UCN-01 (7-hydroxystaurosporine) enhances 5-fluorouracil cytotoxicity through down-regulation of thymidylate synthetase messenger RNA

UCN-01 (7-hydroxystaurosporine) is a newly developed cell cycle inhibitor k nown to have several modes of action, including inhibition of cyclin-depend ent kinase, induction of p21 and suppression of pRb phosphorylation, In ord er to test a combination therapy of UCN-01 and 5-fluorouracil (5-FU), growt h inhibition of CRL 1420 (MIA PaCa-2; undifferentiated pancreatic carcinoma ) by four different treatments was measured using the 3-(4,5-dimethylthiazo l-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, The treatments use d were UCN-01 alone, 5-FU alone, 5-FU followed by UCN-01 (5-FU/UCN-01) and UCN-01 followed by 5-FU (UCN-01/5-FU). We also assessed changes in thymidyl ate synthetase (TS) mRNA levels, TS activity, and 5-FU incorporation by RNA (F-RNA) for each treatment. Although treatment with UCN-01 alone, 5-FU alo ne, and 5-FU/UCN-01 inhibited CRL 1420 growth in a concentration-dependent manner, treatment with UCN-01/5-FU inhibited the growth of CRL 1420 synergi stically at less than 1 mug/ml drug concentration. The down-regulation of T S mRNA by UCN-01 resulted in stable total TS and decreased free TS, and UCN -01/5-FU resulted in enhanced thymidylate synthetase inhibition rate (TSIR) compared to UCN-01 alone and 5-FU/UCN-01, This increased TSIR due to UCN-0 1 pretreatment was accompanied by elevated F-RNA concentrations in the UCN- 01/5-FU treatment. The suppression of TS mRNA and TS activity by UCN-01 may lead to higher sensitivity of tumor cells to 5-FU and may explain the syne rgistic antitumor effect of UCN-01/5-FU, In conclusion, low concentrations of UCN-01 (from 0.01 to 1 mug/ml) may be clinically useful, affording low c ytotoxicity of UCN-01, while enhancing the antitumor effect of 5-FU.