title: Mode of action of methotrexate-albumin in a human T-cell leukemia line and activity against an MTX-resistant clone.
abstract: Limitations of low mol. wt anticancer drugs are short tumor exposure times and toxicity to normal tissue. Methotrexate (MTX) covalently linked to human serum albumin (HSA) as a macromolecular carrier caused tumor regressions concomitant with a favorable toxicity profile in a clinical phase I trial (Hartung et aL, Clin. Cancer Res., 1999, 5, 753). We examined the uptake, intracellular degradation, metabolism and thymidylate synthase (TS) inhibition of MTX-HSA in the T-cell leukemia line CCRF-CEM and the MTX transport resistant clone CCRF-CEM/MTX. The number of MTX molecules per albumin molecule was determined by electrospray mass spectrometry. A loading ratio (LR) of approximately 1.4 mol MTX/albumin revealed intact complexes with one and two MTX molecules/albumin. In the complex with an LR of 5.7, albumin with up to 16 MTX molecules was seen. MTX-HSA was taken up by CCRF-CEM cells via endocytosis and cleaved by lysosomal enzymes. Liberated MTX was a poor substrate of folylpolyglutamate synthetase and was exported into the medium. TS was inhibited and cell survival was impaired by MTX-HSA in a time- and concentration-dependent manner. CCRF-CEM/MTX cells exhibited a growth inhibition of 30-40% after MTX-HSA treatment, but no TS inhibition. The alternative uptake route via endocytosis enables MTX-HSA to overcome transport resistance to MTX.
authors.affiliation: Division of Molecular Toxicology, German Cancer Research Center, Heidelberg.
authors.forename: M
authors.initials: M
authors.lastname: Weigand
authors.forename: G
authors.initials: G
authors.lastname: Hartung
authors.forename: J
authors.initials: J
authors.lastname: Roboz
authors.forename: S
authors.initials: S
authors.lastname: Sieger
authors.forename: M
authors.initials: M
authors.lastname: Wolf
authors.forename: H
authors.initials: H
authors.lastname: Sinn
authors.forename: H H
authors.initials: HH
authors.lastname: Schrenk
authors.forename: M
authors.initials: M
authors.lastname: Wiessler
authors.forename: E
authors.initials: E
authors.lastname: Frei
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002999:Clone Cells; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007120:Immunochemistry; D015458:Leukemia, T-Cell / Q000188:drug therapy* / Q000473:pathology; D008247:Lysosomes / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D008970:Molecular Weight; D011099:Polyglutamic Acid / Q000378:metabolism; D011485:Protein Binding; D012709:Serum Albumin / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D016019:Survival Analysis; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D012709:Serum Albumin; D011099:Polyglutamic Acid; D013940:Thymidylate Synthase; D008727:Methotrexate