Abstract
Background: Acquired chemoresistance to 5-fluorouracil (5-FU) remains one of the obstacles for the success of 5-FU-based cancer chemotherapy, and some molecular mechanisms of acquired 5-FU resistance are still unknown. The main action of 5-FU is the suppression of DNA replication by inhibiting Thymidylate Synthase (TS). Materials and Methods: We analyzed 5-FU resistance mechanisms using the head and neck squamous cell carcinoma cell lines, UM-SCC-23, and two different resistant cell lines, UM-SCC-23/WR and UM-SCC-23/MR, which were procured from UM-SCC-23 cells. To acquire resistantance, the two cells underwent repeated treatment of 5-FU with different durations and frequency. We determined differences in the cell-cycle distribution and the expression of TS proteins in the three cell lines. Moreover, cell-cycle distribution in cells wich acquired resistance after 5-FU treatment, was compared to that of parental cells, using flow cytometric analysis. Results: There was a remarkable increase in TS protein expression levels in UM-SCC-23/WR following 5-FU treatment. S-phase cells of UM-SCC-23 and UM-SCC-23/WR cells were immediately increased after treatment with 5-FU, whereas UM-SCC-23/MR were accumulated to the S-phase slightly later. Conclusion: The cell-cycle perturbation or elevation of TS protein expression may be involved in acquired 5-FU resistance and identifies 5-FU resistance mechanisms in the two different 5-FU treatment regimens.
Head and neck squamous cell carcinoma (HNSCC) is a major health problem, and much effort is being made at the different locations of its presentation. The combination of cisplatin and 5-fluorouracil (5-FU) regimen has been acknowledged as being the gold standard of induction treatment for three decades. Great progress has been made in locoregionally-advanced inoperable disease, mainly with the optimal combination of chemoradiotherapy in the induction-phase of the treatment.
5-FU is an analogue of uracil, and rapidly enters cells using the same facilitated transport mechanism as uracil. After entering the cell a depletion of dTMP resulting in a subsequent depletion of dTTP, which then induces perturbations in the levels of the other deoxynucleotide levels (dATP, dGTP and dCTP) through various feedback mechanisms. Deoxynucleotide pool imbalances (in particular, the dATP/dTTP ratio) may also inhibit DNA synthesis and repair.
We reported that a prolonged 5-FU treatment forced a slower cell-cycle progression. In particular, S-phase progression slowed-down significantly after 5-FU treatment in head and neck carcinoma cells. Whereas, in previous studies, TS expression and polymorphisms are associated with 5-FU chemo-sensitivity or –resistance. (1) We hypothesized that cell-cycle change, as a result of 5-FU treatment and the variation of TS has a corellation with 5-FU resistance.
In the present study, we isolated resistant cells using two different 5-FU exposure protocols; the first was a 1-day treatment with high concentration (1d-high) schedule and the other one was a 5-day treatment with low concentration (5d-low) schedule. These 5-FU treatment protocols were based on clinical administration in HNSCC. Then, we investigated whether TS expression and change of cell-cycle progression are associated with 5-FU resistance. Our results indicated potential solution to 5-FU resistance in HNSCC chemotherapy.
Schedule of isolation of 5-FU-resistant cell line.
Materials and Methods
Cell lines and maintenance. The HNSCC cell lines UM-SCC-23 were maintained in Dulbecco's modified Eagle's medium (DMEM: Sigma, St.Louis, MO, USA) with 10% fetal bovine serum (FBS: Life Technologies, Rockville, MD, USA) at 37°C and 5% CO2. UM-SCC-23 cells were kindly provided by Dr.Thomas Carey of the Department of Otolaryngology/Head and Neck Surgery at the University of Michigan (Ann Arbor, Michigan, USA).
Isolation of 5-FU-resistant cells. The UM-SCC-23 cells were inoculated into a 10-cm dish and cultured for 24 h in DMEM with 10% FBS. Cells were then treated with 5-FU (Kyowa-hakkou, Tokyo, Japan) according to the following two schedules (Figure 1). In the 5-day treatment with low concentration (5d-low) schedule, cells were treated with 5-FU for 120 h and the concentration was increased from 1.0 μg/ml to 64 μg/ml every one month. In the 1-day treatment with high-concentration (1d-high) schedule, cells were treated with 5-FU during 24 h and the concentration was increased from 1.0 μg/ml to 80 μg/ml every one week. In both schedule, cells were harvested and replated into a 10-cm dish and repeat these schedules.
Clonogenic assay. The appropriate number of cells were inoculated into a 6-cm dish, and then treated with each concentration of 5-FU with a 1d-high schedule treatment (24 h) or a 5d-low schedule treatment (120 h). Next, the cells were washed twice with PBS and the culture medium was exchanged for a fresh one. 7-14 days after inoculation colonies were stained with 0.05% crystal violet. Colonies of 50 cells or more were scored as originating from a single clonogenic cell.
Synchronisation of HNSCC cells in the G0/G1 phase and BrdU cell cycle analysis. Synchronisation of HNSCC cells in the G0/G1 phase was achieved by maintaining UM-SCC-23, UM-SCC-23M/R and UM-SCC-23W/R cells for 7 days in 1% FBS medium. Under these conditions, more than 90 % of cells were in the G0/G1 phase. The next stage was a media exchange of 10% FBS medium with 5-FU. The cell-cycle distribution of each cell after 5-FU treatment was described previously. As a short summary, the cell-cycle distribution was determined by a two-parameter flow cytometrical analysis: PI (DNA content) and BrdU uptake. After 5-FU treatment for various durations, cells were labeled with BrdU and harvested. Then, nuclei were isolated and treated with 2 N HCl. Nuclei in 50 ml of PBS-TB were treated with anti-BrdU antibody (Becton Dickinson, San Jose, CA, USA) followed by Alexa fluor 488 goat antimouse antibody (Molecular Probes, Eugene, OR, USA). Nuclei were counterstained with PI, filtered through Cell-Strainer (Becton Dickinson) and then analyzed with a FACS Caliber™ cytometer using ModFit LT™ software after various 5-FU treatment durations.
Western blot analyses. For western blot analysis, cell lysates (30 μg) were solubilized in Laemmli sample buffer by boiling and were then subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The following primary antibodies were used: TS (Thymidylate Synthase; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and Actin (Santa Cruz Biotechnology, Inc.).
Results
Establishment of 5-FU-resistant cells. We isolated the 5-FU-resistant cells by 2 different treatment schedules from UM-SCC-23. The resistant cells from the 5d-low schedule treatment, the cells were named UM-SCC-23/MR cells, and the cells from the 1d-high schedule treatment were named UM-SCC-23/WR. The 5-FU resistance of these cells was confirmed using a clonogenic assay. The results are shown in Figure 2. UM-SCC-23/MR cells were 1.6-times more resistant to 5-FU than UM-SCC-23 cells under the 5d-low schedule. UM-SCC-23/WR cells were 2.0-times more resistant to 5-FU than UM-SCC-23 cells under the 1d-low schedule.
Sensitivities of HNSCC parental cells and two kinds of resistant cells to 5-FU treatment. Treatment durations of a 1-day treatment with high concentration (1d-high) (A) and a 5-day treatment with low concentration (5d-low) (B) schedule are 24 h and 120 h respectively. The UM-SCC-23M/R and UM-SCC-23W/R cells have acquired resistantance through continuous and bolus regimens. Each data point is the mean of three independent experiments. The vertical bars show standard deviations.
Cell-cycle status of 5-FU-resistant and -sensitive cell lines. To ascertain the 5-FU resistance mechanism, we analyzed the cell-cycle distribution after 5-FU treatment of both cells. The 5-FU concentration was fixed at 1.0 μg/ml for each cell. At the indicated time, BrdU was incorporated and flow cytometry was analyzed. S-phase cells were increased at 24 h after treatment with 5-FU in UM-SCC-23 and UM-SCC-23/WR cells, whereas UM-SCC-23/MR cells were accumulated at the S-phase at 48 h. The sub-G1 phase cells increased from 72 h to 120 h in UM-SCC-23/WR, compared to the UM-SCC-23 and UM-SCC-23/MR cells (Figure 3).
Expression of TS proteins in resistant and sensitive cell lines. The expression levels of TS proteins were detected after 5-FU treatment using western blotting analysis. TS protein expression levels of UM-SCC-23 were slightly increased after 5-FU treatment. TS protein expression increased after the treatment of 5-FU in UM-SCC-23/MR from 48 h after exposure to 96 h. There was a remarkable increase in the TS protein expression levels of UM-SCC-23/WR (Figure 4).
Discussion
Several factors of resistance to fluoropyrimidine-based chemotherapy have been demonstrated by clinical and cellular experiments during recent years (1-10). The analysis of TS expression levels or cell-cycle interaction to fluoropyrimidine resistantence is the most common approach (1-5). In previous studies, different responses were reported between bolus and continuous treatment regimens of 5-FU in vitro in gastric cancer cell lines (9). To continue this research and confirm their validity, we established two different 5-FU-resistant cell lines with 5-FU treatment by a 5-day treatment with low concentration (5d-low) schedule or a 1-day treatment with high concentration (1d-high) schedule (UM-SCC-23/MR, WR), and confirmed 5-FU resistantence by clonogenic assay in UM-SCC-23/WR cells with the 1d-high schedule, and in UM-SCC-23/MR cells with the 5d-low treatment schedule.
5-FU is an anticancer drug mainly targeting S-phase cells, and the metabolites of 5-FU can only be incorporated into the DNA and RNA of cycling cells. In addition, TS inhibition, inducing deoxynucleotide inbalance can only damage cells undergoing DNA synthesis. Thus, attenuation of cell cycling may protect resistant cancer cells from 5-FU cytotoxicity. Furthermore, prolonged G1 and S phase may also provide cancer cells with more time-to-DNA damage repair induced by 5-FU. Hence, forcing resistant cancer cells into cell cycle might be another potential strategy to reverse 5-FU chemo-resistance. As stated above, early and middle S-phase cells were increased after 5-FU treatment. These results indicate that 5-FU inhibited DNA synthesis after 5-FU treatment. The cell-cycle distribution of these 5-FU-resistant cells and parental UM-SCC-23 cells were determined by flow cytometrical analysis in this study. The cell-cycle distribution of these cell lines were determined with 5-FU treatments. In UM-SCC-23 cells and UM-SCC-23/WR cells, G1-S progression was delayed by 5-FU treatment but not in the UM-SCC-23/MR cells. According to this result, the resistant cells established by the 5d-low schedule treatment of 5-FU did not show delayed G1-S progression after 5-FU treatment. This result indicated that the resistance due to the 5d-low schedule treatment of 5-FU did not cause inhibition of progression into the S phase as a response after 5-FU treatment, nor have a tolerance to DNA synthesis errors in the S phase.
Cell-cycle distribution of HNSCC parental cells and resistant cells following 5-FU treatment at various durations. The cells were synchronized to G0/G1 phase before 5-FU treatment.
Expression levels of TS of HNSCC parental cells and resistant cells following 5-FU treatment for various durations.
Moreover, TS inhibition by 5-FU treatment causes dNTP imbalance in the nucleotide pool of the cells and then inhibits DNA synthesis. Many reports found that high TS expression correlated with poor prognosis and resistance to fluorouracil based chemotherapy (2-4). In particular, 5-FU-resistant cells showed high TS expression. In our study, TS protein expression was analyzed following various durations of 5-FU treatment. In UM-SCC-23/WR cells, TS expression was increased rapidly, but was not induced in UM-SCC-23 cells and 23/MR cells. These result indicate that the resistance of the 1d-high schedule treatment to 5-FU showed a high TS expression as a response to 5-FU treatment, and did not cause dNTP imbalance or inhibit DNA synthesis. In our previous study, we treated edotolac which is a selective COX-2 inhibitor to 5-FU-resistant cell line. TS expression of these cells was lowered after 5-FU treatment, and, the 5-FU sensitivity of these cells were reversed to the same level of 5-FU sensitive cells.(10) In fact, the mechanism of the sensitization of 5-FU by TS inhibition depended on the resistance by bolus 5-FU treatment.
Resistance mechanisms classified according to bolus and continuous treatments.
We confirmed two resistance responses after the 1d-high and 5d-low 5-FU schedule treatment. The resistant cells with the 1d-high treatment showed that TS expression was induced after 5-FU treatment and the resistance of TS inhibition depended on each resistant cell. In addition, the resistant cells with the 5d-low treatment showed G1/S progression was not delayed after 5-FU treatment. It depended on the resistance to the inhibition of dNTP imbalance in the nucleotide pool of the cells and DNA Synthesis (Figure 5).
Conclusion
In the present study, 5-FU resistance mechanisms were classified according to the 1d-high and 5d-low treatment of 5-FU. Many results showed a higher expression of TS induced the resistance to 5-FU, allowing us to confirm the 1d-high schedule treatment resistance. However, the mechanism of the 5d-low schedule treatment of 5-FU resistance, that showed G1/S progression was not delayed after 5-FU treatment, and the reason why remains unclear. In future experiments, we will analyze sub-G1 cells sorted from UM-SCC-23/MR cells after 5-FU treatment, and attempt to determine their sensitivity to 5-FU.
Footnotes
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Conflicts of Interest
The Authors have no conflicts of interest directly relevant to the content of this article.
- Received February 15, 2014.
- Revision received March 29, 2014.
- Accepted April 1, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
