Vitamin D receptor agonists inhibit pro-inflammatory cytokine production from the respiratory epithelium in cystic fibrosis.

BACKGROUND: 1,25-Dihydroxycholecalciferol (1,25(OH)(2)D(3)) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF. METHODS: CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH)(2)D(3) or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH)(2)D(3). RESULTS: Pretreatment with 1,25(OH)(2)D(3) was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH)(2)D(3) treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH)(2)D(3) was associated with an increase in hCAP18/LL-37 mRNA and protein levels. CONCLUSIONS: Both 1,25(OH)(2)D(3) and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF.

Selective vitamin D receptor modulators and their effects on colorectal tumor growth.

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is an endocrine hormone whose classic role is the maintenance of calcium homeostasis. It is well documented that 1,25(OH)(2)D(3) also has anti-tumor effects on a number of cancers and cancer cell lines including breast, colorectal, gastric, liver, ovarian, prostate, and non-melanoma skin cancers. Included in the anti-tumor activities of 1,25(OH)(2)D(3) are its ability to cause antiproliferation, prodifferentation and decrease angiogenesis. Furthermore, through regulation of the plaminogen activator (PA) system and a class of proteolytic enzymes called matrix metalloproteinases (MMPs), 1,25(OH)(2)D(3) reduces the invasive spread of tumor cells. Because of the calcemic limitations of using 1,25(OH)(2)D(3) as a therapy, we have tested the effects of a novel Gemini vitamin D analogue, Deuterated Gemini (DG), on mouse colorectal cancer. We demonstrated that DG is more potent in reducing tumor volume and mass, compared to control and 1,25(OH)(2)D(3). DG significantly prevented (100% reduction, p<0.05) the invasive spread of colorectal tumor cells into the surrounding muscle, and had no effect on serum calcium levels. Thus, DG acts as a selective vitamin D receptor modulator (SVDRM) by enhancing select anti-tumor characteristic 1,25(OH)(2)D(3) activities, without inducing hypercalcemia. Thus, DG shows promise in the development of colorectal cancer therapies.

Vitamin D and cancer.

The correlation between decreased morbidity and mortality of cancer and exposure to sunlight is known. The many biological functions of vitamin D that contribute to cancer prevention have only recently begun to be appreciated. Once activated 1,25-dihydroxyvitamin D [1,25(OH)2D3] functions as a potent inhibitor of normal and cancer cellular proliferation. Vitamin D deficiency in mice led to a 60% increase in colon tumor growth, compared to vitamin D-sufficient mice. The ligand binding domain of the Vitamin D receptor was shown to accommodate a class of 1,25(OH)2D3-analogs that possess an additional side-arm. These novel Gemini analogs were evaluated in vitro and in vivo. Select Gemini analogs were 100 times or more effective in inhibiting colon tumor growth in mice, compared to their parent compound. Correcting vitamin D deficiency may decrease the risk of developing colon cancer, while the novel Gemini 1,25(OH)2D3-analogs have the potential for therapeutic application in human colon cancer.

Colon cancer and solar ultraviolet B radiation and prevention and treatment of colon cancer in mice with vitamin D and its Gemini analogs.

It has been recognized that people who live at higher latitudes and who are vitamin D deficient are at higher risk of dying from many common cancers including colon cancer. To evaluate the role of vitamin D deficiency on colon tumor growth, Balb/c adult male mice were fed either a vitamin D sufficient or vitamin D deficient diet for 10 weeks. Mice were arranged into groups of six and each animal received subcutaneously 10(4) MC-26 cells in the posterior trunk. The tumor size was recorded daily. By day 9 there was a significant difference in tumor volume between the vitamin D sufficient and vitamin D deficient mice. By day 18 the vitamin D deficient animals had a tumor size that was 56% larger compared to the animals that were vitamin D sufficient. To determine whether treatment with active vitamin D analogs could further decrease colon tumor growth in a vitamin D sufficient state, groups of mice were treated with the novel 19-nor-Gemini compounds. The mice were fed a low calcium diet. Twenty-four hours after tumor implantation, the mice received, three times weekly, one of the vitamin D analogs or the vehicle. The group that received Gemini 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-19-nor-20S-cholecalciferol (3) showed a dose-dependent decrease in tumor volume. On day 19, at the dose level of 0.02microg molar equivalents (E), the tumor volume was reduced by 41% when compared to the control group. At the same time point, the hexadeuterated analog 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero-19-nor-20S-cholecalciferol (4), administered at the 10-fold lower dose of 0.002microgE, showed a 52% reduction in tumor volume (p<0.05), compared to the control group. Animals that received 1,25(OH)(2)D(3) at 0.002 and 0.02microg showed a trend in tumor volume reduction at the highest dose but the changes were not statistically significant. An evaluation of serum calcium concentrations revealed that the calcium levels were normal in all groups, except the group receiving 0.02microgE of 4. The results from these studies demonstrate that vitamin D deficiency may accelerate colon cancer growth and that novel Gemini analogs of 1,25(OH)(2)D(3) may be an effective new approach for colon cancer treatment.

Translational study of vitamin D differentiation therapy of myeloid leukemia: effects of the combination with a p38 MAPK inhibitor and an antioxidant.

Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-dihydroxyvitamin D3 (1,25D3) or its analogs (deltanoids). However, translation of these findings to the clinic is limited by calcemic effects of deltanoids. Strategies to overcome this problem include combination of deltanoids with other compounds to induce differentiation at lower, noncalcemic, deltanoid concentrations. We previously showed that either carnosic acid, an antioxidant, or SB202190, a p38 MAPK inhibitor, increase the potency of 1,25D3 in the HL60 cell line. Here, we report that simultaneous addition of both these agents further increases differentiation potency of deltanoids in this cell line and in freshly obtained leukemic cells ex vivo. Activity of MAPK pathways showed that increased differentiation was associated with enhanced activity of JNK pathway in all responding cell subtypes. Our studies suggest that patients with CML or AML subtypes M2 and M4, but not M1, M3 or M4eo, are particularly suitable for this combination therapy. We conclude that the established cell line HL60 presents a good model for some, but not all, subtypes of myeloid leukemia, and that the JNK pathway plays an important role in monocytic differentiation of human leukemic cells ex vivo, as well as in vitro.

Double bond in the side chain of 1alpha,25-dihydroxy-22-ene-vitamin D(3) is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney.

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1alpha,25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] like 1alpha,25(OH)(2)D(3), is also converted into the same final product, calcitroic acid. This finding indicated that 1alpha,25(OH)(2)D(2) also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1alpha,25(OH)(2)D(2) when compared to the side chain of 1alpha,25(OH)(2)D(3), has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1alpha,25(OH)(2)D(2). We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D(2) related compounds. We compared the metabolism of 1alpha,25-dihydroxy-22-ene-vitamin D(3) [1alpha,25(OH)(2)-22-ene-D(3)], a synthetic vitamin D analog whose side chain differs from that of 1alpha,25(OH)(2)D(3) only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1alpha,25(OH)(2)-22-ene-D(3) and 1alpha,25(OH)(2)D(3) are converted into common metabolites namely, 1alpha,24(R),25-trihydroxyvitamin D(3) [1alpha,24(R),25(OH)(3)D(3)], 1alpha,25-dihydroxy-24-oxovitamin D(3) [1alpha,25(OH)(2)-24-oxo-D(3)], 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D(3). This finding indicates that the double bond in the side chain of 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Along with the aforementioned metabolites, 1alpha,25(OH)(2)-22-ene-D(3) is also converted into two additional metabolites namely, 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Furthermore, we did not observe direct conversion of 1alpha,25(OH)(2)-22-ene-D(3) into 1alpha,25(OH)(2)D(3). These findings indicate that 1alpha,25(OH)(2)-22-ene-D(3) is first converted into 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Then the double bonds in the side chains of 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3) undergo reduction to form 1alpha,24(R),25(OH)(3)D(3) and 1alpha,25(OH)(2)-24-oxo-D(3), respectively. Thus, our study indicates that the double bond in 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1alpha,25(OH)(2)-22-ene-D(3) metabolism indicating that prior C-24 hydroxylation of 1alpha,25(OH)(2)-22-ene-D(3) is required for the double bond reduction to occur.

Gene regulatory potential of 1alpha,25-dihydroxyvitamin D(3) analogues with two side chains.

The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) acts through the transcription factor vitamin D receptor (VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins, and specific DNA binding sites (VDREs). Ligand-mediated conformational changes of the VDR are the core of the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signalling. Studying the interaction of 1alpha,25(OH)(2)D(3) analogues with this molecular switch should allow the characterization of their potential selective biological profile. A 1alpha,25(OH)(2)D(3) analogue with two side chains (Ro27-2310 or Gemini) was found to stabilize functional VDR conformations and VDR-RXR heterodimers on a VDRE with a slightly lower sensitivity than the natural hormone. A 19-nor derivative of Gemini (Ro27-5646) showed similar sensitivity whereas 5,6-trans (Ro27-6462) 3-epi (Ro27-5840) and 1alpha-fluoro (Ro27-3752) derivatives were equal to each other, but approximately 30-times less sensitive than Gemini. A des-C,D derivative of Gemini (Ro28-1909) showed only residual activity at maximal concentrations. In contrast to 1alpha,25(OH)(2)D(3), Gemini and its derivatives showed a differential preference in stabilizing VDR conformations which was found to be modulated by DNA coactivator and corepressor proteins. An analysis of the gene regulatory potential of the VDR agonists in cellular reporter gene systems demonstrated the same ranking as in the in vitro systems, but Gemini and its 19-nor derivative were found to be more sensitive than 1alpha,25(OH)(2)D(3) which indicates that the natural hormone is selectively metabolized. This study used straightforward methods for the in vitro and ex vivo evaluation of the gene regulatory potential of 1alpha,25(OH)(2)D(3) analogues. Gemini was highlighted as an interesting drug candidate which could not be optimized through obvious chemical modifications in its A-ring. J. Cell. Biochem. Suppl. 36: 179-190, 2001.

24-Oxo metabolites of vitamin D3 analogues: disassociation of their prominent antileukemic effects from their lack of calcium modulation.

The seco-steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of malignant cells including those of the hematopoietic system. The 24-oxo metabolite of 1,25(OH)(2)D(3) also has prominent antiproliferative activities against various cancer cells. We chemically synthesized five novel 24-oxo vitamin D(3) analogues and evaluated their abilities both to inhibit clonal growth and induce differentiation of myeloid leukemia cells and to cause hypercalcemia. The 1alpha,25-dihydroxy-16-ene-D(3) [1,25(OH)(2)-16-ene-D(3)] and 1alpha,25-dihydroxy-16-ene-19-nor-D(3) [1,25(OH)(2)-16-ene-19-nor-D(3)] and their 24-oxo metabolites showed greater potency than 1,25(OH)(2)D(3) in their abilities to inhibit clonal proliferation of HL-60, NB4, and U937 leukemic cell lines as measured by methylcellulose soft-gel assay. Their inhibition of clonal growth was irreversible as analyzed by pulse exposure studies. The synthetic analogues also had greater potency than 1,25(OH)(2)D(3) to induce differentiation of HL-60 and NB4 cells as measured by generation of superoxide, nonspecific esterase production, and induction of CD11b and CD14 cell surface antigens and to increase the proportion of these cells in the G(0)-G(1) phase of the cell cycle. For most assays, the 24-oxo metabolite was slightly more potent than the unmodified analogue, and 50% activity was usually found in the nanomolar range. These analogues and their 24-oxo metabolites also inhibited fresh leukemic cell clonal proliferation. Expression of p27(KIP1), a cyclin-dependent kinase inhibitor that plays an important role in blocking the cell cycle, was found by Western blot analysis to be induced by the analogues and their 24-oxo metabolites in both HL-60 and U937 cells, suggesting a possible mechanism by which these analogues inhibit leukemic growth. Notably, the calcemic activity tested by injections of 1alpha,25-dihydroxy-16-ene-24-oxo-19-nor-D(3) in mice was at least 12-fold less than 1alpha,25(OH)(2)-16-ene-19-nor-D(3). Taken together, chemically synthesized 24-oxo metabolites of 1alpha,25(OH)(2)-16-ene-D(3) and 1alpha,25(OH)(2)-16-ene-19-nor-D(3) irreversibly inhibited proliferation and induced differentiation of acute myeloid leukemia cells with minimal toxicity; these compounds may have a role in the maintenance phase of therapy for acute myeloid leukemia.

Novel vitamin D(3) analog, 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3), that modulates cell growth, differentiation, apoptosis, cell cycle, and induction of PTEN in leukemic cells.

The active form of vitamin D(3), 1,25(OH)(2)D(3), inhibits proliferation and induces differentiation of a variety of malignant cells. A new class of vitamin D(3) analogs, having 2 identical side chains attached to carbon-20, was synthesized and the anticancer effects evaluated. Four analogs were evaluated for their ability to inhibit growth of myeloid leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer cells. All 4 analogs inhibited growth in a dose-dependent manner. Most effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3) (Gemini-19-nor), which has 2 side chains and removal of the C-19. Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more potent than 1,25(OH)(2)D(3) in inhibiting 50% clonal growth (ED(50)) of NB4, HL-60, MCF-7, and LNCaP cells, respectively. Gemini-19-nor (10(-8) M) strongly induced expression of CD11b and CD14 on HL-60 cells (90%); in contrast, 1,25(OH)(2)D(3) (10(-8) M) stimulated only 50% expression. Annexin V assay showed that Gemini-19-nor and 1,25(OH)(2)D(3) induced apoptosis in a dose-dependent fashion. Gemini-19-nor (10(-8) M, 4 days) caused apoptosis in approximately 20% of cells, whereas 1,25(OH)(2)D(3) at the same concentration did not induce apoptosis. Gemini-19-nor increased in HL-60 both the proportion of cells in the G(1)/G(0) phase and expression level of p27(kip1). Moreover, Gemini-19-nor stimulated expression of the potential tumor suppressor, PTEN. Furthermore, other inducers of differentiation, all-trans-retinoic acid and 12-O-tetradecanoylphorbol 13-acetate, increased PTEN expression in HL-60. In summary, Gemini-19-nor strongly inhibited clonal proliferation in various types of cancer cells, especially NB4 cells, suggesting that further studies to explore its anticancer potential are warranted. In addition, PTEN expression appears to parallel terminal differentiation of myeloid cells.

Synergistic growth inhibition of prostate cancer cells by 1 alpha,25 Dihydroxyvitamin D(3) and its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A.

Prostate cancer is a major cause of male cancer death. In vitro and in vivo data support a role for 1 alpha,25 Dihydroxyvitamin D(3) (1 alpha,25(OH)(2)D(3)) in regulating the growth and differentiation of the normal prostate gland yet prostate cancer cells appear significantly less sensitive to this action. Vitamin D(3) receptor (VDR) content or mutational status do not correlate clearly with the antiproliferative effects of 1 alpha,25(OH)(2)D(3) and therefore it is unclear why prostate cancer cell lines are significantly less sensitive to this action. We hypothesized that the antiproliferative responses of prostate cancer cells to 1 alpha,25(OH)(2)D(3) are suppressed by a process involving histone deacetylation. Sodium butyrate (NaB) and trichostatin A (TSA) are inhibitors of histone deacetylase (HDAC) activity. Low doses of NaB or TSA (300 microM and 15 nM respectively), which alone were relatively inactive, synergized with 1 alpha,25(OH)(2)D(3) in liquid and semi-solid agar to inhibit the growth of LNCaP, PC-3 and DU-145 prostate cancer cells. Still greater synergy was observed between vitamin D(3) hexafluoride analogs and either NaB or TSA. The mechanism appeared to involve neither the cyclin-dependent kinase inhibitor, p21((waf1/cip1)) nor cell cycle arrest, but rather induction of apoptosis. These data suggest that cells dysregulate the normal pro-apoptotic signals of 1 alpha,25(OH)(2)D(3) during prostate cancer development by a mechanism involving histone deacetylation. Combination therapy with potent vitamin D(3) analogs and clinically approved HDAC inhibitors may overcome this lesion and improve the treatment of both androgen-dependent and independent prostate cancer.

The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation.

We compared the effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analog, 1alpha,25-dihydroxy-16-ene-vitamin D3 [1alpha,25(OH)2-16-ene-D3], as well as their interactions with 17-beta estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than 1alpha,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1alpha,25(OH)2D3. Western blot analysis of 1alpha,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1alpha,25(OH)2D3 was larger than that of 1alpha,25(OH)2-16-ene-D3. These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.

Differential activities of 1alpha,25-dihydroxy-16-ene-vitamin D(3) analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis.

To clarify physiological role of the carbon 3 (C-3) epimerization of 1alpha,25(OH)(2)D(3) and biologic significance of a 3-epi metabolite of 1alpha,25(OH)(2)D(3), we examined biologic activities of the 3-epimers of 1alpha,25(OH)(2)D(3) and 1alpha,25(OH)(2)-16-ene-D(3) analogs in terms of modulation of cell cycle phase distribution and cell-surface CD11b antigen expression of HL-60 cells, transactivation of vitamin D target genes in transfected cells, stimulation of VDR/RXRalpha heterodimer formation in a rabbit reticulocyte lysates transcription/translation system, stimulation of VDR/RXRalpha/VDRE complex formation, and induction of HL-60 cell apoptosis. The analogs tested here were 1) 1alpha,25(OH)(2)D(3), 2) 1alpha,25(OH)(2)-3-epi-D(3), 3) 1alpha,25(OH)(2)-16-ene-D(3), 4) 1alpha,25(OH)(2)-16-ene-3-epi-D(3), 5) 1alpha,25(OH)(2)-16-ene-23-yne-hexafluoro(F(6))-D(3), 6) 1alpha,25(OH)(2)-16-ene-23-yne-hexafluoro(F(6))-3-epi-D(3), 7) 1alpha,25-(OH)(2)-16-ene-20-epi-23-yne-D(3), and 8) 1alpha,25(OH)(2)-16-ene-20-epi-23-yne-3-epi-D(3). When compared to the 3-natural (beta) analogs, the 3-epi (alpha) analogs were biologically significantly less active. The findings support the hypothesis that the C-3 epimerization is an inactivation pathway of 1alpha,25(OH)(2)D(3) and its analogs in vitamin D target tissues. We also found that the 3-epi analogs, but not the 3-natural (beta) analogs, were the potent inducers of apoptosis of HL-60 cells. These results suggest that the analogs could be divided into two groups, in which the 3-epi analogs were the potent inducers of apoptosis of HL-60 cells, and the 3-natural analogs were the potent modulators of HL-60 cell growth and differentiation. This is the first report demonstrating that the 3-epimerization of the hydroxyl group at C-3 of the A-ring of 1alpha,25(OH)(2)D(3) plays an important role to modulate HL-60 cell differentiation and apoptosis.

Metabolites and analogs of 1alpha,25-dihydroxyvitamin D(3): evaluation of actions in bone.

Analogs of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] activate both genomic mechanisms via the nuclear vitamin D(3) receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D(3) receptor (pmVDR). Both of these pathways are normally activated by 1alpha,25(OH)(2)D(3), but as a result of synthesis of numerous analogs of 1alpha,25(OH)(2)D(3) these pathways can be distinguished. We used increasing doses of vitamin D(3) analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D(3) (R) and 1alpha,25(OH)(2)-16,23E-diene-D(3) (A) are 10-fold more potent than 1alpha,25(OH)(2)D(3) for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1alpha-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1alpha,25(OH)(2)D(3) to increase osteocalcin mRNA or protein release. 1alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D(3), (E) is an agent that is 10 fold more potent than 1alpha,25(OH)(2)D(3) at increasing osteocalcin mRNA and protein release, whereas 1alpha,25(OH)(2)-3-epi-D(3) increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1alpha,25(OH)(2)D(3). These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F(6) on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1alpha-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.

Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells.

The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).

Characterization of a novel analogue of 1alpha,25(OH)(2)-vitamin D(3) with two side chains: interaction with its nuclear receptor and cellular actions.

The hormone 1alpha,25(OH)(2)-vitamin D(3) (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1alpha,25(OH)(2)D(3) (20E-125D) increases transcription 200-5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3) (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays: binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells: ED(50) 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED(50)(125D)/ED(50)(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.

1,25-dihydroxyvitamin D3 synthetic analogs inhibit spontaneous metastases in a 1,2-dimethylhydrazine-induced colon carcinogenesis model.

In order to substantiate the role of vitamin D applicability for the prevention of colon cancer and its spontaneous metastases, the effect of 1,25-dihydroxyvitamin D3 and its synthetic analogs, 1, 25-dihydroxy-16,23Z-diene-26,27-hexafluoro-D3 (Ro 25-5317) and 1, 25-dihydroxy-16,23E-diene-26,27-hexafluoro-19-nor-D3 (Ro 25-9022), have been evaluated in a 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis model in Sprague-Dawley rats. In animals maintained on 2.75 nmol/kg 1,25-dihydroxyvitamin D3 diet no statistical difference was seen in tumor incidence when compared with control while in animals on 3.0 nmol/kg 1,25-dihydroxyvitamin D3 diet, the incidence of tumors was significantly lower. In animals maintained on 3.0 nmol/kg Ro 25-5317 diet also no statistical difference was seen in tumor incidence compared with control while in animals on 3. 5 nmol/kg Ro 25-5317 diet the incidence of tumors was significantly lower. The incidence of tumors in the group of animals maintained on 3.0 nmol/kg and 3.5 nmol/kg Ro 25-9022 was significantly lower, at 32.1% and 27.6% respectively, compared to control. In the two groups of animals maintained on the 1,25-dihydroxyvitamin D3 diet no significant difference in the incidence of metastasis was seen. In the group of animals maintained on 3.0 nmol/kg Ro 25-5317 diet only regional metastases were seen. However, no metastases developed in the rats on 3.5 nmol/kg Ro 25-5317 diet. After administration of 3.0 nmol/kg Ro 25-9022 diet, metastases developed in a significantly less number of animals while no metastases occurred in the rats maintained on the 3.5 nmol/kg Ro 25-9022 diet. The above studies will provide a scientific basis for the progression into further clinical trials in the treatment, and/or chemoprevention of human colorectal cancer.

Response element and coactivator-mediated conformational change of the vitamin D(3) receptor permits sensitive interaction with agonists.

The vitamin D receptor (VDR) is the nuclear receptor for 1, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] that acts as a ligand-dependent transcription factor via combined contact with coactivator proteins (steroid receptor coactivator-1, transcriptional intermediary factor 2, and receptor associated coactivator 3) and specific DNA binding sites [vitamin D response elements (VDREs)]. Ligand-mediated conformational changes of the VDR contribute to the key mechanisms in this nuclear hormone signaling process. 1alpha,25(OH)(2)D(3), MC1288 [20-epi-1alpha,25(OH)(2)D(3)], ZK161422 [20-methyl-1alpha,25(OH)(2)D(3)], and Ro27-2310 (also called Gemini, having two side chains at carbon 20) were used as model VDR agonists. The analysis of agonist-induced VDR conformations and coactivator interactions were found to be insufficient for extrapolating in vivo activities. In DNA-independent assays, such as classical limited protease digestions and glutathione S-transferase pull downs, Gemini seemed to be up to 10,000-fold and the other VDR agonists 10- to 100-fold weaker than in functional in vivo assays. A more accurate description of the gene regulatory potential of VDR agonists was obtained with all tested VDR agonists by analyzing VDR conformations in the context of VDRE-bound VDR-retinoid X receptor heterodimers, in such assays as gel supershift, gel shift clipping, and limited protease digestion in the presence of DNA and cofactor. Coactivators were found to shift the ligand sensitivity (by a factor of 4 for Gemini) and the ratio of VDR conformations in the presence of DNA toward the high-affinity ligand binding conformation (c1(LPD)). In conclusion, the induction of response element- and coactivator-modulated VDR conformations appears to be a key step for the gene regulatory function of a VDR agonist. The quantification of these effects would be of central importance for the evaluation of the cell-specific efficacy of systemically applied 1alpha, 25(OH)(2)D(3) analogs.

Effects of long-term administration of vitamin D3 analogs to mice.

This study explores the effects of chronic administration of vitamin D(3) compounds on several biological functions in mice. Knowledge of long-term tolerability of vitamin D(3) analogs may be of interest in view of their potential clinical utility in the management of various pathologies such as malignancies, immunological disorders and bone diseases. Four unique vitamin D(3) analogs (code names, compounds V, EO, LH and LA) and 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) were administered i.p. for 55 weeks to Balb/c mice. Each analog had previously been shown to have potent in vitro activities. After 55 weeks of administration, the mice had a profound decrease in their serum levels of interleukin-2 (IL-2). Likewise, several analogs depressed serum immunoglobulin G concentrations (compounds LH and LA), but levels of blood lymphocytes and splenic lymphocyte subsets (CD4, CD8 and CD19) were not remarkably depressed. The percent of committed myeloid hematopoietic stem cells was 4- to 5-fold elevated in the bone marrow of the mice that received analogs LH and V; nevertheless, their peripheral blood white and red cell counts and platelets were not significantly different in any of the groups. The mice that received 1,25(OH)(2)D(3) had a decrease in bone quantity and quality with a decrease in cross-sectional area and cortical thickness, and a 50% reduction in both stiffness and failure load compared with the control group. In contrast, the cohort that received a fluorinated analog (compound EO) developed bones with significantly larger cross-sectional area and cortical thickness as well as stronger mechanical properties compared with the control group. At the conclusion of the study, body weights were significantly decreased in all experimental mice. Their blood chemistries were normal. Extensive gross and microscopic autopsy analyses of the mice at the conclusion of the study were normal, including those of their kidneys. In conclusion, the vitamin D(3) analogs were fairly well tolerated. They did suppress immunity as measured by serum IL-2 and may provide a means to depress the immune response after organ transplantation and for autoimmune diseases. Use of these analogs prevented the detrimental effects of vitamin D(3) administration on mechanical and geometric properties of bone, while one analog (compound EO) actually enhanced bone properties. These results suggest that long-term clinical trials with the analogs are feasible.

Induction of tissue plasminogen activator secretion from rat heart microvascular cells by fM 1,25(OH)(2)D(3).

We investigated the effects of 1,25-dihydroxyvitamin D(3) [25(OH)(2)D(3)] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)(2)D(3) and several of its analogs. The results showed that 1,25(OH)(2)D(3) induced tPA secretion at 10(-10) to 10(-16) M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca(2+) channels, Ro-24-5531, were approximately 10% as active as 1,25(OH)(2)D(3). An analog that binds the vitamin D receptor poorly but is an effective Ca(2+) channel agonist, Ro-24-2287, required approximately 10(-13) M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)(2)D(3). Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca(2+)]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)(2)D(3) was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)(2)D(3) on two different induction pathways of tPA release.