Thymidylate Synthase In Cancer Cells Research Articles

Abstract 2159: In vitro characterization of PTAU and FU interactions in colon cancer cells

Abstract Background: Fluorouracil (FU) remains a main anticancer therapy for colorectal cancer (CRC) and other solid tumors. The mechanism of action of FU is associated with inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. High toxicity and wide-spread chemoresistance, however, have limited the utilization of FU-based therapies. Five intracellular enzymes, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), thymidine phosphorylase (TP), and uridine phosphorylase (UP) are key determinants of FU sensitivity or resistance. Combination therapies aimed at improving the bioavailability of FU and reducing host toxicity has been evaluated. Recently, our group developed a new uridine phosphorylase inhibitor, 5-phenylthio-acyclouridine (PTAU) that has shown promise in reducing host toxicity and increasing FU efficacy in vivo. The current studies were to characterize interactions of PTAU and FU in vitro and to assess its effects on key metabolic enzymes in human colon cancer cells. Hypothesis: We hypothesized that inhibition of uridine phosphorylase by PTAU would ameliorate the toxic effects of FU and enhance its efficacy by permitting increased doses of FU. Methods: Three colon cancer cell lines with differing p53 status, HCT116-p53wt (wild-type), HCT116-p53null, and HT-29 (p53-mutant); and a normal colonic epithelial cell line, CRL-1790, were used. Doubling times were determined, and IC50 values for FU were derived from kill curves using MTT assay. Cells were treated for 72 hours with IC50 doses of FU, PTAU (100 μM) or uridine (25 μM), alone or in combination, or with the solvent, DMSO. mRNA levels for DPD, TS, TP, and UP were quantitated by qRT-PCR; for intracellular protein expression, cells were fixed and stained with monoclonal antibodies and analyzed by flow cytometry. Results: FU sensitivity correlated with cell doubling times. Basal expression of FU metabolic enzymes differed between CRC and normal cells. mRNAs for DPD and UP were higher in normal relative to cancer cells, but TS mRNA was higher in cancer cells. FU treatment upregulated mRNA expression of DPD, UP, and TS in cancer cells but not in normal cells. Only in cancer cells, DPD induction by FU was potentiated by PTAU. In contrast, treatment with FU decreased protein levels of DPD, TP, and TS in cancer cells. PTAU induced TS protein expression in cancer cells, an effect that was reduced by co-treatment with FU. Conclusions: FU induces mRNA levels of DPD, UP, and TS in cancer cell lines, but protein levels are reduced; the discrepancy between mRNA and protein levels following FU treatment underscores the need for evaluation of colon cancer samples to assess response to FU therapy. This work was supported by a Supplement of the MSM/TU/UABCCC U54 Partnership grant (3U54-CA118948-09S1) from NIH. Citation Format: Esther A. Suswam, Gaurav Kumar, Hyung-Gyoon Kim, Mohamed Osmar, Mahmoud H. el Kouni, Upender Manne. In vitro characterization of PTAU and FU interactions in colon cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2159.

Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase

Background:Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU.Methods:Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5′-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine.Results:Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5′-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments.Conclusions:Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC.

CDK inhibitor enhances the sensitivity to 5-fluorouracil in colorectal cancer cells

Thymidylate synthase (TS) is a dNTP synthetic enzyme and is also a target enzyme of 5-fluorouracil (5-FU). 5-FU is one of the anticancer agents most frequently used for the treatment of colorectal cancers. However, the clinical rate of response to its use as a single agent is not exceptionally high. Therefore, various combination chemotherapies have been devised. The elevated expression of TS in cancer cells is a serious obstacle in the clinical use of 5-FU. In the present study, TS expression was up-regulated by the knockout of the p21WAF1/CIP1 gene in human colorectal cancer HCT116 cells, suggesting that TS expression is mediated through the inhibition of cyclin-dependent kinase (CDK). Based on these findings, we tested whether the CDK inhibitor (CDKI) SU9516, acted as a suppressor of TS. SU9516 effectively reduced the expression of TS in a dose-dependent manner. Furthermore, the reduction of TS expression resulted in enhancement of the sensitivity to 5-FU in human colon cancer DLD-1 cells. Thus, SU9516 might be a promising compound for combination chemotherapy with 5-FU.

Structure of Human Thymidylate Synthase Suggests Advantages of Chemotherapy with Noncompetitive Inhibitors

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.