kh-1060 and seocalcitol

Population studies suggest putative links between vitamin D (VD)-deficiency and risk of cancer and diabetes. The insulin/IGF-I receptor represents a signaling target of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) that is implicated in both diabetes and cancer, therefore we hypothesized that VD actions may be mediated through this adhesion molecule. In this study, we show that 1,25 vitamin D3 and its analogues EB1089 and KH1060 potently inhibit CEACAM1 expression in cancer cells. This effect was associated with significant reductions in mRNA and protein levels, resulting from transcriptional and posttranslational actions respectively. Insulin/IGF-I-mediated IRS-1 and Akt activation were enhanced by VD treatment. Similarly, CEACAM1 downregulation significantly upregulated the insulin and IGF-I receptors and mimicked the effect of VD-mediated enhanced insulin/IGF-I receptor signaling. Despite improved insulin/IGF-I signaling, the anti-proliferative actions of VD were preserved in the absence or presence of forced CEACAM1 expression. Forced CEACAM1, however, abrogated the anti-invasive actions of VD. Our findings highlight CEACAM1 as a target of VD action. The resulting inhibition of CEACAM1 has potentially beneficial effects on metabolic disorders without necessarily compromising the anticancer properties of this vitamin.

Topics: Antigens, CD; Antineoplastic Agents; Blotting, Western; Calcitriol; Cell Adhesion Molecules; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; Receptor, IGF Type 1; Receptor, Insulin; RNA Interference; Signal Transduction; Time Factors; Vitamins

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the hormonal active form of vitamin D3, could represent a potentially therapeutic agent in autoimmune diseases. Cyclosporin A (CsA) shows immunoregulatory properties, which, in many respects, seem to be similar to those of 1,25(OH)2D3. Our aim was to investigate the possible synergistic effect exerted by CsA in combination with 1,25(OH)2D3 or its nonhypercalcemic analogues, EB 1089 and KH 1060, on the proliferative response of T lymphocytes obtained from active ulcerative colitis patients.. The T lymphocyte-enriched population was treated with phytohemagglutinin and CsA (doses from 1 ng to 1000 ng/ml) alone or in association with 1,25(OH)2D3 or EB 1089 or KH 1060 (0.1, 1, 10 nM final concentration). Cell proliferation was determined by [3H]thymidine incorporation and analyzed on day 5 of culture.. After incubation with CsA, T lymphocyte proliferation was significantly inhibited in comparison with the vehicle-treated cultures. However, T lymphocytes from ulcerative colitis patients were significantly more sensitive to CsA than those from healthy controls. The inhibition in T lymphocyte proliferation, after treatment of the cultures with CsA associated with either 1,25(OH)2D3 or EB 1089 or KH 1060, was synergistic at well-defined concentrations.. Taking into account the lowest CsA dose (1 ng/ml), the highest synergistic inhibition in the proliferation of T lymphocytes prepared from ulcerative colitis patients was found combining CsA and 10 nM of 1,25(OH)2D3 or 10 nM of EB 1089 or KH 1060 at the three concentrations. The results obtained, associating the lowest CsA dose and the lowest KH 1060 concentration, may suggest an alternative therapeutic approach in these patients, reducing the dose, and consequently the toxicity, of CsA.

Topics: Adult; Calcitriol; Cell Physiological Phenomena; Colitis, Ulcerative; Cyclosporine; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Female; Humans; Immunosuppressive Agents; In Vitro Techniques; Male; Middle Aged; T-Lymphocytes; Vitamin D

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.

Topics: Alitretinoin; Animals; Biological Transport; Calcitriol; Cell Division; Cells, Cultured; Drug Synergism; Kinetics; Mice; Muscle, Skeletal; Retinoids; Structure-Activity Relationship; Tretinoin; Vitamin D

This study examined the effect exerted by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and two vitamin D analogues, EB 1089 and KH 1060, on the proliferation of T lymphocytes obtained from ulcerative colitis (UC) patients and healthy controls. The proliferative response of T lymphocytes to phytohaemagglutinin treatment was first analyzed on days three, five, and seven of culture. Cell proliferation was significantly lower in UC patients than that observed in healthy controls. The highest proliferation value, in either controls or patients, was registered on day five of culture. On day seven, a decrease in proliferation occurred, less evident in patients with respect to controls, whereas on day three, controls and patients showed the same proliferation value. The response of T lymphocytes of either healthy controls or UC patients to 1,25(OH)2D3, EB 1089, or KH 1060 was then investigated, treating the cells for three, five, and seven days with 10 nM vitamin D derivatives. In the presence of these compounds, cell proliferation was significantly inhibited in both groups, but on day seven, the inhibition of lymphocyte proliferation was remarkable in controls, whereas in patients it was similar to that registered on day five. The highest inhibition values were always obtained in the presence of KH 1060, and the time dependence was continuous in controls, but in the presence of EB 1089 only in patients. T lymphocytes prepared from healthy controls and UC patients were then cultured for five days in the presence of vitamin D derivatives at three different concentrations (0.1, 1, and 10 nM). In the two groups, a dose-dependent inhibition was registered in the presence of 1,25(OH)2D3 or EB 1089, while the inhibition of proliferation exerted by KH 1060 was not dose-dependent. The results obtained suggest an option for the use of the two non-hypercalcemic vitamin D analogues in the therapy of UC patients, perhaps in association with other immunosuppressive drugs.

Topics: Adult; Calcitriol; Calcium Channel Agonists; Cell Division; Colitis, Ulcerative; Dose-Response Relationship, Drug; Drug Interactions; Female; Humans; Immunosuppressive Agents; Lymphocyte Activation; Male; Middle Aged; Mitogens; T-Lymphocytes

The beta-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1alpha,25-dehydroxyvitamin D(3)) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1alpha,25(OH)2D(3) induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of beta-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for beta-catenin binding. Accordingly, 1alpha,25(OH)2D(3) repressed beta-catenin-TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic beta-catenin and reduced by TCF-4. Also, 1alpha,25(OH)2D(3) inhibited expression of beta-catenin-TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor delta, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1alpha,25(OH)2D(3) induces E-cadherin and modulates beta-catenin-TCF-4 target genes in a manner opposite to that of beta-catenin, promoting the differentiation of colon carcinoma cells.

Topics: Active Transport, Cell Nucleus; Adenocarcinoma; Antineoplastic Agents; beta Catenin; Cadherins; Calcitriol; Cell Adhesion Molecules; Cell Differentiation; Cell Membrane; Cholecalciferol; Colonic Neoplasms; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Humans; Ligands; Macromolecular Substances; Phenotype; Protein Binding; Receptors, Calcitriol; RNA, Messenger; Signal Transduction; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Transfection; Tumor Cells, Cultured; Vitamin D

We and others have previously shown that selected vitamin D analogs potentiate the vitamin D receptor (VDR) mediated transcription much more efficiently than the natural hormone itself. Here we show that the transcriptionally active 20-epi analogs, namely KH 1060 and MC 1288, protect VDR against degradation more efficiently than calcitriol at 10(-10) M concentration (VDR t(1/2) > 48 h, 17 h, and 10 h, respectively). The conformationally epi-like analog EB 1089 did not significantly alter the half-life of VDR (10.3 h), but retained the VDR levels over longer periods of time than calcitriol. The transcriptionally weak analog GS 1558, on the other hand, enhanced VDR degradation even more than what was observed with the unliganded receptor (t(1/2) 4.5 h and 5 h, respectively). Inhibition of proteasome activity by the inhibitor MG-132 resulted in a marked increase in the VDR levels in cells treated with the vehicle or GS 1558 (2.5-fold and 2.7-fold, respectively), more than twice the levels observed in the presence of calcitriol or EB 1089 (1.2-fold and 1.1-fold, respectively). MG-132 treatment did not increase the VDR levels in cells treated with KH 1060 or MC 1288. The electrophoretic mobility shift assay (EMSA) with nuclear extracts from MG-132-treated cells revealed formation of a high-molecular-weight RXRbeta-VDR-VDRE complex, which also contained Sug1. In the presence of calcitriol, 34% of total VDR in its DNA binding state was present in this complex. The 20-epi analogs effectively prevented the formation of this complex, since, in this case, only 16% of total VDR was found in this complex. These results suggest that KH 1060 and MC 1288 induce a VDR conformation, which prevents binding of proteins mediating receptor degradation. As a result, the regulation of VDR degradation differs from that found with the calcitriol-VDR complex resulting in superactive transcriptional action of the analogs.

Topics: Calcitriol; Calcium Channel Agonists; Cysteine Endopeptidases; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Leupeptins; Models, Biological; Molecular Conformation; Molecular Structure; Multienzyme Complexes; Nuclear Proteins; Osteoblasts; Osteocalcin; Proteasome Endopeptidase Complex; Protein Binding; Receptors, Calcitriol; Tumor Cells, Cultured; Vitamin D

This study examined the action of 9-cis retinoic acid and 1,25-dihydroxyvitamin D3 analogues (KH 1060, EB 1089 and MC 903) on the release of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in the rat C cell line CA-77. This cell line mainly secretes CGRP. Using radioimmunoassays (RIAs) for CT and CGRP, we measured the release of both peptides in the culture medium as well as the amount of these proteins contained in the CA-77 C cells. 9-cis retinoic acid decreased the release of both CGRP and CT dose-dependently in the range between 1 nM and 1 microM. The half-effective dose was 10 nM. The treatment of CA-77 C cells with 0.1 microM calcitriol alone only slightly decreased the release of both CT and CGRP. The increase in the amount of CT and CGRP released by the action of 1 microM dexamethasone was reduced by 1 microM 9-cis retinoic acid, and this effect was enhanced by the addition of 0.1 microM calcitriol or KH 1060, EB 1089 and MC 903. When the C cells were continuously stimulated by dexamethasone, after 6 days of exposure to the combined treatment with calcitriol analogues + 9-cis retinoic acid, there was a greater decrease in the amount of CGRP contained in the C cells than after treatment with 9-cis retinoic alone. Our data suggested that combined treatment with retinoic acid and calcitriol analogues exerted a stronger inhibition on the amounts of the two peptides either contained in the cells or released in the medium than each hormone alone.

Topics: Alitretinoin; Animals; Calcitonin; Calcitonin Gene-Related Peptide; Calcitriol; Carcinoma, Medullary; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Kinetics; Rats; Thyroid Neoplasms; Tretinoin; Tumor Cells, Cultured

Vitamin D-related compounds can inhibit cancer cell growth, but the biologic mechanism of this inhibition remains to be determined. We investigated the possibility that these compounds interfere with the activity of insulin-like growth factors. Such activity can be suppressed or otherwise modulated by specific insulin-like growth factor-binding proteins.. The human breast cancer cell line MCF-7 was used in this study. The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and two related compounds, EB1089 and KH1060, on cell proliferation were assessed by monitoring cell numbers and by measuring cellular incorporation of [3H]thymidine. Changes in the accumulation of insulin-like growth factor-binding proteins in cell-conditioned media (i.e., culture fluids) were assessed by means of standard protein blotting techniques; ligand blots were probed with [125I]insulin-like growth factor I, and immunoblots were probed with antibodies raised against specific binding proteins. Binding protein messenger RNA levels were determined by use of RNA blotting methods and complementary DNA probes.. At concentrations of 10(-8) M and 10(-9) M, EB1089 and KH1060 exhibited stronger antiproliferative activity than 1,25(OH)2D3. When each of the vitamin D-related compounds was used separately at a concentration of 10(-9) M, a 20- to 25-fold increase in the concentration of insulin-like growth factor-binding proteins in MCF-7 cell-conditioned media was observed; this binding capacity was increased nine-fold, ninefold, and threefold, respectively, in the presence of 10(-10) M EB1089, KH1060, and 1,25(OH)2D3. Immunoblotting experiments demonstrated that all three vitamin D-related compounds induced the accumulation of insulin-like growth factor-binding protein 5 in cell-conditioned media. The accumulation of this binding protein was associated with an increase in cellular expression of its messenger RNA. EB1089 and 1,25(OH)2D3 attenuated the growth-promoting activity of insulin-like growth factor I on MCF-7 cells; however, these compounds did not inhibit the growth-promoting activity of long R3 IGF-I, an insulin-like growth factor I analogue with greatly reduced affinity for insulin-like growth factor-binding proteins.. Our results indicate that vitamin D-related compounds stimulate production of insulin-like growth factor-binding protein 5, thereby indirectly suppressing cell proliferation.

Topics: Antineoplastic Agents; Blotting, Northern; Blotting, Western; Breast Neoplasms; Calcitriol; Cell Division; Female; Humans; Insulin-Like Growth Factor Binding Protein 5; Tumor Cells, Cultured

The ability of the physiologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and two novel vitamin D analogues, EB1089 and KH1060 to induce the differentiation of the U937 and HL-60 leukaemic cell lines was evaluated, alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Studies revealed that following 96 h treatment, the vitamin D derivatives inhibited the proliferation, and induced the differentiation of U937 and HL-60 cells in a dose-dependent manner, as determined by cell counts and nitroblue tetrazolium (NBT) reduction assays, respectively. EB1089 and KH1060 were found to be more effective than 1,25(OH)2D3 in exhibiting their antiproliferative and differentiative effects. In contrast, induction of leukaemic cell differentiation with 1 ng/ml GM-CSF after 96 h was less effective when compared with the vitamin D derivatives used individually. Fluorescence activated cell scanning (FACS) analyses indicated that the vitamin D derivatives readily induced the expression of the monocyte-associated cell surface antigen, CD14, and also the beta2-integrins, CD11b and CD18 in both cell lines after 48 h and 96 h treatment. The ability of EB1089 and KH1060 to induce these antigens was achieved with greater efficacy relative to the native hormone. When U937 and HL-60 cell cultures were cotreated for 48 h with the vitamin D compounds and GM-CSF and analysed by FACS, enhanced effects on CD14 and CD11b induction were observed compared to those of the compounds alone. These co-operative effects may occur as a consequence of molecular events which involve the transcription by vitamin D receptors (VDR) of genes required for the responsiveness of immature cells to factors such as GM-CSF, and place these and other related vitamin D analogues as potential therapeutic agents in the treatment of leukaemia.

Topics: Calcitriol; CD11 Antigens; Cell Differentiation; Cell Division; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia; Lipopolysaccharide Receptors; Tumor Cells, Cultured

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.

Topics: Alitretinoin; Antigens, CD; Antineoplastic Agents; Calcitriol; Cell Differentiation; Dose-Response Relationship, Drug; Drug Interactions; Flow Cytometry; HL-60 Cells; Humans; Leukemia; Tretinoin; Tumor Cells, Cultured

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Dose-Response Relationship, Drug; Female; Humans; Immunosuppressive Agents; Insulin-Like Growth Factor I; Mitosis; Time Factors; Tumor Cells, Cultured

The physiologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol (D3), plays an important role in embryonic development and cell differentiation. Previously, we have demonstrated that D3 significantly induces differentiation and inhibits growth of LA-N-5 human neuroblastoma cells at concentrations of 24 nm and higher. In this study, we compared two D3 analogs, 20-epi-22oxa-25a,26a,27a-tri-homo-1,25-D3 (KH 1060) and 1,25-dihydroxy-22,24-diene, 24,26,27-trihomo (EB 1089), with D3 with respect to their effects on differentiation and growth inhibition. We report an inhibition of growth by 45-55% in cells treated with 0.24 nm EB 1089 and 0.24 nM KH 1060, similar to that seen in cells treated with 24 nM D3. At these concentrations, both EB 1089 and KH 1060 stimulate the differentiation of LA-N-5 neuroblastoma cells as shown by increased neurite outgrowth, decreased N-myc expression and decreased invasiveness in vitro. An increase in acetylcholinesterase activity, a functional measure of differentiation, was also exhibited. Previous reports have shown that treatment doses needed to achieve 24 nM serum concentrations of D3 in patients would result in hypercalcemia. EB 1089 and KH 1060 can cause the same in vitro effects on LA-N-5 human neuroblastoma cells at 1/100 of the concentration required of D3. These data suggest a potential clinical efficacy of EB 1089 and KH 1060 as biological response modifiers.

Topics: Acetylcholinesterase; Antineoplastic Agents; Calcitriol; Cell Differentiation; Cell Division; Cholecalciferol; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neoplasm Invasiveness; Neuroblastoma; Staining and Labeling; Tumor Cells, Cultured

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to inhibit breast cancer cell growth both in vitro and in vivo. A major drawback is that high doses of 1,25-(OH)2D3 are needed which may result in undesirable side effects like the development of hypercalcemia and an increased risk of bone metastases due to the stimulation of bone resorption by 1,25-(OH)2D3. Several newly developed 1,25-(OH)2D3 analogs have a reduced calcemic activity, but their direct effects on bone resorption have not yet been examined. Presently, the antiestrogen tamoxifen is the most important endocrine therapy for breast cancer. Recent studies have demonstrated the benefit of the combination tamoxifen and 1,25-(OH)2D3/analogs for the inhibition of breast cancer cell growth. Besides inhibition of breast cancer growth tamoxifen appeared to have beneficial effects on bone. The purpose of the present study was to investigate the effect of tamoxifen on 1,25-(OH)2D3- and analogs (EB1089 and KH1060)-stimulated bone resorption in an in vitro model. Bone resorption was stimulated by 1,25-(OH)2D3 and analogs in a dose-dependent manner with KH1060 and EB1089 being more potent and 1,25-(OH)2D3. Tamoxifen caused a strong dose-dependent inhibition (70% at 10 microM) of 1,25-(OH)2D3- and EB1089-stimulated bone resorption. KH1060-stimulated bone resorption was also inhibited by tamoxifen but to a lesser extent (36%). Also the pure antiestrogen ICI164,384 but not 17 beta-estradiol inhibited 1,25-(OH)2D3-stimulated bone resorption. Together, this study demonstrates that tamoxifen considerably reduces 1,25-(OH)2D3/analogs-stimulated bone resorption and therefore may be useful to reduce the risk of bone metastases. This together with the observed beneficial effects on breast cancer cell growth indicates that tamoxifen together with 1,25-(OH)2D3/analogs is an interesting combination for the treatment of breast cancer. The mechanism of the bone resorption inhibitory action is not yet known but seems to be independent of the estrogen pathway.

Topics: Animals; Bone Resorption; Calcitriol; Dose-Response Relationship, Drug; Estradiol; Mice; Polyunsaturated Alkamides; Tamoxifen

Although numerous studies have shown potent antiproliferative and differentiation-inducing effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and its analogs on cells not directly related to bone metabolism, only few reports focussed on the effects of these analogs on bone. We compared the action of several recently developed analogs with that of 1,25-(OH)2D3 on human (MG-63) and rat (ROS 17/2.8) osteoblast-like cells and on in vitro bone resorption. In MG-63 cells the analogs EB1089 and KH1060 were about 166,000 and 14,000 times more potent than 1,25-(OH)2D3 in stimulating type I procollagen and 100 and 6,000 times more potent in stimulating osteocalcin production, respectively. Also in ROS 17/2.8 cells EB1089 and KH1060 were most potent in inducing osteocalcin synthesis. In vitro bone resorption was 2.3 and 17.5 times more potently stimulated by EB1089 and KH1060, respectively. In MG-63 cells, 1,25-(OH)2D3 and the analogs inhibited cell proliferation, whereas both 1,25-(OH)2D3 and the analogs stimulated the growth of ROS 17/2.8 cells. Differences in potency could neither be explained by affinity for the vitamin D receptor nor by a differential involvement of protein kinase C in the action of the analogs. Together, these data show that also in bone the analogs EB1089 and KH1060 are more potent than 1,25-(OH)2D3 but that the potency of the analogs compared to 1,25-(OH)2D3 is dependent on the biological response. On the basis of these observations it can be concluded that the reported reduced calcemic effect in vivo is not the result of a decreased responsiveness of bone to these analogs. Lastly, in view of eventual clinical application of 1,25-(OH)2D3-analogs, the observed stimulation of in vitro bone resorption and growth of an osteosarcoma cell line warrant in vivo studies to further examine these effects.

Topics: Animals; Antineoplastic Agents; Binding, Competitive; Bone Resorption; Calcitriol; Cell Division; Cells, Cultured; Cholecalciferol; Enzyme Inhibitors; Glyceryl Ethers; Humans; In Vitro Techniques; Osteoblasts; Osteocalcin; Osteosarcoma; Procollagen; Protein Kinase C; Rats; Receptors, Calcitriol; Structure-Activity Relationship; Tumor Cells, Cultured

We have investigated the effects on bone resorption of two new potent antiproliferative vitamin D3 analogs, EB 1089 and KH 1060, by studying recruitment of osteoclasts in murine bone marrow cultures and 45Ca release from prelabeled neonatal mouse calvarial bones. Binding studies to vitamin D receptor protein, from human osteosarcoma MG-63 cells, demonstrated kd values of 8.5 x 10(-11) for 1 alpha,25(OH)2D3, 6.5 x 10(-11) for KH 1060, and 2.7 x 10(-10) for EB 1089. 1 alpha,25(OH)2D3 and EB 1089 were equipotent stimulators of osteoclast recruitment in murine bone marrow cultures, with EC50 at 10(-10) mol/L, whereas KH 1060 was about tenfold more potent with an EC50 at 10(-11) mol/L. In serum-free media, 1 alpha,25(OH)2D3 enhanced 45Ca release from neonatal mouse calvarial bones with EC50 at 10(-11) mol/L, but in the presence of 10% fetal calf serum (FCS) the stimulatory effect was significantly diminished, with a threshold value at 10(-10) mol/L. EB 1089 stimulated bone resorption with an estimated EC50 at 3 x 10(-11) mol/L, whereas KH 1060 was about tenfold more potent than 1 alpha,25(OH)2D3, and stimulated bone resorption with an EC50 at 10(-12) mol/L. The effects of EB 1089 and KH 1060 on 45Ca release were not significantly affected by the addition of 10% FCS. Addition of vitamin D binding protein to serum-free incubations of neonatal mouse calvarial bones, significantly inhibited the bone resorbing effect of 1 alpha,25(OH)2D3, but did not affect EB 1089 and KH 1060 induced 45Ca release.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bone Marrow; Bone Marrow Cells; Bone Resorption; Calcitriol; Calcium; Cell Differentiation; Cells, Cultured; Femur; Humans; Mice; Osteoclasts; Receptors, Calcitriol; Tumor Cells, Cultured; Vitamin D

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as 1,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CD966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was addictive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Cell Division; Drug Resistance; Drug Screening Assays, Antitumor; Estradiol; Humans; Proto-Oncogene Proteins c-myc; Receptors, Calcitriol; Tamoxifen; Tumor Cells, Cultured

The nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), VDR, belongs to the nuclear receptor superfamily. This ligand-inducible transcription factor mediates the genomic VD signalling pathways by binding to specific response elements in the promoter region of VD regulated genes. Two types of natural VD response elements are used as models for the VDR-mediated transcriptional activation: one is bound by VDR-homodimers and is found in the human osteocalcin gene promoter, and the other is bound by heterodimers of VDR with retinoid X receptors (RXRs) as in the mouse osteopontin promoter. Here, we demonstrate that the VD analogues MC903, EB1089 and KH1060, previously shown to be potent regulators of proliferation and differentiation, are able to act as ligands for VDR and replace VD as a ligand in both nuclear signalling pathways. We found that they have different potency and sensitivity in their ability to stimulate the hormone-dependent promoter element. MC903 and EB1089 provide about 20% higher induction of gene activity than VD in a gene reporter system, whereas KH1060 was more sensitive, inducing transcription at about 100-fold lower doses than VD. Interestingly, VD and its analogues induce VDR homodimer-mediated gene activity at a 3- to 4-fold lower concentration than that of VDR-RXR heterodimers. This suggests that the ligand concentration is an additional regulatory level in the discrimination between signalling pathways involving homo- and heterodimeric hormone receptors.

Topics: Animals; Base Sequence; Calcitriol; DNA; Gene Expression Regulation; Humans; Ligands; Mice; Molecular Sequence Data; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Transfection; Tumor Cells, Cultured