title: Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates.
abstract: Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26-A, C26-10, C26-G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5-fluorouracil (5-FU) alone or in combination with leucovorin (LV) (2-5-fold), to the thymidylate synthase (TS) inhibitors, AG337 (2-7-fold), ZD1694 (3-49-fold), ZD9331 (3-40-fold), LY231514 (2-21-fold) or GW1843U89 (4-29-fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2-50-fold) compared with their HF variants cultured in standard medium containing up to 8 microM folic acid. LV could only increase sensitivity to 5-FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2-7-fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7-fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3-fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5-FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5-FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models.
authors.affiliation: Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: H H
authors.initials: HH
authors.lastname: Backus
authors.forename: H M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: D
authors.initials: D
authors.lastname: Wouters
authors.forename: J M
authors.initials: JM
authors.lastname: Padrón
authors.forename: N
authors.initials: N
authors.lastname: Molders
authors.forename: C L
authors.initials: CL
authors.lastname: van Der Wilt
authors.forename: C J
authors.initials: CJ
authors.lastname: van Groeningen
authors.forename: G
authors.initials: G
authors.lastname: Jansen
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism; D005472:Fluorouracil / Q000494:pharmacology*; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000378:metabolism; D006801:Humans; D002955:Leucovorin / Q000627:therapeutic use; D008175:Lung Neoplasms; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; C548700:Slc19a1 protein, mouse; C492292:Slc19a2 protein, mouse; C056930:thymidylate synthase-dihydrofolate reductase; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D002955:Leucovorin; D005472:Fluorouracil