Clasina L. van der Wilt

The multilayered postconfluent cell culture as a model for drug screening

New drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern - the multilayered postconfluent cell culture model. Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3-5 days and then start to form multilayers. The initial exponential growth of the culture is followed by a plateau phase when cells reach confluence. This produces changes in the morphology of the cells. For some cell lines, it is possible to observe cell differentiation. A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or viability, which allows semiautomation of the experiments. Several experiments were performed to assess the differences and similarities between cells cultured as monolayers and multilayers, and eventually, compared with the results for solid tumours and some other models such as spheroids. Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanied by a decrease in the expression of cell-cycle-related proteins and a decrease in cellular nucleotide pools. Gene and protein expression of topoisomerase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression of DT-diaphorase. P53 expression increased. Multilayer cultures present distinctive properties for drug transport across the membrane, drug accumulation and retention. In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in multilayers when compared with monolayers, which may be related to a decrease in drug penetration to the inner regions of the multilayers. Alteration of these pharmacodynamic parameters is directly related to a decrease in drug activity. The most powerful application of multilayers is in the assessment of cytotoxicity. Solid tumour cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The data show that multilayers are more resistant to the drugs than the corresponding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g. the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated under hypoxic conditions. Gemcitabine was active against ovarian cancer but not against colon cancer, resembling the in vivo situation. This observation was not evident with monolayer experiments. Another interesting application is the possibility to perform drug combination studies. The combination of gemcitabine and cisplatin proved to produce selective cell kill in H322 cells (non-small cell lung cancer cell line). Neither of the drugs was independently able to produce similar effects. In summary, multilayer cultures are relatively simple three-dimensional systems to study the effect of microenvironmental conditions on anticancer drug activity. The model might serve as a base for a more rigorous secondary in vitro screening.

Biochemical modification of the toxicity and the anti-tumour effect of 5-fluorouracil and cis-platinum by WR-2721 in mice

WR-2721 (ethiofos) was tested on Balb/c mice for its chemoprotective capacity against 5-fluorouracil (5FU) monotherapy. In this combination WR-2721 was not active, but WR-2721 pretreatment allowed an elevation of the cisplatin (CDDP) dose in 5FU/CDDP combination therapy in these mice. Thrombocytopenia caused by the 5FU/CDDP (100 and 7 mg/kg, respectively) therapy was prevented by WR-2721 (200 mg/kg) and a partial protection against leukopenia was observed in C57Bl/6 mice. Various WR-2721/CDDP/5FU combinations were tested on two murine colon tumour models. The best antiproliferative effect against Colon 26 (in Balb/c mice) and the lowest toxicity were found with 5FU (100 mg/kg) and CDDP (5.5 mg/kg) delivered together 30 min after WR-2721 (200 mg/kg). The increased efficacy of WR-2721/CDDP/5FU both in Colon 26 and Colon 38 (in C57Bl/6 mice) compared to single 5FU or 5FU/CDDP treatment at the same dose could not be explained by enhanced inhibition of thymidylate synthase (TS), the 5FU target enzyme. The protection by WR-2721 against toxicity of CDDP/5FU might enable the use of high doses of CDDP in this combination.

18F-radiopharmacokinetics of [18F]-5-fluorouracil in a mouse bearing two Colon tumors with a different 5-fluorouracil sensitivity: A study for a correlation with oncological results

The tissue distribution and biodynamics of 18F-labelled 5-fluorouracil (FU) are described and studied for correlation with its in vivo antitumor activity. The in vivo model consisted of Balb/c mice bearing a FU sensitive (Colon 26-10 carcinoma) tumor in the left and a less responsive (Colon 26 carcinoma) tumor in the right abdominal side of the animal. Distribution and efflux of 18F-label from tumor, blood, and other tissues were determined by obduction at 0.5, 1, 2, 4, and 6 h postintravenous injection. For a comparison, the 18F-labeled 5-fluoro-6-hydroxy and cis-5-fluoro-6-ethoxy uracil adducts were studied in the same in vivo model. For 18F-FU it was found that the 18F-label tumor kinetics rapidly fell into a biphasic mode: a relatively short 18F beta phase (18F t1/2 beta 21 +/- 3 min), linked with the total body metabolic capacity and clearance of the animal, and a longer 18F gamma phase, linked with the intrinsic intratumoral FU metabolism (Colon 26-10: 18F t1/2 gamma 10.3 h; Colon 26: 18F t1/2 gamma 5.6 h). It is proposed that the observed faster 18F efflux of the less responsive Colon 26 corresponds to an enhanced breakdown of 5-fluoronucleotides to 5-fluoronucleosides and subsequent elimination from the tumor cells. It is concluded that on PET scanning, measurement of the dynamic 18F t1/2 gamma and 18F t1/2 beta parameter is of prime importance for an insight in the in vivo tumor biology of a patient.

High-Dose 5-Fluorouracil with Uridine-Diphosphoglucose Rescue Increases Thymidylate Synthase Inhibition but Not 5-Fluorouracil Incorporation into RNA in Murine Tumors

5-Fluorouracil (5FU) shows a steep dose response curve in several experimental systems, but the clinical use of high doses is hampered by the toxic side effects of this drug. Uridine diphosphoglucose (UDPG) rescue allows an increase in the maximum tolerated dose of 5FU in mice from 100 (FU100) to 150 mg/kg (5FU150+UDPG) and the higher dose is more effective than the standard treatment against several tumors. In the present paper we report on the effect of high-dose 5FU on thymidylate synthase (TS) levels and on 5FU incorporation into RNA. In the resistant murine tumor (Colon 26A) high-dose 5FU inhibited TS catalytic activity 8 h after treatment (4-fold; p = 0.00041) and the inhibition persisted until day 3 (p < 10–4). Standard-dose 5FU did not significantly inhibit TS activity. In a relatively sensitive tumor (Colon 26-10), there was no difference in the initial extent of TS inhibition by the two 5FU doses, but TS was still inhibited (2-fold) on day 3 after (5FU150+UDPG) while it was within the normal range after 5FU100. In both tumor types TS activity showed an impressive rebound (3-fold) on days 3–7, and this occurred after both 5FU doses. In Colon 26A, however, a new 5FU injection on day 7 was still able to inhibit TS but not as effectively as the first dose. 5FU incorporation into RNA reached similar peak values (8 pmol/µg RNA) after the two 5FU doses, but the clearance was faster in mice receiving UDPG rescue. We conclude that UDPG does not interfere with the extent of TS inhibition by 5FU, but UDPG allows the use of a higher dose of 5FU resulting in enhanced TS inhibition. UDPG, however, increases 5FU clearance from RNA. In this experimental system the inhibition of TS seems essential in order to obtain a good antitumor activity, while 5FU incorporation into RNA does not seem to play a role in the antitumor activity of 5FU. Since preliminary results indicate that UDPG is well tolerated by patients, the use of higher 5FU doses may improve the response rate of human tumors.

5-Ethyl-2'-deoxyuridine, a modulator of both antitumour action and pharmacokinetics of 5-fluorouracil

Abstract The aim of the present studies was to elucidate the effects and optimal modulatory conditions of 5-ethyl-2′-deoxyuridine (EtdUrd) on the antitumour efficacy, pharmacokinetics and catabolism of 5-fluorouracil (5-FU) on Colon-26 and Colon-38 murine tumours. HPLC and GC-MS techniques were used to measure the concentrations of 5-FU, dihydro-5-fluorouracil, EtdUrd, 5-ethyluracil and uridine in the plasma and that of 5-FU and 5-fluoro-2′-deoxyuridine monophosphate (FdUMP) in the tumours. It was shown that EtdUrd, given 1 h before 5-FU, selectively enhanced the antitumour action of 5-FU, without significantly increasing its toxic side-effects, thus resulting in an approximately three times higher therapeutic index. Pharmacokinetic studies revealed that 1 h after 400 mg/kg EtdUrd administration – i.e. at the time of 5-FU treatment – the plasma concentration of EtdUrd was 269 μM, and that of 5-ethyluracil, as the major metabolite of EtdUrd, was 421 μM. It is of interest that EtdUrd pretreatment did not change the maximal plasma concentration of 5-FU; however, the half-life of the terminal elimination increased from 114.5 min to 171.2 min and thus the mean residence time of 5-FU rose significantly (P < 0.05). After the combined treatment, the maximal concentration of dihydro-5-fluorouracil in the plasma decreased from 61.06 μM to 29.70 μM (P < 0.01). The intratumoral concentrations of 5-FU were 34%–158% higher 6–96 h after the combined treatment than after the single 5-FU treatment. EtdUrd also caused a moderate increase in the intratumoral level of FdUMP. It is noteworthy, that EtdUrd increased the endogenous uridine concentration in the plasma from 18 μM to a maximum of 249 μM, and the level remained high for longer than 6 h. The present studies indicate that EtdUrd enhances the therapeutic index of 5-FU by reducing the catabolism, prolonging the plasma and intratumoral concentrations of 5-FU and, at the same time, offering protection to normal organs by increasing the endogenous uridine level.

Thymidylate Synthase Inhibition Induces P53 Dependent and Independent Cell Death

Thymidylate synthase (TS) is an important target for chemotherapy in colon cancer. It is the rate limiting de novo enzyme for synthesis of thymine nucleotides, one of the precursors for DNA synthesis. TS can be inhibited by several clinically active compounds 1,2 such as 5-fluorouracil (5-FU) and the antifolates (AG337 (Thymitaq, Nolatrexed), ZD1694 (Tomudex, Raltitrexed), LY231514 (MTA or pemetrexed). This will lead to inhibition of DNA synthesis and imbalance in dUTP/dTTP pools which will result in DNA damage. DNA damage can trigger downstream events such as p533, bax 4, Fas receptor and caspase-3 to initiate apoptosis 5. It is however not completely known which mechanisms are involved in the onset of apoptosis after TS inhibition. Therefore, we studied the induction of downstream events after exposure of colon cancer cell lines to 5-FU and antifolates and the role that p53 plays during the process of apoptosis initiation.

Effect of Leucovorin on 5-Fluorouracil Induced Inhibition of Thymidylate Synthase in Patients with Colon Cancer

5-Fluorouracil (5FU) is often used in the treatment of patients with advanced colorectal cancer [1], but most tumors show intrinsic or acquired resistance; the objective response rates are below 20%. Currently leucovorin (LV; 5-formyl-tetrahydrofolate) is added to this therapy leading to improved response rates generally between 30–40% [2]. Resistance against 5FU in patients may be related to a reduced inhibition of thymidylate synthase (TS) by FdUMP, the active metabolite of 5FU [1]. TS catalyzes the conversion of dUMP to TMP for which 5,10-methylene-tetrahydrofolate (CH2-THF) is the methyldonor. Inhibition of TS by FdUMP is mediated by the formation of a covalent ternary complex between FdUMP, TS and CH2-THF. The extent and retention of inhibition of TS, depending on the concentrations of FdUMP and CH2THF, are considered to be limiting factors. In addition, TS can have different characteristics in tumors of different patients, determining whether inhibition by FdUMP is effective or not. It has been demonstrated by others [3,4] and ourselves [5] that TS shows a very large heterogeneity in colorectal cancer; this includes a very large variation in enzyme activity (more than 20-fold) and variations in kinetic properties (affinity of FdUMP and of the natural substrate dUMP for TS). Also different affinities for the folate-cofactor have been demonstrated [6]. Spears et al [7] postulated that a number of these factors may determine resistance to inhibition by FdUMP. Several authors determined inhibition of TS in tumors of patients after treatment with 5FU [7–9]. However, generally the time of biopsies was limited to a few hr after 5FU administration, but never longer than 24 hr. Since a long retention may be a crucial factor in determining the antitumor effect we measured the inhibition of TS in tumors and normal tissues of patients 48 hr after treatment with 5FU. In addition we determined whether enhancement of the intra-tumoral folates pools by administration of LV would lead to a more pronounced inhibition of TS. We not only measured TS with the FdUMP ligand bindings assay but also with the catalytic assay, i. e. the conversion of dUMP to TMP.

Comparison of 5-Fluoro-2’-Deoxyuridine and 5-Fluorouracil in the Treatment of Murine Colon Cancer; Effects on Thymidylate Synthase

The fluoropyrimidines 5-fluoro-2’-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) are both applied in the treatment of patients with colorectal cancer. These agents exert their action after metabolization to the nucleotide level (Fig. 1). The nucleotides 5-fluorouridine triphosphate (FUTP) and 5-fluoro-2’-deoxyuridine triphosphate (FdUTP) may mediate antiproliferative effects by their incorporation into cellular RNA and DNA, respectively. Anabolism to 5-fluorodeoxyuridine monophosphate (FdUMP) causes an inhibition of the key-enzyme of the pyrimidine de novo synthesis, thymidylate synthase (TS)1, 2. FdUrd’s main mechanism of action is thought to be inhibition of TS3, while the action of FUra is incorporation into RNA and TS inhibition in different magnitudes, depending on the schedule4, 5.