Mechanism of trifluorothymidine potentiation of oxaliplatin-induced cytotoxicity to colorectal cancer cells.

Oxaliplatin (OHP) is an anticancer agent that acts by formation of Platinum-DNA (Pt-DNA) adducts resulting in DNA-strand breaks and is used for the treatment of colorectal cancer. The pyrimidine analog trifluorothymidine (TFT) forms together with a thymidine phosphorylase inhibitor (TPI) the anticancer drug formulation TAS-102, in which TPI enhances the bioavailability of TFT in vivo. In this in vitro study the combined cytotoxic effects of OHP with TFT were investigated in human colorectal cancer cells as a model for TAS-102 combinations. In a panel of five colon cancer cell lines (WiDr, H630, Colo320, SNU-C4 and SW1116) we evaluated the OHP-TFT drug combinations using the multiple drug-effect analysis with CalcuSyn software, in which the combination index (CI) indicates synergism (CI<0.9), additivity (CI=0.9-1.1) or antagonism (CI>1.1). Drug target analysis was used for WiDr, H630 and SW1116 to investigate whether there was an increase in Pt-DNA adduct formation, DNA damage induction, cell cycle delay and apoptosis. Trifluorothymidine combined with OHP resulted in synergism for all cell lines (all CI<0.9). This was irrespective of schedule in which either one of the drugs was kept at a constant concentration (using variable drug ratio) or when the two drugs were added in a 1 : 1 IC(50)-based molar ratio. Synergism could be increased for WiDr using sequential drug treatment schedules. Trifluorothymidine increased Pt-DNA adduct formation significantly in H630 and SW1116 (14.4 and 99.1%, respectively; P<0.05). Platinum-DNA adducts were retained best in SW1116 in the presence of TFT. More DNA-strand breaks were induced in SW1116 and the combination increased DNA damage induction (>20%) compared with OHP alone. Exposure to the drugs induced a clear cell-cycle S-phase arrest, but was dose schedule and cell line dependent. Trifluorothymidine (TFT) and OHP both induced apoptosis, which increased significantly for WiDr and SW1116 after TFT-OHP exposure (18.8 and 20.6% respectively; P<0.05). The basal protein levels of ERCC1 DNA repair enzyme were not related to the DNA damage that was induced in the cell lines. In conclusion, the combination of TFT with the DNA synthesis inhibitor OHP induces synergism in colorectal cancer cells, but is dependent on the dose and treatment schedule used.

The Hollow Fibre Assay as a model for in vivo pharmacodynamics of fluoropyrimidines in colon cancer cells.

The Hollow Fibre Assay (HFA) is usually applied as an early in vivo model for anti-cancer drug screening, but is potentially an excellent model for short-term in vivo pharmacodynamic studies. We used the model to study the in vivo role of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) in the cytotoxicity and pharmacodynamics of TAS-102 in colon cancer cells. TAS-102 is a new oral drug formulation, which is composed of trifluorothymidine (TFT) and thymidine phosphorylase inhibitor (TPI), which prevents TFT degradation. We compared the activity with Xeloda (capecitabine), which is activated by TP into 5FU. Hollow fibres filled with human Colo320 or Colo320TP1 colorectal cancer cells with deficient or high TP expression, respectively, were implanted subcutaneously (s.c.) at both flanks of BALB/c mice. The mice were treated orally over 5 days with TAS-102, TFT alone, 5'DFUR+/-TPI or capecitabine at their maximum tolerated dose (MTD). The cells were retrieved from the fibres and assayed for growth (MTT assay), cell cycle distribution (flow cytometry) and apoptosis induction (FragEL method). TAS-102 induced considerable growth inhibition (50%, P<0.01) to both cell lines, which was completely abolished in the absence of TPI. Capecitabine and its metabolite 5'DFUR reduced proliferation of Colo320TP1 cells in the fibres significantly (down to 25-40%), but much less in Colo320 cells, whereas addition of TPI reduced the effect of 5'DFUR, although not completely. These differences in cytotoxic effects were reflected in the pharmacodynamic evaluation. TAS-102 induced a G2M-phase arrest (from 25 to 40%) and apoptosis (>8-fold), which was more pronounced in Colo320 than in Colo320TP1. Again, omission of TPI neutralised the effect of TAS-102. Similarly, 5'DFUR and capecitabine induced a significant G2M-phase arrest (up to 45%) in the Colo320TP1 cell line, but less pronounced in the parental Colo320. Addition of TPI to 5'DFUR reduced this effect to control levels. Also induction of apoptosis was reduced in the presence of TPI. The data demonstrated that the HFA is excellently suited for studying short-term pharmacodynamic effects of fluoropyrimidines in vivo. TAS-102 is only effective in inducing cytotoxicity when systemic TPI is present, but acts against both low and high TP expressing colon cancer cells, while 5'DFUR needs cellular TP to exert significant activity.

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.

Intracellular thymidylate synthase inhibition by trifluorothymidine in FM3A cells.

UNLABELLED: Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5-Fluorouracil (5FU). TS activity reduced to 9% (0.1 microM TFT) and 58% (1 microM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK-) were far less sensitive to TFT compared to FM3A cells. CONCLUSION: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.