, combination therapy of TRAIL with low dose gemcitabine markedly enhanced UCB cell response (except in the TRAIL-resistant HT1376 cell line). The enhanced response was both time- and concentration-dependent and asymptotic at gemcitabine concentration >1 µmol/l. To define the mechanisms underlying gemcitabine-augmented TRAIL action, we evaluated the expression of several proteins regulating the apoptotic pathway. Gemcitabine-augmented TRAIL effect was associated with inhibition of the Bcl-2 protein (intrinsic signalling) along with activation of the caspase (extrinsic) cascade. The combined maximal stimulation of both the intrinsic and extrinsic signalling pathways also appeared to overcome the survival (PI3K/Akt) pathway as evident by the lack of response to wortmannin. Our semisolid multicellular-spheroid model showed that TRAIL and gemcitabine selectively caused UCB cells to undergo apoptosis without affecting normal cells, and both appeared to penetrate deeply enough to allow for combination intravesical therapy.

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