Free Thymidylate Synthase Research Articles

Abstract 225: Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model

Abstract The purpose of this study is to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively analyze the treatment outcome of 5-fluorouracil (5-FU) in colon cancer. Predicting the outcome of chemotherapeutic agents plays an essential role in improving dose strategies. To that end, we have developed an integrated mechanistic PK/PD model to analyze the kinetics of tumor response to 5-FU treatment. Compared to the existing PK/PD models, our model is novel in the way it incorporates the 5-FU metabolism pathway and downstream antiproliferative events to provide a comprehensive model of treatment outcome. 5-FU has been widely used to treat cancers, including colon cancer. 5-FU metabolites can be incorporated into RNA, DNA and also interfere with thymidylate synthase (TS) catalytic activity, which is the main mechanism of 5-FU cytotoxicity. TS activity has been shown to be the predictor for treatment efficacy. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate catalyzed by TS is the sole intracellular de novo source of thymidylate. The formation of a ternary complex between fluorodeoxyuridylate (FdUMP) and TS obstructs the access of dUMP to the TS nucleotide-binding site. Resultant thymidine deprivation and dUMP accumulation give rise to the deoxynucleotide triphosphate (dNTP) pool imbalance, which could trigger S-phase specific DNA fragmentation, cell cycle arrest and apoptosis. In our PK model, compartmental analysis is developed to track the distribution of 5-FU from its entry into body to interstitial fluid outside the tumor cells and to its cellular uptake. In the cell model, the connection of TS inhibition and dNTP pool imbalance is implemented. In addition to the central plasma and peripheral tissue compartments, our model includes compartments for: 5-FU concentration in the extracellular fluids; intracellular 5-FU; 5-FU anabolites; genomic 5-FU in RNA, and in DNA; and, TS - FdUMP complex. Posttreatment free TS level is used to compute the perturbed TS catalytic activity. Using the model outcome on TS inhibition, we modeled the kinetics of dNTP pool perturbation. The dNTP pool imbalance leads to double strand break (DSB) formation that initiates DNA damage repair response, kinetics of which are also modeled. Our model uses the law of mass action and Michaelian kinetics and each molecular reaction is expressed as a nonlinear differential equation. Time course data from the literature are used in parameter estimation. Our simulation results successfully captured the kinetics of treatment outcome from the administration of the 5-FU to downstream effects in the tumor. The next step in our effort is to expand the model to include the tumor growth kinetics. Citation Format: Chenhui Ma, Alexandru Almasan, Evren Gurkan-Cavusoglu. Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 225.

Pretreatment with insulin enhances anticancer functions of 5-fluorou-racil in human esophageal and colonic cancer cells

To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anticancer functions and its mechanisms in the human esophageal cancer cell line (Eca 109) and human colonic cancer cell line (Ls-174-t). The effect of insulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After insulin treatment or insulin/5-FU treatment, cell cycle distribution of both cell lines was analyzed by flow cytometry. Western blot assay was used to assess the expression of caspase-3 and thymidylate synthase (TS). Apoptosis was detected by flow cytometry, DNA fragmentation assay, and terminal transferase dUTP nick end labeling assay (TUNEL). Moreover, the changes of 5-FU uptake after insulin pretreatment were detected by HPLC assay and Western blot analysis. We found that insulin enhanced the inhibitory effect of 5- FU on cell proliferation when Eca 109 cells and Ls-174-t cells were pretreated with insulin for the appropriate time. Insulin increased the cell number of the S phase and the uptake of 5-FU. Insulin/5-FU treatment enhanced apoptosis of tumor cells and upregulated the expression of cleaved caspase-3 compared with 5-FU treatment. Moreover, insulin/5-FU treatment induced the changes of free TS and the TS ternary complex level compared with 5-FU treatment in Eca 109 and Ls-174-t cells. These data suggest that insulin enhances anticancer functions of 5- FU when it is treated before 5-FU for the appropriate time in human esophageal and colonic cancer cell lines.

Nuclear Expression of Thymidylate Synthase in Colorectal Cancer Cell Lines and Clinical Samples

Thymidylate synthase (TS) [TYMS; OMIM reference number (188,350)] is normally considered to be a cytoplasmic enzyme. However, a few reports have suggested it may also be present in the nucleus. To explore this in more detail, we used a highly specific polyclonal antibody to TS and a combination of techniques, including immunocytochemistry, confocal microscopy, cell fractionation, and Western blotting. We developed cell line HeLa-55, a HeLa derivative that grossly overexpresses TS. Although the vast majority of TS was in the cytoplasm, some TS also was seen in the nucleus. TS in parental HeLa cells and in normal human fibroblasts was seen exclusively in the cytoplasm. HeLa-55 cells exposed to 5-fluorodeoxyuridine were fractionated and examined by Western blotting. Interestingly, both free TS and the ternary complex of TS were seen in the cytoplasmic fraction but only free TS was detected in the nuclear fraction. Amongst different cell lines examined, HCT-15 and normal fibroblasts showed no nuclear TS, HCC-2998 and SW-620 showed a small amount of nuclear TS, and HT-29, RKO, and HCT-116 showed a strong nuclear TS signal. Nuclear staining was clearly evident in some clinical colorectal specimens, both normal and malignant. This staining was definitively shown to be TS by competition with recombinant TS protein. A putative leucine-rich nuclear export sequence was identified but its function could not be confirmed. We conclude that small amounts of TS protein is present in the nucleus of some cell types but further work is needed to determine the significance of this observation.

Apoptosis and thymidylate synthase inductions by 5-fluorouracil in gastric cancer cells with or without p53 mutation.

We studied apoptosis and thymidylate synthase (TS) inductions by 5-fluorouracil (5-FU) in gastric cancer cells with wild-type p53 (MKN-45 and MKN-74) and with mutated p53 (MKN-28 and KATO-III). Apoptotic inductions in MKN-45 and MKN-74 were stronger than those in MKN-28 and KATO-III, suggesting that wild-type p53 may contribute to the induction of apoptosis. After continuous exposure to 0.1 microg/ml of 5-FU for 96 h, no TS induction was obtained in KATO-III, while approximately twice the amount of TS was observed compared to non-treatment cells in MKN-45, MKN-74, and MKN-28. The results of immunohistochemical staining for TS and p53 showed no relation between these two protein expressions in endoscopic biopsy specimens of 25 patients with advanced gastric cancer. These results indicated that p53 status may not play a pivotal role in regulating TS expression. We found no significantly different effects of 5-FU between intermittent (repeat of 24-h continuous infusion and 24-h drug-free) and continuous treatments in either MKN-28 or stem cells (CD34+ hematopoietic progenitor cells) when the same area under the time-concentration curve of 5-FU was administered. The TS induction in MKN-28 cells by intermittent treatment was significantly higher than that by continuous treatment; however, most TSs in both types of 5-FU treatment cells were of the inactive form, i.e., TS bound to FdUMP, a 5-FU metabolite. Therefore, neither intermittent nor continuous treatment appears to induce 5-FU resistance related to the level of increment free TS. In conclusion, our observations suggested that p53 mutation may be associated with apoptotic induction by 5-FU; however, p53 status may not strongly affect TS induction by 5-FU. Intermittent treatment can be replaced with continuous treatment without causing 5-FU resistance.

Cytotoxic synergism of methioninase in combination with 5-fluorouracil and folinic acid

Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H 4folate) is due to elevation of the methylene tetrahydrofolate (CH 2-H 4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH 2-H 4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H 4folate) and, subsequently, CH 2-H 4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H 4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H 4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC 50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H 4folate, respectively. 5-HCO-H 4folate increased the intracellular concentrations of CH 2-H 4folate and H 4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H 4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H 4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.

Reduction of 5-fluorouracil (5-FU) gastrointestinal (GI) toxicity resulting from the protection of thymidylate synthase (TS) in GI tissue by repeated simultaneous administration of potassium oxonate (Oxo) in rats.

An important cytotoxic effect of 5-fluorouracil (5-FU) is the inactivation of thymidylate synthase (TS) (EC 2.1.1.45) activity by the formation of a ternary complex consisting of covalently bound 5-fluorodeoxyuridine 5'-monophosphate (FdUMP), TS and 5,10-methylenetetrahydrofolate (CH2FH4). The gastrointestinal (GI) toxicity of 5-FU is also caused by its phosphorylation in the GI tract. Potassium oxonate (O(XO)) competitively inhibits pyrimidine phosphoribosyltransferase (EC 2.4.2.10), which converts 5-FU to 5-fluorouridine 5'-monophosphate (FUMP) in vitro. In this study the benefits of combining Oxo and tegafur (FT), which is a masked compound of 5-FU, in reducing the GI toxicity of 5-FU and in protecting the activity of TS in the normal GI tissues were evaluated. We administered orally a preparation of 1 M FT and 0.4 M 5-chloro-2,4-dihydroxypyridine (CDHP) with or without 1 M O(XO) (called S-1 and FT + CDHP, respectively) or vehicle only (control) to rats for ten consecutive days and compared the toxicity, the histopathological findings and the free TS activity in the GI tissues of the treated rats. During the experimental periods, the signs of toxicity, such as a decrease in body weight, diarrhea and death, were only observed in the rats treated with FT + CDHP. The histopathological findings in the ileum and colon samples from rats treated consecutively with S-1 on day 1, day 4, day 7 and day 10 were less frequent and more mild than in the samples from rats treated with FT + CDHP. Furthermore, the free TS activities in the ileum samples of rats given S-1 and FT + CDHP were significantly decreased compared with the activity in samples from the control rats throughout the experimental periods. The free TS activities in GI tissues of rats treated with S-1 were higher than the TS activities in tissues from rats treated with FT + CDHP daily from day 4 to day 10, although activities in S-1-treated rat were decreased to almost same low levels as in FT + CDHP-treated rats on day 1. Our results suggest that repeated simultaneous administration of Oxo and FT can effectively protect the activity of TS by decreasing FdUMP via FUMP from 5-FU in GI tissue, and may lead to a reduction in GI toxicity.

Immunological quantitation of thymidylate synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex with the monoclonal antibody TS 106

Thymidylate synthase (TS) is responsible for the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. One of the principal mechanisms of action of 5-fluorouracil (5-FU) is the inhibition of TS by formation of a ternary covalent complex consisting of TS-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate. We have developed a Western immunoblot assay using the monoclonal antibody TS 106 to measure ternary complex and free TS in intact human carcinoma cells following exposure to either 5-FU alone or 5-FU plus leucovorin. Lysates from cells treated with either 5-FU or 5-FU/leucovorin were resolved in 15% polyacrylamide gel, transferred onto nitrocellulose and immunoblotted using TS 106 antibody. Detection of positive bands was by a chromogenic substrate strain. Immunoblotting detected free TS at 36 kDa and TS in ternary complex at 38.5 kDa which were quantitated by densitometric scanning. This assay was able to detect a ternary complex from intact cells treated with 5-FU or 5-FU/leucovorin up to 96 h after drug removal. The ratio of complex to free TS was up to 2-fold greater in 5-FU/leucovorin-treated cells compared to those treated with 5-FU alone. This assay may be applied to measuring the formation and stability of ternary complex and free TS in patient tissue samples.

Schedule dependent inhibition of thymidylate synthase and tumor growth by 5-fluorouracil in Yoshida sarcoma bearing rats.

Schedule dependent inhibition of thymidylate synthase (TS) and tumor growth by 5-fluorouracil (FUra) was examined in Yoshida sarcoma (YS) bearing Donryu rats. After implantation of YS cells (1 x 10(4), FUra (20 mg/kg/day) was continuously (group C) or daily bolus injection (group B) administered for 6 days. On day 7, tumor weight was 1.57 +/- 0.58 g in group C and 0.45 +/- 0.10 g in group B (P less than 0.01), free TS was 2.23 +/- 83 fmol/mg protein in group B and 96 +/- 55 fmol/mg protein in group C (P less than 0.05), and inhibition rate of TS was 88.3 +/- 5.3% in group C and 94.7 +/- 3.0% in group B (P less than 0.05). A significant correlationship was found between free TS and tumor weight (P less than 0.05). As the next step, continuous infusion (group C) or daily bolus injection (group B) for 6 days was started on day 5 after implantation of YS cells. The relative increase of tumor on day 9 was 256 +/- 111% in group C and 112 +/- 22.1% in group B (P less than 0.05). On day 11, total TS of the resected tumor was 650 +/- 153 fmol/mg protein in group C and 391 +/- 124 fmol/mg protein in group B (P less than 0.05), and inhibition rate of TS was 78.8 +/- 12.4% in group C and 84.4 +/- 8.6% in group B. Daily bolus injection of FUra causes a superior antitumor and antimetabolic effect. The schedule dependent cytotoxicity of FUra should be taken into account when a chemotherapeutic protocol is designed.

Biochemical modulation of 5-fluorouracil with or without leucovorin by a low dose of brequinar in MGH-U1 cells.

Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical anti-tumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU +/- leucovorin (LV) +/- brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC50) for brequinar, FU, and FU+LV (100 microM) were 0.4, 20, and 10 microM, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC10 (0.1 microM) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 microM brequinar and FU +/- LV did not increase the cytotoxicity of FU +/- LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 microM brequinar for 24 h alone or followed by a 2-h exposure to FU (25 microM) +/- LV (100 microM). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU+brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 microM hypoxanthine and 25 microM FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).

Folinic acid modulation of fluorouracil: tissue kinetics of bolus administration

Thymidylate synthase (TS) is the enzyme target of 5-fluorouracil (FUra) that recent laboratory and clinical studies with folinic acid (calcium leucovorin) suggest may mediate important antitumor cytotoxicity. Measurement in carcinoma tissue of parameters related to TS inhibition by 5-fluorodeoxyuridylate (FdUMP), by analogy to hormone receptor analysis, should be useful to determine which patients should receive fluoropyrimidine drug therapy and to evaluate folinic acid requirements. Folinic acid is metabolized to 5,10-methylenetetrahydropteroylglutamine (CH2FH4), which must be present in large excess to effect desired levels of maximal inhibition of TS, by promoting formation and stabilization of TS-FdUMP-CH2FH4 ternary complexes. In patients with metastatic disease, serial biopsies of tumor and normal tissues for studies of pharmacodynamic responses to test-dose FUra or folinic acid are shown to be easily added to routine intraoperative management. A suitable methodologic approach is described and examples given of assays of free TS, FdUMP, dUMP, and CH2FH4 levels after FUra or folinic acid, that may be useful in future studies aimed at improving the cost-effectiveness of FUra-folinic acid combinations.

Inhibition of thymidylate synthase after administration of doxifluridine in a transplantable colon carcinoma in the rat.

Parameters for inhibition of thymidylate synthase (TS) in a DMH-induced transplantable rat colon carcinoma were studied after intraperitoneal administration of bolus doxifluridine (5'-dFUR) 200 mg/kg. Levels of 5'-dFUR, 5-fluorouracil (5-FU), fluorouridinediphosphate (FUDP), and fluorouridinetriphosphate (FUTP) were determined by use of high-performance liquid chromatography. Micromethods for analysis of 5-fluoro-2'-deoxyuridylate (FdUMP) and TS were used to study the in vivo intracellular pharmacokinetics of TS inhibition. Peak values of 5'-dFUR and 5-FU were found at 30 min and showed exponential declines with values close to zero at 5 hr. Substantial levels of FUDP and FUTP were found throughout the 24 h observation time. Peak FdUMP levels were modest compared to those observed after equimolar administration of 5-FU, but FdUMP persisted in amounts well above available binding sites on TS for the 24 h observation time. Reduction of free TS enzyme to undetectable levels (less than 0.05 pmol/g) lasted for 4 h, and at 24 h, there was still almost 70% enzyme inhibition. The total amount of TS (TStot) defined as free [3H]FdUMP-titrable enzyme (TSf) plus TS bound to FdUMP in a ternary complex (TSb) increased as a result of 5'-dFUR bolus injection from 15 to 50 pmol/g during the 24 hr observation time. We conclude from these data that 5'-dFUR is converted to 5-FU and subsequently to FdUMP, and the results suggest that 5'-dFUR exerts its cytotoxic effects through inhibition of TS and incorporation into RNA.