title: Importance of pharmacodynamics in the in vitro antiproliferative activity of the antifolates methotrexate and 10-ethyl-10-deazaaminopterin against human head and neck squamous cell carcinoma.
abstract: The pharmacodynamic profiles of methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) were determined in three head and neck squamous cell carcinoma (HNSCC) cell lines. Cell growth inhibition was tested using a semi-automated 96-well based proliferation assay, the sulforhodamine B (SRB) assay. Drug concentrations ranged from 10(-5) to 10(-9) M, with exposure periods of 4, 24, 48, 72 and 96 hr. The SRB-test was performed after each of these periods of continuous exposure and after an additional period of 24 and 48 hr in drug-free medium. Without a drug-free period the IC50 values strongly depended on the time of exposure. For example, with respect to MTX, IC50 values at 24 hr ranged from 2.9 (UM-SCC-14C) to over 10 microM (UM-SCC-22B and -11B), but when exposed continuously for 96 hr, IC50 values varied between 0.039 and 0.1 microM. 10-EdAM followed a similar sensitivity pattern with 5-20-fold lower IC50 values. The minimal time to achieve significant growth inhibition varied between the cell lines, < 24 hr for UM-SCC-14C, > 24 and > 48 hr for UM-SCC-11B and -22B, respectively. The cell lines also varied with respect to growth behaviour when placed in drug-free medium for an additional period. Growth of UM-SCC-14C cells was recovered significantly after removing the drug, whereas UM-SCC-22B showed a different pattern: when cultured for over 48 hr, cell growth was strongly inhibited, independent of the drug being removed. This variable pattern of sensitivity could be correlated with the capacity of the cells to form polyglutamate derivatives. After 24 hr, drug accumulation was at least three times lower in UM-SCC-14C than in both other cell lines. The low level of antifolate accumulation in UM-SCC-14C is in line with the recovery from growth inhibition at culture in drug-free medium, while the persistent growth inhibition observed in UM-SCC-22B agrees with the intracellular accumulation of higher polyglutamates. In conclusion, these experiments show that the pharmacodynamic profile varies between HNSCC cell lines and plays an important role in the growth inhibition by antifolates. Both exposure time and the intrinsic capacity to synthesize polyglutamates are important factors in the sensitivity of HNSCC to antifolate drugs.
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: B J
authors.initials: BJ
authors.lastname: Braakhuis
authors.forename: G
authors.initials: G
authors.lastname: Jansen
authors.forename: P
authors.initials: P
authors.lastname: Noordhuis
authors.forename: A
authors.initials: A
authors.lastname: Kegel
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
mesh_terms: D000630:Aminopterin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy*; D002455:Cell Division / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006258:Head and Neck Neoplasms / Q000188:drug therapy*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D011099:Polyglutamic Acid / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D005493:Folic Acid Antagonists; D011099:Polyglutamic Acid; C040210:edatrexate; D000630:Aminopterin; D008727:Methotrexate