title: Determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines.
abstract: OBJECTIVE
  Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target.
  
  
  RESULTS
  The cytotoxicity of irinotecan and SN-38 markedly differed in the two cell lines: irinotecan IC(50) values were 15.8 microM for LoVo cells and 5.17 microM for HT-29 cells; SN-38 IC(50) values were 8.25 n M for LoVo cells and 4.50 n M for HT-29 cells. Topoisomerase I expression (at the mRNA and the protein levels) and catalytic activity were similar in the two cell lines. Irinotecan induced similar amounts of cleavable complexes at its IC(50) in both cell lines. SN-38 induced a concentration-dependent formation of cleavable complexes, which was not significantly different in the two cell lines. Expression of the carboxylesterase CES1 was higher in HT-29 than in LoVo cells. Expression of the carboxylesterase gene CES2 was comparable in the two cell lines and much higher than CES1 gene expression. Carboxylesterase activity was extremely low using p-nitrophenylacetate as a substrate (1.45 and 1.84 pmol/min per mg proteins) and could not even be detected using irinotecan as a substrate. Cell accumulation of irinotecan was markedly different, reaching consistently higher levels in HT-29 cells than in LoVo cells.
  
  
  CONCLUSIONS
  Our results indicate that (1) the cytotoxicity of irinotecan was likely due to the drug itself and not to its metabolite SN-38, and (2) that irinotecan uptake was more predictive of its cytotoxicity than topoisomerase I availability and activity in these two cell lines.
authors.affiliation: Université Victor Segalen Bordeaux 2 and Institut Bergonié, 229 cours de l'Argonne, 33076 Bordeaux, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Pavillard
authors.forename: Cécile
authors.initials: C
authors.lastname: Agostini
authors.forename: Sophie
authors.initials: S
authors.lastname: Richard
authors.forename: Virginie
authors.initials: V
authors.lastname: Charasson
authors.forename: Danièle
authors.initials: D
authors.lastname: Montaudon
authors.forename: Jacques
authors.initials: J
authors.lastname: Robert
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology*; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002384:Catalysis; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000473:pathology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D000077146:Irinotecan; D009363:Neoplasm Proteins; D014407:Tumor Cells, Cultured
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000972:Antineoplastic Agents, Phytogenic; D009363:Neoplasm Proteins; D000077146:Irinotecan; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin