Therapeutic enhancement of S-1 with CPT-11 through down-regulation of thymidylate synthase in bladder cancer

Thymidylate synthase (TS), a target enzyme of 5-fluorouracil (5-FU), is significantly associated with prognosis in various cancers. Recently, it has been reported that S-1, a novel 5-FU-based agent has an effect on bladder cancer. However, in cells with high TS level, S-1 did not have significant effects. Therefore, we examined whether down-regulation of TS enhanced effects of S-1 in them. First, we measured TS level in an aggressive bladder cancer cell line, KU-19-19 by enzyme-linked immunosorbent assay (ELISA) and evaluated its sensitivity to 5-FU using a small interfering RNA (siRNA) for TS. Next, we measured TS mRNA after exposure to various agents. Finally, we evaluated enhancement of cytotoxicity of S-1 by CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) which down-regulated TS in in vivo study. The median TS and dihydropyrimidine dehydrogenase (DPD) level was 53.3 ng/mg and 80.3 ng/mg in KU-19-19 cells, respectively. The 5-FU treatment in KU-19-19 cells transfected with siRNA for TS gene (TYMS) inhibited cell growth more significantly than that for nontargeting control. Down-regulation of TS was observed after exposure to SN-38 (7-ethyl-10-hydroxycamptothecin) in a dose-dependent manner. The combination treatment of 5-FU and SN-38 significantly inhibited cell growth, as compared to the single treatment. Meanwhile, in cells transfected with siRNA for TYMS, neither an additive nor a synergistic effect was observed. Also, combined S-1 and CPT-11 dramatically inhibited tumor growth, compared to S-1 or CPT-11 alone in in vivo study. In conclusion, CPT-11 down-regulated TS level and enhanced the effect of S-1. Thus, the combination therapy with S-1 and CPT-11 might be a novel modality for bladder cancer, even with high TS level. This study confirmed that thymidylate synthase (TS) level in an aggressive human bladder cancer cell line, KU-19-19, was relatively higher than that in other cancer and presented that irinotecan (CPT-11) could down-regulate TS. Finally, the combination therapy with S-1 and CPT-11 resulted in significant tumor growth inhibition through down-regulation of TS in KU-19-19. Thus, combined S-1 and CPT-11 might be a novel treatment in bladder cancer, even with high TS.