Epithelial-mesenchymal Transition is Associated with Acquired Resistance to 5-Fluorocuracil in HT-29 Colon Cancer Cells.

5-Fluorouracil (5-FU) is commonly used for the therapy of colon cancer; however, acquired resistance to 5-FU is a critical barrier to successful treatment and the primary cause of chemotherapy failure. Epithelialmesenchymal transition (EMT) is a process whereby cells undergo alterations in morphology and molecular characteristics promoting tumor progression and metastasis. Accumulating evidence shows that transition from epithelial to mesenchymal phenotype in cancer cells is associated with their resistance to chemotherapy. However, it is still poorly understood whether EMT is involved in acquired resistance to 5-FU. In this study, we developed an in vitro cell model, 5-FU-resistant HT-29 colon cancer cells, and characterized the differences in cellular morphology and molecular alterations between parental and resistant cells. In accord with mesenchymal-like morphology of 5-FU-resistant HT-29 cells, the expression of the mesenchymal marker fibronectin was significantly increased in these cells in comparision with that in the parental cells. Of interest, we also found a marked increase in the expression of EMT-inducing transcription factors Twist, Zeb1, and Zeb2. Finally, 5-FU-resistant cells showed enhanced migration in comparison with parental HT-29. Taken together, these results indicate that EMT could be associated with 5-FU resistance acquired by HT-29 cells. A specific role of each transcription factor found in this study will require further investigation.

INTRODUCTION

Colon cancer has a high incidence and is one of the leading causes of cancer-related death in the elderly (1). As a first-line chemotherapeutic agent for colon cancer, 5-fluorouracil (5-FU) is used to increase the probability of survival (2). The structure of 5-FU is similar to those of pyrimidines in DNA and RNA; 5-FU inhibits thymidylate synthase and is incorporated into RNA and DNA, resulting in cytotoxicity and cell death (3). Although the understanding of the mechanism of 5-FU action has greatly increased in recent years, the development of drug-resistant phenotypes remains a critical limitation to its clinical use (4). The molecular mechanisms of therapeutic resistance to 5-FU are poorly understood, and investigation of molecular pathways associated with chemo-resistance could lead to novel strategies for the treatment of patients with 5-FU-resistant colon cancer.

Epithelial-mesenchymal transition (EMT) is a multi-step morphogenetic process in which epithelial cells downregulate their epithelial properties and acquire mesenchymal characteristics, leading to remodeling of the cytoskeleton and enhancement of migration potential, which is a functional hallmark of EMT (5). The EMT phenotype is characterized by the loss of cell-to-cell adhesion (6,7). Another hallmark of EMT is the loss of the epithelial adhesion molecule E-cadherin and gain of mesenchymal markers such as vimentin and/or fibronectin. Suppression of E-cadherin expression is mediated by several genetic factors or direct promoter repression mediated by Snail, Slug, Twist, Zeb1, and Zeb2 (6-9), all of which interact with E-box elements located within the proximal region of the E-cadherin promoter (6). Remarkably, the signaling pathway that involves these EMT-inducing transcription factors overlaps significantly with each other. Specifically, Snail regulates the expression of Zeb factors and also increases the stability of Twist, followed by activation of transcription of the Slug gene. Thus, it is suggested that EMT-inducing transcription factors cooperate with each other and their specificity might affect spatial and temporal regulation of EMT.

Accumulating evidence suggests that EMT is associated with acquired resistance to cancer therapy as well as cancer metastasis (10). Recently, an EMT phenotype has been identified in a number of cancer cells, including gefitinibresistant non-small cell lung cancer cells (NSCLC) (11), paclitaxel-resistant ovarian cancer cells (12), and tamoxifen-resistant breast cancer cells (13). Furthermore, cancer cells surviving in breast cancer patients after conventional therapy possess mesenchymal characteristics and tumor-initiating features (14). In agreement with these findings, restoration of the epithelial phenotype increases sensitivity of tumor cells to chemotherapy (14). Taken together, these studies suggest that EMT not only increases metastasis, but is also involved in drug resistance in cancer cells.

During the establishment of 5-FU-resistant cells, we found that cells with the 5-FU-resistant phenotype showed morphological changes in comparison with parental HT-29 cells. The present study focused on the alterations in the expression levels of the genes related to EMT.

MATERIALS AND METHODS

5-Fluorouracil (5-FU) and crystal violet were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-E-cadherin antibodies were obtained from Cell signaling (Danver, MA, USA). Anti-Fibronectin and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase conjugated antibodies against mouse or rabbit IgG were obtained from Thermo scientific (Waltham, MA, USA).

HT-29 parental cell line was obtained from Korea Research Institute of Bioscience and Biotechnology (KRIBB, Cheongwon, Chungbuk, Korea). HT-29 parental cell line was cultured in McCoy’s 5A Medium (Hyclone, Logan, Utah, USA). It was grown in media supplemented with 10% Fetal Bovine Serum (FBS) and 100 U/mL penicillin and 100 μg/mL streptomycin (GenDEPOT, Barker, TX, USA) at 37℃ in a humidified incubator with 5% CO2. 5-FU resistant phenotype was established by repeated exposure to 5-FU.

Cell viability was assessed by EZCyTox (Daeil Lab Service, Seoul, Korea). 0.5 × 104 cells were plated in 96-well plate. After 24 h, cells were treated with indicated concentrations of 5-FU. EZ-CyTox solution was added to each well and it was measured at a 450 nm. The results were expressed as a percentage compared to the vehicle treated cells.

Total RNA was purified from cells using the NucleoSpin® RNA (MACHEREY-NAGEL, Dueren, Germany). cDNA synthesis was performed with iScriptTM cDNA Synthesis system (Bio-Rad, Hercules, CA, USA), followed by PCR using GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA). PCR products were analyzed by gel electrophoresis on a 1% agarose (w/v) gel. The sequences of human specific primers for E-cadherin, fibronectin, Twist, Zeb1, Zeb2, and 18S are provided in Table 1.

Table 1.

List of human primers used for RT-PCR

Primer	Forward	Reverse
E-cadherin	5'-TCCATTTCTTGGTCTACGCC-3'	5'-CACCTTCAGCCATCCTGTTT-3'
Fibronectin	5'-TCGAGGAGGAAATTCCAATG-3'	5'-CTCTTCATGACGCTTGTGGA-3'
Twist	5'-GGAGTCCGCAGTCTTACGAG-3'	5'-TCTGGAGGACCTGGTAGAGG-3'
Zeb1	5'-GCACAACCAAGTGCAGAAGA-3'	5'-GAACCATTGGTGGTTGATCC-3'
Zeb2	5'-CAACTCCGATGAACTGCTGA-3'	5'-AGCCTGAGAGGAGGATCACA-3'
18S	5'-CAGCCACCCGAGATTGAGCA-3'	5'-TAGTAGCGACGGGCGGTGTG-3'

Cells were lysed with icecold PRO-PREPTM protein extract solution (iNtRON, Sungnam, Gyunggi, Korea) and protein concentration was quantified using the BCA procedure (Thermo scientific, Waltham, MA, USA). Equal amounts of protein samples were separated by SDS-PAGE using 8% or 10% polyacrylamide gel and then transferred onto a polyvinylidene difuoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.07% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with TBST containing 5% milk and the primary antibodies. After three washes with TBST, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The antigen was detected using an Western Bright ECL HRP substrate kit (Advansta, Menlo Park, CA, USA).

24-Transwell migration chamber (Corning Life Sciences, Corning, NY, USA) was used to accomplish cell migration assay. Cells were placed in the upper compartment of the migration chamber and incubated for 24 h at 37℃. The migrating cells at the bottom side of the membranes stained with crystal violet.

All results, expressed as the mean ± SD, were analyzed by a two-tailed Student’s t-test. The acceptable level of significance was established at P < 0.05.

RESULTS

To determine the 5-FU sensitivity in our drug-resistant model, cell viability assay was performed following 5-FU treatment. As we expected, cells with the 5-FU-resistant phenotype had significantly increased viability compared with parental HT-29 cells (Fig. 1A). Phase-contrast microscopy revealed a marked alteration of cellular morphology in 5-FU-resistant HT-29 cells (Fig. 1B). Whereas parental cells maintained epithelial morphology, 5-FU-resistant cells exhibited spindle shape, intercellular spaces, and scattering, suggesting loose cell-cell interaction in comparison with parental HT-29 cells.

Fig. 1.

Acquired 5-FU resistant HT-29 cells show morphological changes compared with parental HT-29 cells. (A) Cell viability was assessed after treatment with increasing concentrations of 5-FU. (B) 5-FU resistant HT-29 cells show significant alteration in cellular morphology (200× magnification

The observed morphological changes implied that the 5-FU-resistant cells had transitioned to a mesenchymal phenotype. To determine whether these morphological changes were associated with EMT, we examined the expression of epithelial and mesenchymal marker proteins. Consistent with morphological changes, 5-FU-resistant cells showed down-regulation of the epithelial marker E-cadherin and up-regulation of the mesenchymal marker fibronectin (Fig. 2). These results suggest that EMT could be associated with acquired drug resistance after long-term exposure to 5-FU.

Fig. 2.

5-FU resistant phenotype exhibits an EMT-like cellular characteristics compared with parental HT-29 cells. (A) Protein expression of E-cadherin and fibronection were determined by western blotting. (B) Density of each blot was quantified using Image J. Each value represents the mean ± SD of three independent experiments. *, **Significantly different from the parental HT-29 cells at p < 0.05, 0.01, respectively (Student’s t-test).

To confirm EMT in 5-FU-resistant cells, we performed RT-PCR analysis of EMT markers and EMT-inducing transcription factors. As shown in Fig. 3A, the level of the E-cadherin transcript was reduced in 5-FU-resistant cells. In contrast, the expression level of fibronectin was dramatically higher in 5-FU-resistant cells than in the parental cells (Fig. 3A). Consistent with the expression of this EMT marker, the expression of the EMT-inducing transcription factors Twist, Zeb1, and Zeb2 was also significantly increased in 5-FU-resistant cells (Fig. 3B).

Fig. 3.

5-FU resistant HT-29 cells induce mesenchymal marker accompanied by EMT-inducing transcription factors. RNA expression of (A) EMT markers such as E-cadherin and fibronectin, and (B) EMT-inducing transcription factors Twist, Zeb1, and Zeb2 were analyzed using RT-PCR. Each value represents the mean ± SD of three independent experiments. *, ***Significantly different from the parental HT-29 cells at p < 0.05, 0.001, respectively (Student’s t-test).

Epithelial cells undergoing EMT lose their adhesive phenotype and concomitantly acquire higher migratory ability (5). We observed that 5-FU-resistant cells were more motile than parental HT-29 cells, as assessed by a transwell migration assay (Fig. 4). These results confirm that acquisition of the 5-FU-resistant phenotype involves EMT, resulting in higher motile capability, which might be critical for tumor progression.

Fig. 4.

5-FU resistant HT-29 cells show higher motility than parental HT-29 cells in transwell migration assay. (A) Cell migration was assessed using 24-transwell migration chamber for 48 hr and cells at the bottom side of the membranes were stained with crystal violet. (B) Migrating cells were counted and plotted as mean ± SD of three independent experiments. ***Significantly different from the parental HT-29 cells at p < 0.001 (Student’s t-test).

DISCUSSION

5-FU is used frequently in chemotherapy for many kinds of solid tumors. However, patients who initially respond to 5-FU ultimately become resistant (15,16). In this study, we have established a 5-FU-resistant cell line derived from parental HT-29 colon cancer cells and characterized the phenotype of 5-FU-resistant cells in terms of EMT. Importantly, we found that the expression of the EMT-promoting transcription factors Twist, Zeb1, and Zeb2, which repress E-cadherin transcription, correlates significantly with the 5-FU-resistant phenotype in HT-29 colon cancer cells.

Development of drug resistance has been a major barrier to successful treatment and the primary cause of chemotherapy failure. Multiple mechanisms responsible for the drugresistant phenotype in cancer cells have been recognized and recent evidence points to the role of EMT. The link between EMT and chemotherapy resistance has been investigated in NSCLC. Interestingly, the sensitivity of NSCLC to EGFR kinase inhibition depends on their EMT phenotype: mesenchymal-like cells are less sensitive to EGFR inhibition than cells with the epithelial phenotype (17). In line with the letter, the expression of EMT-related genes in lung cancer cell lines is related to their resistance to the inhibitors of both EGFR and PI3K/Akt pathways, which has also been confirmed in a small group of patients (18). Furthermore, in a study that used both surgical specimens and cell lines, an EMT-like cellular phenotype was associated with increased resistance to most conventional therapeutic approaches. Of interest, the changes in gene expression during EMT are very similar to the changes in the expression of stem cell markers (19). Notably, residual resistant tumor cells following breast cancer chemotherapy frequently exhibit a stem cell–like phenotype including increased mammosphere formation, which is a characteristic of stem cells (20-22). Recently, the association between EMT-like properties and a stem-cell like phenotype has been reported not only in breast cancer and NSCLC, but also in urinary bladder, head and neck, pancreas, and colorectal carcinoma (23). Thus, the mechanisms of resistance to chemotherapeutic agents might be linked to stem cell– like properties of tumor cells that have undergone EMT via the activation of cellular signaling pathways that are common to both processes, such as TGF-β, Wnt, Notch, and Hedgehog (24).

In summary, cells remaining after long-term exposure to 5-FU showed an EMT phenotype accompanied by acquired drug resistance. Together with other recent studies that suggest the association between EMT and drug resistance, this study might provide a clue for overcoming therapy failure caused by drug resistance. The importance of particular EMT-promoting transcription factors and related signaling pathways in maintaining the EMT phenotype may vary depending on the tissue type (25-28). Therefore, understanding the specific role of each transcription factor found in this study and elucidation of related cellular signaling will require further investigation.