Original article Gene expression profile of 5-fluorouracil metabolic enzymes in laryngeal cancer cell line: Predictive parameters for response to 5-fluorouracil-based chemotherapy Ana Lı ´via Silva Galbiatti a, *, Heloisa Cristina Caldas b , Jose ´ Victor Maniglia c , E ´ rika Cristina Pavarino a , Eny Maria Goloni-Bertollo a a FAMERP- Sa˜o Jose ´ do Rio Preto Medical School Genetics and Molecular Biology Research Unit - UPGEM AV Brigadeiro Faria Lima, 5416 Sa ˜o Jose ´ do Rio Preto, 15090000 Brazil b FAMERP - Laboratory of Experimental Immunology and Transplantation/LITEX, Brazil c FAMERP - Otorhinolaryngology and Head and Neck Surgery Department, Brazil 1. Introduction Head and neck cancers constitute for 12% of all malignancies in the world. Laryngeal cancer is the second most frequent tumor of the head and neck and it is estimated about 151,000 new cases world wild each year and the death incidence for this disease is estimated to be 82 per 100,000 people per year [1,2]. Chemotherapy focused on specific molecular targets and pathways are becoming common for cancer treatments, as antifolate chemotherapeutics that are designed to target key folate-dependent enzymes, therefore leading to inhibition of nucleotide biosynthesis and cell death [3,4]. 5-fluorouracil (5- FU) chemotherapy is an antifolate that may be utilized to head and neck cancer treatment and therapeutic dose range of 20–25 mg hr/ L has been consistently shown to yield optimal results in terms of clinical efficacy and safety [5,6]. It achieves its therapeutic efficacy through inhibition of thymidylate synthase enzyme (TYMS), which is essential for DNA synthesis and repair. It causes cell apoptosis and cell cycle arrest by suppressing the cell’s ability for synthesizing DNA. It is possible that 5-FU chemotherapy may also influence another enzymes involved in folate pathway. These enzymes are codified by genes that have essential role in folate pathway [7–9]. Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx A R T I C L E I N F O Article history: Received 24 February 2014 Accepted 17 March 2014 Keywords: Gene expression Laryngeal neoplasms Fluorouracil A B S T R A C T Background: 5-fluorouracil (5-FU) is an antifolate chemotherapeutic that has become established in many therapeutic regimes, but sensitivity variations and development of resistance are common problems that limit the efficiency of the treatments. Inter-individual variations to 5-FU outcome have been attributed to different expression profiles of genes related to folate metabolism. Methods: To elucidate the mechanisms of variations to 5-FU outcome, the authors investigated MTHFR, DHFR, TYMS and SLC19A1 folate genes expression for 5-FU response in laryngeal cancer cell line (Hep-2). Concentrations of 10, 50, and 100 ng/mL of 5-FU chemotherapeutic were added separately in Hep-2 cell line for 24 hours at 37 8C. Cell sensibility was evaluated with fluorescein isothiocyanate (FITC) label Bcl-2 by flow cytometry. The real-time quantitative PCR (qPCR) technique was performed for quantification of gene expression using TaqMan 1 Gene Expression Assay. ANOVA and Bonferroni’s post hoc tests were utilized to statistical analysis. Results: The numbers of viable Hep-2 cells with 10, 50, and 100 ng/mL concentrations of 5-FU chemotherapy were 15.87, 28.3 and 68.9%, respectively. Statistical analysis showed significant association between control group and increased expression for TYMS gene in cells treated with 100 ng/ mL/5-FU chemotherapy (P < 0.05). Conclusions: The authors found association between the highest 5-FU dose chemotherapy and increased expression levels for TYMS folate gene in laryngeal cancer cell line. Although these experiments were performed in vitro, the results suggest that genetic factors are thought to play an important role in drug metabolism and may be useful for predicting treatment outcomes. ß 2014 Elsevier Masson SAS. All rights reserved. * Tel.: +55 1732015907; fax: +55 1732015700. E-mail address: analivia_sg@yahoo.com.br (A.L.S. Galbiatti). G Model BIOPHA-3389; No. of Pages 5 Please cite this article in press as: Galbiatti ALS, et al. Gene expression profile of 5-fluorouracil metabolic enzymes in laryngeal cancer cell line: Predictive parameters for response to 5-fluorouracil-based chemotherapy. Biomed Pharmacother (2014), http://dx.doi.org/ 10.1016/j.biopha.2014.03.015 Available online at ScienceDirect www.sciencedirect.com http://dx.doi.org/10.1016/j.biopha.2014.03.015 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.