Investigating the involvement of the enzymes MTHFR, and TP in HCT116 colorectal cancer cells resistance to 5-fluorouracil

Abstract

Colorectal cancer (CRC) ranks 3rd in occurrence among men and women worldwide. It is particularly predominant in Western countries. This cancer has been treated for decades with 5-fluorouracil (5FU). However, patients develop resistance to 5FU which represents a limitation to the use of this drug in the clinic. To overcome this resistance, a better understanding of the mechanism of action of 5FU is needed and more importantly a clear elucidation of the resistance to this drug. In order to exert its cytotoxic effects against cancer, 5-FU must first be metabolized to the nucleotide level. Fluoropyrimidine nucleotides exert their cytotoxic effects through different mechanisms. The most important anti-tumor effect of 5-FU can be ascribed to the inhibition of thymidylate synthase (TS) by FdUMP. TS is a crucial enzyme for the de novo synthesis of thymidylate (dTMP) which is needed for DNA synthesis. On another hand, Methylene-tetrahydrofolate reductase (MTHFR) is a key regulatory enzyme involved in the conversion of CH2HF4 which is needed to bind with 5FU to disrupt DNA synthesis by forming a ternary complex. Another pathway of 5FU action is through thymidine phosphorylase (TP) that converts 5FU to FdUMP which is essential to inhibit TS. In this study, we used the HCT116 parental CRC cell line and its 5FU-resistant derivative HCT116-FUR and we compared the effect of 5-FU on their cell growth. Inhibition of proliferation by 5-FU was demonstrated using the trypan blue exclusion assay and the MTT assay. On a molecular level, 5-FU had no effect on protein expression level of both TP, and MTHFR which are 2 key enzymes involved in its metabolism. Similarly, we examined the clinical significance of the MTHFR gene polymorphism C667T and showed that the genotype CT was not associated with resistance to the drug. Our study highlights that key enzymes TP, and MTHFR are not involved in the mechanism of resistance of 5-FU, and that further investigations and newer target protein are needed to understand its mechanism.