Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

Abstract

5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116RF10. HCT116RF10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116RF10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116RF10 cells. Interestingly, HCT116RF10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.

Trapping of 5-Fluorodeoxyuridine Monophosphate by Thymidylate Synthase Confers Resistance to 5-Fluorouracil

“…Recently, we established a 5-FU-resistant cell line, HCT116R F10 cells, from parental human CRC HCT116 cells and analyzed the resistance mechanisms of 5-FU . In previous findings, HCT116R F10 cells were weakly sensitive to SN-38, the active metabolite of irinotecan, and cisplatin compared with the parental HCT116 cells . The sensitivity of SN-38 and cisplatin was 1.4-fold (EC 50 = 3 nM in HCT116R F10 cells; 4.2 nM in HCT116 cells) and 1.2-fold (EC 50 = 4.5 μM in HCT116R F10 cells; 5.2 μM in HCT116 cells) higher in HCT116R F10 cells than in parental HCT116 cells, respectively .…”

“…The concentration that confers 50% efficacy (EC 50 ) of 5-FU in the 5-FU-resistant HCT116R F10 and parental HCT116 cells in the colony formation and WST-8 assays is shown in Table and Figures A,B. We recently hypothesized that 5-FU-resistant CRC cells have upregulated TYMS expression and use a fraction of their TS to trap FdUMP, resulting in 5-FU resistance . Indeed, the protein levels of free-TS, FdUMP-TS-CH2-THF covalent complex, and total TS were significantly higher in HCT116R F10 cells than in HCT116 cells under the passage culture conditions (Figures C,D).…”

“…Recently, we established a 5-FU-resistant cell line, HCT116R F10 cells, from parental human CRC HCT116 cells and analyzed the resistance mechanisms of 5-FU . In previous findings, HCT116R F10 cells were weakly sensitive to SN-38, the active metabolite of irinotecan, and cisplatin compared with the parental HCT116 cells .…”

“…Cancer cells are known to acquire resistance to anticancer drugs through a variety of mechanisms. The common cancer resistance mechanisms include inactivation of drugs, enhancement of drug efflux, alteration of drug target molecules, utilization of bypass pathways, facilitation of DNA damage repair, and escaping cell death. ,, Many studies have examined the mechanisms of resistance to 5-FU and its derivatives. ,, The function and/or expression of TS and other enzymes related to the 5-FU anabolism or catabolism pathways are often altered, accelerating resistance to 5-FU. ,,− In addition, the known mechanisms of 5-FU resistance are perturbance of cell death and autophagy, altered epigenetic repression, and expression/functional changes in drug transporters and noncoding RNA (i.e., microRNA and long noncoding RNA). ,, It is widely considered that TS is part of an important molecular mechanism that enhances 5-FU sensitivity and that targeting TS is an excellent strategy to reverse 5-FU resistance. ,, Indeed, numerous studies have shown that the gene amplification of TYMS , leading to mRNA and protein overexpression is a major mechanism of resistance to 5-FU and its derivatives. − In addition, we have shown that 5-FU-resistant CRC cells increase TYMS expression relative to 5-FU-sensitive CRC cells and use a fraction of TS to trap FdUMP, which results in resistance to 5-FU and its derivatives . We predict that the regulation of TS status, which refers to the balance between the active free-TS form and the inactive FdUMP-TS covalent complex, may confer 5-FU resistance …”

Microsatellite Status and IκBα Expression Levels Predict Sensitivity to Pharmaceutical Curcumin in Colorectal Cancer Cells

“…As various CRC cell lines differ in their response to treatments due to genomic and phenotypic alterations and molecular mechanisms [ 18 , 19 , 20 , 21 , 22 ], we hypothesized that curcumin sensitivity might also be linked to specific molecular features such as mutations or activated molecular signaling pathways.…”