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Annemieke Verstuyf

Publications and source records attributed to Annemieke Verstuyf.

At least 19 recordsLinked to original sources
PubMed2007

The development of CD-ring modified analogs of 1alpha,25-dihydroxyvitamin D.

During a 20-year collaboration the laboratories of UGent and KU Leuven have developed different series of Vitamin D analogs characterized by structural modifications in the central CD-ring system. Modifications have first involved the introduction of substituents at C11 and the epimerization at C14, and subsequently more drastic changes consisting in both ring deletion and enlargement relative to the natural CD-ring system. Lately, the focus has shifted towards the synthesis of analogs featuring a symmetrical CD-ring core. As an illustration two different series are presented.

Calcitriol↗
PubMed2006

Removal of C-ring from the CD-ring skeleton of 1alpha,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly.

It is now well established that 1alpha,25(OH)2D3 is metabolized in its target tissues through the modifications of both side chain and A-ring. The C-24 oxidation pathway is the side chain modification pathway through which 1alpha,25(OH)2D3 is metabolized into calcitroic acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1alpha,25(OH)2D3 is metabolized into 1alpha,25(OH)2-3-epi-D3. During the past two decades, a great number of vitamin D analogs were synthesized by altering the structure of both side chain and A-ring of 1alpha,25(OH)2D3 with the aim to generate novel vitamin D compounds that inhibit proliferation and induce differentiation of various types of normal and cancer cells without causing significant hypercalcemia. Previously, we used some of these analogs as molecular probes to examine how changes in 1alpha,25(OH)2D3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1alpha,25(OH)2D3 with unique biological activity profiles were synthesized. Two of the analogs, SL 117 and WU 515 lack the C-ring of the CD-ring skeleton of 1alpha,25(OH)2D3. SL 117 contains the same side chain as that of 1alpha,25(OH)2D3, while WU 515 contains an altered side chain with a 23-yne modification combined with hexafluorination at C-26 and C-27. Presently, it is unknown how the removal of C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1alpha,25(OH)2D3 in both the isolated perfused rat kidney, which expresses only the C-24 oxidation pathway and rat osteosarcoma cells (UMR 106), which express both the C-24 oxidation and C-3 epimerization pathways. The results of our present study indicate that SL 117 is metabolized like 1alpha,25(OH)2D3, into polar metabolites via the C-24 oxidation pathway in both rat kidney and UMR 106 cells. As expected, WU 515 with altered side chain structure is not metabolized via the C-24 oxidation pathway. Unlike in rat kidney, both SL 117 and WU 515 are also metabolized into less polar metabolites in UMR 106 cells. These metabolites displayed GC and MS characteristics consistent with A-ring epimerization and were putatively assigned as C-3 epimers of SL 117 and WU 515. In summary, we report that removal of the C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 does not alter its target tissue metabolism significantly.

Animals↗
PubMed2006

The Vitamin D analogue TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients with Crohn's disease.

Crohn's disease (CD) is an inflammatory disease characterized by the activation of the immune system in the gut. Since tumor necrosis factor (TNF-alpha) plays an important role in the initiation and perpetuation of intestinal inflammation in CD, we investigated whether TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)], a Vitamin D analogue, could affect peripheral blood mononuclear cells (PBMC) proliferation and exert an immunosuppressive effect on TNF-alpha production in CD patients, and whether this immunosuppressive action could be mediated by NF-kappaB down-regulation. TX 527 significantly decreased cell proliferation and TNF-alpha levels. On activation, NF-kappaB, rapidly released from its cytoplasmatic inhibitor (IKB-alpha), transmigrates into the nucleus and binds to DNA response elements in gene promoter regions. The activation of NF-kappaB, stimulated by TNF-alpha, and its nuclear translocation together with the degradation of IKB-alpha were blocked by TX 527. At the same time, NF-kappaB protein levels present in cytoplasmic extracts decreased in the presence of TNF-alpha and increased when PBMC were incubated with TX 527. The results of our studies indicate that TX 527 inhibits TNF-alpha mediated effects on PBMC and the activation of NF-kappaB and that its action is mediated by Vitamin D receptor (VDR), which is activated when the cells are stimulated with TX 527.

Adult↗
PubMed2006

Immune regulation of 25-hydroxyvitamin D-1alpha-hydroxylase in human monocytic THP1 cells: mechanisms of interferon-gamma-mediated induction.

CONTEXT: 25-Hydroxyvitamin D can be activated to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] by the rate-limiting enzyme 1alpha-hydroxylase in cells of the immune system under control of immune stimuli, such as interferon-gamma (IFNgamma). In pathological situations, such as sarcoidosis, this can lead to systemic excess of 1,25(OH)(2)D(3) and hypercalcemia. OBJECTIVE: The aim of this study was to elucidate the intracellular pathways used by the immune system to tightly regulate 1,25(OH)(2)D(3) production in monocytes and macrophages. DESIGN: Human monocytic THP1-cells were differentiated and activated by IFNgamma and a secondary stimulus, such as lipopolysaccharide or phorbol myristate acetate. 1alpha-Hydroxylase mRNA levels were quantified by real-time RT-PCR. The involvement of different signaling pathways in the regulation of this enzyme was investigated using specific pharmacological inhibitors, whereas phosphorylation of signal transducer and activator of transcription 1alpha and CCAAT/enhancer binding protein beta was investigated by Western blotting. RESULTS: In undifferentiated monocytic THP1 cells, IFNgamma needs to be combined with a second stimulus, such as lipopolysaccharide, to induce 1alpha-hydroxylase. In contrast, in phorbol myristate acetate-differentiated THP1 macrophages, IFNgamma alone induces 1alpha-hydroxylase and to much higher levels. Many different signaling pathways need to be activated concurrently to allow immune-mediated 1alpha-hydroxylase up-regulation. We show involvement of the Janus kinase-signal transducer and activator of transcription, MAPK, and nuclear factor-kappaB pathways, with a crucial role for the transcription factor CCAAT/enhancer binding protein beta. Furthermore, histone remodeling involving histone deacetylases and histone acetylase p300 is required. CONCLUSION: The present findings indicate that IFNgamma-mediated 1,25(OH)(2)D(3) production, as observed in granulomatous diseases such as sarcoidosis, will take place only under conditions where the necessary other signaling pathways are also activated.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2006

Vitamin D resistance.

Vitamin D is a secosteroid of nutritional origin but can also be generated in the skin by ultraviolet light. After two hydroxylations 1,25-(OH)2 vitamin D avidly binds and activates the vitamin D receptor (VDR), a nuclear transcription factor, hereby regulating a large number of genes. The generation of VDR deficient mice has expanded the knowledge on vitamin D from a calcium-regulating hormone to a humoral factor with extensive actions. The effects of the vitamin D system on calcium and bone homeostasis are largely mediated by promoting active intestinal calcium transport via the induction of the epithelial calcium channel TRPV6. Although VDR is redundant in bone, it may regulate the differentiation and function of several bone cells. In skin, VDR expression in keratinocytes is essential in a ligand-independent manner for the maintenance of the normal hair cycle. Therefore, VDR but not vitamin D deficiency results in alopecia. Moreover, 1,25-(OH)2 vitamin D impairs the proliferation not only of keratinocytes but also of many cell types by regulating the expression of cell cycle genes, leading to a G1 cell cycle arrest. In addition, VDR inactivation in mice results in high renin hypertension, cardiac hypertrophy and thrombogenesis. Finally, a dual effect of vitamin D was observed in the immune system where it stimulates the innate immune system while tapering down excessive activation of the acquired immune system. Taken together, the vitamin D endocrine system not only regulates calcium homeostasis but affects several systems mainly by altering gene expression but also by ligand-independent actions.

Animals↗
PubMed2006

Vitamin D and cancer.

1,25-dihydroxy Vitamin D [1,25-(OH)(2)D] exerts its effects via the vitamin D receptor (VDR) that belongs to the steroid/thyroid hormone receptor superfamily leading to gene regulation which results in various biological responses. Within the last two decades, the receptor has been shown to be present not only in classical target tissues such as bone, kidney and intestine but also in many other non-classical tissues. Besides the almost universal presence of VDRs, some cell types (e.g. keratinocytes, monocytes, bone, placenta) are capable of metabolizing 25-hydroxyvitamin D to 1,25(OH)(2)D by the enzyme 1alpha-hydroxylase (CYP27B1). The combined presence of 25(OH)D-1alpha-hydroxylase as well as the specific receptor in several tissues introduced the idea of a paracrine role for 1,25(OH)(2)D. Moreover, it has been demonstrated that 1,25(OH)(2)D can induce differentiation and inhibit proliferation of a wide variety of cell types. The molecular mechanisms behind this antiproliferative action is thoroughly explored but the whole picture is still difficult to understand. Important cell cycle regulators are involved such as cyclins, cyclin dependent kinases and their corresponding inhibitors as well as E2F transcription factors and accompanying pocket proteins. However the precise hierarchical structure of this wide diversity of actions of 1,25(OH)(2)D on genes influencing cell cycle progression is not firmly established nor do we understand which pathways are essential and which redundant. The antiproliferative action makes 1,25-(OH)(2)D and its analogs a possible therapeutic tool to treat hyperproliferative disorders, among which different types of cancer. This review focuses on the effects of 1,25(OH)(2)D and its analogs on cell proliferation, the results in in vivo experiments in Vitamin D deficient or resistant animals to cancer and the current epidemiological and intervention studies linking Vitamin D status or treatment and human cancer.

Animals↗
PubMed2005

Versatile synthesis and biological evaluation of 1,3-diamino-substituted 1alpha,25-dihydroxyvitamin D3 analogues.

A concise route to 1alpha,3beta-diamino-25-hydroxy-3-deoxyvitamin D(3) (5) and 1beta,3alpha-diamino-25-hydroxy-3-deoxyvitamin D(3) (6) has been developed starting from (R)- or (S)-carvone for the construction of the modified A-ring fragments. The conversion of the hydroxyl group to amine function with complete inversion of the configuration was efficiently accomplished by Mitsunobu reaction using phthalimide as nucleophile or activation of the hydroxyl group as mesylate followed by reaction with NaN(3). Diamino 5 and 6 as well as monoamino 3, 4, 30, and 31 vitamin D(3) derivatives have shown poor binding to VDR compared with 1alpha,25-dihydroxyvitamin D(3). The most active compound in the inhibition of MCF-7 cell proliferation and HL 60 cell differentiation was 1alpha-amino analogue 3. Also, very low in vivo calcemic effects of derivatives 3 and 4 were found.

Amination↗
PubMed2005

Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has potent antiproliferative effects characterized by a hampered G(1)/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)(2)D(3). Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)(2)D(3) and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)(2)D(3)-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)(2)D(3) was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)(2)D(3) depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) failed to repress these E2F target genes and lost its antiproliferative action in p107(-/-);p130(-/-) cells but not in pRb(-/-) cells.

Animals↗
PubMed2005

Mechanisms for the selective action of Vitamin D analogs.

The non-classical effects of 1,25(OH)(2)D(3) create possible therapeutic applications for immune modulation (e.g. auto-immune diseases and graft rejection), inhibition of cell proliferation (e.g. psoriasis, cancer) and induction of cell differentiation (e.g. cancer). The major drawback related to the use of 1,25(OH)(2)D(3) is its calcemic effect, which prevents the application of pharmacological concentrations. Several analogs are now available that show modest to good selectivity with regard to specific effects (e.g. anticancer or immune effects or bone anabolism versus hypercalcemia) when tested in appropriate in vivo models. The molecular basis for this selectivity is only partially understood and probably a variable mixture of mechanisms.

Animals↗
PubMed2005

Altered Vitamin D receptor-coactivator interactions reflect superagonism of Vitamin D analogs.

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH)(2)D(3) in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH)(2)D(3) namely 19-nor-14-epi-23-yne-1,25-(OH)(2)D(3) (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)(2)D(3) (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH)(2)D(3). Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR-coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)(2)D(3). TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR-DRIP205 interaction induced by 1,25-(OH)(2)D(3) at 10(-8)M. Evaluation of additional 1,25-(OH)(2)D(3)-analogs and their impact on VDR-coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR-coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.

Cell Line↗
PubMed2005

Superagonistic action of 14-epi-analogs of 1,25-dihydroxyvitamin D explained by vitamin D receptor-coactivator interaction.

Two 14-epi-analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)(2)D(3)], 19-nor-14-epi-23-yne-1,25-(OH)2D3 (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)2D3 (TX527), show enhanced antiproliferative (at least 10-fold) and markedly lower calcemic effects both in vitro and in vivo, compared with 1,25-(OH)2D3. This study aimed to evaluate their superagonistic effect at the level of interaction between the Vitamin D receptor (VDR) and coactivators. Mammalian two-hybrid assays with VP16-fused VDR and GAL4-DNA-binding-domain-fused steroid receptor coactivator 1 (SRC-1), transcriptional intermediary factor 2 (Tif2), or DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)2D3 (up to 16- and 20-fold stronger induction of VDR-SRC-1 interaction for TX522 and TX527 at 10(-10) M). Similar assays in which metabolism of 1,25-(OH)2D3 was blocked with VID400, a selective inhibitor of the 1,25-(OH)2D3-metabolizing enzyme CYP24, showed that the enhanced potency of these analogs in establishing VDR-coactivator interactions can only partially be accounted for by their increased resistance to metabolic degradation. Crystallization of TX522 complexed to the ligand binding domain of the human VDR demonstrated that the epi-configuration of C14 caused the CD ring of the ligand to shift by 0.5 angstroms, thereby bringing the C12 atom into closer contact with Val300. Moreover, C22 of TX522 made an additional contact with the CD1 atom of Ile268 because of the rigidity of the triple bond-containing side chain. The position and conformation of the activation helix H12 of VDR was strictly maintained. In conclusion, this study provides deeper insight into the docking of TX522 in the LBP and shows that stronger VDR-coactivator interactions underlie the superagonistic activity of the two 14-epi-analogs.

Animals↗
PubMed2005

1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage.

Exposure to UVB irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused damage. Photocarcinogenesis is caused largely by mutations at sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, we demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against the induction of CPDs by UVB. This protection required pharmacologic doses 1,25(OH)2D3 and an incubation period of at least 8 h before irradiation. Furthermore, we provided arguments indicating that the anti-proliferative capacity of 1,25(OH)2D3 underlies its protective effect against UVB-induced DNA damage. Finally, we showed that 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), two low-calcemic analogues of 1,25(OH)2D3, were even 100 times more potent than the parent molecule in inhibiting UVB-caused DNA damage. These molecules are therefore promising candidates for the chemoprevention of UVB-induced skin cancer.

Cell Proliferation↗
PubMed2004

Chemoenzymatic synthesis and biological evaluation of C-3 carbamate analogues of 1alpha,25-dihydroxyvitamin D3.

The synthesis of new analogues of 1alpha,25-dihydroxyvitamin D3 containing a carbamate function at the A-ring fragment has been described using the cross-coupling approach. The carbamate group was selectively introduced at the C-3 position by regioselective enzymatic alkoxycarbonylation of A-ring enyne 3 and subsequent treatment with ammonia, amines, amino alcohols, and amino acids. Biological studies to evaluate the potency of all five of these carbamate analogues were performed and demonstrated very low binding affinity for the vitamin D receptor compared with 1alpha,25-dihydroxyvitamin D3. Moreover, all the carbamate analogues were less active than 1alpha,25-dihydroxyvitamin D3 in inhibiting cell proliferation or stimulating cell differentiation. Of all the five analogues, the 3-O-carbamoyl-1alpha,25-(OH)2-D3 analogue 10a was the most potent one in vitro. However, all investigated carbamate analogues demonstrated lower calcemic effects in vivo than the parent compound.

Antineoplastic Agents↗
PubMed2004

Synthesis and biological activity of 22-oxa CD-ring modified analogues of 1alpha,25-dihydroxyvitamin D3: spiro[5.5]undecane CF-ring analogues.

The synthesis and biological activity of novel CD-ring modified analogues of 22-oxa-1alpha,25-dihydroxyvitamin D(3), lacking the D-ring and featuring a connection between C-18 and C-21 (spiro[5.5]undecane CF-ring analogues), is described. The central ring system is conveniently synthesised from an achiral intermediate. The analogues have marginal binding affinity for the nVDR and possess low to moderate genomic activity.

Calcitriol↗
PubMed2004

Synthesis and biological activity of 22-oxa CD-ring modified analogues of 1alpha,25-dihydroxyvitamin D3: cis-perhydrindane CE-ring analogues.

The synthesis and biological activity of novel CD-ring modified analogues of 22-oxa-1alpha,25-dihydroxyvitamin D(3), lacking the D-ring and featuring a connection between C-12 and C-21 (cis-perhydrindane CE-ring analogues), is described. The synthesis of the CE-ring system follows Meyers' methodology for the preparation of enantiomerically pure hydrinden-2-ones. The analogues show a complete lack of binding affinity for the vitamin D receptor (pig nVDR) and of antiproliferative activity (MCF-7 cells), as compared to calcitriol.

Breast Neoplasms↗
PubMed2004

Two 14-epi analogues of 1,25-dihydroxyvitamin D3 protect human keratinocytes against the effects of UVB.

In search of photoprotective agents, we recently demonstrated a protective effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] against different events mediated by ultraviolet B (UVB) in human keratinocytes. Pharmacological doses of 1,25(OH)(2)D(3) were required to obtain significant UVB protection; however, these doses cannot be used in vivo due to the calcemic properties of 1,25(OH)(2)D(3). Therefore, we evaluated the photoprotective capacities of two low-calcemic 14-epi analogues of 1,25(OH)(2)D(3), 19-nor-14-epi-23-yne-1,25(OH)(2)D(3) (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3) (TX 527). Using cultured human keratinocytes, we investigated the influence of TX 522 and TX 527 on two hallmark events in UVB-irradiated keratinocytes: the induction of apoptosis and the production of interleukin-6 (IL-6). Treatment of the keratinocytes with TX 522 or TX 527, 24 h before irradiation, resulted in a significant and dose-dependent reduction of both UVB-induced apoptosis and IL-6 production. Both analogues were equally efficient in their anti-UVB effects and at least 100 times more potent than 1,25(OH)(2)D(3). We further demonstrated that metallothionein (MT) mRNA expression was clearly induced by 1,25(OH)(2)D(3) and both analogues. MT acts as a radical scavenger in oxygen-mediated UVB injury and its induction may therefore be relevant for the anti-UVB effects of 1,25(OH)(2)D(3) and both analogues. Taken together, these findings create new perspectives for the use of active vitamin D analogues as photoprotective agents.

Apoptosis↗