title: In Vitro schedule-dependent interactions between the multitargeted antifolate LY231514 and gemcitabine in human colon adenocarcinoma cell lines.
abstract: OBJECTIVE
  Multitargeted antifolate (MTA) and gemcitabine (GEM) have shown preclinical and clinical activity in tumor histotypes such as colon, renal, small and non-small cell lung cancers, hepatomas and carcinoid tumors. In our study, we investigated the cytotoxic activity of MTA alone or in combination with GEM using different exposure schedules in three different colon cancer cell lines (LoVo, WiDr, and LRWZ).
  
  
  METHODS
  Cytotoxic activity was evaluated by sulforhodamine B assay, cell cycle perturbations and apoptosis were evaluated by flow cytometry, and thymidylate synthase expression was evaluated by immunohistochemical method.
  
  
  RESULTS
  A 48-h exposure to MTA caused a minimal and no-dose-response effect on the three cell lines used. Flow cytometric analysis showed a cell accumulation in S phase that completely resolved in LoVo and LRWZ cell lines and persisted in WiDr cells after a 48-h washout. Moreover, a significant increase in thymidilate synthase expression was observed in all of the cell lines after MTA exposure. Among the different combinations tested, the highest synergistic interaction, assessed using Kern's method and expressed as the synergistic ratio index, was produced by pretreatment with GEM followed by MTA (ratio index: 1.3- 6.7). It is possible that the depletion of nucleotide pools induced by MTA and required for DNA synthesis prevented cells from repairing DNA damage caused by GEM. The type and degree of drug interactions were not paralleled by apoptosis, which was almost always negligible, or by the type and persistency of the cell cycle perturbations.
  
  
  CONCLUSIONS
  Our results indicate that the sequential administration of GEM --> MTA provides the greatest benefit in the clinical treatment of colon cancer.
authors.affiliation: Istituto Oncologico Romagnolo, Forlì, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Tesei
authors.forename: Luca
authors.initials: L
authors.lastname: Ricotti
authors.forename: Franca
authors.initials: F
authors.lastname: De Paola
authors.forename: Dino
authors.initials: D
authors.lastname: Amadori
authors.forename: Giovanni L
authors.initials: GL
authors.lastname: Frassineti
authors.forename: Wainer
authors.initials: W
authors.lastname: Zoli
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000473:pathology; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D004273:DNA, Neoplasm / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D004347:Drug Interactions; D019008:Drug Resistance, Neoplasm; D004359:Drug Therapy, Combination; D005434:Flow Cytometry; D005493:Folic Acid Antagonists / Q000627:therapeutic use*; D005971:Glutamates / Q000627:therapeutic use*; D006147:Guanine / Q000031:analogs & derivatives / Q000627:therapeutic use*; D006801:Humans; D020287:In Situ Nick-End Labeling; D000068437:Pemetrexed; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D005493:Folic Acid Antagonists; D005971:Glutamates; D000068437:Pemetrexed; D003841:Deoxycytidine; D006147:Guanine; D013940:Thymidylate Synthase; D000093542:Gemcitabine