Energy Metabolism and Stemness and the Role of Lauric Acid in Reversing 5-Fluorouracil Resistance in Colorectal Cancer Cells

Abstract

While 5-fluorouracil (5FU) plays a central role in chemotherapy for colorectal cancer (CRC), resistance to 5FU remains a major challenge in CRC treatment, and its underlying mechanisms remain unclear. In this study, we investigated the relationship between 5FU resistance acquisition, stemness, and energy metabolism. Among the two CRC cell lines, HT29 cells exhibited glycolytic and quiescent properties, while CT26 cells relied on oxidative phosphorylation (OXPHOS) for energy. In contrast, the 5FU-resistant sublines (HT29R and CT26R), developed through continuous exposure to low concentrations of 5FU, demonstrated enhanced stemness. This was associated with glycolytic dominance, low proliferation, and reduced reactive oxygen species (ROS) production. However, treatment with the medium-chain fatty acid lauric acid shifted the cells to OXPHOS, reducing stemness, increasing ROS levels, and inducing cell death, therefore reversing 5FU resistance. These findings suggest that an enhancement in stemness and the reprogramming of energy metabolism play key roles in acquiring 5FU resistance in CRC. While lauric acid reversed 5FU resistance, further clinical studies are required.

Keywords: 5-fluorouracil; cancer stemness; colorectal cancer; drug resistance; energy metabolism; oxidative stress.

Figure 1

Differences in proliferation, differentiation, and energy metabolism between two CRC cell lines. HT29 and CT26 cells were cultured in a regular medium. (A) Cell growth. (B) Protein levels during colonocyte-associated differentiation. (C) Mitochondrial stress test results. (D) OXPHOS parameters. (E) Glycolytic stress test results, including the maximum ECAR. (F) Energy metabolism phenotypes. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; ALP, alkaline phosphatase; MUC2, mucin 2; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; Max, maximum; OXPHOS, oxidative phosphorylation; ANOVA, analysis of variance.

Figure 2

Differences in oxidative stress between two CRC cell lines. HT29 and CT26 cells were cultured in a regular medium. (A) Assessment of MtVol with mitogen. (B) MMP assessed using TMRE. (C) H₂O₂ levels assessed using DHR123. (D) MtSOX. (E) 4HNE. Scale bar: 50 μm. Right panel: semi-quantification of fluorescence images. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; MtVol, mitochondrial volume; MMP, mitochondrial membrane potential; TMRE, tetramethylrhodamine ethyl ester; DHR123, dihydrorhodamine 123; mtSOX, mitochondrial superoxide; 4HNE, 4-hydroxynonenal; ANOVA, analysis of variance.

Figure 3

Differences in stemness between two CRC cell lines. HT29 and CT26 cells were cultured in a regular medium. (A) Expression of stemness marker genes LGR5 and NS. Right panel: semi-quantification of RT-PCR signals. (B) Sensitivity to 5FU. (C) Expression of naïve/prime transition-associated genes KLF4, PRODH, LIN28a, and DNMT3B. Right panel: semi-quantification of RT-PCR signals. (D) Sphere areas. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; NS, nucleostemin; ACTB, β-actin; RT-PCR, reverse transcription polymerase chain reaction; 5FU, 5-fluorouracil; KLF4, Krüppel-like factor 4; PRODH, proline dehydrogenase; DNMT3B, DNA methyltransferase 3B.

Figure 4

Characterization of 5FU-resistant cell lines derived from two CRC cell lines. The HT29R and CT26R cell lines resistant to 5FU were established from HT29 and CT26 cells, respectively, by continuous treatment with low-dose 5FU (IC₅) for 50 passages. (A) 5FU sensitivity, indicated by IC₅₀. (B) Cell growth in regular medium. (C) Expression of 5FU-resistance-related genes TS, DPD, MTHFR, and TYMP. (D–F) OXPOHS parameters: basal OCR (D), maximum OCR (E), and ATP (F). (G) Max ECAR. (H) Energy metabolism phenotype. (I) MtVol assessed using MitoGreen. (J) MMP assessed using TMRE. (K) Mitochondrial H₂O₂ levels were assessed using DHR123. (L) MtSOX. (M) 4HNE. (N–P) HT29R and CT26R treated with 5FU (5 μg/mL for 48 h). H₂O₂ (N), mtSOX (O), 4HNE (P). Error bars: standard deviation of three independent trials Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; 5FU, 5-fluorouracil; IC, inhibitory concentration; TS, thymidylate synthase; DPD, dihydropyrimidine dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; TYMP, thymidine phosphorylase; OXPHOS, oxidative phosphorylation; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; MtVol, mitochondrial volume; MMP, mitochondrial membrane potential; TMRE, tetramethylrhodamine ethyl ester; DHR123, dihydrorhodamine 123; mtSOX, mitochondrial superoxide; 4HNE, 4-hydroxynonenal; ANOVA, analysis of variance.

Figure 5

Stemness of 5FU-resistant cell lines derived from two CRC cell lines. 5FU-resistant HT29R and CT26R cell lines were established from HT29 and CT26 cells, respectively, by continuous treatment with low-dose 5FU (IC₅) for 50 passages. (A) Apoptosis. (B) 5FU (5 μg/mL for 48 h). (C) Mitophagy. Scale bar: 50 μm. Right panel: semi-quantification of fluorescence images. (D) Expression of stemness-associated genes HIF1α and ME1. Right panel: semi-quantification of RT-PCR signals. (E) Sphere areas. (F) Expression of the naïve/prime transition-associated genes PRODH and LIN28a. Right panel: semi-quantification of RT-PCR signals. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; 5FU, 5-fluorouracil; ACTB, β-actin; RT-PCR, reverse transcription polymerase chain reaction; PRODH, proline dehydrogenase; HIF1A, hypoxia-inducible 1α; ME1, cytosolic NADPH dehydrogenase 1 (malic enzyme 1); ANOVA, analysis of variance.

Figure 6

Effect of lauric acid (LAA) on stemness in 5FU-resistant CRC cell lines. The 5FU-resistant cell lines HT29R and CT26R were treated with 5FU (10 μg/mL) with or without LAA (40 μg/mL) for 40 h. (A) Cell growth. (B) 4HNE. (C) Apoptosis. (D) ATP. (E) Lactate levels in the culture medium. (F,G) Protein levels of HIF1α (F) and ME1 (G). (H) Sphere areas. Error bars: standard deviation of three independent trials. Asterisk, p < 0.05. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. CRC, colorectal cancer; 5FU, 5-fluorouracil; LAA, lauric acid; 4HNE, 4-hydroxynonenal; HIF1α, hypoxia inducible 1α; ME1, cytosolic NADPH dehydrogenase 1 (malic enzyme 1); ANOVA, analysis of variance.