Yutaka Tanaka

Cytokines induce thymidine phosphorylase expression in tumor cells and make them more susceptible to 5′-deoxy-5-fluorouridine

The present study shows that various cytokines such as tumor necrosis factor (TNFα), interleukin-1α (IL-1α), and interferon-γ (IFNγ) make tumor cells much more susceptible to the cytostatic 5′-deoxy-5-fluorouridine (5′-dFUrd) than to 5-fluorouracil (5-FUra) and other cytostaties. These three cytokines increased the susceptibility of human cancer cell lines (COLO201, MKN45 and WiDr) but did not affect that of normal fibroblast WI38 cells. The cytokine mixture induced a 50-fold increase in the susceptibility of COLO201 to 5′-dFUrd, whereas a 12-fold increase and a less than 5-fold enhancement in the susceptibility to 5-FUra and other cytostatics, respectively, were observed. The increased susceptibility would be a result of the induction of thymidine phosphorylase (TdR Pase), which is the essential enzyme for the conversion of 5′-dFUrd to 5-FUra. The cytokine mixture increased TdR Pase activity by up to 47 times and greatly induced its mRNA expression in the cancer cell lines. These results suggest that the therapeutic benefit of 5′-dFUrd would be improved by its use in combination with the cytokines.

Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N4‐pentyloxycarbonyl‐5′‐deoxy‐5‐fluorocytidine, Xeloda™) and its intermediate metabolite doxifluridine [5′‐deoxy‐5‐fluorouridine (5′‐dFUrd, Furtulon®)] to 5‐fluorouracil (5‐FUra) in tumors. In a previous study, we found that several cytostatics were able to up‐regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up‐regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up‐regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5′‐dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up‐regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX‐1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside phosphorylases (PyNPase) including dThdPase and uridine phosphorylase. The preferential up‐regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5′‐dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5‐FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5′‐dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted. Int. J. Cancer 83:127–134, 1999.

Antitumor activity of combinations of anti-HER-2 antibody trastuzumab and oral fluoropyrimidines capecitabine/5′-dFUrd in human breast cancer models

Abstract. The anti-HER-2 antibody, trastuzumab (Herceptin), and the oral fluoropyrimidine, capecitabine (Xeloda), are both effective in breast cancer with different modes of action and toxicity profiles. Therefore, the efficacy in combination therapy of these agents for the treatment of HER-2-overexpressing breast cancer was of interest. An antagonistic interaction in vitro between trastuzumab and 5-fluorouracil (5-FUra) in combination has previously been reported. In the same study, the in vivo antitumor activity of this combination was investigated. However, the results were inconclusive since 5-FUra at the dose used had sufficient antitumor efficacy as a single agent and therefore it was not possible to clarify the potential difference between 5-FUra alone and the combination. In the present study, we investigated the efficacy of trastuzumab in combination with capecitabine or its intermediate metabolite 5′-dFUrd in HER-2-overexpressing human breast cancer cell lines and xenograft models. In vivo antitumor activity of the combination was at least additive, in terms of tumor growth inhibition and tumor growth delay, in human breast cancer models KPL-4 and BT-474. The combination treatment in vivo was superior to the treatment with either single agent alone, even though in vitro treatment with trastuzumab and 5′-dFUrd/5-FUra showed antagonistic antiproliferative activity in these cell lines. The reason for the discrepancy between the in vivo and in vitro results was not clarified. However, observed additive in vivo antitumor activity clearly indicates that the clinical efficacy of the combination of trastuzumab and capecitabine/5′-dFUrd against HER-2-overexpressing breast cancer is worth investigating.

Cytokines induce uridine phosphorylase in mouse colon 26 carcinoma cells and make the cells more susceptible to 5'-deoxy-5-fluorouridine

The antiproliferative activity of 5‐fluorouracil (5‐FUra) and 5′‐deoxy‐5‐fluorouridine (5′‐dFUrd), used in combination with typical cytokines and growth factors, was investigated in mouse colon 26 carcinoma cells. Tumor necrosis factor α (TNFα), interleukin‐1a (IL‐1α), and interferon γ (IFNγ) at low doses showing < 50% inhibition of cell growth by themselves enhanced the susceptibility of the cells to the activity of 5′‐dFUrd. In particular, a mixture of these cytokines greatly enhanced the activity of 5′‐dFUrd and 5‐FUra by up to 12.4‐ and 2.7‐fold, respectively, whereas the activity of other cytostatics was only slightly changed (< 1.5‐fold). Basic fibroblast growth factor also increased the susceptibility, but only to 5′‐dFUrd. This preferential enhancement of the activity of 5′‐dFUrd would be due to induction by the cytokines of uridine phosphorylase (Urd Pase), by which 5′‐dFUrd is converted to 5‐FUra. TNFα, IL‐1α, IFNγ, and a mixture of these factors increased the enzyme activity by up to 3.7‐fold in colon 26 cells. Consequently, the anabolism of 5′‐dFUrd to fluoronucleotides and the incorporation of 5‐FUra into RNA in colon 26 cells were increased by TNFα treatment. In addition, the increase by the cytokine mixture in the susceptibility to 5′‐dFUrd was abolished by an inhibitor of Urd Pase, 2,2′‐anhydro‐5‐ethyluridine. These results indicate that induction of Urd Pase activity by cytokines is a critical event that increases the susceptibility to 5′‐dFUrd.

Murine interleukin‐12 prevents the development of cancer cachexia in a murine model

Murine colon 26 carcinoma causes cachexia even when the tumor burden is small. In this tumor model, murine IL‐12 suppressed the induction of cancer cachexia and also inhibited tumor growth. IL‐12 reduced the serum levels of IL‐6, a cachexia mediator in this model, and alleviated the body weight loss and other abnormalities associated with cachexia, such as adipose tissue wasting and hypoglycemia. The anticachectic activity was observed even at low doses of IL‐12, insufficient to inhibit tumor growth. IL‐12 greatly increased levels of IFN‐γ in the tumor tissue and, to a lesser extent, in the circulation. IFN‐γ given intraperitoneally also prevented cancer cachexia, although it did not reduce IL‐6 levels either in the tumor or in the circulation. In athymic mice bearing the same colon 26 tumor, IL‐12 was no longer anticachectic and did not induce IFN‐γ. These results indicate that the anticachectic activity of IL‐12 is T‐cell‐dependent and results from at least 2 mechanisms, the down‐regulation of IL‐6 and the up‐regulation of IFN‐γ.

Structure of a novel phospholipase C inhibitor, vinaxanthone (Ro 09-1450), produced by penicillium vinaceum

Abstract Vinaxanthone is a novel phospholipase C inhibitor produced by Penicillium vinaceum Gilman and About NR6815. Its structre (MW 576, C28H16O14) has been elucidated as a polycyclic xanthone with poly acidic functional groups based on various NMR studies including HMBC, COLOC, 2D-INADEQUATE and selective 1D-INADEQUATE.

Antitumor and antimetastatic activities of human recombinant interferon alpha A/D

Human recombinant interferon alpha A/D (αA/D) was examined for its antitumor activity in several mouse tumor models using metastatic tumors, such as B16 melanoma F1, BL6 and F10, UV2237m fibrosarcoma, and K1735m melanoma. Therapeutic treatment with αA/D reduced the incidence of pulmonary metastasis and inhibited the tumor growth resulting in an increase of the mean survival time. Since αA/D also showed a prophylactic activity against the metastasis, its antitumor activity was suggested to be due to augmentation of the host defense systems. This was confirmed by the fact that αA/D inhibited thein vivo growth and incidence of pulmonary metastasis of B16 F1 sublines regardless of their sensitivity to the direct antiproliferative activity of the IFNin vitro.

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.

5'-Deoxy-5-fluorouridine improves cachexia by a mechanism independent of its antiproliferative action in colon 26 adenocarcinoma-bearing mice.

SummaryThe cytostatic agent 5′-deoxy-5-fluorouridine (5′-dFUrd) improves cachexia and prolongs survival, suppressing tumor growth in mice bearing large burdens of colon 26 adenocarcinoma. To investigate the mode of this anticachectic action, we isolated colon 26 variants that were resistant to the anticachectic activity in vivo in tumorbearing mice that initially responded to 5′-dFUrd in terms of tumor growth and cachexia but again became cachectic and refractory to the drug after prolonged treatment. The original line and variants were equally susceptible to the antiproliferative action of 5′-dFUrd, and their growth was stopped. However, 5′-dFUrd given to cachectic mice exhibiting large burdens of these variants could not reverse wasting and only slightly prolonged the survival period. These results indicate that the anticachectic activity of 5′-dFUrd is independent of its antiproliferative action and that the survival of colon 26-bearing mice is shorter when the size of the tumors is not reduced to levels below those that cause cachexia.

Restoration by recombinant interferon alpha A/D of host defense systems against tumor in immunosuppressed mice

Recombinant human interferon alpha A/D (αA/D) restored or augmented host defense systems against tumors in immunosuppressed mice. In normal C57BL/6 mice, inoculation of B16 melanoma F1 cells caused few pulmonary metastasis, whereas in mice pretreated with cyclophosphamide (CY) it caused a high incidence of pulmonary metastasis, leading to earlier death than in the normal mice inoculated with the same dose of the tumor. αA/D given after the CY treatment counteracted the deleterious effects of the CY treatment. Since such restorative activity was seen even against the subline of B16 F1 which had been made resistant to its direct antiproliferative effect, αA/D seems to exert its effect indirectly through host defense systems. However, this activity of αA/D in the mice pretreated with CY was abrogated by inoculation of anti-asialo GM1 serum but not by i-carrageenan. The CY treatment reduced NK activity, while α A/D given after the CY treatment restored or augmented the NK cell activity in lung cells and peripheral blood mononuclear cells, but not in spleen cells. These findings suggest that the restoration or augmentation of NK activity in the lung and/or peripheral blood might be the major factor leading to the antimetastatic activity of αA/D in the mice treated with CY.