title: Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation.
abstract: The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Singh
authors.forename: A A
authors.initials: AA
authors.lastname: Fouladi-Nashta
authors.forename: D
authors.initials: D
authors.lastname: Li
authors.forename: N
authors.initials: N
authors.lastname: Halliday
authors.forename: D A
authors.initials: DA
authors.lastname: Barrett
authors.forename: K D
authors.initials: KD
authors.lastname: Sinclair
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D000241:Adenosine / Q000378:metabolism / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D000242:Cyclic AMP / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007042:Hypoxanthines / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000187:drug effects / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism; D011687:Purines / Q000378:metabolism*; D015398:Signal Transduction; D013763:Tetrahydrofolates / Q000494:pharmacology; D013936:Thymidine / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; D007042:Hypoxanthines; D011073:Polyamines; D011687:Purines; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D008715:Methionine; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D000469:Alkaline Phosphatase; D000241:Adenosine; D013936:Thymidine; D008727:Methotrexate