Exploring additional mechanisms of cell injury by fluoropyrimidines in the treatment of cancer: an investigation of novel protide NUC-3373

Abstract

5-Fluorouracil (5-FU) has been the backbone of chemotherapy for solid tumours such as colorectal cancer (CRC) for over 60 years. Despite advances in combinatorial treatments, most patients experience innate or acquired resistance to 5-FU as well as deleterious side effects. NUC-3373 is designed to overcome chemo-resistance by addition of a phosphoramidate moiety to the 5-FU molecule. Currently in Phase I clinical trials, NUC-3373 has a pharmacokinetic profile superior to 5-FU. The primary mode of action of both NUC-3373 and 5-FU is to inhibit thymidylate synthase (TS); resulting in a thymine-depleted state and impaired DNA replication and repair. In view of the current contradicting literature regarding the value of TS as a predictive biomarker for 5-FU, this study sought to elucidate the dynamics of TS in response to NUC-3373. Two CRC cell lines were selected from a panel; one more sensitive (HCT116) and one less sensitive (SW480) to NUC-3373 treatment. TS protein expression and localisation were investigated by Western blot and immunocytochemistry. Endoplasmic reticulum (ER) stress was assessed on a biochemical level in addition to examining ultrastructure by Transmission Electron Microscopy (TEM). Baseline TS was not predictive of response to NUC-3373 in cell lines. NUC-3373 treatment resulted in the formation of TS ternary complexes, a net increase in TS and a nuclear to cytoplasmic translocation of TS. NUC-3373 induced ER stress, detected by an upregulation of BiP protein and CHOP gene expression. Stereological assessment of TEM images found that, in response to NUC-3373, HCT116 cells undergo dilation of ER whereas in SW480 cells it proliferates. These studies propose an additional mechanism of action for fluoropyrimidines that is independent of DNA damage. The ability of a cell to overcome ER stress may be a determinant of its resistance to fluoropyrimidines and a potential target for combination therapies.