A importância da seqüência de administração do irinotecan e 5-fluorouracil na inibição da proliferação do carcinoma de cólon humano in vitro

Colorectal cancer is one of the most frequent human malignancies, and the third leading cause of cancer death in the world. Notwithstanding important improvements in therapeutic strategies, results in advanced disease remain unsatisfactory. This is illustrated by the fact that the most widely used drug in this malignancy, the antimetabolite 5-fluorouracil (5-FU), was more than 40 years ago developed, and produces response rates of only 10-15%. Recently, the topoisomerase I inhibitor irinotecan (CPT-11) was found to elicit comparable responses rates in both untreated and 5-FU-pretreated, relapsed patients suffering from colon carcinoma. This prompted the evaluation of the 5-FU/CPT-11 combination in this disease. Although the obtained results in early clinical studies are promising, the best sequence of administration has yet to be established. In this study, the CPT-11/5-FU combination has been evaluated for increased cell growth inhibition with respect to that by either agent alone in the SW620, HT-29, and SNU-C4 human colon carcinoma cell lines. For this purpose, cells were exposed to either drug alone or to various combinations and sequences of fixed, low doses (IC20) of one drug, with higher, varying doses of the other. Then assessed for cell growth inhibition by the sulforhodamine B assay, either immediately, and/or after culturing for 2 additional days in drug-free medium. The interactions between CPT-11 and 5-FU were evaluated using a computer program for multiple drug effect analysis, which enables calculation of combination indices (CIs) referring to synergism, additivity, or antagonism when 1, respectively. Cellular responses were related to thymidylate synthase, topoisomerase I, and carboxyl esterase activities, assessed by a ligand-binding and a H-release assay, a DNA decatenation assay, and a spectrophotometric method, respectively. As DNA damage formation is implicated in the mechanism of action of both drugs, cellular responses were also related to the introduction of frank and/or alkali-labile DNA strand-breaks using a fluorescence-enhancement assay for DNA unwinding. To better understand the molecular basis of the drug interactions, we furthermore examined the effects of 2 h or 24 h IC20, IC50, and/or IC80 of 5-FU or CPT-11 alone on cellular determinants likely relevant to drug cytotoxicity. These included DNA excision repair capacity, cell cycle phase distribution, plasma membrane integrity, and DNA-topoisomerase I complex formation. For these purposes we made use of a [H-methyl]thymidine incorporation