levoleucovorin and nolatrexed

Cancer is related to the cellular proliferative state. Increase in cell-cycle regulatory function augments cellular folate pool. This pathway is therapeutically targeted. A number of drugs influences this metabolism, that is, folic acid, folinic acid, nolatrexed, and methotrexate. Our previous study showed methotrexate influences on rat/human sulfotransferases. Present study explains the effect of nolatrexed (widely used in different cancers) and some micronutrients on the expressions of rat/human sulfotransferases. Female Sprague-Dawley rats were treated with nolatrexed (01-100 mg/kg) and rats of both sexes were treated to folic acid (100, 200, or 400 mg/kg) for 2-weeks and their aryl sulfotransferase-IV (AST-IV; β-napthol sulfation) and sulfotransferase (STa; DHEA sulfation) activities, protein expression (western blot) and mRNA expression (RT-PCR) were tested. In human-cultured hepatocarcinoma (HepG2) cells nolatrexed (1 nM-1.2 mM) or folinic acid (10 nM-10 μM) were applied for 10 days. Folic acid (0-10 μM) was treated to HepG2 cells. PPST (phenol catalyzing), MPST (dopamine and monoamine), DHEAST (dehydroepiandrosterone and DHEA), and EST (estradiol sulfating) protein expressions (western-blot) were tested in HepG2 cells. Present results suggest that nolatrexed significantly increased sulfotransferases expressions in rat (protein, STa, F = 4.87, P < 0.05/mRNA, AST-IV, F = 6.702, P < 0.014; Student's t test, P < 0.01-0.05) and HepG2 cells. Folic acid increased sulfotransferases activity/protein in gender-dependant manner. Both folic and folinic acid increased several human sulfotransferases isoforms with varied level of significance (least or no increase at highest dose) in HepG2 cells pointing its dose-dependent multiphasic responses. The clinical importance of this study may be furthered in the verification of sulfation metabolism of several exogenous/endogenous molecules, drug-drug interaction and their influences on cancer pathophysiological processes. Further studies are necessary.

Topics: Animals; Antimetabolites, Antineoplastic; Arylsulfotransferase; Blotting, Western; Cell Cycle; Dose-Response Relationship, Drug; Female; Folic Acid; Hep G2 Cells; Humans; Leucovorin; Male; Methotrexate; Micronutrients; Quinazolines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sex Factors; Sulfotransferases

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.

Topics: Antimetabolites, Antineoplastic; Biological Transport; Blotting, Northern; Blotting, Southern; Blotting, Western; Carrier Proteins; Cell Division; Cell Membrane; Chlorides; DNA Mutational Analysis; DNA, Complementary; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Inhibitors; Exons; Folic Acid; Folic Acid Antagonists; Humans; Indoles; Inhibitory Concentration 50; Isoindoles; Kinetics; Leucovorin; Leukemia; Membrane Proteins; Membrane Transport Proteins; Methotrexate; Mutagenesis, Site-Directed; Mutation; Polymorphism, Single-Stranded Conformational; Protein Structure, Secondary; Protein Structure, Tertiary; Quinazolines; Recombinant Proteins; Reduced Folate Carrier Protein; Thymidylate Synthase; Time Factors; Transfection; Trimetrexate; Tumor Cells, Cultured

Effects of lipophilic thymidylate synthase (TS) inhibitor AG337 on human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (MTX) resistance were assayed using continuous or intermittent drug exposure. During 120-h continuous exposure, HNSCC cell lines A253 and FaDu are equally MTX sensitive (EC50 equals approximately 15nM); AG337 is less potent (EC50 approximately equals 1 microM). A253 is intrinsically resistant to 24-h intermittent MTX exposure (EC50 equals approximately 17 microM; FaDu, EC50 equals approximately 0.3 microM); both HNSCC cell lines are resistant to 24-h AG337 exposure (EC50 >100 microM). CCRF-CEM shows MTX (EC50 =14 nM) and AG337 (EC50 equals approximately 0.6 microM) sensitivity in continuous exposure similar to HNSCC; however, AG337 retains potency against CCRF-CEM cells in intermittent exposure (24-h, EC50 equals approximately 2 microM; 6-h, EC50 equals approximately 48 microM). The reduced folate leucovorin (LV) at > or = 0.1 microM fully protects from growth inhibition by continuous MTX exposure, but growth inhibition by AG337 is reversed only slightly by < or = 100 microM LV. Thymidine fully protects A253 and FaDu against growth inhibition by AG337, while hypoxanthine alone is without effect, suggesting inhibition is TS-specific. CCRF-CEM sublines with acquired MTX-resistance resulting from DHFR overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG337 in continuous exposure (all EC50 =0.4 microM). These data indicate that AG337 may be useful in therapy of tumors that have acquired resistance to MTX by most common mechanisms.

Topics: Cell Cycle; Cell Division; Culture Media; Drug Resistance; Enzyme Inhibitors; Fetal Blood; Head and Neck Neoplasms; Humans; Hypoxanthine; Inhibitory Concentration 50; Leucovorin; Leukemia; Methotrexate; Quinazolines; Thymidine; Thymidine Phosphorylase; Thymidylate Synthase; Tumor Cells, Cultured