Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels. Implications for metabolic bone disease
Journal of Clinical Endocrinology and Metabolism (USA), 1997, 82/1 (209-212)
Serum vitamin D metabolites and PTH were measured in seven subjects with a history of previous partial gastrectomy (PGX) and metabolic bone disease. The elimination tone-quarter of (3H)25-hydroxyvitamin D3 ((3H)25OHD3) in serum was assessed after an iv pulse dose of 5 microCi (26,27-3H)25OHD3. Median serum 25OHD3 was 37.5 (27.5-101.3) nmol/L, (normal range (NR) 10.8-58.5 nmol/L), mean serum 1,25-dihydroxyvitamin D (1,25-(OH)2D3) was raised at 175 + or - 72 pmol/L, (NR 48-120 pmol/L) and mean PTH was also high, 67 + or - 27 ng/L, (NR 10- 60 ng/L). Serum tone-quarter (3H)25OHD3 ranged from 10.9-21.2 days. A strong negative correlation existed between tone-quarter (3H)25OHD3 and serum 1,25- (OH)2D3 (Spearman's rank correlation coefficient (r = -0.82, P = 0.002)) and PTH) Spearman's rank correlation coefficient (r = -0.81, P = 0.001)). Four subjects who had high initial PTH concentrations (60-115 ng/L) and elevated 1,25-(OH)2D levels (162300 pmol/L) were reassessed after calcium supplementation to suppress secondary hyperparathyroidism (2degreeHPT). In this subgroup, after-treatment PTH fell from 82 + or - 24 to 52 + or B 24 ng/L (mean plus or minus SD), not significant; 1,25-(OH)2D fell from 210 plus or minus 61 to 116 plus or minus 28 pmol/L, P = 0.015; and tone-quarter (3H)25OHD3 increased from 13.2 + or - 1.9 to 18.9 plus or minus 3.1 days, P = 0.012. Patients with PGX and evidence of 2degreeHPT with elevated 1,25(OH)2D have a reduced tone-quarter (3H)25OHD3, and this may explain the increased susceptibility of the subjects to osteomalacia. Calcium supplementation suppresses 2degreeHPT, increases tone-quarter (3H)25OHD3 and may protect against PGX osteoporosis and osteomalacia.
The effect of season and latitude on in vitro vitamin D formation by sunlight in South Africa
South African Medical Journal (South Africa), 1996, 86/10 (1270-1272)
Aims. To assess the effect of season and latitude on the in vitro formation of previtamin D3 and vitamin D3 from 7-dehydrochloresterol (7-DHC) by sunlight in two cities in South Africa, Cape Town and Johannesburg. Methods. An in vitro study utilising vials containing 7-DHC, which were exposed to sunlight for a period of 1 hour between 8:00 and 17:00 on 1 day a month for a year. Previtamin D3 and vitamin D3 were separated from 7-DHC by high-performance liquid chromatography, and the amounts formed were calculated with the use of external standards. Results.A marked seasonal Variation in vitamin D3 production was noted in Cape Town, with very little being formed during the winter months of April through September. In Johannesburg, in vitro formation changed little throughout the year, and was similar to that found in Cape Town during the summer. During sunlit hours, vitamin D3 production was maximal at midday and small quantities were still being formed between 8:00 and 9:00, and between 16:00 and 17:00 during the summer. During winter in Cape Town, peak formation at midday was less than one-third of that in Johannesburg, and negligible amounts were formed before 10:00 and after 15:00. Conclusions. The previously documented seasonal variation in serum 25-hydroxyvitamin D recorded in patients in Johannesburg is probably a consequence of the increased clothing worn and the decreased time spent out of doors during winter, rather than decreased ultraviolet radiation reaching the earth. The limited in vitro formation of vitamin D3, during winter in Cape Town may have clinical implications insofar as the management of metabolic bone diseases like rickets and osteoporosis is concerned. Breast-fed infants resident in the area are likely to suffer from vitamin D deficiency rickets unless vitamin D supplements are provided, or the mothers are encouraged to take their children out of doors.
Effects of 2 years' treatment of osteoporosis with 1alpha-hydroxy vitamin D3 on bone mineral density and incidence of fracture: A placebo-controlled, double-blind prospective study
Endocrine Journal (Japan), 1996, 43/2 (211-220)
A two-year double-blind study monitored and evaluated the effects of 1alpha- hydroxy vitamin D3 (1alpha(OH)D3) on the lumbar (L2-4BMD) and total body bone mineral densities (TBBMD) and occurrence of fracture in 113 female osteoporotic patients receiving 0.75 microg/day of 1alpha(OH)D3 (n=57) or a placebo (n=56) with calcium supplementation in both groups. L2-4BMD increased 1.81% and 2.32% after one and 2 years in the 1alpha(OH)D3 group, but decreased 1.89% (P<0.05) and 0.28% in the placebo group. A significant difference (P<0.01) existed between the two groups after one year. TBBMD decreased significantly in the placebo group by 3.34% (P<0.01) and 3.52% after one and 2 years. Six new fractures occurred in the control group, but only two in the 1alpha(OH)D3 group (Odd's ratio=0.343, 95% confidence range; 0.0648-1.815). There were no serious adverse effects of the 1alpha(OH)D3 treatment. It was concluded that two-year treatment with 1alpha(OH)D3 increased the lumbar BMD and inhibited the decrease in TBBMD. Although it was not significant, new fracture occurrence in the 1alpha(OH)D3 group was around 1/3 of that in the control group.
1,25-Dihydroxyvitamin D3 enhances the enzymatic activity and expression of the messenger ribonucleic acid for aromatase cytochrome P450 synergistically with dexamethasone depending on the vitamin D receptor level in cultured human osteoblasts
Endocrinology (USA), 1996, 137/5 (1860-1869)
Not every postmenopausal woman with a low level of estrogen suffers from osteoporosis, and no correlation of bone density with serum estrogen level, but a significant correlation with adrenal androgens, is often noted. Vitamin D3 has been reported to be osteoclastic in vitro, whereas the effectiveness of vitamin D3 for the treatment of osteoporosis is clinically relevant. To study the roles of these factors in the development of osteoporosis, we characterized aromatase activity converting androgens to estrogens in human osteoblasts, because postmenopausal women maintain considerable levels of adrenal androgens. Glucocorticoids at 10-9-10-7 M transiently induced the expression and enzymatic activity of aromatase cytochrome P450 (P450(AROM)) in primary cultured osteoblasts, and the K(m) value for androstenedione (4.7 + or -2.9 nM) was lower than that in adipose tissue and skin. Human osteoblasts showed a promoter specificity different from that found in other tissues. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) alone did not induce aromatase activity, but enhanced and maintained glucocorticoid-induced P450(AROM) gene expression. This synergistic effect was not observed by other sex steroids or retinoic acids. The enhancement of P450(AROM) activity by 1,25(OH)2D3 varied from 0.94-fold (no enhancement) to 2.40-fold (maximal enhancement) among the individual human osteoblasts examined, but the magnitude of the enhancement was significantly correlated with the level of vitamin D receptor messenger RNA (P < 0.05). Cycloheximide did not abolish the synergistic effect of 1,25(OH)2D3, suggesting that de hove protein synthesis is not required for the synergism with 1,25-(OH)2D3. These results suggest that bone tissue can synthesize estrogen from adrenal androgens by a unique aromatase activity depending on the level of vitamin D receptor expressed.
1,25-dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis
Proceedings of the National Academy of Sciences of the United States of America (USA), 1996, 93/15 (7861-7864)
Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP). EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 (1,25- (OH)2D3). 1,25-(OH)2D3 could also prevent the progression of EAE when administered at the appearance of the first disability symptoms. Withdrawal of 1,25-(OH)2D3 resulted in a resumption of the progression of EAE. Thus, the block by 1,25-(OH)2D3 is reversible. A deficiency of vitamin D resulted in an increased susceptibility to EAE. Thus, 1,25-(OH)2D3 or its analogs are potentially important for treatment of MS.
All-trans and 9-cis retinoic acid enhance 1,25-dihydroxyvitamin D3-induced monocytic differentiation of U937 cells.
Leuk Res (ENGLAND) Aug 1996, 20 (8) p665-76
Retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (D3) are well known for inducing differentiation in many leukemic cell lines. The nuclear signalling pathways of RA and D3 are mediated through their cognate receptors, the retinoic acid receptor (RAR) and vitamin D3 receptor (VDR), respectively. Retinoid X receptor (RXR) is an auxiliary factor that forms a heterodimer with RAR and VDR, enabling their efficient transcriptional activation. 9-cis RA, a high-affinity ligand for RXR, greatly enhanced D3-induced CD14 expression in U937 cells, while RA alone did not induce CD14 expression. 9-cis RA also resulted in morphological changes of U937 cells to macrophage-like cells when combined with D3, while RA alone resulted in granulocyte-like cells. RA and D3 together enhanced c-fms expression, phagocytic activity, and acted synergistically to promote nitroblue tetrazolium reduction activity and inhibit proliferation. Northern analysis showed that U937 cells constitutively expressed RAR-alpha, VDR and RXR-alpha mRNAs. RA or D3 alone or in combination did not affect RAR-alpha and VDR expression, while 9-cis RA and 9-cis RA plus all-trans RA significantly reduced RXR-alpha expression. Interestingly, D3 could restore the down-regulation of RXR-alpha mRNA by 9-cis RA. These findings suggest that there is crossover of the nuclear signalling pathways of RA and D3. This may have clinical implications in that RA and D3 may be used in combination for differentiation-inducing therapy in acute myelogenous leukemia and myelodysplastic syndrome.
Expression of Retinoid X Receptor alpha is increased upon monocytic cell differentiation.
Biochem Biophys Res Commun (UNITED STATES) Mar 18 1996, 220 (2) p315-22
1 alpha, 25-Dihydroxyvitamin D3 (VD) is a potent inducer of monocytic differentiation of both normal and leukemic cells. Its effects are mediated by its nuclear receptor (VDR). Efficient gene activation requires the heterodimerization of VDR with Retinoid X Receptors (RXR). In the present study using specific antibodies, we analyzed the expression of the RXR alpha protein in blood mononuclear cells from acute myeloid patients (AML) (10 cases) and from myelomonocytic cell lines arrested at different stages of differentiation. We observed that the RXR alpha expression increased during myelomonocytic differentiation, since the highest levels were found in AML samples and in myelomonocytic cell lines having the highest amounts of monocytic precursors. We also demonstrated that fresh leukemic cells, whatever their stage of differentiation, as well as myelomonocytic cell lines, respond to VD by an increase in RXR alpha levels. Combinations of all-trans retinoic acid (RA) and VD, in some cases, increased this effect. This response suggests the involvement of RXR alpha in monocytic differentiation upon VD treatment.
Combination of a potent 20-epi-vitamin D3 analogue (KH 1060) with 9- cis-retinoic acid irreversibly inhibits clonal growth, decreases bcl-2 expression, and induces apoptosis in HL-60 leukemic cells
Cancer Research (USA), 1996, 56/15 (3570-3576)
All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with acute promyelocytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiation- and apoptosis-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) inhibits proliferation and induces differentiation of myeloid leukemic cells.The 9-cis-RA, unlike all-trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D3 analogue, 20-epi-22-oxa- 24a,26a,27a-tri-homo-1alpha,25(OH)2D3 (KH 1060), which belongs to the family of potent 20-epi-1,25(OH)2D3 analogues, with 9-cis-RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro. Our data show that KH 1060 alone is a very potent inhibitor of clonal proliferation of HL-60, but this effect is reversible, and that 9-cis-RA alone is a weak inhibitor of clonal proliferation of HL-60 cells. In contrast, the combination of KH 1060 and 9- cis-RA synergistically and irreversibly inhibited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation. This combination also affected the expression of apoptosis-related genes. The bcl-2 protein became nearly undetectable, and expression of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 cells was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for alpha-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentiation markers with two-color immunofluorescence. Treatment with the combination of KH 1060 and 9-cis-RA was a potent inducer of differentiation of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, induced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl-2 ratio. This drug combination may have important therapeutic significance.
Monocytic differentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor alpha in human monoblast U937 cells
Journal of Leukocyte Biology (USA), 1996, 60/6 (778-783)
Interferon-gamma (IFN-gamma), vitamin D3 (VD), and retinoic acid (RA) induce differentiation of human monoblastic leukemia U937 cells to macrophage-like cells with potential superoxide anion-generating activity upon further stimulation. Here we report that U937 cells thus differentiated show various responses to apoptotic induction with a cytotoxic anti-Fas antibody and tumor necrosis factor (TNF). VD-or RA-treated U937 cells acquired resistance against Fas- or TNF receptor (TNFR)-mediated apoptosis, whereas apoptotic cell death was accelerated in IFN-gamma-treated cells. By flow cytometric analyses, no decrease in expression of surface Fas antigen or p55 TNFR was observed in differentiated U937 cells. Cell surface expression of CD11b was seen only when differentiation was induced with VD or RA but not with IFN-gamma. The growth of VD- or RA-treated cells was retarded but IFN-gamma-treated cells were prolific. These findings suggest that the differentiation state differs with the inducer and that the cellular response to apoptotic induction is closely related to the state including the cell cycle.
Myeloma cell growth arrest, apoptosis, and interleukin-6 receptor modulation induced by EB1089, a vitamin D3 derivative, alone or in association with dexamethasone
Blood (USA), 1996, 88/12 (4659-4666)
We have previously shown that malignant plasma cells expressed the specific receptor for 1,25-dihydroxyvitamin D3 and that this derivative could significantly inhibit the proliferation of such malignant cells. More recently, new vitamin D3 derivatives have been generated with extraordinarily potent inhibitory effects on leukemic cell growth in vitro. These new data prompted us to (re)investigate the capacity of such new vitamin D3 derivatives to inhibit myeloma cell growth in comparison with that of dexamethasone, a potent antitumoral agent in multiple myeloma. In the current study, we show that EB1089, a new vitamin D3 derivative, (1) induces G1 growth arrest of human myeloma cells, which is only partially reversed by interleukin-6 (IL-6); (2) induces apoptosis in synergy with dexamethasone, IL-6, leukemia-inhibitory factor, and Oncostatin M, with an agonistic anti- gp130 monoclonal antibody being unable to prevent this apoptosis; (3) downregulates both the gp80 (ie, the alpha chain of the IL-6 receptor (IL- 6Ralpha)) expression on malignant plasma cells and the production of soluble IL- 6Ralpha, and finally (4) inhibits the deleterious upregulation of gp80 expression induced by dexamethasone while limiting the dexamethasone-induced upregulation of gp130 expression. Considering that these in vitro effects of EB1089 have been observed at doses obtainable in vivo (without hypercalcemic effects), our present data strongly suggest that EB1089 could have a true interest in the treatment of multiple myeloma, especially in association with dexamethasone.
Mutation in the ligand-binding domain of the retinoic acid receptor alpha in HL-60 leukemic cells resistant to retinoic acid and with increased sensitivity to vitamin D3 analogs
Leukemia Research (United Kingdom), 1996, 20/9 (761-769)
Even though retinoic acid can induce complete remissions in patients with acute promyelocytic leukemia, the duration of response is short and further therapy with this agent is less effective, suggesting the development of drug resistance. One possible way to overcome this problem is to use retinoic acid in combination with another agent that can induce differentiation, such as vitamin D3 or its analogs. In order to understand the mechanism of drug resistance to retinoic acid, we have isolated a clone of human HL-60 myeloid leukemic cells that is resistant to all-trans retinoic acid by continuous exposure to this agent. We have observed that the resistant cell line was also resistant to 9-cis-retinoic acid and more sensitive to the antileukemic action of the vitamin D3 analog, 1,25-dihydroxy-16-ene-23-yne-26,27-F6-cholecalciferol. In addition, this combination showed synergistic antileukemic action against the wild type HL-60 leuke mic cells. DNA sequence analysis revealed a mutation in the ligand binding region of retinoic acid receptor alpha in the HL-60/RA cells in which a glycine was replaced by an aspartic acid. Using gel retardation assays, we observed a large reduction in the formation of RXR-RAR heterodimers in the HL-60/RA cell line as compared to the parental cell line. This mutation in the retinoic acid receptor alpha of the HL-60/RA cells may be responsible for drug resistance to ATRA and 9-cis-retinoic acid and increased sensitivity to vitamin D3 analogs.
1,25-dihydroxyvitamin D3 primes acute promyelocytic cells for TPA-induced monocytic differentiation through both PKC and tyrosine phosphorylation cascades.
Exp Cell Res. 1996 Jan 10. 222(1). P 61-9
NB4 cells are the only in vitro model of differentiation in acute promyelocytic leukemia (APL). Although these cells respond to all-trans-retinoic acid to form neutrophils, our group has recently shown that these cells are capable of terminal monocytic differentiation in response to combined treatment with 1,25-dihydroxyvitamin D3 (1,25 D3) and 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that the agents need not be present simultaneously, but may be added sequentially. TPA treatment prior to 1,25 D3 led to the appearance of adherent cells; however, when 1,25 D3 treatment preceded TPA treatment cells expressed all differentiation markers reflective of terminal differentiation. This priming effect of 1,25 D3 was both dose and time dependent. Increasing the interval between 1,25 D3 and TPA treatment caused a decrease in this priming potential indicative of limited commitment inducing capacity of 1,25 D3. In order to characterize the mechanism of action of 1,25 D3 and TPA, chemical inhibitors of phosphorylation were used. Staurosporine and bisindolymaleimide GF 109203X treatment prior to and during 1,25 D3 treatment or TPA treatment caused attenuation of the differentiation response. Experiments utilizing tyrosine kinase and phosphatase inhibitors supported the hypothesis that 1,25 D3 signaling was mediated by both serine/threonine and tyrosine phosphorylation cascades. Results from this study provide evidence to support the hypothesis that 1,25 D3 signaling occurs via nongenomic mechanisms which when combined with the signaling effects of TPA, allow for the terminal differentiation of APL cells. This model should be used to develop new differentiation therapies for APL and other leukemias.
[Synthesis of retinoids with a modified polar group and their antitumor activity. Report I]
Bioorg Khim. 1995 Dec. 21(12). P 941-9
ISeveral retinoids with modified polar group were synthesized. Biological screening using HL-60 promyelocyte leukemia cells showed that the free carboxyl in the retinoid molecules is not the only group responsible for exhibiting the differentiating activity.
Induction of differentiation in murine erythroleukemia cells by 1 alpha,25-dihydroxy vitamin D3.
Cancer Lett. 1995 Apr 14. 90(2). P 225-30
The Friend murine erythroleukemia (MEL) cells can be stimulated to differentiate in response to a variety of chemical inducing agents. In the present study, the effect of 1 alpha,25-dihydroxyvitamin D3 on differentiation of MEL cells was investigated. Vitamin D3 induced differentiation of MEL cells in culture as determined by elevated hemoglobin content, a rise in the number of benzidine-positive cells and increase in acetylcholine esterase activity. The optimum concentration of the vitamin required to induce differentiation of MEL cells was found to be 750 nM. The pattern of induction of differentiation was similar to that observed with DMSO and the induction of differentiation by vitamin D3 was inhibited by dexamethasone.
Synergistic differentiation of U937 cells by all-trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3 is associated with the expression of retinoid X receptor alpha.
Biochem Biophys Res Commun. 1994 Aug 30. 203(1). P 272-80
Among the nuclear hormone receptors, the retinoid X receptors (RXRs) play a central role through their ability to heterodimerize with other members of this family of transcription factors, including retinoic acid (RA) and vitamin D (VD3) receptors. We have previously found that all-trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3 cooperate to induce monocytic differentiation of U937 human leukemic cells. Here the expression of RXR alpha protein in myelomonocytic cells was studied by immunodetection using polyclonal antibodies. RXR alpha was detected upon exposure of cells to VD3 and higher levels were found in cells treated by combinations of RA and VD3 under conditions where both agents synergized for inducing monocytic properties.
1,25(OH)2-16ene-vitamin D3 is a potent antileukemic agent with low potential to cause hypercalcemia.
Leuk Res (1994 Jun) 18(6):453-63
Compounds that induce cancer cells to differentiate are clinically effective for several types of malignancies. The 1,25- dihydroxyvitamin D3[1,25(OH)2D3(C)] induces leukemic cells, including HL-60, to differentiate and/or no longer proliferate, but it causes hypercalcemia. Development of vitamin D analogs that are more potent in their abilities to affect leukemic cells without causing greater hypercalcemia, may be useful therapeutically. A novel analog [1,25(OH)2-16ene-D3(HM)] has a double bond between C-16 and C-17; it appears to be an extremely effective antileukemic agent with the same or fewer effects on serum calciums. We define the potency of this compound and compare it with seven, previously reported, potent analogs of 1,25(OH)2D3. HM inhibited clonal growth of HL-60 cells by 50% at 1.5 x 10(-11) M. This was about equipotent to 1,25(OH)2-16ene- 23yne-D3(V), about 100-fold more potent than many of the other analogs, and 1000-fold more potent than 1,25(OH)2D3. The rank order of leukemic inhibitory activity was: 1,25(OH)2-16ene-D3(HM) > or = 1,25(OH)2- 16ene-23yne-D3(V) > 1,25(OH)2-23ene-D3(EX) = 1,24(OH)2- 22ene-24-cyclopropyl-D3(BT) = 22-oxa- 1,25(OH)2D3(EU) = 1,25(OH)2-24- homo-D3(ER) > 1,25(OH)2D3(C) > 1,25(OH)2-24- dihomo-D3(ES). The rank order of their effects on induction of differentiation of HL-60 cells, as measured by superoxide production and nonspecific esterase activity, was similar to their antiproliferative activities. In contrast, each analog slightly stimulated proliferation of normal human myeloid clonal growth. Serum calcium levels were the same or slightly less when either 1,25(OH)2-16ene-D3(HM) or 1,25(OH)2D3 (0.0625, 0.125, or 0.25 microgram) was given intraperitoneally to mice for 5 weeks. HM bound to 1,25(OH)2D3 receptors about 1.5-fold more avidly than 1,25(OH)2D3. In fact, this vitamin D3 appears to be the most avid binder to 1,25(OH)2D3 receptors that has been identified to date. In contrast, HM had a greater than 50-fold lower affinity for the D-binding proteins as compared with 1,25(OH)2D3, thus increasing the availability of the compound for target tissues. Further differentiation experiments showed that HM was more potent than 1,25(OH)2D3 in the presence of serum, but was equipotent in serum-free conditions. Taken together, our experiments suggest that 1,25(OH)2-16ene-D3(HM) may be more potent than 1,25(OH)2D3(C) because of its higher affinity to the 1,25(OH)2D3 receptors and its low affinity to the D-binding protein present in serum. HM is an ideal compound for clinical studies including patients with preleukemia and other neoplasia, as well as several skin disorders, such as psoriasis.
Inhibition of breast cancer cell growth by combined treatment with vitamin D3 analogues and tamoxifen.
Cancer Res (UNITED STATES) Nov 1 1994, 54 (21) p5711-7
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.
The anti-proliferative effect of vitamin D3 analogues is not mediated by inhibition of the AP-1 pathway, but may be related to promoter selectivity.
Oncogene (ENGLAND) Nov 2 1995
The hormone 1,25-dihydroxyvitamin D3 (VD) is able to induce cellular differentiation and to inhibit cellular proliferation, which provides it with an interesting therapeutic potential in cancer. However, side effects of VD on homeostasis (eg hypercalcemia) had made the need for the development of VD analogues with low calcemic effect. On the human breast cancer cell line MCF-7 we obtained with the VD analogue EB1089 an about 100-fold higher anti-proliferative effect than with VD. We found that this difference in biological activity is neither related to increased functional affinity to the VD receptor nor to repression of AP-1 activity. The physiologically most prominent complex of the VD receptor is a heterodimer with the retinoid X receptor that binds VD response elements formed two hexameric core binding motifs being arranged either as direct repeats spaced by 3 nucleotides (DR3s) or as inverted palindromes spaced by 9 nucleotides (IP9s). We observed that EB1089 stimulates transcriptional activation from IP9-type elements at clearly lower concentrations than from DR3-type elements. It is possible that IP9-type response elements play an important role in or contribute to the control of cell proliferation, so that promoter-selectivity may explain the high anti-proliferative effect of EB1089.
1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.
J Clin Invest (UNITED STATES) Apr 1 1996, 97 (7) p1577-88
1,25(OH)2 Vitamin D3 (VD3) and retinoic acid (RA) function as ligands for nuclear receptors which regulate transcription. Though the cardiovascular system is not thought to represent a classical target for these ligands, it is clear that both cardiac myocytes and vascular smooth muscle cells respond to these agents with changes in growth characteristics and gene expression. In this study we demonstrate that each of these ligands suppresses many of the phenotypic correlates of endothelin-induced hypertrophy in a cultured neonatal rat cardiac ventriculocyte model. Each of these agents reduced endothelin-stimulated ANP secretion in a dose-dependent fashion and the two in combination proved to be more effective than either agent used alone (VD3: 49%; RA:52%; VD3 + RA:80% inhibition). RA, at concentrations known to activate the retinoid X receptor, and, to a lesser extent, VD3 effected a reduction in atrial natriuretic peptide, brain natriuretic peptide, and alpha-skeletal actin mRNA levels. Similar inhibition (VD3:30%; RA:33%; VD3 + RA:59% inhibition) was demonstrated when cells transfected with reporter constructs harboring the relevant promoter sequences were treated with VD3 and/or RA for 48 h. These effects were not accompanied by alterations in endothelin-induced c-fos, c-jun, or c-myc gene expression, suggesting either that the inhibitory locus responsible for the reduction in the mRNA levels lies distal to the activation of the immediate early gene response or that the two are not mechanistically coupled. Both VD3 and RA also reduced [3H]leucine incorporation (VD3:30%; RA:33%; VD3 + RA:45% inhibition) in endothelin-stimulated ventriculocytes and, once again, the combination of the two was more effective than either agent used in isolation. Finally, 1,25(OH)2 vitamin D3 abrogated the increase in cell size seen after endothelin treatment. These findings suggest that the liganded vitamin D and retinoid receptors are capable of modulating the hypertrophic process in vitro and that agents acting through these or similar signaling pathways may be of value in probing the molecular mechanisms underlying hypertrophy.
Inhibitory effect of 220-oxa-1,25-dihydroxyvitamin D3 on the proliferation of pancreatic cancer cell lines.
Gastroenterology; 110(5):1605-13 1996
BACKGROUND & AIMS: Effective chemotherapy for pancreatic cancer is urgently needed. The aim of this study was to compare the anti-proliferative activity of a new vitamin D3 analogue, 22-oxa-1,25-dihydroxyvitamin D3 (22-oxa-calcitriol), on pancreatic cancer cells lines with that of 1,25-dihydroxyvitamin D3 (calcitriol) with analysis of vitamin D receptor status. METHODS: Antiproliferative effects of both agents were compared using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and by measuring the tumor size of xenograft inoculated into athymic mice. Vitamin D receptor contents by Scatchard analysis and mutational analysis of receptor complementary DNA were performed. RESULTS: In vitro, 22-oxa-calcitriol and calcitriol markedly inhibited the proliferation (3 of 9 cell lines) and caused a G1 phase cell cycle arrest by appearance of numerous domes. In vivo, 22-oxa-calcitriol inhibited the growth of BxPC-3 xenografts more significantly than calcitriol without including hypercalcemia. Hs 766T, showing no response to either agent, had the second highest receptor contents with no abnormalities in its primary structure deduced by receptor complementary DNA. CONCLUSIONS: 22-oxa-calcitriol may provide a more useful tool for the chemotherapy of pancreatic cancer than calcitriol. Also, the susceptibility of the cell lines to both agents is not well determined by evaluating either the contents or the mutation of vitamin D receptor.
Antiproliferative responses to two human colon cancer cell lines to vitamin D3 are differently modified by 9-cis-retinoic acid.
Cancer Res; 56(3):623-32 1996
1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] exerts antiproliferative actions in colorectal cancer, but their underlying molecular mechanisms have not been determined. 1,25(OH)2D3 regulates target gene transcription via a specific nuclear vitamin D receptor (VDR), which mediates hormone action preferentially as a heterodimer with 9-cis-retinoic acid receptors (RXRs). We investigated the actions of 1,25(OH)2D3 and 9-cis-retinoic acid (RA) in two human colon cancer cell lines, HT-29 and Caco-2. Both expressed mRNAs encoding VDR, RXR alpha, and RXR gamma, and VDR was regulated posttranscriptionally in Caco-2 cells. There was an antiproliferative response of both cell lines to 1,25(OH)2D3. 9-cis-RA exerted antiproliferative effects on Caco-2 cells but blocked 1,25(OH)2D3 actions in HT-29 cells. The 1,25(OH)2D3-responsive gene 25-hydroxyvitamin D3 24-hydroxylase was induced in both cell lines b 1,25(OH)2D3 but in only HT-29 cells by 9-cis-RA. 1,25(OH)2D3 and 9-cis-RA cotreatment enhanced 24-hydroxylase expression in HT-29 cells only. The 24-hydroxylase enzyme is known to result in catabolism of 1,25(OH)2D3 and attenuation of its actions. Increased 24-hydroxylase activity in HT-29 cells, but not in Caco-2 cells, in response to 9-cis-RA may account for some of the complex cell-specific responses demonstrated in these studies.
Vitamin D: a modulator of cell proliferation and differentiation
J Steroid Biochem Mol Biol; 37(6):873-6 1990
1,25-Dihydroxyvitamin D3, [1,25(OH)2D3], the biologically most active metabolite of vitamin D3, is involved in the regulation of calcium homeostasis and bone metabolism. Recently, receptors for 1,25(OH)2D3 have also been shown in cells and tissues not directly related to calcium homeostasis. Experimental data obtained with leukaemic and cancer cell lines, both in vitro and in vivo, showed the effects of 1,25(OH)2D3 on cell differentiation and proliferation. However, high doses of the sterol have to be used to observe these effects. Additional studies are needed to establish whether 1,25(OH)2D3 or suitable analogues have a therapeutic potential in malignant diseases without unacceptable toxicity like the development of hypercalcemia.
Vitamin D3 analogs inhibit growth and induce differentiation in LA-N-5 human neuroblastoma cells
Clinical and Experimental Metastasis (United Kingdom) , 1996, 14/3 (239-245)
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-D 3 (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.