Response of bladder cancer cells and xenografts to treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and gemcitabine

A53 Introduction : Approximately 80% of newly diagnosed bladder cancers are superficial transitional cell carcinomas (TCC), which usually recur and can progress to higher stages after surgical resection. Current topical (intravesical) therapeutics used to prevent recurrence have significant limitations. TRAIL induces apoptosis selectively in variety of tumor cells, but not in normal cells. Moreover, in combination with other DNA-damaging agents, TRAIL has synergistic antitumor activity. Combination therapy with TRAIL and gemcitabine has not been explored. We hypothesize that TRAIL and gemcitabine will have an additive effect on TCC.
 Methods : Gemcitabine (Eli Lilly) and TRAIL/Apo2L (PeproTech Inc.) were dissolved in saline. Human TCC cell lines as monolayers in appropriate media under standard conditions (37°C, 5% CO 2 ) were tested. Cells in log phase growth were seeded in 96-well microtiter (5 x 10 4 cells/well) or in 60-mm culture plates (5 x 10 5 ). After overnight incubation, the medium was replaced with one containing gemcitabine at a concentration of 10 -8 to 10 -4 M (without or with 300 ng/mL TRAIL). After 4-hr incubation at 37 o C, the medium containing the therapeutic agents was replaced with fresh drug-free medium. Cells were cultured for further 72 hrs. Cytotoxicity was analyzed by standard MTT of the 96-well microtiter plates and expression of apoptotic proteins determined by SDS-PAGE and western blot analysis on whole-cell lysates from 60-mm plates. Athymic nude mice were injected subcutaneously with human TCC cells (UMUC14) at their left flank. When tumors reached ~3 mm in diameter, animals (4 per group) were treated with a), TRAIL intratumor injection (i.t.); b), TRAIL intraperitoneal injection (i.p.); c), gemcitabine i.p.; and d), saline i.t. (control). The dose of TRAIL was 20 µg daily, and that of gemcitabine was 2 mg twice weekly, for a total of 6 times.
 Results : The IC 50 for gemcitabine was 1 µM for RT112. Both CRL1472 and UMUC14 cell lines were less sensitive with IC 50 >10 µM. In the presence of TRAIL there was a marked increase (p Conclusions: Low dose gemcitabine in combination with TRAIL enhanced TCC cell killing even in TRAIL resistant cell lines. Inhibition of BCL2 protein expression appeared to mediate this response with maximal BCL2 inhibition observed at doses