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01-12-2013 | Research Article

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

Authors: Ana Lívia Silva Galbiatti, Rodrigo Castro, Heloisa Cristina Caldas, João Armando Padovani Jr, Érika Cristina Pavarino, Eny Maria Goloni-Bertollo

Published in: Tumor Biology | Issue 6/2013

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.

Chu EA, Kim YJ. Laryngeal cancer: diagnosis and preoperative work-up. Otolaryngol Clin North Am. 2008;41(4):673–95.PubMedCrossRef

Sapkota A, Hsu CC, Zaridze D, Shangina O, Szeszenia-Dabrowska N, Mates D, et al. Dietary risk factors for squamous cell carcinoma of the upper aerodigestive tract in central and eastern Europe. Cancer Causes Control. 2008;19(10):1161–70.PubMedCrossRef

Torrente MC, Rodrigo JP, Haigentz Jr M, Dikkers FG, Rinaldo A, Takes RP, et al. Human papillomavirus infections in laryngeal cancer. Head & Neck. 2011;33(4):581–6.CrossRef

Lorch JH, Posner MR, Wirth LJ, Haddad RI. Induction chemotherapy in locally advanced head and neck cancer: a new standard of care? Hematol Oncol Clin North Am. 2008;22(6):1155–63.PubMedCrossRef

Gonen N, Assaraf YG. Antifolates in cancer therapy: structure, activity and mechanisms of drug resistance. Drug Resist Updat. 2012;15(4):183–210.PubMedCrossRef

Visentin M, Zhao R, Goldman ID. The antifolates. Hematol Oncol Clin North Am. 2012;26(3):629–48.PubMedCrossRef

Taguchi T, Tsukuda M, Mikami Y, Matsuda H, Horiuchi C, Yoshida T, et al. Concurrent chemoradiotherapy with cisplatin, 5-fluorouracil, methotrexate, and leucovorin in patients with advanced resectable squamous cell carcinoma of the larynx and hypopharynx. Acta Otolaryngol. 2006;126(4):408–13.PubMedCrossRef

Doe J. Methotrexate. Mylan Pharmaceuticals Inc. 2012. http://www.drugs.com/pro/methotrexate.html. Accessed 11 Jun 2013.

Kraljevic Pavelic S, Cacev T, Kralj M. A dual role of p21 ^waf1/cip1 gene in apoptosis of HEp-2 treated with cisplatin or methotrexate. Cancer Gene Ther. 2008;15(9):576–90.PubMedCrossRef

10.

Brockstein BE. Management of recurrent head and neck cancer: recent progress and future directions. Drugs. 2011;71(12):1551–9.PubMedCrossRef

11.

Price KA, Cohen EE. Current treatment options for metastatic head and neck cancer. Curr Treat Options Oncol. 2012;13(1):35–46.PubMedCrossRef

12.

Bertino JR. Cancer research: from folate antagonism to molecular targets. Best Pract Res Clin Haematol. 2009;22:577–82.PubMedCrossRef

13.

Sáez-Ayala M, Fernández-Pérez MP, Montenegro MF, Sánchez-del-Campo L, Chazarra S, Piñero-Madrona A, et al. Melanoma coordinates general and cell-specific mechanisms to promote methotrexate resistance. Exp Cell Res. 2012;318(10):1146–59.PubMedCrossRef

14.

Morales C, Garcia MJ, Ribas M, Miro R, Muñoz M, Caldas C, et al. Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells. Mol Cancer Ther. 2009;8:424–32.PubMedCrossRef

15.

Moura JA, Valduga CJ, Tavares ER, Kretzer IF, Maria DA, Maranhão RC. Novel formulation of a methotrexate derivative with a lipid nanoemulsion. Int J Nanomedicine. 2011;6:2285–95.PubMed

16.

Hider SL, Bruce IN, Thomson W. The pharmacogenetics of methotrexate. Rheumatology. 2007;46:1520–4.PubMedCrossRef

17.

Katori H, Tsukuda M, Taguchi T. Analysis of efficacy and toxicity of chemotherapy with cisplatin, 5-fluorouracil, methotrexate and leucovorin (PFML) and radiotherapy in the treatment of locally advanced squamous cell carcinoma of the head and neck. Cancer Chemother Pharmacol. 2007;59:789–94.PubMedCrossRef

18.

Celtikci B, Leclerc D, Lawrance AK, Deng L, Friedman HC, Krupenko NI, et al. Altered expression of methylenetetrahydrofolate reductase modifies response to methotrexate in mice. Pharmacogenet Genomics. 2008;18:577–89.PubMedCrossRef

19.

Specenier PM, Vermorken JB. Current concepts for the management of head and neck cancer: chemotherapy. Oral Oncol. 2008;45:409–15.PubMedCrossRef

20.

Pai RB, Lalitha RM, Pai SB, Kumaraswamy SV, Lalitha N, Bhargava MK. Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate. Exp Oncol. 2009;31:118–20.PubMed

21.

Celtikci B, Lawrance AK, Wu Q, Rozen R. Methotrexate-induced apoptosis is enhanced by altered expression of methylenetetrahydrofolate reductase. Anticancer Drugs. 2009;20(9):787–93.PubMedCrossRef

22.

Askari BS, Krajinovic M. Dihydrofolate reductase gene variations in susceptibility to disease and treatment outcomes. Curr Genomics. 2010;11(8):578–83.PubMedCrossRef

23.

Erčulj N, Kotnik BF, Debeljak M, Jazbec J, Dolžan V. Influence of folate pathway polymorphisms on high-dose methotrexate-related toxicity and survival in childhood acute lymphoblastic leukemia. Leuk Lymphoma. 2012;53(6):1096–104.PubMedCrossRef

24.

Obuchi W, Ohtsuki S, Uchida Y, Ohmine K, Yamori T, Terasaki T. Identification of transporters associated with etoposide sensitivity of stomach cancer cell lines and methotrexate sensitivity of breast cancer cell lines by quantitative targeted absolute proteomics. Mol Pharmacol. 2013;83(2):490–500.PubMedCrossRef

25.

Lee YL, Xu X, Wallenstein S, Chen J. Gene expression profiles of the one-carbon metabolism pathway. J Genet Genomics. 2009;36:277–82.PubMedCrossRef

26.

Linhart HG, Troen A, Bell GW, Cantu E, Chao WH, Moran E, et al. Folate deficiency induces genomic uracil misincorporation and hypomethylation but does not increase DNA point mutations. Gastroenterology. 2009;136:227–35.PubMedCrossRef

27.

Bertino JR, Gorlick R. Mechanisms of methotrexate resistance in osteosarcoma. Clin Cancer Res. 1999;5:621–7.PubMed

28.

Guardiola E, Peyrade F, Chaigneau L, et al. Results of a randomized phase II study comparing docetaxel with methotrexate in patients with recurrent head and neck cancer. Eur J Cancer. 2004;40:2071–6.PubMedCrossRef

29.

Abali EE, Ercikan E, Skacel NE, Celikkaya H, Hsieh YC. Regulation of human dihydrofolate reductase activity and expression. Vitam Horm. 2008;79:267–92.PubMedCrossRef

30.

Parle-McDermott A, Pangilinan F, Mills JL, Kirke PN, Gibney ER, Troendle J, et al. The 19-bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR) may decrease rather than increase risk for spina bifida in the Irish population. Am J Med Genet A. 2007;143:1174–80.CrossRef

31.

Sowers R, Toguchida J, Qin J, Meyers PA, Healey JH, Huvos A, et al. mRNA expression levels of E2F transcription factors correlate with dihydrofolate reductase, reduced folate carrier, and thymidylate synthase mRNA expression in osteosarcoma. Mol Cancer Ther. 2003;2(6):535–41.PubMed

32.

Serra M, Reverter-Branchat G, Maurici D, Benini S, Shen JN, Chano T, et al. Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells. Ann Oncol. 2004;15:151–60.PubMedCrossRef

33.

Mishra PJ, Humeniuk R, Mishra PJ, Longo-Sorbello GS, Banerjee D, Bertino JR. A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. Proc Natl Acad Sci USA. 2007;104(33):13513–8.PubMedCrossRef

34.

Trinh BN, Ong CN, Coetzee GA, Yu MC, Laird PW. Thymidylate synthase: a novel genetic determinant of plasma homocysteine and folate levels. Hum Genet. 2002;111:299–302.PubMedCrossRef

35.

Kremer JM. Toward a better understanding of methotrexate. Arthritis Rheum. 2004;50:1370–82.PubMedCrossRef

36.

Chiusolo P, Reddiconto G, Farina G, Mannocci A, Fiorini A, Palladino M, et al. MTHFR polymorphisms’ influence on outcome and toxicity in acute lymphoblastic leukemia patients. Leuk Res. 2007;31:1669–74.PubMedCrossRef

37.

Mattia E, Toffoli G. C677T and A1298C MTHFR polymorphisms, a challenge for antifolate and fluoropyrimidine-based therapy personalization. Eur J Cancer. 2009;45:1333–51.PubMedCrossRef

38.

Robien K, Boynton A, Ulrich CM. Pharmacogenetics of folate-related drug targets in cancer treatment. Pharmacogenomics. 2005;6:673–89.PubMedCrossRef

39.

Ma D, Huang H, Moscow JA. Down-regulation of reduced folate carrier gene (RFC1) expression after exposure to methotrexate in ZR-75-1 breast cancer cells. Biochem Biophys Res Commun. 2000;279(3):891–7.PubMedCrossRef

40.

Abdel-Haleem AM, El-Zeiry MI, Mahran LG, Abou-Aisha K, Rady MH, Rohde J, et al. Expression of RFC/SLC19A1 is associated with tumor type in bladder cancer patients. PLoS One. 2011;6(7):e21820.PubMedCrossRef

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
Authors: Ana Lívia Silva Galbiatti
Rodrigo Castro
Heloisa Cristina Caldas
João Armando Padovani Jr
Érika Cristina Pavarino
Eny Maria Goloni-Bertollo
Publication date: 01-12-2013
Publisher: Springer Netherlands
Published in: Tumor Biology / Issue 6/2013
Print ISSN: 1010-4283
Electronic ISSN: 1423-0380
DOI: https://doi.org/10.1007/s13277-013-0960-3

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