5’-脱氧氟尿苷联合干扰素对转染TP基因结肠癌细胞株裸鼠实验模型的影响

背景与目的：人结直肠癌细胞基本无胸苷磷酸化酶(thymidine phosphorylase,TP)表达。本实验主要探讨人结肠癌细胞株LS174T转染TP基因后,在裸鼠肿瘤组织中对抗癌药物5’-脱氧氟尿苷(5’-deoxy-5-fluorouridine,5’-DFUR)、氟尿嘧啶(5-fluorouracil,5-FU)和干扰素-α2a(interferon-α2a,INF-α2a)干预效果的影响。方法：以慢病毒为载体转染TP基因至结肠癌细胞株LS174T,得到稳定传代的高TP表达株。将96只BALB/c裸鼠分成2组,分别应用野生型及转染TP基因后的LS174T细胞株接种裸鼠背部皮下,制成结肠癌TP低、高表达模型。每组再随机分为6组,分别应用0.9%NaCl溶液、INF-α2a、5-FU、5-FU+INF-α2a、5’-DFUR和5’-DFUR+INF-α2a干预5 d,2周后处死裸鼠,分离肿瘤称重后,分别应用鼠抗人CD34、血管内皮生长因子(vascular endothelial growth factor,VEGF)和抗PD-ECGF单克隆抗体进行免疫组织化学染色。判定是否有TP表达及对微血管进行计数。结果：在野生型LS174T裸鼠荷瘤模型中,使用抗肿瘤药物的3~6组肿瘤质量与对照组相比均明显降低,抑癌率分别为48%、27%、48%和57%,差异有统计学意义(P<0.05)。其中5’-DFUR组(48%)与5’-DFUR+INF-α2a组(57%)、5-FU+INF-α2a组与5’-DFUR+INF-α2a组(27%与57%)相比,抑癌率差异有统计学意义(P<0.05)。在LS174T-TP细胞株裸鼠模型中,5’-DFUR组和5’-DFUR+INF-α2a组抑癌率分别为27%和48%,而使用5-FU的两组未显示出良好的抗肿瘤效果(7%和3%)。免疫组织化学染色显示,野生型LS174T接种形成的肿瘤组织中TP表达阴性,微血管数目较少,而转染TP基因后,肿瘤组织中TP表达明显上升,微血管密度明显增多(P<0.05)。结论：结肠癌细胞株LS174T转染TP基因后,在裸鼠实验肿瘤模型中,TP表达明显增加,使细胞内激活抗癌药物5’-DFUR的抗癌作用明显增强,联合应用INF-α2a能进一步增强这种抗癌作用。而转染TP基因或联合应用INF-α2a对常规抗癌药物5-FU的抗癌效果无明显影响。     

γ干扰素通过诱导 PD-ECGF/TP表达增强去氧氟尿苷和 5-FU对膀胱癌细胞 RT4的杀伤作用

背景与目的:血小板衍化内皮细胞生长因子 /胸苷磷酸化酶 (platelet-derived endothelial cell growth factor/thymidine phosphorylase,PD-ECGF/TP)是去氧氟尿苷和 5 氟尿嘧啶在体内转化成活性抗癌物质的重要酶,它受到白细胞介素-1、γ干扰素等细胞因子的调节.本研究旨在探讨γ干扰素对 RT4膀胱癌细胞 PD-ECGF/TP表达的调节作用及其与去氧氟尿苷和 5 氟尿嘧啶抗癌作用的关系.方法:分别用 RT-PCR和 Western blot检测膀胱癌细胞 RT4中 PD-ECGF/TP mRNA和蛋白质的表达, MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetho-xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]法测定去氧氟尿苷和 5 氟尿嘧啶对 RT4膀胱癌细胞的细胞毒作用.结果:γ干扰素能增强 RT4膀胱癌细胞 PD-ECGF/TP mRNA和蛋白质的表达,γ干扰素与去氧氟尿苷和 5 氟尿嘧啶分别合用,能使去氧氟尿苷和 5 氟尿嘧啶的 IC50分别从( 9.7± 0.2) mmol/L和 (130.0± 21.2)μ mol/L降至 (3.7± 0.9)mmol/L和 (49.3± 18.4)μ mol/L(P< 0.01).结论:γ干扰素通过诱导 RT4膀胱癌细胞表达 PD-ECGF/TP增强去氧氟尿苷和 5 氟尿嘧啶的抗癌作用,提示联合应用γ干扰素和去氧氟尿苷或 5 氟尿嘧啶可能提高膀胱癌的化疗效果.     

Overexpression of pyrimidine nucleoside phosphorylase enhances the sensitivity to 5'-deoxy-5-fluorouridine in tumour cells in vitro and in vivo

5-Fluorouracil (5-FU) and 5'-deoxy-5-fluorouridine (5'-DFUR), a prodrug of 5-FU, are representative of the chemotherapeutic agents for colorectal adenocarcinomas. Pyrimidine nucleoside phosphorylase (PyNPase) catalyses the conversion of 5'-DFUR to 5-FU, the activated form. Murine adenocarcinoma CT26 cells were transfected with human PyNPase cDNA. The engineered transfectants producing PyNPase augmented the response to 5'-DFUR in vitro and in vivo. Animals were administered by means of intraperitoneal (i.p.) injection, and not orally, in order to obtain a better efficiency of absorption. The tumours of the transfected cells nearly all disappeared, even following treatment with quite a small amount of the anticancer agent. The animals injected with the tranfected cells were protected against subsequent challenge with the parental tumour cell line. These findings demonstrate that PyNPase gene transfection increases the sensitivity to 5'-DFUR, and thereby decreases the toxicity of the agent.     

Thymidine phosphorylase expression correlates with malignant potential and anti-tumor effect of doxifluridine on gastric cancer: multivariate analysis for adjuvant chemotherapy doxifluridine vs. 5-fluorouracil

Doxifluridine (5'-DFUR) is an anticancer drug converted to 5-fluorouracil (5-FU) by thymidine phosphorylase (TP). TP is an angiogenetic and platelet-derived endothelial cell growth factor. We evaluated the relation between TP expression and chemotherapeutic efficacy and prognosis for gastric cancer. Advanced gastric cancer patients given oral adjuvant chemotherapeutics either 5'-DFUR; 163 patients or 5-FU; 162 patients were examined. TP expression was assessed with immunohistochemical staining. Multivariate analysis for influencing survival was done, employing variables such as gender, age, procedure, tumor size, location, Borrmann type, histologic factors [type, depth of invasion, lymph node metastasis (n), lymphatic invasion (ly), and venous invasion (v)], drug administered, and TP expression. In the patients with serosal invasion, 5'-DFUR in TP positive was an independent prognostic factor (risk ratio, 4.450; 95% confidence limit, 2.099-9.436), indicating significantly improved prognosis over the 5-FU group. In TP negative, n and ly were independent prognostic factors, but the survival curves of the two chemotherapeutic groups were not significantly different. TP expression was not prognostic factor in 5'-DFUR group, while, in 5-FU group, TP expression was an independent prognostic factor (2.834, 1.467-5.476). In conclusion, it was suggested that TP positive gastric cancer with serosal invasion increased malignant potential of the tumor and 5'-DFUR efficacy.     

Thymidine phosphorylase expression is useful in selecting adjuvant chemotherapy for stage III gastric cancer

Thymidine phosphorylase (TP) is an enzyme which converts doxifluridine (5'-DFUR) to 5-fluorouracil (5-FU). To assess whether TP expression is useful for selecting adjuvant chemotherapy in advanced gastric cancer, we compared effects of oral 5'-DFUR and 5-FU and assessed correlation between drug efficacy and TP expression level. We examined TP expression in 286 patients. TP expression was assessed with immunohistochemical staining. When we compared prognosis in two chemotherapy groups with high TP expression, better survival was observed in 5'-DFUR than in 5-FU group (p=0.0413). Especially in stage III, patients with high TP had better survival in 5'-DFUR than in 5-FU group.     

Thymidine phosphorylase expression and efficacy of adjuvant doxifluridine in advanced colorectal cancer patients

To clarify the correlation between the expression level of thymidine phosphorylase (TP) and efficacy of doxifluridine (5'-DFUR) and 5-fluorouracil (5-FU), samples from 177 colorectal cancer patients who underwent curative resection were evaluated by immunohistochemical staining using a newly developed monoclonal antibody 1C6-203. Patients were randomly given either oral 5'-DFUR or 5-FU as postoperative adjuvant chemotherapy. In Dukes' C staged colon cancer patients treated with 5'-DFUR, better survival was observed in the high TP patients than the low TP patients (P=0.025 by the log-rank test). The observed 5-year survival rates were 91.2 and 74.8%, respectively. No correlation between TP expression and patient prognosis was detected in the 5-FU group. In Dukes' C stage colon patients with high TP expression, the 5'-DFUR group had slightly better survival than the 5-FU group. These findings suggest that TP may be a chemosensitive marker for 5'-DFUR as postoperative adjuvant chemotherapy for advanced colon cancer patients.     

Comparative Antitumor Activity of 5-Fluorouracil and 5'-Deoxy-5-fluorouridine in Combination with Radiation Therapy in Mice Bearing Colon 26 Adcnocarcinoma

The present study compared the antitumor activities of chemotherapy with 5-fluorouracil (5-FU) and with its prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in combination with radiotherapy on a solid colon 26 adenocarcinoma in the mouse. A single administration of 5'-DFUR immediately after local irradiation on day 10 after tumor inoculation produced more than additive antitumor effects, while only an additive effect was observed in the combined treatment with 5-FU and radiation. This over-additive effect of 5'-DFUR was more obvious in a fractionated-dose treatment schedule, where the same combined modality treatment was given three times on days 6, 10 and 14 after inoculation of the tumor cells. 5'-DFUR enhanced the radiation effects on the tumor in terms of the delay in tumor growth as well as the increase in the survival time. 5-FU produced only a marginal additive antitumor effect. Furthermore, radiation damage to normal tissues (skin damage by local irradiation and bone marrow and spleen damage by whole-body irradiation) was not enhanced by 5'-DFUR, though radiation damage to the thymus was additive. On the other hand, 5-FU produced toxic effects that were additive for all normal tissues tested. Thus, at doses that were the most effective against tumors, relative therapeutic gain factors (the ratio of the effect on tumors to that on the bone marrow) of 5'-DFUR and 5-FU were 1.24 and 0.49, respectively. These results suggest that 5'-DFUR will have a greater potential than 5-FU in combined modality treatment of cancer patients.     

Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.

Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5''-deoxy-5-fluorouridine (5''-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2''-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5''-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line (1 microM dThd increased the IC50 value 1000-fold), but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 (1 microM dThd raised the IC50 value 3-fold). We observed a substantial ''bystander'' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5''-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5''-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.     

Thymidine phosphorylase and dihydropyrimidine dehydrogenase levels in primary colorectal cancer show a relationship to clinical effects of 5'-deoxy-5-fluorouridine as adjuvant chemotherapy

Thymidine phosphorylase (TP) converts 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine), an intermediate metabolite of capecitabine, to 5-fluorouracil (5-FU). While dihydropyrimidine dehydrogenase (DPD) catalyzes 5-FU to inactive molecules. We investigated TP and DPD levels in tumor tissue to assess their clinical significance as indicators for selecting colorectal cancer patients for 5'-DFUR adjuvant chemotherapy. A total of 88 colorectal cancer patients were classified into Dukes' B and C groups and treated for 2 years with oral 5'-DFUR (800 mg/body/day). During the follow-up period, 20 of the 88 patients developed a recurrence. All the patients were examined retrospectively for primary tumor TP and DPD levels and clinical response to 5'-DFUR. Results showed that: a) median levels of TP and DPD in the primary tumor, measured by enzyme-linked immunosorbent assay (ELISA), were, respectively, 43.6 and 32.3 U/mg protein. Primary tumor TP levels of the 20 patients who had a recurrence were lower than those of the 68 patients with no recurrence (p=0.07); b) although there were no significant differences in clinicopathologic features between high and low median TP level groups, disease-free survival was better in the high TP than in the low TP group (89% vs. 64%, at year 4); and c) of patients classified into 4 groups such as high TP/DPD, high TP but low DPD, low TP but high DPD, and low TP/DPD, patients with high TP but low DPD had the best disease-free survival, whereas the low TP but high DPD group had the worst survival. These results suggest that TP and DPD levels in primary colorectal tumors may be a useful indicator for selecting patients likely to respond to 5'-DFUR adjuvant chemotherapy and probably capecitabine, a prodrug of 5'-DFUR.     

Prognostic and predictive value of thymidine phosphorylase activity in early-stage breast cancer patients

The antitumor effects of 5-fluorouracil (5-FU) and its derivatives depend upon the activity of nucleoside metabolic enzymes in tumor tissues. Thymidine phosphorylase (TP) converts 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine, to 5-FU. The relationship between TP expression in tumor tissues and patient survival was retrospectively examined in early-stage breast cancer patients treated with either oral 5'-DFUR administered for 6 months or surgery alone in a prospective randomized controlled trial. Thymidine phosphorylase expression in tumor cells and tumor-associated stromal (TAS) cells was examined by immunohistochemistry in 650 tissue samples from patients in this trial (n = 1217). Eight-year follow-up data showed that high TP expression in tumor cells was a significant prognostic indicator of a favorable outcome only for the patients in the 5'-DFUR group. Thus, TP expression was shown to be a predictive factor of 5'-DFUR efficacy. Conversely, a low TP expression in TAS cells was also a potent favorable prognostic indicator. These results on TP status in 2 tumor cell types could provide novel information for predicting prognosis for a patient subgroup, which would receive a probable therapeutic effect from 5'-DFUR, and presumably, from adjuvant therapy of capecitabine in early-stage breast cancer. Determination of TP status might also identify a patient subgroup whose prognosis is quite favorable even without adjuvant therapy. Further investigations on prognostic and predictive implications of TP activity in a clinical setting are warranted.     

Comparative Antitumor Activity and Intestinal Toxicity of 5'-Deoxy-5-fluorouridine and Its Prodrug Trimethoxybenzoyl-5'-deoxy-5-fluorocytidine

N⁴-Trimethoxybenzoyl-5'-deoxy-5-fluorocytidine (Ro 09–1390), a prodrug of the cytostatic 5'-deoxy-5-fluorouridine (5'-DFUR), was synthesized with the aim of reducing of the dose-limiting toxicity of 5'-DFUR, which is diarrhea. In mice bearing Lewis lung carcinoma, 5'-DFUR given po produced a substantial amount of 5-fluorouracil (5-FU) in the intestinal tract as well as in tumors, where the enzyme pyrimidine nucleoside phosphorylase, essential for conversion of 5'-DFUR to 5-FU, is predominantly located. With the oral administration of Ro 09–1390 only a small amount of 5-FU was formed in the intestine; however, the administration of Ro 09–1390 and 5'-DFUR at the same dose produced similar amounts of 5-FU in tumor tissues. These differences in metabolism were reflected in their toxicity and antitumor efficacy. The administration of 5'-DFUR resulted in damage to the intestinal mucosal membrane and diarrhea in normal mice, whereas Ro 09–1390 was much less toxic to the intestinal tract. As regards antitumor activity, Ro 09–1390 and 5'-DFUR at equivalent doses inhibited the growth of Lewis lung carcinoma to similar extents. Since Ro 09–1390 was much less toxic to the intestinal tract than 5'-DFUR, mice bearing Lewis lung carcinoma could be given Ro 09–1390 daily over a longer period and at a higher dose, resulting in a longer survival time.     

Mechanism and possible biochemical modulation of capecitabine (Xeloda), a newly generated oral fluoropyrimidine

A new 5-FU analog, Capecitabine (Xeloda; N-[1-(5-deoxy-b-D-ribofuranosyl)-5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidyl]-n-penyl carbamate), was generated to decrease the incidence of GI toxicity and to increase the efficacy. Capecitabine is designed as a prodrug of 5'-deoxy-5-fluorouridine (5'-DFUR), which is clinically used for gastric, breast and colorectal cancer patients undergoing single or combination chemotherapy in Japan. Capecitabine was converted to 5'-DFUR by either human carboxyestelase or cytidine deaminase, which were mainly localized in human liver. 5'-DFUR was converted to the active form of 5-FU by thymidine phosphorylase (dThdPase) in human tumors. The expression of dThdPase was higher in malignant tumors than in noninvolved normal tissues. In this regard, a high concentration of either 5'-DFUR or 5-FU in malignant tumors may be obtained by oral administration of Capecitabine. In addition, in vivo study showed synergistic or additive effects of Capecitabine combined with anti-cancer agents (Taxanes, Mitomycin C or Cyclophosphamide), cytokines, growth factors and hormonal agents. Capecitabine may be biochemically modulated by those agents in vivo. In the results of an early phase II study on breast cancer patients in Japan, a high efficacy rate and low toxicity were observed. Also, Capecitabine was already registered as 2nd- or 3rd-line treatments for breast cancer patients by the Food & Drug Administration of the USA. Capecitabine is one of the most promising orally administered 5-FU analogs.     

In-vitro effect of a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) on human gastric cancer cell lines: timing of cisplatin treatment

Abstract. Background: To date, we have few effective chemotherapeutic agents against advanced and recurrent gastric cancer. 5-Fluorouracil (5-FU) and cisplatin (CDDP) are the most widely used drugs, and their combination has demonstrated favorable outcomes. However, the method (especially the timing) of CDDP administration is not well established. Methods: We examined the in-vitro effect of a combination of 5-FU and CDDP on four human gastric cancer cell lines: MKN-1, MKN-28, MKN-45, and MKN-74. The cell lines were exposed to 50% of the inhibitory concentrations of 5-FU and CDDP for 72 h and 8 h, respectively. CDDP was applied before, simultaneously with, and after the start of treatment with 5-FU. Results: When CDDP was applied after 5-FU, the cytotoxic activity against MKN-28, MKN-45, and MKN-74 was significantly potentiated. Against MKN-1, the earlier the initiation of CDDP treatment, the stronger was the cytotoxic effect. Conclusion: Our results suggest that the cytotoxicity of a combination of 5-FU and CDDP against human gastric cancer cells is both cell-line- and schedule-dependent and is especially affected by the timing of the CDDP treatment. Received: August 22, 2001 / Accepted: December 7, 2001     

Antitumor effects of 5'-deoxy-5-fluorouridine (5'-DFUR) against various murine tumors in combination with recombinant interferon alpha or cytostatics

Antitumor effects of oral fluorinated pyrimidines including 5'-DFUR and their combination with recombinant interferon (rIFN) alpha A/D (Bgl) or cytostatics were investigated using various tumor models in mice. 5'-DFUR was more effective against Colon 26 than tegafur and 5-FU as a single agent, and their therapeutic indices were 14.7, 2.3 and 1.2, respectively. In combination with various cytostatics, 5'-DFUR as well as 5-FU and tegafur showed better efficacy against mouse Colon 26, Meth A fibrosarcoma and A755 adenocarcinoma than any other single agents. Combination therapy with 5'-DFUR and rIFN-alpha A/D also showed better efficacy against all seven murine tumors tested than their single therapy. In these combination therapies, increased toxicities measured by a body weight change and the incidence of toxic death were also observed to some extent. Among combinations with fluorinated pyrimidines, those with 5'-DFUR showed the toxicities at a lesser degree than those with 5-FU or tegafur. These results indicate that 5'-DFUR would be useful for the multiple combination chemotherapy like 5-FU and tegafur.     

胸苷磷酸化酶表达与结直肠癌关系研究进展

胸苷磷酸化酶(thymidine phosphorylase, TP)是嘧啶核苷合成与分解过程中的一个关键酶。目前认为TP与血小板源性内皮细胞生长因子(platelet-derived endothelial cell growth factor, PD-ECGF)具有同源性, 其诱导血管形成和抗凋亡的作用与结直肠癌的生长、转移密切相关。同时TP也是使5'-脱氧氟尿苷(5'-deoxy-5-fluorouridine, 5'-DFUR)等(5-fluorouracil, 5-FU)前体药物转化为5-FU的关键酶, 其活性与结直肠癌细胞对氟尿嘧啶类药物的敏感性及靶向治疗密切相关。因TP在肿瘤的生长、转移、治疗和预后方面均有重要作用, 阐明其表达机制具有重要意义。本文将对近年TP在结直肠癌中的表达与肿瘤血管新生及与激活5'-DFUR发挥细胞毒作用、治疗结直肠癌的研究进展进行综述。      

Enhancement of antitumor activity of 5'-deoxy-5-fluorouridine (Furtulon) by taxane in human gastric cancer xenografts

5'-deoxy-5-fluorouridine (5'-DFUR, Furtulon) is activated to 5-fluorouracil (5-FU) by thymidine phosphorylase (dThdPase) highly expressed in many types of tumors. In previous studies, we demonstrated that taxanes (paclitaxel or docetaxel) up-regulated the tumor levels of dThdPase and enhanced the efficacy of 5'-DFUR in human colon and mammary xenograft models. In the present study, combination therapy of 5'-DFUR with taxanes in human gastric cancer xenograft models also showed, at the least, additive anti-tumor activity without significant augmentation of toxicity. Furthermore, paclitaxel up-regulated dThdPase expression in the tumor tissues as confirmed with ELISA and immunohistochemistry. These results suggest taxanes would potentiate the efficacy of 5'-DFUR by up-regulating the tumor levels of dThdPase in gastric xenograft models. Clinical trials of 5'-DFUR in combination with taxane against gastric cancer are warranted.     

The mechanism underlying resistance to 5-fluorouracil and its reversal by the inhibition of thymidine phosphorylase in breast cancer cells

Fluoro-deoxyuridine monophosphate (FdUMP) is an active metabolite of 5-fluorouracil (5-FU) synthesized through two hypothesized pathways: The orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. In the present study, the mechanism underlying 5-FU resistance was investigated, focusing on changes in 5-FU metabolism using MCF-7, 5-FU-resistant MCF-7/5-FUR, MDA-MB-231 and 5-FU-resistant MDA-MB-231/5-FUR breast cancer cells. The amount of FdUMP present following treatment with 5-FU was determined by the density of the upper band of thymidylate synthase detected by western blotting, and its changes were investigated. MCF-7/5-FUR cells exhibited 5-FU resistance (36.6-fold), and showed decreased OPRT (−69.3%) and TK (−42.6%) levels. MDA-MB-231/5-FUR cells also exhibited 5-FU resistance (15.8-fold), and showed decreased TP (−79.0%) and increased TK (+184%) levels. MCF-7/5-FUR and MDA-MB-231/5-FUR cells both showed decreased synthesis of FdUMP by 91 and 86%, respectively. In MCF-7 and MCF-7/5-FUR cells, the synthesis of FdUMP was decreased when 5-FU was combined with an RR inhibitor, indicating that FdUMP was synthesized through the OPRT-RR pathway. The synthesis of FdUMP was decreased when 5-FU was combined with a TP inhibitor in MDA-MB-231 cells and combined with an RR inhibitor in MDA-MB-231/5-FUR cells, indicating that the synthesis pathway of FdUMP was changed from the TP-TK pathway to the OPRT-RR pathway on acquiring resistance to 5-FU. Notably, the synthesis of FdUMP was increased and the resistance to 5-FU was reversed in MCF-7/5-FUR cells (half maximal inhibitory concentration (IC₅₀): 219.9 to 0.093 µM) and MDA-MB-231/5-FUR cells (IC₅₀: 157.3 to 31.0 µM) when 5-FU was combined with a TP inhibitor. In conclusion, the metabolism of 5-FU and the mechanism underlying the resistance to 5-FU differed among cell lines, and inhibition of TP reversed resistance to 5-FU, thus suggesting that the combination of 5-FU and a TP inhibitor may be considered a promising cancer therapy.