Combination studies of Trifluorothymidine with Oxaliplatin in colon cancer cell lines

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Trifluorothymidine (TFT) is part of the formulation TAS-102 which currently undergoes clinical trials and also contains the thymidine phosphorylase inhibitor TPI. TFT is activated by thymidine kinase to TFT-MP which inhibits thymidylate synthase (TS) leading to dTTP depletion in the cell causing dUTP (and also TFT-TP) to be misincorporated into DNA, resulting in DNA damage and possibly cell death. This may enhance the effect of DNA damaging agents, such as oxaliplatin (OHP). We studied the effect of TFT on OHP induced cytotoxicity in colon cancer cells, using growth inhibition experiments in which TFT was combined with OHP in different combination schedules. A panel of 5 colon cancer cell lines (H630, WiDr, Colo320, SNU-C4, SW1116) was used for the experiments. To evaluate the effects multiple drug effect analysis was performed using Calcusyn software and expressed as mutually nonexclusive combination index (CI): synergism, CI<0.9; additivity, 0.9<CI<1.1; antagonism, CI>1.1. For H630, WiDr and SW1116 we also investigated whether there was an increase in DNA damage induction, Pt-DNA adduct formation and/or cell cycle delay when the drugs were combined. The effect of the combinations of TFT-OHP showed synergism in all cell lines when the drugs were combined simultaneously (0.4 < CI < 1.0). This was not enhanced when the cells were preincubated 24 hr with one of the drugs. Most DNA strand breaks were induced after exposure to OHP compared to TFT, and this was most significant in SW1116, the cell line most resistant to TFT (IC₅₀=7.45 μM) compared to H630 or WiDr (IC₅₀<2.1 μM). At least 20% more DNA damage was induced when cells were exposed to [IC₁₀₀] of TFT and OHP combined compared to one of the drugs alone. The formation of Pt-adducts was 1.8-2.6 pmol/ug DNA in these cell lines when exposed to OHP for 24 hr. Pre-incubating 4 hr with 10 μM TFT decreased Pt-adduct formation with 13% or more. In a simultaneous combination with TFT a significant increase in Pt-adduct formation was seen in H630 and SW1116 (13% and 99% respectively; p<0.05), but not in WiDr. Pt-adducts were best retained in SW1116 cells (81.6%) and could be increased when TFT was added (> 10%). Exposure to [IC₇₅] of TFT or OHP for 48 hr induced a clear cell cycle S-phase arrest, although dose-schedule and cell line-dependent. More decrease in G1-population was observed after TFT exposure compared to OHP alone. After exposure to TFT-OHP at their [IC₇₅]s a stronger S-phase arrest was seen, for all cell lines (> 6.2%), but was hardly influenced by a 4 hr TFT pre-incubation period. We currently determine basal protein levels of DNA repair enzymes to be evaluated in relation to DNA damage that was induced in these cell lines. In conclusion, the combination of TFT with the DNA synthesis inhibitor OHP can induce synergistic effects when the drugs are given simultaneously and that DNA damage induction by OHP can be enhanced when combined with TFT but is dependent on the dose-schedule used.