nolatrexed and Colonic-Neoplasms

Trifluorothymidine (TFT) is a fluoropyrimidine that is part of the novel combination metabolite TAS-102, in which TFT is combined with a potent thymidine phosphorylase inhibitor (TPI). TAS-102 is currently tested as an orally chemotherapeutic agent in different schedules in a phase I study. In its monophosphate form, TFT can inhibit thymidylate synthase (TS) activity after binding to the TS-nucleotide binding site leading to dTTP depletion, and in its triphosphate form TFT is incorporated into DNA, eventually leading to DNA damage. In this in vitro study, we investigated whether TFT could potentiate cytotoxicity of the antifolate-based TS inhibitors AG337 (Nolatrexed), ZD1694 (Raltitrexed) and GW1843; and whether increased TS inhibition or DNA damage would be related to this result.. The drug combinations were studied in colon cancer cell lines either grown at low or high folate conditions. Multiple drug effect analysis was performed after measuring growth inhibition when the drugs were combined (MTT Assay) and expressed as Combination Index (CI), where CI<0.9 indicates synergism, CI=0.9-1.1 indicates additivity and CI>1.1 indicates antagonism. Drug target analysis was performed using the TS in situ inhibition assay and the FADU DNA-damage assay. Cells were exposed to either the drugs alone or in combination to determine the effect on TS activity and DNA damage induction, respectively.. Three experimental procedures were used to test the interaction of the drugs: either one of the drugs was kept at a constant concentration (IC25) or two drugs were added in a 1:1 IC50-based molar ratio. The combinations of TFT with one of the antifolates in which one of the drugs was kept at a constant concentration were synergistic for all antifolates in WiDr/F cells, which grow in low folate medium (CI=0.6-0.8), but only additive to antagonistic for the cell lines growing in high folate medium: TFT-AG337: CI=0.9-2.3; TFT-ZD1694: CI=0.9-1.3; TFT-GW1843: CI=0.8-1.7. The procedure in which the two drugs were added in a 1:1 IC50-based molar ratio showed antagonism for all three combinations in all cell lines (CI>2.7). TS inhibition (14.3%) and DNA damage (8%) were more pronounced than expected (P<0.05) when TFT was combined with GW1843 in WiDr/F cells, in contrast to AG337 and ZD1694, which showed inhibiting effects as expected (additive).. The combination of TFT with the antifolates AG337, ZD1694 and GW1843 is mainly additive when the drugs are given simultaneously and this is mediated by an additive TS inhibition and DNA damage. The drug interaction may partly be dependent on the folate homeostasis since WiDr/F cells growing at low folate conditions show pronounced synergism in growth inhibition, two-sided TS inhibition and DNA damage, especially when TFT is combined with the tight-binding TS inhibitor GW1843.

Topics: Antimetabolites; Colonic Neoplasms; DNA Damage; Drug Interactions; Folic Acid; Folic Acid Antagonists; Homeostasis; Humans; Indoles; Isoindoles; Quinazolines; Thiophenes; Trifluridine; Tumor Cells, Cultured

We recently showed an increase in tumour uptake of 2-[(11)C]thymidine in patients with gastrointestinal malignancies after thymidylate synthase (TS) inhibition. To understand the phenomenon in more detail, we investigated whether TS inhibition by different TS inhibitors leads to a dose- and time-dependent change in the uptake of radiolabelled thymidine, and whether radiotracer uptake is related to changes in cell viability resulting from treatment.. RIF-1 and HT29 cells were treated with the TS inhibitors 5-fluorouracil (5-FU) and AG337 (nolatrexed dihydrochloride), as well as cisplatin as control. The cell viability and net accumulation of [(3)H]thymidine after a 1-h pulse was determined at different times after drug treatment.. In both cell lines, [(3)H]thymidine uptake increased after a 2-h treatment with 5-FU, in a dose- and time-dependent manner. [(3)H]thymidine uptake decreased at 24 and 48 h post treatment. AG337 also produced a similar effect. In contrast to the TS inhibitors, cisplatin decreased [(3)H]thymidine uptake in RIF-1 and HT29 cells at all time points. Cell viability was compromised only after 24 h.. Using two types of TS inhibitor, we have shown an increase in [(3)H]thymidine uptake, in a dose-dependent manner, a few hours after TS inhibition when the cell viability was not compromised. This effect was not seen with a non-TS inhibitor. These findings suggest that 2-[(11)C]thymidine positron emission tomography can be used to study TS inhibition in vivo at early time points when cell viability is not compromised and may therefore be helpful in the development of new TS inhibitors and in differentiating between patients with tumours sensitive to TS inhibitors and those unlikely to respond.

Topics: Adenocarcinoma; Biological Transport, Active; Cell Line, Tumor; Cell Survival; Cisplatin; Colonic Neoplasms; Enzyme Inhibitors; Fibrosarcoma; Fluorouracil; HT29 Cells; Humans; Quinazolines; Radionuclide Imaging; Radiopharmaceuticals; Thymidine; Thymidylate Synthase; Time Factors; Tritium

Thymidylate synthase (TS) is an important target for chemotherapy and can be inhibited by 5-fluorouracil (5-FU) and the antifolates, AG337 (Nolatrexed) and multitargeted antifolate (MTA or Pemetrexed). In addition, 5-FU can be incorporated into RNA and DNA, and MTA can inhibit two other enzymes. It is, however, unclear to what extent these differences in drug action will influence activation of downstream mechanisms mediated via TS inhibition. Therefore, two human colon cancer cell lines, WiDr and Lovo, with a different clonogenic origin, were treated with equitoxic concentrations of 5-FU, AG337, and MTA to determine the induction of DNA damage, cell cycle arrest, downstream protein expression, and cell death. At these concentrations, the specific TS inhibitor AG337 induced more DNA damage (up to 20%) than MTA and 5-FU. FACS analysis showed that all drugs induced S phase arrest in Lovo and WiDr that was most pronounced after 5-FU and AG337 exposure (50-70%). Western blotting showed that p53 induction was not detectable in mutant (mt) p53 WiDr and increased much earlier in wild-type (wt) Lovo cells after 5-FU and MTA (24 h) than after AG337 exposure (72 h). In contrast to 5-FU-treated Lovo cells, the bcl-2/bax ratio decreased after antifolate exposure. Nevertheless, both 5-FU and antifolates induced similar amounts of cell death (up to 60%). These results demonstrate that in human colon cancer cells differences in downstream events between AG337 and 5-FU or MTA are related to the additional effects of 5-FU and MTA, which are not associated with TS inhibition.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Cell Cycle; Colonic Neoplasms; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Fluorometry; Fluorouracil; Folic Acid Antagonists; Humans; Hydroxymethyl and Formyl Transferases; Phosphoribosylglycinamide Formyltransferase; Quinazolines; RNA, Neoplasm; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase; Tumor Cells, Cultured

Topics: Antimetabolites, Antineoplastic; Apoptosis; Caspase 3; Caspases; Colonic Neoplasms; DNA Damage; Enzyme Inhibitors; Fas Ligand Protein; Fluorouracil; Folic Acid Antagonists; Glutamates; Guanine; Humans; Membrane Glycoproteins; Pemetrexed; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The combined cytotoxic effects of the thymidylate synthase (TS) inhibitors 5-fluorouracil (5FU) and different antifolates were studied in seven colon cancer cell lines. Growth inhibition of the antifolates, Nolatrexed, Raltitrexed, GW1843U89, or MTA in combination with 5FU, was determined and multiple drug effect analysis showed that the drugs acted mostly additively. The only synergistic interaction was found for 5FU and Nolatrexed in the LS174T cell line. Also Raltitrexed and 5FU were slightly synergistic in WiDr/F cells grown at low folate levels, but for the other cell lines grown at high folate levels this combination was more antagonistic. GW1843U89 and 5FU were mainly additive, while 5FU and MTA showed antagonism in WiDr and additivity in LS174T. The effect of the drugs at their target was evaluated by in situ TS inhibition. We observed lower TS activity in all cells when two drugs were used instead of one. Statistical analysis revealed that none of the values of the combinations was higher or lower than could be expected from the product of the effect of single drugs. We concluded that the effects on TS inhibition were additive for all 5FU/antifolate combinations in all cell lines. DNA strand break formation, as a result of TS inhibition, was measured by means of a fluorometric analysis of DNA unwinding. Raltitrexed-induced DNA damage was significantly increased by 5FU in WiDr cells [single agent: 67% double stranded (ds) DNA, combination: 39% ds DNA, P<0.0001]. In LS174T a trend for antagonistic effects was observed for combinations of MTA, GW1843U89, or Raltitrexed and 5FU. The combinations showed additive effects in WiDr/F cells. The overall conclusion of the three assays in each of the cell lines indicated that 5FU and antifolate combinations were predominantly additive in colon cancer cells.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Colonic Neoplasms; DNA Damage; Fluorouracil; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

AG337 is the recent non-classical thymidylate synthase inhibitor with promising activity and manageable toxicity in phase I clinical trials. In this study, we investigated the cytotoxic activity of AG337 alone and in combination with cisplatin in cultured human colon (HT29) and ovarian (2008) cancer cell lines and their derived counterparts selected for their resistance to 5-fluorouracil (5-FU) (HT29-5-FU) and cisplatin (2008C13). We observed that AG337 had potent cytotoxic effects in colon (IC50 = 0.17 MicroM) and ovarian cancer cells (IC50 = 0.65 microM). The cytotoxic activity of AG337 was higher than that of 5-FU in the two models. The Activity of AG337 was not significantly affected in 5-FU-resistant HT29-5-FU colon cancer cells characterized by an amplification of the thymidylate synthase gene (IC50 = 0.27 microM, p = 0.15). Combinations of cisplatin and AG337 exert synergistic activity in both ovarian and colon cancer cells. Interestingly, this synergism was maintained in 5-FU- and cisplatin-resistant cells. Therefore, our data encourage further examination of combinations of AG337 with cisplatin in cancer chemotherapy.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Colonic Neoplasms; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Female; Fluorouracil; Humans; Ovarian Neoplasms; Quinazolines; Tumor Cells, Cultured