Tumor Biology

, Volume 34, Issue 6, pp 3765–3771 | Cite as

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

  • Ana Lívia Silva Galbiatti
  • Rodrigo Castro
  • Heloisa Cristina Caldas
  • João Armando PadovaniJr
  • Érika Cristina Pavarino
  • Eny Maria Goloni-Bertollo
Research Article


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.


Antifolate Chemotherapy treatment Gene expression Methotrexate Cell-line Laryngeal carcinoma 



The Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) is highly appreciated for the financial support (grant nos.: 2010/12930-4 and 2010/12932-7). The authors thank Prof. Dr. Moacir F. Godoy for the help in statistical analysis and Profa. Dra Eloiza Helena Tajara for providing the cell line. The authors are also grateful to the Oncology Department, Hospital de Base, São José do Rio Preto for providing methotrexate; National Counsel of Technological and Scientific Development (CNPQ) and FAMERP/FUNFARME are highly acknowledged.

Conflicts of interest



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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2013

Authors and Affiliations

  • Ana Lívia Silva Galbiatti
    • 1
  • Rodrigo Castro
    • 1
  • Heloisa Cristina Caldas
    • 2
  • João Armando PadovaniJr
    • 3
  • Érika Cristina Pavarino
    • 1
  • Eny Maria Goloni-Bertollo
    • 1
  1. 1.Genetics and Molecular Biology Research Unit (UPGEM)São José do Rio Preto Medical School (FAMERP)São José do Rio PretoBrazil
  2. 2.Laboratory of Experimental Immunology and Transplantation (LITEX)São José do Rio Preto Medical School (FAMERP)São José do Rio PretoBrazil
  3. 3.Otorhinolaryngology and Head and Neck Surgery DepartmentSão José do Rio Preto Medical School (FAMERP)São José do Rio PretoBrazil

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