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

Abstract

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 (CIo0.9), additivity (CI ¼ 0.9 -1.1) or antagonism (CI41.1). 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 CIo0.9). 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; Po0.05). 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 (420%) 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; Po0.05). 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.

Therapeutic potential of TAS-102 in the treatment of gastrointestinal malignancies

“…The sensitivity of colon cancer cells to oxaliplatin could be increased by simultaneous exposure to TFT [Temmink et al 2007d], resulting in increased Pt-DNA-adduct formation, and subsequent increased DNA damage induction and apoptosis induction. The TFT-oxaliplatin combination was dose-schedule dependent, since TFT pre-incubation decreased oxaliplatin-induced cytotoxicity to colorectal cancer cells.…”

“…In in vitro studies potential TAS-102 combinations were extensively investigated by combinations with other anticancer agents (Table 1). TFT was combined with other TS inhibitors [Temmink et al 2006b], oxaliplatin [Temmink et al 2007d], or irinotecan (CPT-11) [Temmink et al 2007c], in different schedules in order to determine their interactions. At low folate conditions TFT and folate-based TS inhibitors (e.g.…”

[18F]FDG and [18F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

“…Lysates were analysed with 12% SDS -PAGE followed by blotting onto a polyvinylidene difluoride membrane. Thymidine kinase-1 was detected by overnight incubation with 1 : 150 dilution of an anti-TK-1 monoclonal antibody (QED Bioscience, San Diego, CA, USA) followed by 60 min incubation with a 1 : 2000 dilution of secondary antibody (horseradish peroxidase-conjugated rabbit anti-mouse antibody, DakoCytomation, Glostrup, Denmark) as described previously (Temmink et al, 2007). b-actin was used to control for loading.…”

“…For cell cycle analysis, cells were exposed to IC 50 concentrations of the various drugs for 4, 24, 48 or 72 h, fixed in 70% ethanol (1 Â 10 6 cells per ml) and stored at 41C until analysis, as described previously (Temmink et al, 2007) according to a slightly modified protocol.…”

TAS-102: A Novel Antimetabolite for the 21st Century

“…TFT preincubation of colorectal cancer cell lines enhanced cytotoxicity induced by SN38, the active metabolite of irinotecan [52]. The combination of TFT with the third-generation platinum agent, oxaliplatin, resulted in a dose-and scheduledependent synergism between these drugs as well [53]. TFT was also synergistic with erlotinib in EGFR expressing colon cancer cells, even in the presence of K-Ras mutation [54].…”