Potent antiproliferative effects of 25-hydroxy-16-ene-23-yne-vitamin D3 that resists the catalytic activity of both CYP27B1 and CYP24A1.

The potency of 25-hydroxyvitamin D3 (25(OH)D3) is increased by several fold through its metabolism into 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by cytochrome P450 27B1 (CYP27B1). Thus, the pivotal role of 1α-hydroxylation in the activation of vitamin D compounds is well known. Here, we examined the metabolism of 25-hydroxy-16-ene-23-yne-vitamin D3 (25(OH)-16-ene-23-yne-D3), a synthetic analog of 25(OH)D3 in a cell-free system and demonstrated that 25(OH)-16-ene-23-yne-D3 is neither activated by CYP27B1 nor inactivated by cytochrome P450 24A1 (CYP24A1). These findings were also confirmed in immortalized normal human prostate epithelial cells (PZ-HPV-7) which are known to express both CYP27B1 and CYP24A1, indicating that the structural modifications featured in 25(OH)-16-ene-23-yne-D3 enable the analog to resist the actions of both CYP27B1 and CYP24A1. To provide intelligible structure-function information, we also performed molecular docking analysis between the analog and CYP27B1. Furthermore, 25(OH)-16-ene-23-yne-D3 was found to suppress the growth of PZ-HPV-7 cells with a potency equivalent to 1α,25(OH)2D3. The antiproliferative activity of 25(OH)-16-ene-23-yne-D3 was found to be vitamin D receptor (VDR)-dependent as it failed to inhibit the growth of mammary tumor cells derived from VDR-knockout mice. Furthermore, stable introduction of VDR into VDR-knockout cells restored the growth inhibition by 25(OH)-16-ene-23-yne-D3. Thus, we identified 25-hydroxy-16-ene-23-yne-vitamin D3 as a novel non-1α-hydroxylated vitamin D analog which is equipotent to 1α,25(OH)2D3 in its antiproliferative activity. We now propose that the low potency of the intrinsic VDR-mediated activities of 25(OH)D3 can be augmented to the level of 1α,25(OH)2D3 without its activation through 1α-hydroxylation by CYP27B1, but by simply preventing its inactivation by CYP24A1.

Novel Gemini vitamin D3 analogs: large structure/function analysis and ability to induce antimicrobial peptide.

We have synthesized 39 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] analogs having two side chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anticancer activity. One of these active analogs, BXL-01-0126, was more potent than 1,25(OH)2D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 when compared to 1,25(OH)2D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of acute myeloid leukemia (AML) cells to the Gemini analog, BXL-01-126, in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG-immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients to provide them with protection against various microbial infections through CAMP induction.

A novel Gemini vitamin D analog represses the expression of a stem cell marker CD44 in breast cancer.

CD44 is a multifunctional transmembrane protein involved in cell proliferation, angiogenesis, invasion, and metastasis. CD44 is identified as a cancer stem cell marker, and the CD44-positive breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 may be an important target for cancer prevention and therapy. Therefore, we investigated for the inhibitory effect of a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), on mammary tumor growth and CD44 expression in MCF10DCIS.com human breast cancer in vitro and in vivo. MCF10DCIS.com cells were injected into mammary fat pads in immunodeficient mice, and BXL0124 was then administered intraperitoneally (0.1 µg/kg body weight) or orally (0.03 or 0.1 µg/kg body weight) 6 days a week for 5 weeks. At necropsy, mammary tumors and blood were collected for evaluating tumor growth, CD44 expression, and serum calcium level. BXL0124 suppressed mammary tumor growth and markedly decreased the expression of CD44 protein in MCF10DCIS xenograft tumors without causing hypercalcemic toxicity. BXL0124 also inhibited the expression of CD44 protein and mRNA as well as the transcriptional activity of the CD44 promoter in cultured MCF10DCIS.com cells. The repression of CD44 expression induced by BXL0124 was blocked by siRNA vitamin D receptor (VDR), indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. The novel Gemini vitamin D analog, BXL0124, represses CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of a Gemini vitamin D derivative on breast cancer stem cells.

A new insight into the role of rat cytochrome P450 24A1 in metabolism of selective analogs of 1α,25-dihydroxyvitamin D3.

We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D3 (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D3 (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D3 (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D3 (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.

Silibinin can induce differentiation as well as enhance vitamin D3-induced differentiation of human AML cells ex vivo and regulates the levels of differentiation-related transcription factors.

Induction of terminal differentiation is a conceptually attractive approach for the therapy of neoplastic diseases. Although vitamin D derivatives (deltanoids) can induce differentiation of AML cells in vitro, so far deltanoids have not been successfully brought to the clinic, due to the likelihood of life-threatening hypercalcemia. Here, we incubated freshly obtained blood cells from patients with AML with a plant antioxidant (PAOx), silibinin (SIL), alone or together with a deltanoid. Twenty patients with AML (all subtypes except M3) were available for this study, and in 14 (70%), SIL (60 µM) either induced differentiation ex vivo, or enhanced differentiation induced by deltanoids, or both. Interestingly, SIL acting alone induced differentiation only in cases in which chromosome aberrations could not be detected. In eleven samples sufficient material was available for a limited analysis of the underlying events. Quantitative RT-PCR showed that differentiation markers were upregulated at the mRNA level by both SIL and deltanoids, suggesting that intracellular signaling pathways upstream of transcription factors (TFs) were activated by these agents. Western analysis for proteins which function as TFs in deltanoid-induced monocytic differentiation, such as members of Jun and C/EBP families, surprisingly demonstrated that SIL upregulated all these TFs in the cases tested. This suggests that although the presence of SIL may not always be sufficient to induce differentiation, it can serve as a differentiation enabling factor for blasts obtained from a large proportion of patients with AML. Thus, SIL/deltanoid combinations warrant further consideration as preventive/therapeutic regimens in human leukaemia.

Selective inhibition of cyclooxygenase-2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog.

Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor.

Synergistic antileukemic activity of carnosic acid-rich rosemary extract and the 19-nor Gemini vitamin D analogue in a mouse model of systemic acute myeloid leukemia.

OBJECTIVE: Differentiation therapy with the hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D(3) induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D(3) analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML. METHODS: Proliferation and differentiation of WEHI-3B D- (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice. RESULTS: 19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D(3), in the induction of differentiation (EC(50) = 0.059 +/- 0.011, 0.275 +/- 0.093 and 0.652 +/- 0.085 nM, respectively) and growth arrest (IC(50) = 0.072 +/- 0.018, 0.165 +/- 0.061 and 0.895 +/- 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 +/- 2.5 days in untreated mice to 66.5 +/- 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D(3) combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures. CONCLUSION: Combined effectiveness of 1,25D(3) analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.

Novel Gemini vitamin D(3) analogs have potent antitumor activity.

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.

Calcitriol derivatives with two different side chains at C-20 III. An epimeric pair of the gemini family with unprecedented antiproliferative effects on tumor cells and renin mRNA expression inhibition.

The searches for drugs that exhibit antineoplastic activity and regulate blood pressure are among the most prevalent and compelling research activities today. Amazingly, there is ample precedence for the antiproliferative action of vitamin-D-related compounds and their role as endocrine suppressors of renin biosynthesis. We have recently synthesized a number of novel calcitriol analogs of the gemini family and originally selected for further studies an epimeric pair related to 19-nor-calcitriol whose 21-methyl group was replaced by a 5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl group. While maintaining the acceptable calcemic responses, the IC50 concentrations of interferon-gamma release were reduced and the antiproliferative activity and inhibition of renin mRNA expression enhanced. Replacing the geminal methyl groups on the calcitriol-related side chain of these gemini compounds with trideuteriomethyl moieties further boosted the potency in the colon cancer model in mice some 10-fold, reduced NMU-induced breast cancer carcinogenesis in rats and decreased the IC(50) values for renin mRNA inhibition into the pM range.

Inhibition of prostate growth and inflammation by the vitamin D receptor agonist BXL-628 (elocalcitol).

The prostate is a target organ of vitamin D receptor (VDR) agonists and represents an extra-renal site of 1,25-dihydroxyvitamin D(3) synthesis, but its capacity to respond to VDR agonists has, so far, been almost exclusively probed for the treatment of prostate cancer. We have analyzed the capacity of VDR agonists to treat benign prostatic hyperplasia (BPH), a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic one responsible for urinary irritative symptoms, and an inflammatory component. Preclinical data demonstrate that VDR agonists, and notably BXL-628 (elocalcitol), reduce the static component of BPH by inhibiting the activity of intra-prostatic growth factors downstream of the androgen receptor, and the dynamic component by targeting bladder cells. In addition, BXL-628 inhibits production of proinflammatory cytokines and chemokines by human BPH cells. These data have led to a proof-of-concept clinical study that has successfully shown arrest of prostate growth in BPH patients treated with BXL-628, with excellent safety. We have documented the anti-inflammatory effects of BXL-628 also in animal models of autoimmune prostatitis, observing a significant reduction of intra-prostatic cell infiltrate following administration of this VDR agonist, at normocalcemic doses, in mice with already established disease. These data extend the potential use of VDR agonists to novel indications that represent important unmet medical needs, and provide a sound rationale for further clinical testing.

Altered nuclear receptor corepressor expression attenuates vitamin D receptor signaling in breast cancer cells.

PURPOSE: We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1alpha,25-dihydroxyvitamin D(3). EXPERIMENTAL DESIGN: Profiling, transcriptional, and proliferation assays were undertaken in 1alpha,25(OH)(2)D(3)-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21). RESULTS: Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G(1) of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor alpha-negative (n = 7) tumors. 1alpha,25(OH)(2)D(3) resistance in cancer cell lines was targeted by cotreatments with either 1alpha,25(OH)(2)D(3) or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D(3) compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells. CONCLUSIONS: Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1alpha,25(OH)(2)D(3) resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D(3) compounds and low doses of TSA.

The synthetic triterpenoid CDDO-imidazolide induces monocytic differentiation by activating the Smad and ERK signaling pathways in HL60 leukemia cells.

Synthetic triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) or CDDO-imidazolide [2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid imidazolide (CDDO-Im)], induce cell differentiation in myeloid leukemia cells but their mechanism of action is not known. CDDO-Im induces monocytic differentiation markers, CD14, and nonspecific esterase in HL60 leukemia cells. We show that CDDO-Im activates the extracellular signal-regulated kinase (ERK) signaling pathway and up-regulates CCAAT/enhancer-binding protein beta, a transcription factor critical for monocytic differentiation. The monocytic differentiation induced by CDDO-Im was partially blocked by the mitogen-activated protein kinase/ERK kinase 1 inhibitor PD98059, suggesting that the mitogen-activated protein kinase-ERK1/2 pathway plays a role in the differentiation induced by CDDO-Im. Furthermore, CDDO-Im activates the transforming growth factor beta (TGF-beta)/Smad signaling pathway. CDDO-Im enhanced the phosphorylation of the receptor-regulated Smads, phospho-Smad3, and phospho-Smad1/5, but not phospho-Smad2, and induced the expression of Smad4. Monocytic differentiation induced by CDDO-Im was blocked by both TGF-beta antibody and the bone morphogenetic protein (BMP) antagonist Noggin. This indicates that activation of the Smad signaling pathway by triterpenoids is an important mechanism of monocytic differentiation. CDDO-Im induced the synthesis of mRNA for TGF-beta2, BMP6, TGF-beta type II receptor, and BMP type II receptor. CDDO-Im synergized with members of the TGF-beta superfamily or with 1alpha,25(OH)2vitamin D3 (D3) in monocytic differentiation, and the synergistic effect was particularly striking in combination with D3. The combination of triterpenoids and D3 may have a practical use in differentiation therapy of myeloid leukemia as well as for promoting the formation of bone and cartilage.

Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3.

Analogs of 1α,25-dihydroxyvitamin D3 (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D3 (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs. For example, 1α,25-dihydroxy-16-ene-20-epi-vitamin D3 (S4), but not 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR-106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3-epi-S4, a metabolite of S4 produced through C-3 epimerization pathway. Using GC/MS, ESI-MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25-hydroxy-16-ene-20-epi-vitamin D3-1-stearate and 25-hydroxy-16-ene-20-epi-vitamin D3-1-oleate and their respective C-3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR-106 cells in which both 1α,25(OH)2-16-ene-20-epi-D3 and 1α,25(OH)2-16-ene-20-epi-3-epi-D3 undergo C-1 esterification into stearic and oleic acid esters.

C-20 cyclopropyl vitamin D3 analogs.

The formal C-20 methylation of 1,25-dihydroxy vitamin D3 (calcitriol) and bridging of two methyl groups produces spiro[cyclopropane-1, 20'-calcitriol], colloquially referred to as C-20 cyclopropylcalcitriol, which is much more active in MLR for suppression of interferon-gamma release than calcitriol, and hypercalcemia in mice is elicited at a ten-fold lower dose when compared to calcitriol. Introduction of the Delta16,17-double bond, modification of the side chain by 23-unsaturation and replacement of the methyl groups at C-26 and C-27 with trifluoromethyl moieties create a highly active series of vitamin D analogs. As previously observed in the calcitriol series, the presence of the C-16 double bond in the cyclopropyl analogs also arrests metabolic side-chain oxidation in the at the C-24 oxo level in UMR 106 cells. The enhanced biological activity is ascribed, at least in part, to the improved resistance toward metabolic degradation.

Gene expression profiling changes induced by a novel Gemini Vitamin D derivative during the progression of breast cancer.

We investigated gene expression changes induced by a novel Gemini Vitamin D(3) analog, RO-438-3582 (1alpha,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-cholecalciferol, Ro3582), in a unique human breast MCF10 model. We used two breast epithelial cell lines from this model, namely MCF10AT1 (Ha-ras oncogene transfected MCF10A, early premalignant) and MCF10CA1a (fully malignant and metastatic derived from the MCF10AT1 line). We analyzed gene expression changes induced by Ro3582 using GeneChip technology, quantitative RT-PCR, Western blot analysis, or a gene transcription assay. Interestingly, we found distinct gene expression profile differences between Ro3582-induced response of the early premalignant MCF10AT1 and the malignant and metastatic MCF10CA1a cell lines. Moreover, while the Gemini Vitamin D(3) analog Ro3582 modulated the expression of several Vitamin D target genes such as the 24-hydroxylase, CD14, osteocalcin, and osteopontin in both cell lines, Ro3582 regulated many genes involved in cell proliferation and apoptosis, cell adhesion, invasion, angiogenesis as well as cell signaling pathways, such as the BMP and TGF-beta systems, differently in the two cell lines. The Gemini Vitamin D(3) analog Ro3582 induced more significant gene changes in the early premalignant MCF10AT1 cells than in the malignant metastatic MCF10CA1a cells, suggesting that Gemini Vitamin D(3) analogs may be more effective in preventing the progression of an early stage of breast carcinogenesis than in treating late stage breast cancer.

Novel Gemini-vitamin D3 analog inhibits tumor cell growth and modulates the Akt/mTOR signaling pathway.

We have shown previously that 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3 (Gemini) compounds, which have two side chains attached to carbon-20, had increased anti-tumor activities against breast, prostate and leukemia cell lines in comparison to 1,25(OH)2 vitamin D3. This prompted us to synthesize additional Gemini compounds with further modifications and evaluate their anticancer effects. Most effective in this series was 1,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-vitamin D3 [Gemini-23-yne-26,27-hexafluoro-D3]. This analog was approximately 10-fold more potent than previously characterized Gemini compounds in inhibiting the clonal growth of HL-60, MCF-7 and LNCaP cell lines. Also in MCF-7 cells, Gemini-23-yne-26,27-hexafluoro-D3 caused dephosphorylation of the oncogenic kinase, Akt, resulting in dephosphorylation of the Akt target proteins, Forkhead transcription factor and mammalian target of rapamycin (mTOR). Downstream effectors of mTOR were also inhibited by the analog as demonstrated by decreased phosphorylation of both S6 kinase, and the translation inhibitor, 4E-BP1. The mTOR pathway regulates mRNA translation; exposure of MCF-7 cells to Gemini-23-yne-26,27-hexafluoro-D3 decreased their rate of protein synthesis and increased the association of 4EBP-1 with the translation initiation factor, eIF4E. Inhibition of the Akt-mTOR pathway represents a novel mechanism by which vitamin D3 analogs may modulate the expression and activity of proteins involved in cancer cell proliferation.

1alpha,25-(OH)(2)-D(3) and its synthetic analogue decrease tumor load in the Apc(min) Mouse.

Both calcium and vitamin D are thought to be able to inhibit colon carcinogenesis. To better define the effects of vitamin D, we studied 1alpha,25-(OH)(2)-D(3) and a noncalcemic synthetic analogue of vitamin D(3) (VD(3)) in the Apc(min) mouse. Female Apc(min) mice 4-5 weeks old were randomized to four groups: a VD(3)-treated group (n = 11) were given injections of 0.01 microg of 1alpha,25-(OH)(2)-D(3) i.p. three times per week; an analogue-treated group (n = 10) received 5 microg of 1alpha,25-(OH)(2)-16-ene-19-nor-24-oxo-D(3) i.p. three times per week; and a control group (n = 12) received sham injections of PBS. A sulindac-treated group (n = 10) was used as a positive control. Doses of these compounds were chosen based on previous toxicity studies in mice and rats. After 10 weeks of treatment, mice were killed and two observers (S. H., R. W. I.), blinded to treatment, scored polyp number and size. Tumor number was not affected with 1alpha,25-(OH)(2)-D(3) or vitamin D analogue administration. A significant decrease in total tumor load (sum of all polyp areas) over the entire gastrointestinal tract was seen in the analogue (36% decrease; P < 0.05) and the VD(3) groups (46%; P < 0.001). There was a significant decrease in polyp number (49%; P < 0.001) and polyp area (70%; P < 0.001) in the sulindac group. Reverse transcription-PCR of the total RNA derived from intestinal tissue revealed expression of the vitamin D receptor throughout the small intestine and the colon. Serum calcium levels in the analogue group were not elevated at week 4 of treatment and only moderately elevated (22%) by week 8 (P < or =0.001). In contrast, serum calcium in the VD(3) group was significantly elevated (P < or =0.001) at weeks 4 (23%) and 8 (45%). Food intake and growth rate were significantly lower in the VD(3) group (26%, P < 0.001, and 27%, P < 0.001, respectively) at week 10. In contrast, food intake and growth rate were similar for the control, sulindac, and analogue groups. Our results indicate that a noncalcemic analogue of vitamin D can significantly decrease intestinal tumor load in Apc(min) mice without severe toxic side effects and suggest that these compounds may have utility as chemopreventive agents in groups at high-risk for colon cancer.

A 1alpha,25-dihydroxyvitamin D(3) analog enhances regulatory T-cells and arrests autoimmune diabetes in NOD mice.

Type 1 diabetes is a chronic progressive autoimmune disease characterized by mononuclear cell infiltration, dominated by interleukin-12 (IL-12)-dependent Th1 cells, of the pancreatic islets, with subsequent destruction of insulin-producing beta-cells. Here, we demonstrate that treatment of adult nonobese diabetic (NOD) mice with an analog of 1alpha,25-dihydroxyvitamin D(3), an immunomodulatory agent preventing dendritic cell maturation, decreases lipopolysaccharide-induced IL-12 and gamma-interferon production, arrests Th1 cell infiltration and progression of insulitis, and inhibits diabetes development at nonhypercalcemic doses. Arrest of disease progression is accompanied by an enhanced frequency in the pancreatic lymph nodes of CD4(+)CD25(+) regulatory T-cells that are able to inhibit the T-cell response to the pancreatic autoantigen insulinoma-associated protein 2 and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells. Thus, a short treatment of adult NOD mice with an analog of 1,25-dihydroxyvitamin D(3) inhibits IL-12 production, blocks pancreatic infiltration of Th1 cells, enhances CD4(+)CD25(+) regulatory cells, and arrests the progression of type 1 diabetes, suggesting its possible application in the treatment of human autoimmune diabetes.

Analogs of 1alpha,25-dihydroxyvitamin D(3) as novel inhibitors of renin biosynthesis.

The renin-angiotensin system (RAS) plays a central role in the pathogenesis of hypertension. Recently, we discovered that 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] functions as a negative endocrine regulator of renin biosynthesis, which provides a molecular basis to explore the potential of Vitamin D analogs as renin inhibitors to control the RAS. To search for renin-inhibiting Vitamin D analogs, we screened 20 Vitamin D analog compounds using As4.1-hVDR cell (a juxtaglomerular cell line) culture by Northern blot and luciferase reporter assays. We found that the Gemini compounds, which have two side-chains at carbon-20 position, were particularly active in suppressing renin expression. Eight Gemini compounds were identified that were equally or 10- to 100-times more potent than 1,25(OH)(2)D(3) in renin inhibition. These Gemini compounds also potently stimulate 25-hydroxyvitamin D 24-hydroxylase expression in As4.1-hVDR cells. Administration of compound RO-27-5646 [1,25-dihydroxy-21-(3-methyl-3-hydroxy-butyl)-19-nor-cholecalciferol] in mice caused a marked reduction in renal renin mRNA expression without invoking severe hypercalcemia as seen in 1,25(OH)(2)D(3) treatment. These data establish in principle that Vitamin D analogs can indeed inhibit renin expression in vitro and in vivo, and support the notion that low calcemic Vitamin D analogs can potentially be used as therapeutic agents to control the RAS.

Effect of cellular environment on the selective activation of the vitamin D receptor by 1alpha,25-dihydroxyvitamin D3 and its analog 1alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D3 (Ro-26-9228).

The vitamin D analog, 1alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228) is tissue selective, with a gene regulation preference for bone over duodenum in vivo. In the human osteoblast-like cells, hFOB, the vitamin D receptor (VDR)-mediated transcriptional potencies of Ro-26-9228 and 1,25-dihydroxyvitamin D(3) (1,25D(3)) were similar, but in the intestinal cells, Caco-2, transcriptional potency of Ro-26-9228 was 10-50 times lower. We hypothesized that transcriptional activation of the VDR by Ro-26-9228 in the two cell types is regulated differently, and compared VDR extracted from hFOB or Caco-2 cells for their abilities to interact with a p160 coactivator [glucocorticoid receptor-interacting protein (GRIP)] and with retinoid X receptor (RXR) by pull-down assays. 1,25D(3) had similar potencies to induce interactions of VDR from the two cell types with these partners of transcription. In contrast, Ro-26-9228 induced interaction of osteoblastic VDR with RXR and GRIP but did not induce these interactions with VDR from Caco-2 cells. Further studies revealed that in hFOB cells the unoccupied VDR was cytoplasmic and proteasome sensitive, and that ligand treatment caused a rapid accumulation of the VDR in the chromatin. Both cytoplasmic and chromatin-associated ligand-bound VDR from hFOB cells had the abilities to interact with GRIP. In contrast, in Caco-2 cells, unoccupied VDR was localized in both the cytoplasm (70%) and the chromatin (30%). In Caco-2 cells, the cytoplasmic VDR was proteasome resistant, and neither 1,25D(3) nor Ro-26-9228 induced its binding to GRIP. Only a small fraction of the chromatin-associated VDR was proteasome sensitive, and this fraction was distinguishable by a faster electrophoretic mobility. 1,25D(3) induced an accumulation of the proteasome-sensitive VDR in the chromatin of Caco-2 cells and binding to GRIP. Ro-26-9228 failed to induce accumulation of the proteasome-sensitive VDR in the chromatin or binding to GRIP, but a coincubation of Caco-2 cells with the analog and a proteasome inhibitor restored these abilities. These results suggest that Ro-26-9228 has poor ability to promote the accumulation of a proteasome-sensitive, transcriptionally active VDR isoform in Caco-2 cells, whereas it does not have this limitation in hFOB cells.