Enhancement of the antitumour activity of 5-fluorouracil (5-FU) by inhibiting dihydropyrimidine dehydrogenase activity (DPD) using 5-chloro-2,4-dihydroxypyridine (CDHP) in human tumour cells

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Abstract

The purpose of this study was to evaluate the use of 5-chloro-2,4-dihydroxypyridine (CDHP), a potent inhibitor of dihydropyrimidine dehydrogenase (DPD), to enhance the antitumour activity of the fluoropyrimidines. In an in vitro study, CDHP did not influence cell proliferation by itself. However, CDHP did inhibit 5-fluorouracil (5-FU) degradation and enhanced 5-FU cytotoxicity in a concentration-dependent manner in two human tumour cell lines (MIAPaCa-2 and HuTu80) with relatively high basal DPD activity. CDHP exhibited a maximum effect at a molar ratio (CDHP:5-FU) of more than 0.2. However, CDHP did not have any effect on 5-FU cytotoxicity in the CAL27 tumour cell line, which has a relatively low basal DPD activity, even at concentrations where the DPD activity is almost completely inhibited. In an in vivo study, the maximal tolerable doses (MTD) of tegafur (FT) and a combination of FT and CDHP at a molar ratio of 1:0.4 (FT/CDHP) for nude mice were determined by oral administration for 14 consecutive days. After a single oral administration of either FT or FT/CDHP at the MTD, the 5-FU serum concentration–time profiles were almost the same for both treatment strategies. When nude mice bearing subcutaneous (s.c.) MIAPaCa-2 cells were treated with either FT or FT/CDHP at the MTD, the FT/CDHP treatment showed a significantly higher antitumour effect than the FT treatment (tumour growth inhibition: FT/CDHP, 51±12%; FT, 21±25%; P<0.05). However, the host-body weight suppression induced by FT/CDHP and FT was equivalent. These findings suggest that the combination of fluoropyrimidine and CDHP for the treatment of tumours with a high basal DPD elicits a greater antitumour effect than treatment with fluoropyrimidines alone and we suggest that CDHP inhibits the degradation of 5-FU in the tumour.

Introduction

In mammalian systems, 5-fluorouracil (5-FU) is catabolised to α-fluoro-β-alanine (FBAL), losing its cytotoxicity in the process. Catabolism occurs mainly in the liver via three enzymes: dihydropyrimidine dehydrogenase (DPD, EC1.3.1.2), dihydropyrimidinase (EC3.5.2.2), and ß-ureidopropionase (EC3.5.1.6). DPD is the rate-limiting enzyme in the catabolic process. Recently, encouraging clinical results have led to the development of a new generation of oral fluoropyrimidines, commonly referred to as DPD inhibitory fluoropyrimidines (DIF), that specifically target DPD 1, 2.

Recently, we have developed S-1, a DIF consisting of tegafur (FT), 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate (Oxo) in a molar ratio of 1:0.4:1 [3]. FT, which is a prodrug of 5-FU, functions as an effector. Both CDHP and Oxo, which do not exhibit antitumour activities by themselves, act as modulators. CDHP competitively inhibits DPD approximately 180 times more effectively than uracil in vitro[4], leading to the retention of 5-FU in the blood for a prolonged period 3, 5, 6, 7. Oxo, which competitively inhibits the conversion of 5-FU to 5-fluorouridine 5′-monophosphate by orotate phosphoribosyltransferase (EC2.4.2.10), is mainly distributed in the gastrointestinal (GI) tract after oral administration in rats, leading to the relief of the GI toxicity induced by 5-FU 3, 8. In Japan, S-1 is used clinically for the treatment of gastric and head and neck tumours.

Etienne and colleagues were the first to discuss the clinical relationship between intratumoral DPD levels and the antitumour effect of 5-FU [9]. They treated 62 head and neck cancer patients with biomodulated 5-FU. In each case, a tumoral and non-tumoral biopsy sample was obtained before treatment, and cytosolic DPD activities were measured. Patients with a complete response to the treatment exhibited a significantly lower tumoral/non-tumoral DPD activity ratio than partial or non-responding patients. Moreover, several clinical studies have indicated that the tumoral expression level of DPD is associated with the tumour's response to 5-FU in colorectal [10], gastric 11, 12, and non-small cell lung [13] cancer patients.

Some in vitro studies have discussed the regulation of tumoral DPD activity as a way to biochemically modulate 5-FU. Eniluracil (EU), which is a potent irreversible DPD inhibitor 14, 15, enhanced 5-FU cytotoxicity in five human cancer cell lines with a high basal DPD activity [16]. We previously demonstrated that uracil inhibited 5-FU degradation and enhanced 5-FU cytotoxicity in a concentration-dependent manner in two human tumour cell lines with high basal DPD activities [17]. However, whether the regulation of intratumoral DPD activity can enhance 5-FU antitumour activity in vivo remains uncertain. Thus, we firstly measured basal DPD activities in 31 human cell lines in culture to select a suitable cell line with high basal DPD activity, because downregulation of DPD appears to occur in culture [18]. Only three cell lines had DPD activities greater than 100 pmol/min/mg protein, including MIAPaCa-2 and HuTu80. We used these cell lines to examine the effects of CDHP on cytosolic 5-FU catabolism and the enhancement of 5-FU cytotoxicity in human cancer cell lines and the relationship between these two effects. Furthermore, we investigated the effect of CDHP on the antitumour activity of fluoropyrimidines in a nude mice model by comparing the maximum antitumour effects of FT and FT/CDHP treatments.

Section snippets

Drugs

5-FU was purchased from Wako Pure Chemical Industries (Osaka, Japan); FT and CDHP were synthesised by Taiho Pharmaceutical Co. (Tokyo, Japan); all other chemicals were of the highest standard grade commercially available.

Tumour cell lines

The human pancreas carcinoma line MIAPaCa-2 [19] was purchased from Dainippon Pharmaceutical Co. (Osaka, Japan); the human duodenum adenocarcinoma line HuTu80 (ref. HTB-40) and the human tongue squamous cell carcinoma line CAL27 (ref. CRL-2095) [20] were obtained from the

Inhibitory effect of CDHP on 5-FU catabolism in human tumour cells

We determined the DPD activity of human tumour cells in vitro. As shown in Table 1, the basal DPD activities in both the MIAPaCa-2 and HuTu80 cells were comparatively high, while those in CAL27 cells were comparatively low. The inhibitory effect of CDHP on 5-FU degradation in vitro was investigated using cytosol samples obtained from these cells. Fig. 1 shows the ratio of 5-FU degradation in a DPD reaction mixture treated with various concentrations of CDHP and compared with an untreated

Discussion

We previously evaluated uracil as a modulator that enhances the cytotoxicity of 5-FU using MIAPaCa-2 and HuTu80 cells [17]. Uracil enhanced 5-FU cytotoxicity in a concentration-dependent manner similar to that of CDHP. The maximum effect of uracil was almost the same as that of CDHP, although the uracil treatment required a 100-fold higher concentration than that of CDHP. Fischel and colleagues reported that 1 μM of EU enhanced 5-FU cytotoxicity 1.6–3.0-fold in five human cancer cell lines with

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