Total Thymidylate Synthase Research Articles

The role of thymidylate synthase in non-small cell lung cancer treated with pemetrexed continuation maintenance therapy

Pemetrexed continuation maintenance therapy has been proven to be beneficial for patients with advanced non-squamous non-small cell lung cancer (NSCLC). However, the eligibility criteria for maintenance treatment are too simple. This study sought to evaluate thymidylate synthase (TS) as a predicting biomarker for pemetrexed continuation maintenance treatment in NSCLC. Specimens were collected from 87 patients treated with pemetrexed continuation maintenance therapy before and after four-cycle induction chemotherapy. Real-time quantitative PCR was used to detect TS expression in tissues. The TS expression level was correlated with characteristic clinical data, radiographic response, progression-free time (PFS) and overall survival (OS). Low total TS expression (<8.47) was associated with improved PFS (median: 4.7 months vs. 3.5 months, p = 0.034) and improved OS (time from random assignment: 16.4 months vs. 11.7 months, p = 0.026; time from induction: 19.7 months vs. 14.8 months, p = 0.022). Our results indicate that in NSCLC patients treated with pemetrexed continuation maintenance therapy, low TS expression is associated with improved PFS and OS.

1283P - The role of thymidylate synthase in non-small-cell lung cancer treated with pemetrexed continuation maintenance therapy

Pemetrexed continuation maintenance therapy has been proven to be beneficial for patients with advanced non-squamous non-small cell lung cancer (NSCLC). However, the eligibility criteria for maintenance treatment are too simple. This study sought to evaluate thymidylate synthase (TS) as a predicting biomarker for pemetrexed continuation maintenance treatment in NSCLC. Specimens were collected from 87 patients treated with pemetrexed continuation maintenance therapy before and after four-cycle induction chemotherapy. Real-time quantitative PCR was used to detect TS expression in tissues. The TS expression level was correlated with characteristic clinical data, radiographic response, progression-free time (PFS) and overall survival (OS). Low total TS expression (<8.47) was associated with improved PFS (median: 4.7 months vs. 3.5 months, p = 0.034) and improved OS (time from random assignment: 16.4 months vs. 11.7 months, p = 0.026; time from induction: 19.7 months vs. 14.8 months, p = 0.022). Our results in...

Significance of Folate Receptor Alpha and Thymidylate Synthase Protein Expression in Patients with Non–Small-Cell Lung Cancer Treated with Pemetrexed

Folate receptor alpha (FRA) regulates cellular uptake of folates and antifolates. Information about FRA protein expression in metastatic non-small-cell lung cancer (NSCLC) is limited. We investigated FRA as a biomarker for pemetrexed-based chemotherapy and compared it with thymidylate synthase (TS), the main target of pemetrexed. Pretreatment tumor specimens from 207 patients with advanced NSCLC were assessed for FRA and TS protein expression by immunohistochemistry using the H-score (range, 0-300) and correlated to patients' clinicopathological data, radiographic response, progression-free survival (PFS), and overall survival (OS). Low total (cytoplasmic and nuclear) TS protein expression (H-score < 210) was associated with improved PFS (median: 5.6 versus 3.5 months; hazard ratio [HR] = 0.6379, p = 0.0131) and prolonged OS (median: 22.5 versus 11.5 months; HR = 0.5680,p = 0.0107). An association between lower TS levels and response to pemetrexed-based therapy was found-mean H-score 187 ± 5, median 180 for responders versus mean H-score 201 ± 4, median 210, for non-responders, p = 0.0244. High intracellular FRA expression (H-score ≥110) was associated with prolonged OS (28.9 versus 11.7 months, HR = 0.5316, p = 0.0040) and a trend for association with PFS (5.6 versus 4.1 months, HR = 0.7395, p = 0.0801) was noted. Membranous FRA expression was seen in 83% of patients, moreover, high membranous expression (H-score ≥20) was associated with improved PFS (5.6 versus 3.7 months, HR = 0.6445, p = 0.0306) and OS (22.1 versus 11.5 months, HR = 0.5378, p = 0.0131). A large number of NSCLC patients have high expression of FRA and/or a low level of TS expression. Expression levels of FRA and TS were associated with clinical benefit from pemetrexed therapy.

Abstract 1668: Effect of folate and γ-glutamyl hydrolase modulation on in vivo chemosensitivity of colon cancer cells to 5-fluorouracil

Abstract Background: γ-Glutamyl hydrolase (GGH) removes terminal glutamates of polyglutamylated folates, thereby facilitating hydrolysis and efflux of folates from the cell. GGH overexpression may affect chemosensitivity of colon cancer cells to 5-fluorouracil (5FU) by changing intracellular retention of a folate cofactor (5,10-methylenetetrahydrofolate) necessary for the cytotoxic effects of 5FU and by changing intracellular total and polyglutamylated folate concentrations. We have previously reported that the in vitro chemosensitivity of human HCT116 colon cancer cells overexpressing GGH to 5FU+leucovorin (LV) is decreased at high, whereas it is increased at physiologic, media concentrations of folate compared with the control. We investigated whether GGH overexpression and folate status would affect the in vivo chemosensitivity of HCT116 cells to 5FU+LV in nude mice. Methods: Six-week-old male Balb/c nu/nu mice received s.c. injections in each flanks of HCT116 cells expressing endogenous GGH and sense GGH. When xenografts reached a volume of 80-200 mm3, mice were assigned to 2 (control) or 8 (supplemented) folic acid (FA) mg/kg diet and treated with 5FU (20 mg/kg/day) + LV (1 mg/kg/day) or saline for 5 consecutive days. The growth rate of xenografts was determined. At necropsy, plasma folate and homocysteine concentrations and xenograft folate concentrations, thymidylate synthase (TS) activity, epithelial proliferation and apoptosis were determined. Results: Plasma folate concentration of the FA supplemented mice was significantly higher than that of mice on the control diet (p&amp;lt;0.001) while plasma homocysteine concentrations did not differ between two diets groups. Total and polyglutamylated folate concentrations and TS activity of the xenografts generally reflected dietary FA levels but they were modulated by both 5FU+LV treatment and GGH status. The growth rate of xenografts expressing endogenous GGH was faster than those overexpressing GGH at both FA diets when treated with saline. The growth rate of colon cancer cells overexpressing GGH was inhibited less effectively by 5FU+LV at 8 mg FA supplemented diet and inhibited more effectively by 5FU+LV at 2 mg FA control diet compared with those expressing endogenous GGH (p&amp;lt;0.05). For xenografts treated with 5FU+LV on the control diet, those expressing endogenous GGH had lower Ki-67 % and higher TUNEL % while those overexpressing GGH showed higher Ki-67 % and lower TUNEL % (p&amp;lt;0.05). Conclusions: The in vivo chemosensitivity results support the in vitro observations that both GGH modulation and folate status may increase chemosensitivity of colon cancer cells to 5FU-based chemotherapy. We provide proof-of-principle that GGH and folate status may be important clinical determinants for providing rational and effective tailored colon cancer chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1668. doi:10.1158/1538-7445.AM2011-1668

Antisense Targeting of Thymidylate Synthase (TS) mRNA Increases TS Gene Transcription and TS Protein: Effects on Human Tumor Cell Sensitivity to TS Enzyme-Inhibiting Drugs

Thymidylate synthase (TS) catalyses the only de novo pathway to produce thymidylate for DNA replication and repair and is an important target for cancer chemotherapy. Preexisting or acquired drug resistance in tumor cells limits clinical efficacy of TS-targeting drugs. Cells selected for higher TS protein activity have decreased sensitivity to TS-targeting chemotherapeutic agents (5-FUdR and raltitrexed). New therapeutic strategies are required to overcome treatment resistance. Among these, upregulation of drug resistance mediators in normal, nontarget cells and/or antisense downregulation of those mediators (alone or in combination with protein-targeting drugs) are candidate strategies. We have targeted human TS mRNA with antisense oligodeoxynucleotides (AS ODNs), complementary to the translation start site (TSS), the coding region, and the 3' untranslated region. We report here that, in response to treatment with a novel TSS-targeting AS ODN 791, TS gene transcription in a human cervical carcinoma cell line (HeLa) was unexpectedly increased by 70%. Interestingly, the increased TS gene transcription and nuclear TS RNA did not elevate levels of total cellular TS mRNA, but did increase TS protein activity by 35% and TS protein level by 150%. Increased TS protein activity and level did not alter proliferation rate or sensitivity to TS-targeting drugs (5-FUdR or raltitrexed). To assess concentration-dependent effects of TS on sensitivity to TS-targeting drugs, incremental increases of TS protein levels were generated by transfection of a mammalian TS expression vector. Increases in TS protein of less than approximately 400% did not significantly affect sensitivity to TS-targeting drugs, while greater TS protein levels did. These data indicate that AS ODNs targeting TS mRNA can upregulate TS expression and activity in a manner dependent on the sequence being targeted, and that there exists a threshold increase (greater than approximately 400-700% in HeLa cells), required to initiate resistance to TS-targeting drugs.

Biochemical modulation of 5‐fluorouracil with interferon α/β and γ on murine renal cell carcinoma

AbstractBackground: Conventional therapy for renal cell carcinoma (RCC) using interferon (IFN) has shown limited antitumor action. The purpose of the present study was to investigate the synergistic antitumor effects of IFN and 5‐fluorouracil (5‐FU) and to elucidate the mechanism underlying antitumor effects.Methods: The antitumor effects and biochemical modulation of murine IFN and 5‐FU were determined using murine renal cell carcinoma (RENCA). The activity of thymidylate synthase, thymidine kinase and the concentration of 5‐FU incorporated into RNA was measured using cytosolic extracts of tumors.Results: Triple combination therapy (5‐FU, IFN α/β and IFN γ) showed a synergistic antitumor effect on RENCA tumors, because triple combination therapy suppressed growth significantly compared to combination therapy (IFN α/β and IFN γ, P = 0.0258) and 5‐FU (P &lt; 0.0001). Total thymidylate synthase was decreased by triple combination therapy (5‐FU, IFN α/β and IFN γ, P = 0.0019) and combination therapy (5‐FU and IFN γ, P = 0.0018) compared to 5‐FU alone. Thymidine kinase activity was decreased by triple combination therapy (5‐FU, IFN α/β and IFN γ, P &lt; 0.0001) and combination therapy (5‐FU and IFN α/β, P &lt; 0.0001) compared to 5‐FU alone. The concentration of 5‐FU incorporated into RENCA tumors was increased by triple combination therapy (P = 0.0132) and combination therapy (5‐FU and IFN α/β, P = 0.0124) compared to 5‐FU alone.Conclusions: Interferons α/β and γ showed different biochemical modulation for 5‐FU. Therefore, combination therapy using 5‐FU and IFN showed synergistic antitumor effects on murine RCC.

Changes in thymidylate synthase and its inhibition rate and changes in dihydropyrimidine dehydrogenase after the administration of 5-fluorouracil with cisplatin to nude mice with gastric cancer xenograft SC-1-NU.

Although 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum (cisplatin) in combination have synergistic cytotoxicity against both murine and human neoplasms, the precise mechanism of the synergism, and the effects on thymidylate synthase and its percent inhibition, and the effects on dihydropyrimidine dehydrogenase (DPD) remained to be elucidated. Experimental chemotherapy was performed using SC-1-NU, a human gastric carcinoma xenograft. SC-1-NU was maintained by serial transplantation in male BALB/c nude mice. The nude mice received various chemotherapeutic regimens consisting of 5-FU and/or cisplatin, with different dosages and periods of administration. After the treatment, we examined the in vivo effects of 5-FU and cisplatin in each regimen on thymidylate synthase and its percent inhibition, and the effects on DPD, in addition to the observation of tumor growth inhibition. The combined use of 5-FU (20 mg/kg per day) and cisplatin (either 1.5 or 7.5 mg/kg per day) showed a synergistic antitumor effect, regardless of the different doses of cisplatin. The long-term administration of 5-FU significantly increased both total thymidylate synthase and the percent thymidylate synthase inhibition rate. The short-term administration of 5-FU significantly decreased DPD. Nevertheless, these changes showed no relation to the combined use of cisplatin. Combined administration of cisplatin with 5-FU did not further increase thymidylate synthase inhibition over that occurring with 5-FU alone, which does not support the hypothesis that cisplatin combined with 5-FU modulates thymidylate synthase inhibition in enhancing the anticancer effect of 5-FU. Changes in DPD after the administration of 5-FU may provide an insight into tumor sensitivity and resistance to 5-FU.

Biochemical and molecular effects of UCN-01 in combination with 5-fluorodeoxyuridine in A431 human epidermoid cancer cells.

Concurrent and pre-exposure of A431 human epidermoid cancer cells to UCN-01, an investigational anticancer drug, with 5-fluoro--2'-deoxyuridine (FdUrd), which targets thymidylate synthase, produced more than additive cytotoxicty. A 24-h exposure to 10 nM FdUrd led to inhibition of TS, a 2.5-fold increase in total thymidylate synthase protein content, profound dTTP depletion and a 6.3-fold increase in the ratio of dATP to dTTP, but did not cause single-strand breaks in DNA. However, FdUrd enhanced UCN-01-associated DNA strand breaks. Concurrent thymidine exposure led to repletion of dTTP pools, and cytoprotection against FdUrd alone and with UCN-01. UCN-01 arrested cells in G1, decreased the percentage of FdUrd-treated cells in S phase and reduced FdUrd-DNA incorporation, suggesting the latter was not important for cytotoxicity. Delayed induction of high molecular mass DNA fragmentation and poly(ADP-ribose) polymerase cleavage was observed with the combination of UCN-01 and FdUrd. These findings suggest that while FdUrd-mediated deoxynucleotide imbalance alone was insufficient to induce apoptosis in this p53-mutant cell line, it magnified UCN-01's effects, most likely by interfering with DNA repair. The clinical evaluation of UCN-01 combined with 5-fluoropyrimidines may be of interest.

Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: implications for treatment with fluorouracil.

To determine the time-dependence of fluorouracil (5FU)-induced thymidylate synthase (TS) inhibition in colon cancer patients, the effect of leucovorin (LV), and the relation to response. A 5FU injection (500 mg/m2) was given to 47 patients with advanced colorectal cancer; tumor biopsy specimens were obtained 1 to 72 hours after laparotomy. Eleven patients received LV (2-hour infusion of 500 mg/m2) with 5FU midinfusion; biopsies were obtained after 45 hours. TS inhibition was evaluated by comparing the number of total and free 5-fluoro-2'-deoxy-uridine-5'- monophosphate (UMP) (FdUMP) binding sites and the total and residual catalytic activity of TS. The total catalytic TS activity varied from 0 to 621 pmol/h/mg protein and the total number of FdUMP binding sites varied from 0 to 976 fmol/mg protein. The residual catalytic TS activity after 2, 23, and 45 hours was 41%, 65%, and 74% of the total catalytic activity; the number of free FdUMP binding sites was 12%, 27%, and 49% of the total number, respectively. LV enhanced TS inhibition after 45 hours; the residual catalytic activity decreased from 74% to 49%, and the number of free FdUMP binding sites from 49% to 24%. Eleven of 19 patients treated with hepatic arterial infusion of 5FU had a partial response (PR). In the nonresponding patients, total TS activity was significantly higher (P < .05) than in responding patients. A high TS activity with a poor inhibition correlated with no response. Residual and total TS activity are predictive for response to 5FU. The findings may be applicable for treatment of patients with advanced disease and TS should be evaluated as a prognostic factor in adjuvant chemotherapy studies.

Schedule-dependent inhibition of thymidylate synthase by 5-fluorouracil in gastric cancer

An optimal treatment schedule of 5-fluorouracil (5-FU) remains to be clarified. A randomized study was conducted to investigate schedule-dependent thymidylate synthase (TS) inhibition by 5-FU in 16 patients with gastric cancer who underwent surgical resection. Surgical specimens of tumor, normal gastric mucosa, and regional lymph nodes were obtained 12 hours after administration of 5-FU either as a continuous infusion (1000 mg/m2 for 48 hours) or as a bolus injection (500 mg/m2 x 2 in 48 hours). The total TS activity (567.8 +/- 294 fmol/mg protein) and the rate of TS inhibition (74.7 +/- 23.1%) in cancer tissues were significantly higher in the continuous-infusion group than in the bolus-injection group (228.5 +/- 104.6 fmol/mg protein and 48.8 +/- 12%, respectively). Likewise, the total TS activity (807.4 +/- 440.3 fmol/mg protein) and the rates of TS inhibition in lymph nodes (72.3 +/- 17.1%) and in normal gastric mucosa (85.1 +/- 12.2%) were significantly higher in the continuous-infusion group than those in the bolus-injection group (232.4 +/- 142.3 fmol/mg protein and 53.6 +/- 17.0% in lymph nodes and 46.5 +/- 14.3% in normal gastric mucosa, respectively). There was a significant correlation between the total TS activity and TS inhibition. Continuous infusion of 5-FU provides a superior antimetabolic effect in the treatment of gastric cancer, which may lead to a superior antitumor effect.

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.

Mechanism of the cytotoxic synergism of fluoropyrimidines and folinic acid in mouse leukemic cells.

We have investigated the mechanism by which reduced folates, such as folinic acid, enhance the cytotoxicity of fluoropyrimidines in L1210 mouse leukemic cells. Exposure of L1210 cells to folinic acid resulted in expansion of intracellular pools of 5,10-CH2-H4PteGlun, delayed the reappearance of catalytically active thymidylate synthase (TS) following 5-fluoro-2'-deoxyuridine exposure, and stabilized inhibited TS complexes over the same concentration range that augmented the cytotoxic effects of fluorodeoxyuridine and 5-fluorouracil. The data showed that, in intact L1210 cells, fluorodeoxyridylate behaves as an inhibitor whose complexes with TS dissociated with a biologically significant rate. However, these complexes become functionally irreversible in cells incubated with high levels of folinic acid. It was also found that bound and total TS levels increased in cells treated with fluorodeoxyuridine to an extent that substantially exceeded the increase in protein content per cell under the same conditions. These results are in accord with the concept that folinic acid augments the effects of the fluoropyrimidines by expansion of cellular 5,10-CH2-H4PteGlun pools with subsequent stabilization of ternary complexes among 5-fluoro-2'-deoxyuridine 5'-monophosphate, TS, and 5,10-CH2-H4PteGlun. In light of the accumulation of TS that occurs following exposure to fluoropyrimidines, this stabilization may be needed for efficient tumor cell killing by these agents.