title: Alterations in the expression pattern of MTHFR, DHFR, TYMS, and SLC19A1 genes after treatment of laryngeal cancer cells with high and low doses of methotrexate.
abstract: Inter-individual variations to methotrexate (MTX) outcome have been attributed to different expression profiles of genes related to folate metabolism. To elucidate the mechanisms of variations to MTX outcome, we investigated MTHFR, DHFR, TYMS, and SLC19A1 gene expression profiles by quantifying the mRNA level of the genes involved in folate metabolism to MTX response in laryngeal cancer cell line (HEP-2). For this, three different concentrations of MTX (0.25, 25, and 75 μmol) were added separately in HEP-2 cell line for 24 h at 37 °C. Apoptotis quantification was evaluated with fluorescein isothiocyanate-labeled Bcl-2 by flow cytometry. Real-time quantitative PCR technique was performed by quantification of gene expression with TaqMan® Gene Expression Assay. ANOVA and Bonferroni's post hoc tests were utilized for statistical analysis. The results showed that the numbers of apoptotic HEP-2 cells with 0.25, 25.0, and 75.0 μmol of MTX were 14.57, 77.54, and 91.58%, respectively. We found that the expression levels for MTHFR, DHFR, TYMS, and SLC19A1 genes were increased in cells with 75.0 μmol of MTX (p < 0.05). Moreover, SLC19A1 gene presented lower expression in cells treated with 0.25 μmol of MTX (p < 0.05). In conclusion, our data suggest that MTHFR, DHFR, TYMS, and SLC19A1 genes present increased expression after the highest application of MTX dose in laryngeal cancer cell line. Furthermore, SLC19A1 gene also presents decreased expression after the lowest application of MTX dose in laryngeal cancer cell line. Significant alterations of expression of these studied genes in cell culture model may give support for studies in clinical practice and predict interesting and often novel mechanisms of resistance of MTX chemotherapy.
authors.affiliation: Genetics and Molecular Biology Research Unit (UPGEM), São José do Rio Preto Medical School (FAMERP), Avenida Brigadeiro Faria Lima, n.5416, São José do Rio Preto, SP, Brazil, analivia_sg@yahoo.com.br.
authors.forename: Ana Lívia Silva
authors.initials: AL
authors.lastname: Galbiatti
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Castro
authors.forename: Heloisa Cristina
authors.initials: HC
authors.lastname: Caldas
authors.forename: João Armando
authors.initials: JA
authors.lastname: Padovani
authors.forename: Erika Cristina
authors.initials: EC
authors.lastname: Pavarino
authors.forename: Eny Maria
authors.initials: EM
authors.lastname: Goloni-Bertollo
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics / Q000473:pathology; D008727:Methotrexate / Q000494:pharmacology; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics*; D058978:Reduced Folate Carrier Protein / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D059467:Transcriptome / Q000187:drug effects*
chemical_list: D000964:Antimetabolites, Antineoplastic; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C573423:MTHFR protein, human; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008727:Methotrexate