cholecalciferol and seocalcitol

Promotion of apoptosis (which is frequently dependent on functional p53) is thought to be critical for the effectiveness of chemotherapy or radiotherapy. Studies in this as well as other laboratories have demonstrated that breast tumor cells are relatively refractory to apoptosis in response to modalities that induce DNA damage. This report describes our efforts to understand the basis for the absence of an apoptotic response to adriamycin and ionizing radiation in the breast tumor cell based on alterations in cell-cycle and apoptotic regulatory proteins. We also report on the permissive effects of Vitamin D3 and the Vitamin D3 analog EB 1089 in the promotion of apoptosis in p53-wild-type cells. Our studies suggest that regulation of apoptosis in the breast tumor cell may require modulation of signaling events other than or in addition to the p53-dependent DNA damage response.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcitriol; Cell Cycle; Cell Division; Cholecalciferol; DNA Damage; Enzyme Inhibitors; Female; Humans; Radiation, Ionizing; Topoisomerase II Inhibitors; Tumor Cells, Cultured

Vitamin D receptor (VDR) activities have been noted for a number of B cell malignancies which showed varying sensitivities to vitamin D3 (1,25-dihydroxyvitamin D3, VD3, calcitriol) and its synthetic analogs. The objective of this study was to address the potential effects of VD3 and vitamin D3 analogs (VDAs) on the growth of Hodgkin's lymphoma (HL), a malignant pathology of B cell origin, in vitro.. Immunofluorescence staining showed the expression of VDR by primary Hodgkin's (H) and Reed-Sternberg (RS)-HRS-tumor cells in HL histological sections. Western blot analyses revealed expression of VDR in the HL cell lines Hs445, HDLM2, KMH2, and L428. One-way analysis of variance (ANOVA) on data obtained from water-soluble tetrazolium 1 (WST-1) cell proliferation assay showed decreased cell growth in HDLM2 and L428, 72 h after treatment with 10 µM of either VD3 of VDAs. Western blot analyses showed that treatment of L428 cells with the VDAs (calcipotriol and EB1089) resulted in modest increases in nuclear accumulation of VDR (nuVDR) compared to either dimethyl sulfoxide (DMSO) or VD3 treatments. nuVDR for DMSO control and VD3 was comparable. These results suggest that VD3 or VDAs may affect growth of HL.

Topics: Calcitriol; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Hodgkin Disease; Humans; Receptors, Calcitriol; Vitamin D

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.

Topics: Autophagy; Breast Neoplasms; Calcitriol; Cell Line, Tumor; Cholecalciferol; Cytoprotection; Drug Screening Assays, Antitumor; Female; Gene Silencing; Humans; Phagosomes; Radiation Tolerance; Radiation, Ionizing; Receptor, ErbB-2; Transfection; Vacuoles

The effects of administration of vitamin D₃ and Seocalcitol on MNU-induced carcinogenesis of mammary gland in Sprague-Dawley rats have been investigated. Administration of both substances in a weekly dose of 7 μg/kg caused prolonged latency of mammary gland tumors. The latency of tumors was markedly prolonged for 30-40 days by Seocalcitol. Using PET analysis, reduction in [¹⁸F]2-fluoro-2-deoxy-d-glucose (FDG) uptake or tumor volume in tumors chemopreventively treated with vitamin D₃ were detected in MNU-induced tumors, vitamin D₃ reduced expression of 25-hydroxylase (25OHase) (p<0.01) and 24-hydroxylase (24OHase) (p<0.01) and Seocalcitol 24OHase. Positive regulation of 25OHase mRNA level after the treatment with vitamin D₃ was observed in liver, while in kidney, vitamin D₃ and Seocalcitol induced expression of 24OHase was significant. Our observations indicate a cross talk between respective pathways of VDR, RARs/RXRs, TRs and ERs in carcinogenesis process.

Topics: Animals; Calcitriol; Cell Transformation, Neoplastic; Cholecalciferol; Female; Histocytochemistry; Mammary Neoplasms, Experimental; Methylnitrosourea; Mixed Function Oxygenases; Positron-Emission Tomography; Rats; Rats, Sprague-Dawley; Receptors, Calcitriol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

1alpha,25-dihydroxyvitamin D3 and its analogue, Seocalcitol (EB1089), are able to reverse or slow the process of carcinogenesis in experimental models and cell cultures. The aim of this study was to investigate the effect of administration vitamin D or Seocalcitol to female Sprague-Dawley rats with 1-methyl-1-nitrosourea (MNU)-induced carcinogenesis of mammary glands on binding characteristics and mRNA levels of thyroid hormone receptors (TRs). Chemopreventive administration of vitamin D caused significant reduction of animal body weight. The expression of TRalpha mRNA was significantly higher in liver of animals treated with vitamin D after detection of first tumour. In our experiment, administration of vitamin D or Seocalcitol significantly reduced KA (group MNU+Seo; MNU+D) and increased Bmax (group MNU+Seo) of thyroid receptors in liver when compared to healthy animals. We show that the activity type I 5'-deiodinase was significantly decreased in livers of animals treated with vitamin D. The data from our in vivo experiment has clearly shown, for the first time, that vitamin D but not Seocalcitol i) may affect the body weight of animals, ii) can cause an increase in the expression of TRalpha in rat liver, remaining the functionality of the TRs unaffected, and iii) is responsible for type I iodothyronine 5'-deiodinase activity decrease in rat liver, remaining the expression of the enzyme unaffected.

Topics: Animals; Body Weight; Breast Neoplasms; Calcitriol; Cholecalciferol; DNA; Female; Gene Expression Regulation; Iodide Peroxidase; Liver; Methylnitrosourea; Organ Size; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thyroid Hormone Receptors alpha

In estrogen receptor (ER) positive breast cancer cells such as MCF-7 cells, the anti-tumor effects of 1,25(OH)(2)D(3) (1,25D(3)) may be secondary to disruption of estrogen mediated survival signals. If so, then sensitivity to 1,25D(3) mediated growth arrest could be reduced in estrogen independent breast cancer cells. The aim of these studies was to determine the effects of 1,25D(3) and EB1089 on the ER negative, invasive human breast cancer cell line SUM-159PT. 1,25D(3) and EB1089 reduced SUM-159PT cell growth subsequent to elevation of p27 and p21 levels. 1,25D(3) mediated apoptosis of SUM-159PT cells was associated with an enrichment of membrane bound bax, a redistribution of cytochome c from the mitochondria to the cytosol and PARP cleavage. 1,25D(3) and EB1089 also inhibited SUM-159PT cell invasion through an 8 microM Matrigel membrane. In pre-clinical studies, EB1089 dramatically reduced the growth of SUM-159PT xenografts in nude mice. The decreased size of tumors from EB1089 treated mice was associated with decreased proliferation and increased DNA fragmentation. Our data support the concept that Vitamin D(3) compounds trigger apoptosis by mechanisms independent of estrogen signaling. These studies indicate that Vitamin D(3) based therapeutics may be beneficial, alone or in conjunction with other agents, for the treatment of estrogen independent breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biocompatible Materials; Blotting, Western; Breast Neoplasms; Calcitriol; Cell Division; Cholecalciferol; Collagen; Cytosol; Dose-Response Relationship, Drug; Drug Combinations; Electrophoresis, Polyacrylamide Gel; Estrogen Receptor alpha; Estrogens; Humans; Laminin; Ligands; Mice; Mice, Nude; Mitochondria; Neoplasm Invasiveness; Neoplasm Transplantation; Poly(ADP-ribose) Polymerases; Protein Binding; Proteoglycans; Receptors, Calcitriol; Receptors, Estrogen; Subcellular Fractions; Time Factors; Tumor Cells, Cultured; Vitamin D

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases the growth and enhances the osteolytic effects of prostate cancer cells, it is important to control the level of PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-calcemic analogue, EB1089, suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines PC-3 and LNCaP. The human PTHrP gene contains a sequence element homologous to the negative vitamin D response element within the parathyroid hormone gene. This DNA sequence (nVDRE(hPTHrP)) bound the vitamin D receptor (VDR) present in nuclear extracts from both PC-3 and LNCaP cells. However, when cloned upstream of the SV40 promoter and transiently transfected into PC-3 and LNCaP cells, nVDRE(hPTHrP) downregulated promoter activity in response to 1,25(OH)2D3 or EB1089 treatment in LNCaP, but not in PC-3, cells. These results may help to explain why some prostate cancers appear to be refractory to treatment with vitamin D analogues.

Topics: Base Sequence; Calcitriol; Cell Line, Tumor; Cholecalciferol; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Male; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; RNA, Messenger; Sequence Homology, Nucleic Acid; Vitamin D Response Element

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.

Topics: Biomarkers, Tumor; Calcitriol; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Division; Cholecalciferol; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immune System; Ketoconazole; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step. Furthermore, the unique structure of the EB 1089 side-chain also permits facile determination of its side-chain metabolism. Use of PTAD derivatization and detection of metabolite-specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds.

Topics: Calcitriol; Cholecalciferol; Hydroxylation; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Triazoles

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)2D3] inhibits growth of cells derived from a variety of tumors in vitro and in vivo. Proliferation in vitro of human SCC25 cells, derived from a primary squamous cell carcinoma (SCC) of the tongue, was blocked by 1,25(OH)2D3 and its analog EB1089. A similar effect was observed with 13-cis retinoic acid (RA), which has been used in chemoprevention of SCC. We identified amphiregulin, a member of the epidermal growth factor family, as a 1,25(OH)2D3 target gene in SCC25 cells. Induction of amphiregulin mRNA by 1,25(OH)2D3 was rapid and sustained over 48 h, and was unaffected by cycloheximide. 1,25(OH)2D3 also induced amphiregulin mRNA in estrogen receptor-positive and -negative human breast cancer cell lines, but not in LNCaP human prostate cancer cells. RAR- or RXR-specific retinoids did not affect amphiregulin mRNA levels in SCC25 cells; however, 13-cis RA partially blocked the response to 1,25(OH)2D3. Amphiregulin partially inhibited growth of SCC25 cells in culture. Our data show that amphiregulin is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH)2D3.

Topics: Amphiregulin; Antineoplastic Agents; Breast Neoplasms; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cholecalciferol; Dose-Response Relationship, Drug; EGF Family of Proteins; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Isotretinoin; RNA, Messenger; Tumor Cells, Cultured

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

In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent.

Topics: Activin Receptors, Type I; Antibodies, Blocking; Antineoplastic Agents; Autocrine Communication; Breast; Breast Neoplasms; Calcitriol; Cell Division; Cell Line; Cholecalciferol; DNA-Binding Proteins; Dose-Response Relationship, Drug; Female; Gene Expression; Genes, Reporter; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Calcitriol; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Thymidine; Trans-Activators; Transfection; Transforming Growth Factor beta

Novel side-chain diene sulfones 5, analogues of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (calcitriol, 1), were designed to incorporate some of the therapeutically most favorable structural features of the Leo Pharmaceutical Company's drug candidate diene EB 1089 (seocalcitol, 4) and of the Hopkins' non-calcemic side-chain sulfone analogues 2 and 3. Synthesis of diene sulfones 5 features selective Swern oxidation of a primary silyl ether in the presence of a secondary silyl ether (9-->10) and Horner-Wadsworth-Emmons aldehyde addition by a 1-phosphonyl-3-sulfonyl stabilized carbanion regiospecifically at the 1-position to form E,E-diene sulfone 11. Sulfone diene analogue 5a with natural 1alpha,3beta-diol functionality, but not its diastereomer 5b with unnatural A-ring stereochemistry, is antiproliferative in vitro toward murine keratinocytes and malignant melanoma cells, as well as toward MCF-7 human breast cancer cells. Combining diene sulfone 5a with the currently used anticancer drug adriamycin (ADR) caused a noteworthy 3-fold enhancement of ADR antiproliferative potency in MCF-7 cells. Sulfone diene analogue 5a is weakly active transcriptionally in MCF-7 and ROS 17/2.8 cells, binds poorly but measurably to the vitamin D receptor (VDR), and desirably is non-calcemic in vivo at a daily dose (7 days) of 10 microg/kg of rat body weight.

Topics: Animals; Antineoplastic Agents; Calcitriol; Cell Division; Cholecalciferol; Dose-Response Relationship, Drug; Doxorubicin; Drug Synergism; Humans; Mice; Rats; Receptors, Calcitriol; Structure-Activity Relationship; Sulfones; Transcription, Genetic; Tumor Cells, Cultured

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 x 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.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Calcitriol; Caspase 3; Caspases; Cholecalciferol; Enzyme Activation; Humans; Mitogen-Activated Protein Kinases; Multiple Myeloma; p38 Mitogen-Activated Protein Kinases; Tumor Cells, Cultured

In view of the tumor suppressor role of the transforming growth factor-beta (TGFbeta) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFbeta growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFbeta, was not affected by these two compounds. TGFbeta-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFbeta activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3-4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Cholecalciferol; DNA Replication; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

1. 1alpha,25-dihydroxyvitamin3 (VD) is a nuclear hormone that has important cell regulatory functions but also a strong calcemic effect. EB1089 is a potent antiproliferative VD analogue, which has a modified side chain resulting in increased metabolic stability and a selective functional profile. Since EB1089 is considered for potential systemic application, it will be investigated to what extent its recently identified metabolites (hydroxylated at positions C26 and C26a) contribute to biological profile of the VD analogue. 2. Limited protease digestion analysis demonstrated that EB1089 is able to stabilize the high affinity ligand binding conformation of the VDR, starting at concentrations of 0.1 nM and affecting up to 80% of all receptor molecules. The metabolites EB1445 and EB1470 showed to be 100 fold less potent than EB1089, whereas the remaining three metabolites (EB1435, EB1436 and EB1446) showed a clearly reduced ability to stabilize the high affinity ligand binding conformation. Interestingly, at pharmacological concentrations all EB1089 metabolites stabilized a second, apparently lower affinity conformation to a much higher extent than EB1089. 3. In reporter gene assays all metabolites showed lower potency than EB1089. Moreover, the preference of EB1089 for activation of VDR binding to sites formed by inverted palindromic arrangements spaced by nine nucleotide (IP9-type VD response elements) appeared to be reduced (with EB1445 and EB1470) or completely lost (with EB1435, EB1436 and EB1446). The ranking of EB1089 and its metabolites that was obtained by limited protease digestion and reporter gene assays was confirmed by an analysis of their antiproliferative effect in breast cancer cells. . The potency and selectivity of the EB1089 metabolites in mediating gene regulatory effects was found to be drastically reduced in comparison to the parent compound suggesting that the contribution of the metabolites to the biological effect of EB1089 is minor. However, the compounds showed to be interesting tools for understanding the selective biological profile of EB1089.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Carcinogens; Cholecalciferol; Female; Humans; Ligands; Protein Conformation; Receptors, Calcitriol; Transfection; Tumor Cells, Cultured

Examination of a panel of ER positive breast cancer cell lines showed that they were differentially growth inhibited by vitamin D3 and its analogue EB1089. EB1089 treatment of the breast cancer cell lines MCF-7 E, BT20, T47D, and ZR75 demonstrated a correlation between a reduction in Cdk2 kinase activity towards phosphorylation of histone H1 and a decrease in DNA synthesis, while no modulation of Cdk2 activity was observed in the vitamin D3 and EB1089 resistant cell line MCF-7 L. This was accompanied by a time dependent decrease in the percentage of S phase cells in the responsive lines. Characterization of the expression levels of Cdk2 and its related cell cycle proteins in MCF-7 E cells showed that after EB1089 treatment, there was a concentration and time dependent up-regulation of p21 as well as a decrease in cyclin A proteins. Paradoxically, cyclin E levels were increased as a function of treatment. Analysis of cyclin-Cdk2-Cdki complex formation showed that in EB1089 treated MCF-7 E cells, Cdk2, cyclin A and cyclin E immunoprecipitates contained an increased abundance of p21. In contrast to MCF-7 E cells, increases in both p21 and p27 as well as their complex formation with Cdk2 were observed in BT20 and ZR75 cells. These findings indicate that up-regulation of p21 as well as p27 in some cell types may account for the inactivation of Cdk2 activity and a G1 block of the cell cycle following EB1089 treatment.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cholecalciferol; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; DNA, Neoplasm; Humans; Microtubule-Associated Proteins; Protein Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

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

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