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Effects of a novel vitamin D analogue MC 903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo

MC 903 is a novel vitamin D analogue which has been tested for its effects on cell differentiation and cell proliferation in vitro using the human histiocytic lymphoma cell line U937, and on calcium metabolism in rats in vivo. In the present investigation MC 903 was compared to the natural metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3] and to its synthetic analogue 1 alpha-hydroxycholecalciferol [1 alpha (OH)D3]. MC 903 was found to be a potent inducer of cell differentiation and to inhibit cell proliferation and DNA-synthesis in concentrations comparable to those observed with 1,25(OH)2D3. 1 alpha (OH)D3, which is only active after metabolic conversion to 1,25(OH)2D3, was more than 100 times less potent. Oral or intraperitoneal administration of MC 903 to rats showed that the compound was at least 100 times less active than 1,25(OH)2D3 and 1 alpha (OH)D3 in causing hypercalciuria, hypercalcemia and bone calcium mobilisation. The low vitamin D activity of MC 903 was further confirmed by administration of the compound to rachitic rats. The strong direct effects of MC 903 on cell proliferation and cell differentiation, coupled with its decreased activity as a classical vitamin D makes this compound an interesting candidate for studies in human proliferative disorders such as psoriasis.

20-epi-vitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses.

The 20-epi-vitamin D3 analogues are a novel class of vitamin D3 derivatives, structurally related to 1 alpha,25-dihydroxycholecalciferol (1 alpha,25(OH)2D3). They are characterized by an altered stereochemistry at carbon 20 in the side-chain. In vitro, these new analogues were found to be considerably more potent as regulators of growth and differentiation in the human histiocytic lymphoma cell line U 937 than 1 alpha,25(OH)2D3, despite a practically unchanged calcemic activity in vivo. The most potent analogue, KH 1060, inhibited cell proliferation by 50% at 10(-12) M (14,000 times more active than 1 alpha,25(OH)2D3). At the same time, KH 1060 induced cell differentiation at concentrations as low as 10(-14)M. In addition, the 20-epi-vitamin D3 analogues were found to be very potent inhibitors of T-lymphocyte proliferation induced by interleukin-1 or alloantigen. In this respect, they were several orders of magnitude more active than the potent immunosuppressive agent cyclosporin A (CyA). KH 1060, the most potent analogue, inhibited interleukin-1-induced mouse thymocyte proliferation by 50% at 3 x 10(-16) M and allogeneic stimulation of mouse spleen lymphocytes at 5 x 10(15) M. These effects were considered to be mediated by inhibition of interleukin-2 release from activated T-lymphocytes. The new analogues are of potential interest in the prevention of graft rejection and in the treatment of psoriasis, cancer and auto-immune diseases.

EB1089 : a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro

EB1089 is a novel vitamin D analogue which has been tested for its effects on breast cancer cell growth in vitro, using the established human breast cancer cell line MCF-7, and in vivo on the growth of established rat mammary tumours. Both EB1089 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited MCF-7 cell proliferation with the synthetic analogue being at least an order of magnitude more potent than the native hormone. In vivo anti-tumour effects were investigated using the N-methyl-nitrosourea-induced rat mammary tumour model. Oral treatment with EB1089 was tested at three doses. With the lower dose, significant inhibition of tumour growth was seen in the absence of a rise in serum calcium. The same dose of 1,25-(OH)2D3 had no effect on tumour growth but caused hypercalcaemia. With the higher dose of EB1089, striking tumour regression was seen although serum calcium rose. This report demonstrates that EB1089 possess enhanced anti-tumour activity coupled with reduced calcaemic effects relative to 1,25-(OH)2D3 and thus may have therapeutic potential as an anti-tumour agent.

EB 1089, a novel vitamin D analogue, has strong antiproliferative and differentiation inducing effects on cancer cells

EB 1089 is a novel vitamin D analogue which in vitro strongly inhibits the proliferation of U937 histiocytic lymphoma cells and MCF-7 breast cancer cells, with a potency of 50 to 100 times that of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Studies of c-myc and c-fos expression in MCF-7 cells and of differentiation markers in U937 cells show that growth inhibition by EB 1089 is accompanied by induction of differentiation. The ability of EB 1089 to affect calcium metabolism in vivo in rats is decreased, compared to 1,25(OH)2D3. This low calcemic effect combined with the strong biological effect on cancer cells in vitro, makes EB 1089 an interesting candidate for treatment of cancer.

EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro

1 Effects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea‐induced rat mammary tumours in vivo were investigated. 2 At a dose of 0.5 μg kg−1 body weight, EB1089 caused significant inhibition of tumour progression over the 28 day treatment period in the absence of a significant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 μg kg−1) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death. 3 Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3′ DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 μg kg−1) for 14 days. 4 Effects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF‐7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089. 5 The effects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl‐2 and bax were examined by Western analysis. In MCF‐7 cell cultures treated with 1,25(OH)2D3 or EB1089 (1×10−8 M), bcl‐2 protein levels were decreased in a time‐dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl‐2/bax ratio favouring increased susceptibility of MCF‐7 cells to undergo apoptosis. 6 These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death.

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase.

EB1089, a novel 1,25‐dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI‐H929 human myeloma cells. EB1089 inhibited cell growth of NCI‐H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose‐dependent manner. We could also detect apoptosis in NCI‐H929 cells exposed to EB1089 (1 × 10−7 m for 72 h) using the sub‐G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down‐regulation of the Bcl‐2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089‐treated NCI‐H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose‐ and time‐dependent manner. In addition, EB1089 caused the down‐regulation of p44 extracellular signal‐related kinase (ERK) activity and up‐regulation of the p38 kinase activity during apoptosis of NCI‐H929 cells. These results suggest that EB1089 inhibits growth of NCI‐H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.

Calcipotriol (MC 903): Pharmacokinetics in rats and biological activities of metabolites: A comparative study with 1,25(OH)2D3

Calcipotriol (MC 903) is a novel analogue of the physiologically active metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3]. MC 903 and 1,25(OH)2D3 have similar effects on cell proliferation and cell differentiation in vitro using the human histiocytic lymphoma cell line U 937, but in vivo MC903 has 100-200 times less effect on calcium metabolism. To elucidate this difference, the pharmacokinetic profiles after a single intravenous dose (50 micrograms/kg) of the two compounds to rats were compared. The area under the serum level/time curve (AUC) was more than 100 times higher for 1,25(OH)2D3 than for MC903 and the rate of clearance was more than 100 times higher for MC903 than for 1,25(OH)D3. Serum from MC903 or 1,25(OH)2D3 dosed rats (i.v. 10 micrograms/kg) was investigated for biological activities by incubation of U 937 cells with serum collected 0-24 hr after drug administration. Serum from MC903 dosed rats had an effect only when collected shortly after dosing, whereas serum from 1,25(OH)2D3 dosed rats had an effect when collected up to 4 hr after dosing. The biological effects on the U937 cells of the two major metabolites of MC903 (MC 1046 and MC 1080) were investigated. The metabolites had effects that were more than 100 times weaker than those of the parent compound. The effect of MC903 on proliferative disorders, its fast elimination and the formation of inactive metabolites makes MC903 suitable for topical treatment of psoriasis.

Immunological properties of vitamin D analogues and metabolites.

This commentary has attempted to describe some of the new aspects of our knowledge of the immunological properties of 1 alpha,25(OH)2D3, the physiologically active metabolite of vitamin D3, and its new analogues. These analogues will, in the future, serve as tools to increase our understanding of the role of vitamin D in immunobiology, not only in basal research but also, hopefully, in the therapy of immune-mediated diseases.

Novel cyanoguanidines with potent oral antitumour activity

4-Pyridyl cyanoguanidines with hydrophobic aromatic side chains showed potent antiproliferative activity in the human breast and lung cancer cell lines MCF-7, NYH and H460. In vivo, treatment with N-(6-chlorophenoxyhexyl)-N′-cyano-N″-4-pyridylguanidine (18, 20 mg/kg/day po.), gave a complete remission of tumours in a model of NYH inoculated nude mice.

25-Hydroxyvitamin D3 1α-hydroxylase expression in breast cancer and use of non-1α-hydroxylated vitamin D analogue

IntroductionThe cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1α-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1α-hydroxylated vitamin D analogues to target tissues where 1α-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1α-hydroxylase in breast cancer and to investigate whether a non-1α-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.MethodsThe expression of 1α-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 μm essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells.Results1α-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1α-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1α-hydroxylase, 1 ± 0.07 versus 0.7 ± 0.05, respectively (p < 0.001); 24-hydroxylase, 1 ± 0.08 verus 2.1 ± 0.2, respectively (p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 ± 0.09 and 1.4 ± 0.12 (p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1α-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1α-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.ConclusionThese results demonstrate nearly normal expression levels of 1α-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1α-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1α-hydroxylase is expressed.