Antiproliferative effects of 1alpha,25-dihydroxyvitamin D(3) and vitamin D analogs on tumor-derived endothelial cells

Although there is abundant evidence that 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] inhibits the growth of several cancer cell types, inhibition of angiogenesis may also play a role in mediating the antitumor effects of 1,25-(OH)(2)D(3.) We examined the ability of 1,25-(OH)(2)D(3) to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)(2)D(3) inhibited the growth of TDECs from two tumor models at nanomolar concentrations, but was less potent against normal aortic or yolk sac endothelial cells. The vitamin D analogs Ro-25-6760, EB1089, and ILX23-7553 were also potent inhibitors of TDEC proliferation. Furthermore, the combination of 1,25-(OH)(2)D(3) and dexamethasone had greater activity than either agent alone. 1,25-(OH)(2)D(3) increased vitamin D receptor and p27(Kip1) protein levels in TDECs, whereas phospho-ERK1/2 and phospho-Akt levels were reduced. These changes were not observed in normal aortic endothelial cells. In squamous cell carcinoma and radiation-induced fibrosarcoma-1 cells, 1,25-(OH)(2)D(3) treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2. In conclusion, 1,25-(OH)(2)D(3) and its analogs directly inhibit TDEC proliferation at concentrations comparable to those required to inhibit tumor cells. Further, 1,25-(OH)(2)D(3) modulates cell cycle and survival signaling in TDECs and affects angiogenic signaling in cancer cells. Thus, our work supports the hypothesis that angiogenesis inhibition plays a role in the antitumor effects of 1,25-(OH)(2)D(3).     

The duodenal 1 alpha,25-dihydroxyvitamin D3 receptor in rats with experimentally induced diabetes

Studies in the rat with streptozotocin-induced diabetes of short and long duration revealed decreased circulating 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D] levels and an intact 1,25-(OH)2D3 duodenal cytosolic receptor with a sedimentation coefficient of 3.3S. Whereas no significant alterations in the equilibrium dissociation constant (Kd) were observed in the diabetic animals, the number of 1,25-(OH)2D3-binding sites was increased in the animals with short term (235 +/- 48 vs. 100 +/- 15 fmol/mg protein) and long term (521 +/- 60 vs. 119 +/- 15 fmol/mg protein) diabetes. The data are consistent with the hypothesis that alterations in intestinal calcium absorption previously observed in the diabetic state are due, at least in part, to dynamic relationships between circulating 1,25-(OH)2D concentrations and the number of intestinal 1,25(OH)2D3-binding sites.     

1 alpha,25-dihydroxyvitamin D(3) inhibits angiogenesis in vitro and in vivo

Modulation of angiogenesis is now a recognized strategy for the prevention and treatment of pathologies categorized by their reliance on a vascular supply. The purpose of this study was to evaluate the effect of 1 alpha,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)], the active metabolite of vitamin D(3), on angiogenesis by using well-characterized in vitro and in vivo model systems. 1,25(OH)(2)D(3) (1 x 10(-9) to 1 x 10(-7) mol/L) significantly inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting and elongation in vitro in a dose-dependent manner and had a small, but significant, inhibitory effect on VEGF-induced endothelial cell proliferation. 1, 25(OH)(2)D(3) also inhibited the formation of networks of elongated endothelial cells within 3D collagen gels. The addition of 1, 25(OH)(2)D(3) to endothelial cell cultures containing sprouting elongated cells induced the regression of these cells, in the absence of any effect on cells present in the cobblestone monolayer. Analysis of nuclear morphology, DNA integrity, and enzymatic in situ labeling of apoptosis-induced strand breaks demonstrated that this regression was due to the induction of apoptosis specifically within the sprouting cell population. The effect of 1,25(OH)(2)D(3) on angiogenesis in vivo was investigated by using a model in which MCF-7 breast carcinoma cells, which had been induced to overexpress VEGF, were xenografted subcutaneously together with MDA-435S breast carcinoma cells into nude mice. Treatment with 1,25(OH)(2)D(3) (12.5 pmol/d for 8 weeks) produced tumors that were less well vascularized than tumors formed in mice treated with vehicle alone. These results highlight the potential use of 1,25(OH)(2)D(3) in both the prevention and regression of conditions characterized by pathological angiogenesis.     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1 alpha,25-dihydroxyvitamin D3-differentiated HL60 cells

Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([ 3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1 alpha,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12 x 10(6) cells/incubation. The optimum substrate concentration for its synthesis was 125 nM, giving an apparent Michaelis constant of 360 nM. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nM 1 alpha,25-(OH)2D3 for 4 days synthesized 2.17 +/- 0.07 (S.E.M.) pmol 24,25-(OH)2D3/10(6) cells per h.(ABSTRACT TRUNCATED AT 250 WORDS)     

1alpha,25-Dihydroxy-3-epi-vitamin D3 a physiological metabolite of 1alpha,25-dihydroxyvitamin D3: its production and metabolism in primary human keratinocytes

Recent studies of metabolism using pharmacological substrate concentrations of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] in several tissues including primary cultures of human keratinocytes, bovine parathyroid cells and bone cells led to the identification of 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)(2)-3-epi-D3] as a major natural metabolite of 1alpha,25(OH)(2)D3. In the present study, we demonstrate that human keratinocytes incubated with 25-hydroxy[26,27-(3)H] vitamin D3 produce 1alpha,25(OH)(2)-3-epi-D3 along with 1alpha,25(OH)(2)D3. The production of 1alpha,25(OH)(2)-3-epi-D3 is also identified in human keratinocytes incubated with physiological substrate concentrations of 1alpha,25(OH)(2)D3. Unlike 24-hydroxylase, the major enzyme involved in the further metabolism of 1alpha,25(OH)(2)D3 in human keratinocytes, the enzyme(s) responsible for the production of 1alpha,25(OH)(2)-3-epi-D3 is constitutive and is not inhibited by ketoconazole. It is also noted that 1alpha,25(OH)(2)-3-epi-D3 is further metabolised in human keratinocytes into several as yet unidentified metabolites, the production of which is inhibited to a great extent by SDZ 89-443, an inhibitor of 24-hydroxylase. This finding indicates that the 24-hydroxylase like in the case of 1alpha,25(OH)(2)D3, also plays a major role in the metabolism of 1alpha,25(OH)(2)-3-epi-D3. The results obtained from the metabolism studies performed in parallel among 25OHD3, 1alpha,25(OH)(2)D3 and 1alpha,25(OH)(2)-3-epi-D3 indicate that 1alpha,25(OH)(2)-3-epi-D3 and its metabolites exhibit higher metabolic stability. In summary, we demonstrate for the first time that 1alpha,25(OH)(2)-3-epi-D3 is a physiological metabolite of 1alpha,25(OH)(2)D3 in human keratinocytes. Also, 1alpha,25(OH)(2)-3-epi-D(3) is further metabolised in human keratinocytes mainly through the activity of 24-hydroxylase. Furthermore, our finding of the relative metabolic stability of 1alpha,25(OH)(2)-3-epi-D3 and especially its metabolites when compared to 1alpha,25(OH)(2)D3 and its metabolites provides an important explanation for its previously observed potent inhibitory effect on keratinocyte growth in spite of its low affinity to vitamin D receptor.     

1 alpha,25-dihydroxyvitamin D3 corrects osteomalacia in hypoparathyroidism and pseudohypoparathyroidism

Deficiency of circulating 1 alpha-25-dihydroxyvitamin D3 (1 alpha,25(OH)2D) regularly occurs in hypoparathyroidism (HP) and pseudohypoparathyroidism (PHP). Osteomalacia is occasionally found in the two diseases. Two patients, one with HP and the other with PHP, both with symptomatic and biopsy-proven osteomalacia, were studied before and after treatment with 1 alpha,25(OH)2D3. Laboratory values before treatment were as follows: serum immunoreactive parathyroid hormone was undetectable in the patient with HP and was elevated in the patient with PHP. Serum 25-hydroxyvitamin D, measured by binding assay, was 131.5 and 61.9 nmol/l (normal: 69.1 +/- 15.9 nmol/l); serum 24,25-dihydroxyvitamin D, measured by binding assay, was 13.9 and 3.8 nmol/l (normal: 3.4 +/- 1.4 nmol/l); serum 1 alpha,25(OH)2D, measured by bioassay, was 28.6 and 29.0 pmol/l (normal: 77.3 +/- 22.8 pmol/l) and, measured by receptor assay, was 36.2 and 41.0 pmol/l (normal: 71.8 +/- 35.8 pmol/l) in the HP and PHP patients, respectively. Serum calcium was low and serum inorganic phosphate was high in both cases. Treatment with 1 alpha,25(OH)2D3 (3-5 micrograms per day for 10-12 months) restored serum calcium and inorganic phosphate to normal, alleviated bone pain and healed the osteomalacia as shown on repeat bone biopsy. Our results provide further evidence that isolated deficiency of 1 alpha,25(OH)2D may cause osteomalacia or rickets.     

The effect of temperature on the vitellogenic response in Atlantic salmon post-smolts (Salmo salar)

Effects of low (3 degrees) and high (10 degrees) temperatures on the vitellogenic response to estradiol treatment were analyzed in female and male salmon post-smolts. Vitellogenic response as indicated by levels of circulating vitellogenin, hepatic protein, RNA, and nuclear count depends on the ambient temperature during hormone treatment but not on the previous 1 month acclimation temperature. Thus, the level of vitellogenin in the serum or hepatic RNA did not increase above the levels of the untreated controls in previously warm-acclimated smolts if the fish were exposed to low temperature water during treatment with estradiol. Conversely, cold-acclimated smolts showed increased levels of hepatic RNA and circulating vitellogenin only when transferred to 10 degrees water during treatment. No such response could be elicited if salmon were kept at 3 degrees during estradiol treatment. The negative influence of low ambient temperature on the vitellogenic response could not be eliminated by using higher doses of estradiol. Salmon post-smolts, long-term acclimated to 3 degrees revealed a significant vitellogenic response to estradiol only if the temperature during treatment was increased to 10 degrees. Estradiol treatment at 10 degrees stimulated the growth of liver as determined by the increase in liver somatic index and amounts of total hepatic protein, RNA, and nuclear count. On a per unit liver wet weight basis, however, only RNA increased above the level of the untreated control fish. As the nuclear count per gram of liver is similar in estradiol-treated and untreated groups, estradiol-induced liver growth in the salmon post-smolts may be accounted for by an increase in cell number (hyperplasia) rather than an increase in cell size (hypertrophy).     

Interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with RNA and synthetic polyribonucleotides.

The interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with RNA and synthetic polynucleotides has been examined by using receptor from rachitic chicken intestine. Total intestinal RNA inhibited the binding of receptor to calf thymus DNA-cellulose with an efficiency equivalent to single-stranded DNA. A comparison of the inhibitory activity of several polyribonucleotides gave the following order of activities: poly(I) = poly(G) greater than or equal to double-stranded DNA greater than single-stranded DNA = poly(U). Poly(A), poly(C), and the ribonucleoside monophosphates AMP, GMP, CMP, and UMP had minimal activity. A preference for single-stranded homopolymers was observed [i.e., poly(I) and poly(U) were active, whereas poly(I):poly(C) and poly(U):poly(A) were not]. The ability of nucleic acids to displace receptor from DNA-cellulose was also measured. Both poly(G) and poly(I) were more active than double-stranded DNA in this assay. Furthermore, differences were noted between intestinal RNA fractions separated on the basis of poly(A) content. Receptor also bound to immobilized intestinal RNA and polynucleotides. The KCl concentration necessary to disrupt binding to a given polynucleotide generally paralleled the activity of that molecule in DNA-cellulose inhibition and displacement assays. These results suggest that the 1 alpha,25-dihydroxyvitamin D3 receptor can interact with RNA as well as DNA.     

Radioimmunoassay of cortisone in the adult Atlantic salmon, Salmo salar L

A radioimmunoassay (RIA) was developed to measure cortisone in Atlantic salmon plasma. Diatomaceous earth microcolumn chromatography was used to separate the cortisone from other steroids. The RIA method following chromatography was validated by analyzing each of six samples of blood by double isotope derivative assay (DIDA) and RIA. The RIA gave significantly lower plasma cortisone concentrations (P less than 0.05) than the DIDA: the correlation coefficient was 0.84. Accuracy of the RIA was also demonstrated by addition of radioinert cortisone and measurement of increasing volumes of plasma. Specificity of the antisera was very good. Sensitivity of the RIA was 11 pg/RIA tube or 0.025 micrograms cortisone/dl plasma corrected for recovery and fraction assayed. Plasma cortisone concentrations in adult Atlantic salmon ranged from 2 to 26 micrograms/dl. The mean plasma cortisone concentration of salmon in fresh water was not significantly different from that in sea water. The mean cortisone/cortisol ratio in both adult and juvenile Atlantic salmon was between 1.40 and 1.92 indicating that cortisone and not cortisol may be the principal corticosteroid in Atlantic salmon.     

Direct C-1 hydroxylation of vitamin D compounds: convenient preparation of 1alpha-hydroxyvitamin D3, 1alpha, 25-dihydroxyvitamin D3, and 1alpha-hydroxyvitamin D2.

An efficient procedure for the direct C-1 hydroxylation of vitamin D compounds has been developed. The method involves conversion of vitamin D3 tosylates to 3,5-cyclovitamin D derivatives, allylic oxidation with selenium dioxide, and acid-catalyzed solvolysis to the 1 alpha-hydroxyvitamin D analogs. When applied to vitamin D3,25-hydroxyvitamin D3, and vitamin D2, this sequence give the corresponding 1alpha-hydroxylated derivatives in 10-15% yield.     

Purification of chicken intestinal receptor for 1 alpha, 25-dihydroxyvitamin D3 to apparent homogeneity.

The chicken intestinal 1 alpha, 25-dihydroxyvitamin D3 receptor-like protein has been purified to apparent homogeneity as determined by sodium dodecyl sulfate gel electrophoresis. The techniques employed for the purification include selective precipitation of the receptor by Polymin P (polyethyleneimine) and (NH4)2SO4 and batch adsorption to and selective elution from hydroxylapatite, followed by gel exclusion and DEAE-cellulose chromatography. Finally, the labeled receptor was eluted at a pH of approximately 6.0 on a chromatofocusing column. The protein was purified 6100-fold and the receptor was obtained in 9% yield.     

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1alpha,25-dihydroxyvitamin D3

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK₁ kidney cells. To induce 24-hydroxylase expression, 1,25(OH)₂D₃ (10⁻⁷ M) and dexamethasone were added simultaneously to the medium of LLC-PK₁ cells, and 24 h before dexamethasone treatment, 1,25(OH)₂D₃ was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells. Maximal stimulation was observed with 10⁻⁶ M dexamethasone in both cell lines. The addition of 10⁻⁶ M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10⁻⁶ M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)₂D₃-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)₂D₃-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)₂D₃ enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.