1,25-Dihydroxyvitamin D has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of EB 1089, a low calcemic analogue of vitamin D, on the development of osteolytic bone metastases after intracardiac injection of the human breast cancer cell line MDA-MB-231 in nude mice. Animals injected with tumor cells were implanted simultaneously with osmotic minipumps containing either EB 1089 or vehicle. Both groups remained normocalcemic for the duration of the experiment. The total number of bone metastases, the mean surface area of osteolytic lesions, and tumor burden within bone per animal were markedly decreased in EB1089-treated mice. Furthermore, longitudinal analysis revealed that mice treated with EB1089 displayed a marked increase in survival and developed fewer bone lesions and less hind limb paralysis over time as compared with untreated animals. These results suggest that EB1089 may be beneficial in the prevention of metastatic bone lesions associated with human breast cancer.
The purpose of this research was to evaluate theinfluence of the combination of the vitamin D(3) analogue EB 1089 with fractionated radiation on growth and apoptosis of MCF-7 tumor xenografts in athymic mice.Four to six-week-old ovariectomized mice were injected s.c. with MCF-7 tumor cells suspended in Matrigel. When tumors reached a size of approximately 150-200 mm(3), animals were exposed to EB 1089 (45 pmols/day) for 8 days, whereas mice that were to be irradiated in the absence of EB 1089 received solvent (Solutol HS15). After the termination of EB 1089 and solvent administration, tumors were irradiated (3 x 5 Gy) over a period of 3 days using a 300 KV Pantax Therapax irradiator. Tumor growth was monitored for 25-30 days after the last dose of irradiation in a double-blind manner; tumor cellularity was assessed by H&E and trichrome staining, cell proliferation by Ki-67 staining, and apoptosis by terminal deoxynucleotidyltransferase-mediated nick end labeling assay. Rates of tumor regression were assessed using a mixed effects statistical model.A significantly higher rate of decline in tumor volume (7.5% per day) was observed in mice exposed to radiation subsequent to EB 1089 compared with animals treated with radiation alone (5.6% per day). Final tumor volumes in animals irradiated after EB 1089 were approximately 50% lower than in the group that received radiation alone. Loss of cellularity, a marked reduction in the fraction of proliferating cells, and the promotion of apoptosis confirmed that the combination of EB 1089 with radiation was significantly more effective than radiation alone in blocking tumor cell growth and promoting tumor cell death.This work demonstrates that EB 1089 can improve local tumor control by fractionated radiation, in part through the promotion of apoptotic cell death.
Patients with acute promyelocytic leukemia (APL) usually relapse after all-trans retinoic acid (RA) treatment because this therapy fails to eradicate the malignant clone. Our data showed that KH 1060 and other 20-epi vitamin D3 analogs alone were potent inhibitors of clonal growth of NB4 cells, an APL cell line (ED50, approximately 5 x 10(-11) M). The combination of KH 1060 and 9-cis-RA synergistically and irreversibly enhanced this effect. Neither KH 1060 nor 9-cis-RA (10(-6) M, 3 d) were strong inducers of differentiation of NB4 cells. However, 98% of the cells underwent differentiation to a mature phenotype with features of both granulocytes and monocytes after exposure to a combination of both compounds. Apoptosis only increased after incubation of NB4 cells with 9-cis-RA alone (28%) or with a combination of 9-cis-RA plus KH1060 (32%). Immunohistochemistry showed that the bcl-2 protein decreased from nearly 100% of the wild-type NB4 cells to 2% after incubation with a combination of KH 1060 and 9-cis-RA, and the bax protein increased from 50% of wild-type NB4 cells to 92% after culture with both analogs (5 x 10(-7) M, 3 d). Western blot analysis paralleled these results. Studies of APL cells from one untreated individual paralleled our results with NB4 cells. Taken together, the data demonstrated that nearly all of the NB4 cells can be irreversibly induced to differentiate terminally when exposed to the combination of KH 1060 and 9-cis-RA.
Abstract The effects of different antirheumatic drugs administered according to various dosing regimes on tuberculin hypersensitivity in rats have been assessed quantitating the changes of exudate volume and mononuclear cells immigration at the site of challenge. Dosing at the time of sensitization with aurothiomalate and D‐penicillamine enhanced the cell immigration which was decreased by similar treatment with levamisole. Cyclophosphamide increased the exudate formation. Indomethacin had no effect. Dosing at the time of challenge caused a marked reduction of both the parameters by aurothiomalate and cyclophosphamide and enhancement by levamisole. D‐penicillamine increased only the cellular immigration and indomethacin the exudate formation. Long treatment with aurothiomalate and cyclophosphamide suppressed the responses. Similar treatment with D‐penicillamine and levamisole produced a significant enhancement. Indomethacin had no effect. The relevance of these findings to the testing and mode of action of antirheumatic drugs is discussed.
An experimental rat model, the Subcutaneous Air Sac (SAS) model, was developed to provide an animal model in which neo-vascularization can be easily assessed in situ and quantified using a radiolabelled plasma marker. The SAS model was designed to replace a previous model where neovascularization was induced by chemical injury of rat or rabbit cornea or by implantation of tumour cells intracorneally, a methodology which is believed to cause severe pain to the animals. In the SAS model the air sac replaces the cornea as a transparent avascular substratum in which vascularization can be observed. The air sac is induced by injection of air subcutaneously on the back of the animal. After 8 to 10 days a sufficient air sac has been established. The animal is anaesthesized and by a minor operation the cellulose sponge is implanted upon the air sac under the skin. The vasoproliferative effect of the cellulose sponge causes formation of new vessels which are macroscopically visible 10 days after implantation. The ability of the in vivo SAS model to show an antiangiogenic effect of a systemically applied test compound was investigated using the fumagillin analogue TNP-470 (ochloro-acetylcarbamoyl)-fumagillol) as a positive control at dose levels of 0, 1, 2.5, 5 and 10 mg/kg/day given subcutaneously for 10 days. The neo-angiogenesis was scored both in situ using a subjective point system and by measuring the 125I-activity of the implant and the membrane after an intravenous injection of 125I-labelled antibodies. The neo-angiogenesis was reduced by approximately 45-50% in animals treated with 5 or 10 mg/kg/day of TNP-470 compared to animals treated with the vehicle. The animals treated with 10 mg/kg/day TNP-470 showed signs of toxicity. The SAS model is considered highly relevant for in vivo testing of potential antiangiogenic drugs on humane grounds. The high reproducibility, the low cost and the technical simplicity of the method makes it attractive.
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