Establishment and characterization of two 5-fluorouracil-resistant hepatocellular carcinoma cell lines

Abstract

5-Fluorouracil (5-FU) chemotherapy is the first choice treatment for advanced hepatocellular carcinoma (HCC), and resistance is the major obstacle to successful treatment. Recent studies have reported that epithelial-to-mesenchymal transition (EMT) is associated with chemoresistance in cancers. We speculated that EMT and 5-FU metabolism are related to the mechanism of 5-FU resistance. First, two 5-FU-resistant cell lines, HLF-R4 and HLF-R10, were established from the HLF undifferentiated human HCC cell line. Whereas cell growth was similar in the HLF and HLF-R cell lines, HLF-Rs are about 4- and 10-fold more resistant compared with the HLF cells; thus, we named these cell lines HLF-R4 and HLF-R10, respectively. The terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay also showed a dramatically decreased number of apoptotic cells in the HLF-Rs after treatment with 5-FU. We next assessed the characteristics of the HLF, HLF-R4 and HLF-R10 cells. Consistent with our hypothesis, the HLF-Rs had typical morphologic phenotypes of EMT, loss of cell-cell adhesion, spindle-shaped morphology and increased formation of pseudopodia. Real-time quantitative reverse transcriptase polymerase chain reaction data showed downregulated E-cadherin and upregulated Twist-1 and also indicated that EMT changes occurred in the HLF-Rs. We also found decreased ribonucleotide reductase and increased multidrug resistance protein 5 genes in the HLF-R cells. Our results suggested that the metabolism of EMT and 5-FU has important roles in 5-FU chemoresistance in the HLF-R cells, and that the HLF-R cells would be useful in vitro models for understanding the 5-FU-resistant mechanisms in HCC.

Figure 1

Cell growth assay. The experiments were carried out for 168 h, with counting of the number of cells every 24 h. The proliferation rates of the HLF-R4 (●) and HLF-R10 (▴) cells are similar to that of the HLF cells (○). The results are expressed as the means ± standard error of the mean values from three assays.

Figure 2

Chemosensitivity assay. IC₅₀ values of 5-FU for the HLF (○), HLF-R4 (●), and HLF-R10 (▴) cells are 17.92 μM, 69.80 μM and 193.47 μM, respectively. Therefore, the 5-FU resistance of the HLF-R4 cells is 3.9-fold, and that of the HLF-R10 cells is 10.8-fold greater than that of the HLF cells. The results are expressed as the means ± standard error of the mean values from three assays.

Figure 3

TUNEL assay. To evaluate the effect of 5-FU on the HLF, HLF-R4, and HLF-R10 cells, a TUNEL assay was used to confirm the apoptotic changes. TUNEL staining shows a dramatic increase in the number of cells that were stained green, which indicates that apoptosis occurred in HLF cells exposed to 5-FU. 5-FU-induced apoptosis is inhibited in the HLF-R4 and HLF-R10 cells. The experiments were repeated three times with similar results. Scale bar = 50 μm.

Figure 4

Cell morphology. 5-FU-resistant cells (HLF-R4 and HLF-R10) are morphologically distinct from their parental cell line, HLF. The resistant cells have loss of cell-cell adhesion, spindle-shaped morphology, and increased formation of pseudopodia (arrow). Scale bar, 20 μm.

Figure 5

mRNA expression analysis. qRT-PCR was performed to investigate mRNA levels of RNR-R1, RNR-R2, MRP5, E-cadherin, and Twist-1. PCR shows up-regulation of MRP5 and Twist-1 and down-regulation of RNR-R1, RNR-R2, and E-cadherin dependent on the level of 5-FU chemoresistance. Data are expressed as the means ± standard error of the mean values from three assays (^*p<0.05 by the Mann-Whitney U test).