K. Smid

5-Fluorouracil induced Fas upregulation associated with apoptosis in liver metastases of colorectal cancer patients

BACKGROUND In vitro, thymidylate synthase (TS) inhibition by 5-fluorouracil (5-FU) induces thymineless apoptosis possibly via Fas receptor Fas ligand interactions and cell-cycle arrest. In colorectal cancer patients we evaluated whether 5-FU administration also resulted in apoptosis and cell-cycle arrest and which proteins might be involved. PATIENTS AND METHODS Biopsy specimens were taken from 36 patients 2, 22 or 46 hours after administration of 500 mg/m2 5-FU, and from 12 control patients who did not receive 5-FU. In frozen tissue-sections from liver metastases immunohistochemistry was performed with antibodies directed against p53, p21, E2F2, Rb, Ki67 and TS (cell-cycle related) and bax, BCL-2, BCL-x, mcl-1, PARP, caspase-3, Fas receptor and Fas ligand (apoptosis related). Apoptosis was determined by M30 immunostaining, which recognises a cleavage product of cytokeratin 18. RESULTS Fas receptor expression was 50% higher (P = 0.036) 46 hours after 5-FU administration compared to the control group. This was associated with a 12% increase (P < 0.02) in M30 positive tumour cells and with elevation of caspase-3 and PARP expression. The expression of Ki67 and E2F2 was 30% lower after 46 hours compared to the control group, whereas TS was 56% lower after 2 hours and 32% higher again after 46 hours. No differences in the expression of the other proteins were found. CONCLUSIONS These results suggest that 5-FU decreases proliferation status and induces apoptosis possibly via the Fas pathway. Since Fas mediated cell killing is important for cytotoxic T cells this indicates that clinical studies combining immunotherapy for activation of T cells and chemotherapy using 5-FU might be very effective.

The effect of different routes of administration of 5-fluorouracil on thymidylate synthase inhibition in the rat

A rat colon tumour model of liver metastases was used to administer 5-fluorouracil (5FU) by intraperitoneal (i.p.) bolus injection (50 mg/kg), isolated liver perfusion (ILP, 150 mg/kg) and hepatic artery infusion (HAI, 50 mg/kg). The biochemical effect of 5FU, delivered by different routes, on its target enzyme thymidylate synthase (TS) was studied in both tumour and normal tissues of the rat. In tumour tissue, only small differences were observed in the extent of TS inhibition. A pronounced inhibition of TS was observed 3 h after 5FU administration by all routes, but was followed by a recovery of TS activity within 24 and 48 h. Effects of 5FU on normal tissues were diverse. In liver, TS activity increased 6-fold after ILP and HAI administration of 5FU, and a 2-fold increase of FdUMP binding to TS was seen for all routes of administration. In intestinal mucosa, both induction of TS activity (by ILP) and inhibition of TS activity (by HAI) were observed, while i.p. injection did not cause major changes. TS activity and FdUMP binding to TS in bone marrow was strongly inhibited after administration of 5FU by all routes, but administration by ILP seemed slightly advantageous, since a smaller extent of TS inhibition was observed compared to the other routes of administration. 5FU given by ILP had a small antitumour effect in this colon tumour model, while HAI administration had no antitumour activity. Since this difference in antitumour activity could not be related to differences in TS inhibition in the tumour, the RNA-directed mechanism of action of 5FU could be involved. Focusing on the effects of TS, we may conclude that the ILP administration of 5FU offered the important advantage of a lack of severe TS inhibition in normal tissues, which corresponds with the low systemic toxicity observed.

Evaluation of immunohistochemical staining and activity of thymidylate synthase in cell lines

Levels of the enzyme thymidylate synthase (TS) in tumour tissue play an important role in the outcome of chemotherapy with drugs directed against TS. High TS levels may be predictive for resistance against these drugs1. It has been shown, both in vitro and in vivo that high intrinsic TS levels correlate with resistance to 5-fluorouracil (5FU)2,3. Moreover exposure of several neoplastic cells and tissues to 5FU cause a marked increase of TS levels4,5. The common methods to measure TS in cells and tissues are enzyme assays on crude extracts6. The FdUMP binding assay determines the number of free FdUMP binding sites, while the tritium release assay, based on the conversion of [5-3H]-dUMP into dTMP and 3H2O, provides data about the catalytic activity of TS. These assays require about 2.107 cells. With a polyclonal antibody, raised against recombinant human TS (ahTS)7 the detection of TS protein levels in smaller numbers of cells is possible, either with ELISA or immunohistochemical (IHC) staining methods

Potentiation of 5-fluorouracil induced inhibition of thymidylate synthase in human colon tumors by leucovorin is dose dependent.

The schedule and dose dependency of leucovorin (LV) administration in order to potentiate the antitumor activity of 5-fluorouracil (5FU) is still a unclear issue.1,2,3 Traditionally two schedules for single agent of 5FU were used; weekly administration at 500 mg/m2 and daily (x 5) administration at 300-500 mg/m2, which was repeated every 4 weeks. These two schedules of 5FU have been combined with a variety of LV schedules, which can roughly be divided in high dose (2 hr infusion of LV at 500 mg/m2 with 5FU injected mid-infusion),4 intermediate dose (2 hr infusion of LV at 200 mg/m2)5,6 and low-dose LV (bolus injection of LV at 20-25 mg/m2 injected just before 5FU).6,7 LV has also been administered at similar doses but in different schedules. The response rates at the high and intermediate doses was almost always higher (about 30%) than for single agent 5FU, but for the low dose (as well as for oral LV) a larger variation in response rates was observed.6,7 At a recent symposium on modulation of 5FU8 a concensus was achieved that exposure to LV should be 2 hrs or longer in order to facilitate accumulation of polyglutamates of the reduced folate cofactor. The dose of LV remains un unresolved issue, although it was clear from preclinical studies that at least a concentration of 1 μM should be present in the culture medium in order to achieve modulation of 5FU.9 Although both the intermediate and the high dose of LV result in plasma concentrations of /-LV above this levels10 this does not provide information on the intra-tumoral concentration of LV and the reduced folate cofactor 5,10-methylene-tetrahydrofolate (CH2-THF).

Comparison of Continuous Infusions and Bolus Injections of 5.Fluorouracil with or without Leucovorin: Implications for Inhibition of Thymidylate Synthase

Although 5-fluorouracil (5FU) is in clinical use for more than three decades, it is not clear what is the most efficacious schedule of 5FU administration [1, 2]. For systemic treatment with 5FU a number of different schedules are in clinical use (Table 1). The two major schedules for bolus injections do not show significant differences in either toxicity or antitumor activity. For the prolonged administration schedules, however, the pattern of toxicity changes. For weekly bolus injections, myelotoxicity (mainly leukopenia) is usually dose-limiting; however, at the prolonged administration schedules the pattern of toxicity changes with mucositis and diarrhea as serious site-effects for continuous infusions. In addition, the handfoot syndrome is frequently observed at protracted infusions of several weeks, as well as other skin lesions (see ref. in Table 1). Interestingly the addition of LV to both the bolus injections and the continuous infusions, caused a marked increase in gastrointestinal toxicity. In several schedules this increase of toxicity was the only result of the modulatory agent, with no apparent effect on the antitumor activity.

Role of genomic factors beyond thymidylate synthase in the prediction of response to 5-fluorouracil

ABSTRACT 5-Fluorouracil (5FU) is still a major drug in combinations regimens for the treatment of colorectal cancer (CRC) both in the adjuvant and palliative setting. 5FU or its oral prodrug capecitabine is usually combined with irinotecan/oxaliplatin and the novel agents bevacizumab/cetuximab. Although this improved the outcome, the overall prognosis in patients with metastasized disease is still relatively poor. Although the target for 5FU, thymidylate synthase was shown to have a predictive value, this could only predict response in a subset of patients. Given the heterogeneous and complex nature of CRC, it is likely that other aberrations can affect therapeutic response. As an alternative, we investigated Copy number alterations using oligonucleotide-based high-throughput array-comparative-genomic-hybridization (aCGH) to obtain an unbiased screening of the whole genetic spectrum. Chromosomal aberrations have been identified in 85% of CRC patients and include genomic regions harboring copy number alterations in the DNA. These alterations may change the expression of many genes and might explain the differential response to therapy as shown in recent studies with several 5FU combinations. In order to clarify new predictive parameters for 5FU, we used aCGH in a historical cohort of patients, which received treatment with single agent 5FU, and an unsupervised clustering analysis showed a statistical (p < 0.05) difference between responding and nonresponding patients. We also find that several regions showed differences between responders/non-responders, such as losses in 12p12.3–12q15 and in 18p (where TS is located) in responding patients. Genome-wide analysis may provide an additional tool to discriminate between responders and nonresponders.

5-Fluorouracil Induction of Fas and Apoptosis in Colon Cancer Patients: Relation of Clinical Outcome with Thymidylate Synthase, Mcl-1 and Rb

Thymidylate synthase (TS), catalyses the methylation of dUMP to dTMP with 5,10-methylene-tetrahydrofolate (CH2-THF) (1, 2). The active metabolite of 5-fluorouracil (5-FU), FdUMP forms a ternary complex with TS and CH2-THF leading to TS inhibition, growth arrest and cell death (1), which is stimulated by leucovorin (LV) (3, 4). Resistance to 5-FU is not only related to high TS levels, but also to mechanisms involved in cell growth inhibition or programmed cell death (3, 4, 5). Cell cycle progression is controlled by p53 (6), p21 (7), Rb and E2F (8). Drug treatment induces DNA damage, and p53 activates p21 to initiate cell cycle arrest. The tumor suppressor gene Rb forms a complex with E2F inhibiting their transcriptional activity (8). After phosphorylation of Rb by specific kinases, cell cycle progression can be activated by release of E2F1 and E2F2.