tretinoin and seocalcitol

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, or calcitriol), is a potent mitogen for fibroblasts cultured from rat lungs at postnatal day 4 (P4), during the peak of septation (P3 to P7). In light of the key role of fibroblasts in alveolar septation, the authors conducted studies to measure the extent to which 1,25-(OH)2D3 affects lung maturation in vivo, as well as its ability to influence the stimulatory activity of all-trans retinoic acid (RA). To identify a calcitriol analogue with maximal mitogenic activity and low systemic toxicity, two compounds with reduced calcemic activity (EB1089 and CB1093) and a superagonist (MC1288) were evaluated in neonatal rat lung fibroblast cultures. All 3 analogues were more potent mitogens than 1,25-(OH)(2)D3 itself (MC1288 approximately CB1093 > EB1089 > 1,25-(OH)2D3). In addition, each was more effective than 1,25-(OH)2D3(EB1089 > CB1093 > MC1288 > 1,25-(OH)2D3) in the activation of a vitamin D response element from the platelet-derived growth factor (PDGF)-A gene, whose expression is essential for normal alveolarization. Daily administration of EB1089 to rats 4 to 12 days of age caused an increase in mean alveolar chord length (P < .0001), and also elicited prominent regions of fibroblast hypercellularity, as defined in terms of a vimentin-positive, factor VIII-negative phenotype. EB1089 and RA each induced the expression of 2 important lung structural proteins, collagen and elastin. Regions of fibroblast hypercellularity induced by EB1089 were strongly positive for expression of the alveolarization-relevant growth factors, PDGF-AA and vascular endothelial growth factor (VEGF). These studies demonstrate that 1,25-(OH)2D3 disrupts the overall alveolarization process in the neonatal lung, although it stimulates expression of some proteins associated with lung morphogenesis.

Topics: Animals; Animals, Newborn; Calcitriol; Cell Growth Processes; Cells, Cultured; Collagen; Drug Synergism; Elastin; Factor VIII; Fibroblasts; Lung; Mitogens; Platelet-Derived Growth Factor; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Tretinoin; Vascular Endothelial Growth Factor A; Vimentin; Vitamin D Response Element

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDREhPTHrP) within the human PTHrP gene and involves an interaction between nVDREhPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRalpha forms part of the nuclear protein complex that interacts with nVDREhPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9-cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9-cis-retinoic acid enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-retinoic acid. Promoter activity driven by nVDREhPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH)2D3 and EB1089 in both cell lines. Co-treatment with these compounds and 9-cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell type-specific manner in prostate cancer cells.

Topics: Alitretinoin; Calcitriol; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Male; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; Receptors, Calcitriol; Retinoid X Receptors; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured; Vitamin D Response Element

Previous research has shown that the retinoid 9-cis retinoic acid (RA) promotes apoptosis in pancreatic cancer cells. The vitamin D analog EB1089 does not. Furthermore, cotreatment of cells with 9-cis RA and EB1089 abrogates apoptosis. To explain this, we studied the regulation of proteins involved in apoptotic signaling pathways in pancreatic cancer cells.. The pancreatic adenocarcinoma cell line T3M4 was used. Cell proliferation was measured using the SRB protein dye assay. Induction of apoptosis was evaluated using an ELISA assay. Caspase activation was detected using a colorimetric assay based on cleavage of a caspase-associated substrate. Regulation of protein levels and posttranslational events were detected using immunoblotting.. We confirm that EB1089 diminishes apoptosis induced by 9-cis RA in T3M4 cells. We extend the study to show that EB1089 abrogates increases, induced by 9-cis RA, in caspase activation, p27Kip1 protein levels, Bim and Bax protein levels and in Bax/Bcl2 ratio. In addition, the CDKI p21Waf1 and CAII, a differentiation marker for pancreatic cancer cells are also differentially regulated.. These results suggest that the inhibitory effects of EB1089 on 9-cis RA-induced apoptosis lie upstream of caspase activation and could be associated with reduction of p27Kip1 protein levels.

Topics: Adenocarcinoma; Alitretinoin; Antineoplastic Agents; Apoptosis; Calcitriol; Caspases; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Enzyme-Linked Immunosorbent Assay; Humans; Pancreatic Neoplasms; Tretinoin

1,25-Dihydroxyvitamin D(3) (1,25D(3)), the active metabolite of vitamin D(3), inhibits breast cancer cell growth in vivo and in vitro. To examine mechanisms of 1,25D(3) induced growth arrest and apoptosis, cell lines were established from DMBA induced mammary tumors derived from vitamin D(3) receptor knockout (VDRKO) and wild type (WT) mice. Two VDRKO (KO240, KO288) and two WT (WT145, WT276) cell lines were selected and characterized. All four cell lines express cytokeratins indicative of an epithelial origin, as well as vimentin, which is expressed in many transformed cell lines. The tumorigenicity of the cells was confirmed in vivo as all four cell lines form estrogen responsive tumors in nude mice. Both WT cell lines express the VDR protein and are sensitive to growth inhibition by 1,25D(3) at doses as low as 1 nM. Flow cytometric analysis indicated that 1,25D(3) induces G(0)/G(1) arrest and apoptosis in the WT cell lines. In contrast, both cell lines established from tumors that developed in VDRKO mice lack VDR mRNA and protein. Cells from WT mice exhibit 1,25D(3) inducible transcriptional activity, as measured by reporter gene assays, but cells from VDRKO mice do not. Cells from VDRKO mice are also completely resistant to 1,25D(3) mediated growth arrest and apoptosis over the range of 0.01-100 nM 1,25D(3). VDRKO cells are also resistant to the synthetic vitamin D(3) analogs EB1089 and CB1093 that are more potent growth inhibitors than 1,25D(3) in WT cells. This data conclusively demonstrate that the induction of cell cycle arrest and apoptosis in breast cancer cells by 1,25D(3), EB1089 and CB1093 is dependent on the nuclear VDR. Cells lacking VDR remain sensitive to growth arrest mediated by 9-cis retinoic acid, a ligand for the retinoid x receptor which can heterodimerize with the VDR. Sensitivity to apoptosis induced by the DNA damaging agent etoposide is not altered in VDRKO cells, indicating that VDR ablation does not impair apoptotic pathways in general. All four cell lines display equal sensitivity to tamoxifen induced growth arrest. These estrogen responsive, transformed cell lines which differentially express the VDR provide a novel model system for identification of the mechanisms by which 1,25D(3) regulates proliferation and apoptosis in breast cancer cells.

Topics: Alitretinoin; Animals; Apoptosis; Calcitriol; Cell Line, Tumor; Estrogens; Ethanol; Etoposide; Female; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Mice, Nude; Pregnancy; Receptors, Calcitriol; Tamoxifen; Tretinoin

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

Retinoids and vitamin D are known to exert important anti-tumour effects in a variety of cell types. In this study the effects of 9-cis-retinoic acid (9cRA) the vitamin D analogues EB1089 and CB1093 on three pancreatic adenocarcinoma cell lines were investigated. All compounds caused inhibition of in vitro growth but the vitamin D analogues were generally the more potent growth inhibitors. They were also more effective on their own than in combination with 9cRA. Growth arrest correlated with an increased proportion of cells in the G0/G1 phase. Apoptosis was induced in the three cell lines by 9cRA, whereas neither EB1089 nor CB1093 had this effect. Furthermore, addition of EB1089 or CB1093 together with 9cRA resulted in significantly reduced apoptosis. Our results show that retinoic acids as well as vitamin D analogues have inhibitory effects on pancreatic tumour cells but different and antagonistic mechanisms seem to be employed.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Calcitriol; Cell Division; Drug Screening Assays, Antitumor; Humans; Pancreatic Neoplasms; Tretinoin; 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

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

Retinoids and vitamin D are important factors that regulate cellular growth and differentiation. An additive growth-inhibitory effect of retinoids and vitamin D analogues has been demonstrated for human myeloma, leukaemic and breast cancer cells. We set out to study the effects of the vitamin D analogue EB1089 and the retinoids all-trans- and 9-cis-retinoic acid on the human pancreatic adenocarcinoma cell lines Capan 1 and Capan 2 and the undifferentiated pancreatic carcinoma cell line Hs766T. The cell lines investigated expressed vitamin D receptor, retinoic acid receptor (RAR)-alpha and gamma as determined by polymerase chain reaction after reverse transcription. RAR-beta was expressed only in Hs766T cells. Addition of all-trans-retinoic acid increased the amount of RAR-alpha mRNA in the three cell lines and induced RAR-beta mRNA in Capan 1 and Capan 2 cells. All-trans-retinoic acid at a concentration of 10 nM inhibited the growth of Capan 1 and Capan 2 cells by 40% relative to controls. 9-cis-Retinoic acid was less effective. Neither all-trans-retinoic acid nor 9-cis-retinoic acid affected the growth of Hs766T cells. EB1089, if added alone to the cells, did not significantly inhibit growth. However, the combination of 1 nM EB1089 with 10 nM all-trans-retinoic acid exerted a growth-inhibitory effect of 90% in Capan 1 cells and of 70% in Capan 2 cells. Our data suggest that vitamin D analogues together with retinoids inhibit the growth of human pancreatic cancer cells. However, in vivo studies are necessary to examine the potential use of retinoids and vitamin D analogues on pancreatic cancer.

Topics: Animals; Antineoplastic Agents; Base Sequence; beta 2-Microglobulin; Calcitriol; Cell Division; Cell Line; DNA Primers; Kidney; Molecular Sequence Data; Pancreatic Neoplasms; Polymerase Chain Reaction; Rats; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The effects of the novel vitamin D analogue, EB1089 alone, or in combination with the retinoid, 9-cis retinoic acid (9-cis RA) on indices of apoptosis in MCF-7 breast cancer cells have been examined. EB1089 was capable of reducing bcl-2 protein, a suppressor of apoptosis, and increasing p53 protein levels in MCF-7 cell cultures following 96h treatment. In the presence of 9-cis RA, EB1089 acted to further enhance the down-regulation and up-regulation of bcl-2 and p53 respectively. Furthermore, EB1089 induces DNA fragmentation in MCF-7 cells, a key feature of apoptosis, alone and in combination with 9-cis RA in situ. The observation that EB1089 and 9-cis RA act in a cooperative manner to enhance induction of apoptosis in these cells may have therapeutic implications.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Calcitriol; DNA Damage; DNA, Neoplasm; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53