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 known to have several modes of action, including inhibition of cyclin‐dependent kinase, induction of p21 and suppression of pRb phosphorylation. In order to test a combination therapy of UCN‐01 and 5‐fluorouracil (5‐FU), growth inhibition of CRL 1420 (MIA PaCa‐2; undifferentiated pancreatic carcinoma) by four different treatments was measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. The treatments used 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 thymidylate synthetase (TS) mRNA levels, TS activity, and 5‐FU incorporation by RNA (FRNA) for each treatment. Although treatment with UCN‐01 alone, 5‐FU alone, 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 synergistically at less than 1 μg/ml drug concentration. The down‐regulation of TS 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‐01 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 synergistic antitumor effect of UCN‐01/5‐FU. In conclusion, low concentrations of UCN‐01 (from 0.01 to 1 μg/ml) may be clinically useful, affording low cytotoxicity of UCN‐01, while enhancing the antitumor effect of 5‐FU.