Schedule dependency of antitumor activity in combination therapy with capecitabine/5'-deoxy-5-fluorouridine and docetaxel in breast cancer models.

Docetaxel and capecitabine are being prescribed for the treatment of breast cancer. In this study, we tried to identify the optimal administration schedule in combination therapy with these anticancer drugs in human cancer xenograft models. Capecitabine was given p.o. daily for 2 weeks (days 1-14), whereas docetaxel was given i.v. on day 1, day 8, or day 15 in a 3-week regimen to the mice bearing MX-1 human breast cancer xenograft. The combination showed better antitumor efficacy than the monotherapy of either agent in either dosing regimen. However, the most potent and synergistic activity was observed when docetaxel was given on day 8. This potent effect appears to be characteristic of the combination of docetaxel with capecitabine or its intermediate metabolite 5'-deoxy-5-fluorouridine (doxifluridine; 5'-dFUrd). Docetaxel given on day 8 showed a potent effect in combination with 5'-dFUrd, but a much weaker effect was observed in combination with 5-fluorouracil or UFT, a fixed combination of tegafur and uracil. Better efficacy was also observed in the MAXF401 human breast cancer xenograft and in the mouse A755 mammary tumor when docetaxel was given at the middle of the capecitabine or 5'-dFUrd treatment rather than other dosing regimens. In contrast, the efficacy in WiDr human colon cancer xenograft was somewhat better when docetaxel was given on day 1. One possible explanation for the synergy is that docetaxel up-regulates tumor levels of thymidine phosphorylase, the enzyme essential for the activation of capecitabine and 5'-dFUrd to 5-fluorouracil. In fact, docetaxel up-regulated the thymidine phosphorylase levels 4.8- and 1.9-fold in the WiDr and MX-1 models, respectively. However, it did not significantly up-regulate in the MAXF401 and A755 models in which a potent combination effect was observed as well. Other mechanisms, particularly those for the synergy with docetaxel given at the middle during capecitabine/5'-dFUrd administration, would also exist. Based on these observations, clinical studies on the day 8 combination regimen with docetaxel and capecitabine/5'-dFUrd are warranted.

Tumour inoculation site-dependent induction of cachexia in mice bearing colon 26 carcinoma.

Murine colon 26 carcinoma growing at either subcutaneous (s.c.) or intramuscular (i.m.) inoculation sites causes cachexia in mice. Such animals show extensive loss of body weight, wasting of the muscle and adipose tissues, hypoglycaemia, and hypercalcaemia, even when the tumour weight comprises only about 1.9% of carcass weight. In contrast, the same tumour when inoculated into the liver does not cause any sign of tumour-related cachexia even when the tumour becomes much larger (6.6% of carcass weight). Interleukin 6 (IL-6), a mediator associated with cachexia in this tumour model, is detected at high levels both in the tumour tissues and in the circulating blood of mice bearing colon 26 tumour at the s.c. inoculation site. In contrast, only minute levels of IL-6 are detected in the tumour grown in the liver. The colon 26 tumour grown in the liver does not lose its ability to cause cachexia, because the tumour when re-inoculated s.c. is able to cause extensive weight loss and produce IL-6 as did the original colon 26 cell line. Histological studies revealed differences in the composition of tumour tissues: the tumours grown in the subcutis consist of many polygonal tumour cells, extended-intercellular space, and high vascular density, whereas those grown in the liver consist of spindle-shaped tumour cells. Thus, the environment where tumour cells grow would be a critical factor in determining the cachectic phenotype of cancer cells, including their ability to produce IL-6.

[Efficacy of combination chemotherapy of cyclophosphamide and 5'-deoxy-5-fluorouridine in a mammary tumor xenograft model, MX-1].

Efficacy of long-term combination chemotherapy of cyclophosphamide (CPA) and 5'-deoxy-5-fluorouridine (5'-DFUR), both of which have been widely used as chemotherapeutics against breast cancer patients, was examined in a mammary tumor xenograft model, MX-1. 5'-DFUR suppressed the tumor growth over a long period and prolonged the survival, although it did not reduce the initial tumor burden, CPA induced the disappearance of the tumor burden temporarily. However, CPA became inaffective despite continuation of treatment, and induced the recurrence of the tumor. The combination of these two drugs dramatically reduced the tumor burden, and suppressed the recurrence of the tumor over a long period. The tumor recurring during CPA monotherapy was resistant to CPA but susceptible to 5'-DFUR, which could be a reason for the long-lasting activity of the combination therapy. These results indicate that CPA and 5'-DFUR monotherapies have different modes of antitumor activities in the long-term therapy model, and that these drugs in combination would have better therapeutic advantage than each drug given individually.

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-gamma in the tumor tissue and, to a lesser extent, in the circulation. IFN-gamma 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-gamma. 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-gamma.

Establishment and characterization of cachexia-inducing and -non-inducing clones of murine colon 26 carcinoma.

To investigate the mechanism causing cachexia and its association with shorter patient survival, we cloned weight-loss-inducing and -non-inducing sublines of murine colon 26 carcinoma (colon 26). One clone, clone 20, induced substantial weight loss, wasting of adipose tissue and muscle, and hypoglycemia in mice with a minimum tumor burden of 0.3 g (2 g per 100 g body weight). Clone-20-bearing mice had a median survival of 24.5 days with average tumor weight of 0.4 g. In contrast, clone 5 induced neither severe weight loss, wasting of adipose tissue and muscle, nor hypoglycemia; clone-5-bearing mice survived for a median of 70 days with average tumor weight of 12 g. These results clearly indicate that shorter survival is associated with the degree of cachexia and is independent of tumor size. Using this pair of colon 26 clones, we examined mediators of cachexia. Neither TNF alpha, IL-1 alpha nor IFN gamma was detected in the serum of mice bearing either clone, while IL-6 was detected in mice bearing both clones by ELISA and a bioassay. Administration of anti-IL-6 monoclonal antibody (MAb) partially but significantly suppressed cachexia induction in clone-20-bearing mice. These results point to the involvement of IL-6 in experimental cachexia. However, our finding of the presence of IL-6 in the serum of mice bearing clone 5, which does not induce weight loss, clearly indicates that IL-6 is not solely responsible for the induction of cachexia.

Molecular analysis of the cytokine network involved in cachexia in colon 26 adenocarcinoma-bearing mice.

Two clones, one cachexigenic (clone 20) and the other noncachexigenic (clone 5), from a murine colon adenocarcinoma, colon 26 cells, were used to analyze the involvement of immune reactions as well as the cytokine network in cachexia. Clone 20 induced cachexia in nude and SCID mice as well as in normal BALB/c mice, suggesting that lymphocytes played little, if any, role in the process. Both clones failed to express mRNA of interleukin (IL) 1 alpha, IL-1 beta, IL-6, and tumor necrosis factor alpha in vitro with or without the coculture of NIH3T3 cells or spleen cells. However, IL-6 mRNA was selectively detected at the tumor site of clone 20 but not at that of clone 5-bearing mice. In contrast, tumor necrosis factor alpha mRNA was detected at tumor sites and in spleens of only clone 5-bearing mice, suggesting a potential role of IL-6, but not tumor necrosis factor alpha, in inducing cachexia. Anti-IL-6 antibody partially reversed the weight loss induced by clone 20, whereas the continuous infusion of IL-6 failed to cause weight loss, despite being associated with an elevation of a serum acute phase protein. These results suggest that IL-6 is necessary but not sufficient for the induction of cachexia. Both clones expressed IL-6 mRNA in the presence of IL-1 in vitro, and mice bearing either clone expressed IL-1 beta mRNA at the tumor site. Moreover, IL-1 receptor antagonist (IL-1Ra) mRNA was detected at the tumor site of clone 5-bearing mice but not at that of clone 20-bearing mice, suggesting that IL-1Ra might block IL-1 activity to reduce IL-6 production in clone 5-bearing mice. However, the transfection of clone 20 with IL-1Ra cDNA failed to abolish its capacity to produce IL-6 and to cause cachexia. Collectively, additional factor(s) besides IL-1Ra and IL-1 beta may control IL-6 and some other cachexigenic factor production, thereby causing cachexia in this model.

[Antitumor activity of various cytostatics in mice bearing murine advanced tumors].

Antitumor activities of cytostatics such as 5-FU, 5'-DFUR, cyclophosphamide (CPA), ACNU, CDDP, mitomycin C (MMC) and doxorubicin (DXR) were compared in mice bearing four different murine-tumor models at two different stages of the tumor growth. All cytostatics tested suppressed the tumor growth in most of the four tumors, colon 26 carcinoma, UV 2237 fibrosarcoma, Ehrlich carcinoma and Meth A fibrosarcoma, when the tumor sizes were small (early transplant). When given to mice bearing advanced tumors, 5-FU, CDDP, MMC and DXR were effective only at the higher doses, showing toxicity. In contrast, 5'-DFUR was equally effective against both most of early and advanced tumors except for advanced Meth A against which higher doses of 5'-DFUR were needed. CPA and ACNU equally suppressed the growth of early transplant and advanced tumors of Meth A, although higher doses were needed against advanced tumors of three others. 5'-DFUR was also effective against tumor cachexia (colon 26) and spontaneous metastasis (Lewis lung carcinoma), which are characteristically observed in mice bearing advanced tumors. CPA also showed an anticachectic activity, though the activity was weaker than that of 5'-DFUR. These results suggest that 5'-DFUR and CPA can be used in both intensive and adjuvant chemotherapies.