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1,25-Dihydroxycholecalciferol, the hormonally active vitamin D 3 metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24 R )-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH) 2 D 3 analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR.

Plant polyphenols and vitamins D exhibit chemopreventive and therapeutic anticancer effects. We first evaluated the biological effects of the plant polyphenol resveratrol (RESV) and vitamin D active metabolite PRI-2191 on lung cancer cells having different genetic backgrounds. RESV and PRI-2191 showed divergent responses depending on the genetic profile of cells. Antiproliferative activity of PRI-2191 was noticeable in EGFRmut cells, while RESV showed the highest antiproliferative and caspase-3-inducing activity in KRASmut cells. RESV upregulated p53 expression in wtp53 cells, while downregulated it in mutp53 cells with simultaneous upregulation of p21 expression in both cases. The effect of PRI-2191 on the induction of CYP24A1 expression was enhanced by RESV in two KRASmut cell lines. The effect of RESV combined with PRI-2191 on cytokine production was pronounced and modulated. RESV cooperated with PRI-2191 in regulating the expression of IL-8 in EGFRmut cells, while OPN in KRASmut cells and PD-L1 in both cell subtypes. We hypothesize that the differences in response to RESV and PRI-2191 between EGFRmut and KRASmut cell lines result from the differences in epigenetic modifications since both cell subtypes are associated with the divergent smoking history that can induce epigenetic alterations.

5-Fluorouracil (5-FU) is an anticancer drug that is most frequently used to treat colorectal cancer (CRC) patients, but unfortunately it shows limited efficacy. We recently demonstrated that vitamin D analogs (VDAs), particularly tacalcitol (coded as PRI-2191), potentiate its anticancer activity in an in vivo mouse and human CRC model. The purpose of this study was to explain the mechanism underlying the enhancement of 5-FU efficacy by PRI-2191 towards human HT-29 CRC cells. We showed that PRI-2191 induces the CDKN1A (gene encoding p21 Waf1/Cip1 ) expression directly through vitamin D receptor (VDR) in a p53-independent manner and thus decreases the thymidylate synthase expression both at the mRNA and protein level. It is the main mechanism by which PRI-2191 improves the anticancer efficacy of 5-FU towards HT-29 cells. Additionally, we indicated that the VDR also participates in 5-FU mechanism of action. 5-FU significantly increased TYMS (gene encoding thymidylate synthase (TS)) and BIRC5 (gene encoding survivin) level in HT-29 cells with silenced VDR. Furthermore, PRI-2191 induced E-cadherin and ZO-1 expression and thus reduced the level of BIRC5 in HT-29 cells. The induction of E-cadherin expression may also contribute to the reduction of c-Myc level and consequently the downregulation of TS. Our results also indicate that calcium-sensing receptor (CaSR) plays a role in the activity of PRI-2191 but has no influence on the 5-FU mechanism of action. In conclusion, we suggest that both VDR and CaSR might be useful as molecular markers for predicting treatment outcomes and identifying the CRC patient subgroups who might benefit from 5-FU-based chemotherapy combined with vitamin D analog., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Conventional cytotoxic drugs preferentially eliminate differentiated cancer cells but spare relatively more resistant stem-like cancer cells capable to initiate recurrence. Due to cancer cell plasticity, the stem-like phenotype can be also acquired by cancer cells refractory to treatment with cytotoxic drugs. We investigated whether drugs inhibiting receptor tyrosine kinases could be used to target human colon cancer cells initiating cancer regrowth following conventional cytotoxic treatment. The moderately differentiated cell line HT-29 and poorly differentiated cell line HCT-116 were exposed to 5-fluorouracil (5-FU). Cells that resisted the exposure to 5-FU were subsequently treated with imatinib or sunitinib. Both drugs reduced clonogenicity of 5-FU-refractory cells under normoxic and hypoxic culture conditions. The expression of numerous stemness-related genes was upregulated in cancer cells following the exposure to 5-FU, and remained at a high level in 5-FU-refractory cells undergoing renewal under normoxia, but decreased spontaneously under hypoxia. Imatinib downregulated the expression of stemness-related genes in cells undergoing renewal under normoxia. A combination of imatinib with PRI-2191, an analogue of 1,25-dihydroxyvitamin D 3 , downregulated stemness-related genes in HCT-116/5-FU cells more efficiently than imatinib alone. A synthetic analogue of 1,25-dihydroxyvitamin D 2 (PRI-1906) abolished the effect of imatinib on gene expression in HCT-116/5-FU cells undergoing renewal under normoxia. Sunitinib promoted shift of phenotype of HT-29/5-FU cells undergoing renewal toward stem-like one. It suggests that the phenotype shift toward stemness induced by sequential sunitinib treatment following 5-FU treatment could increase a risk of cancer recurrence. In contrast to sunitinib, imatinib could be used both to interfere with cancer regrowth after conventional chemotherapy and to downregulate the expression of stemness-related genes in residual colon cancer cells capable to initiate cancer recurrence. The findings suggest that imatinib could also be combined with vitamin D analogue PRI-2191 to prevent recurrence more efficiently than imatinib alone and to compensate for vitamin D deficiency resulting from imatinib treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Vitamin D plays an important role in maintaining skeletal development and bone homeostasis. Although vitamin D has been extensively researched, the direct effect of 1,25(OH) 2 D 3 on osteoblasts is unclear. To explore the 1,25(OH) 2 D 3 action on murine osteoblasts, we performed tandem mass tag experiments on MC3T3-E1 cells treated with and without 1,25(OH) 2 D 3 . Three up-regulated proteins (MRC2, WWTR1 and RASSF2) related to bone metabolism were confirmed in this study. 1,25(OH) 2 D 3 up-regulated the expression of MRC2 through vitamin D receptor. MRC2 affects collagen metabolism in osteoblasts. Combined with bioinformatics and parallel reaction monitoring analysis, we inhibited the expression of MRC2 to explore the relationship between MRC2 and collagens. Then we found MRC2 down-regulated COL5A2 and up-regulated MMP13. This study provides a protein profile of 1,25(OH) 2 D 3 -treated murine osteoblasts, reveals a newly discovered signaling axis (1,25(OH) 2 D 3 /VDR/MRC2/COL5A2 and MMP13), and explains the effect of 1,25(OH) 2 D 3 on bone metabolism from a new perspective., (Copyright © 2019 Elsevier B.V. All rights reserved.)

MiR-125b belongs to the class of microRNAs, which are short endogenous non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Recently, it was reported that miR-125b was found to promote migration and invasion of MCF-7 cells and was involved in chemotherapeutic resistance. Decreasing miR-125b expression would have potential therapeutic significance in preventing dissemination of breast cancer cells. The objective of this study was to evaluate miR-125b expression levels in MCF-7 cells following treatment with 1,25-dihydroxyvitamin D 3 (calcitriol) and 1,24-dihydroxyvitamin D 3 (tacalcitol), active metabolite and synthetic analog of vitamin D 3 , respectively. We found that treatment with both calcitriol and tacalcitol caused a decrease in miR-125b expression. In addition, treatment with calcitriol and tacalcitol resulted in an increase in the level of pro-apoptotic BAK1 protein encoded by the target gene of miR-125b. We are discussing the putative mechanism of inhibition of the miR-125b expression by vitamin D receptor (VDR) agonists and we suggest that calcitriol and tacalcitol may be used as a miR-125b inhibitor in breast cancer cells expressing VDR., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)₂D3 or classical 1,25(OH)₂D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina's HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)₂D3- and 20,23(OH)₂D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)₂D3. In contrast, the top canonical pathway induced by 20,23(OH)₂D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)₂D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)₂D3, as well as with its downstream metabolite, 17,20,23(OH)₃D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)₂D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)₂D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.

The active form of vitamin D (1α,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1α,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 μM. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

In our previous study, calcitriol and its analogs PRI-2191 and PRI-2205 stimulated 4T1 mouse mammary gland cancer metastasis. Therefore, we aimed to analyze the inflammatory response in 4T1-bearing mice treated with these compounds. Gene expression analysis of the splenocytes and regional lymph nodes demonstrated prevalence of the T helper lymphocytes (Th2) response with an increased activity of regulatory T (Treg) lymphocytes in mice treated with these compounds. We also observed an increased number of mature granulocytes and B lymphocytes and a decreased number of TCD4⁺, TCD4⁺CD25⁺, and TCD8⁺, as well as natural killer (NK) CD335⁺, cells in the blood of mice treated with calcitriol and its analogs. Among the splenocytes, we observed a significant decrease in NK CD335⁺ cells and an increase in TCD8⁺ cells. Calcitriol and its analogs decreased the levels of interleukin (IL)-1β and IL-10 and increased the level of interferon gamma (IFN-γ) in the plasma. In the tumor tissue, they caused an increase in the level of IL-10. Gene expression analysis of lung tissue demonstrated an increased level of osteopontin ( Spp1 ) and transforming growth factor β (TGF-β) mRNA. The expression of Spp1 was also elevated in lymph nodes. Calcitriol and its analogs caused prevalence of tumor-conducive changes in the immune system of 4T1 tumor-bearing mice, despite the induction of some tumor-disadvantageous effects.

Numerous in vitro and in vivo studies have demonstrated that calcitriol [1,25(OH)2D3] and different vitamin D analogs possess antineoplastic activity, regulating proliferation, differentiation and apoptosis, as well as angiogenesis. Vitamin D compounds have been shown to exert synergistic effects when used in combination with different agents used in anticancer therapies in different cancer models. The aim of this study was to evaluate the mechanisms of the cooperation of the vitamin D compounds [1,24(OH)2D3 (PRI‑2191) and 1,25(OH)2D3] with tyrosine kinase inhibitors (imatinib and sunitinib) together with cytostatics (cisplatin and docetaxel) in an A549 non-small cell lung cancer model. The cytotoxic effects of the test compounds used in different combinations were evaluated on A549 lung cancer cells, as well as on human lung microvascular endothelial cells (HLMECs). The effects of such combinations on the cell cycle and cell death were also determined. In addition, changes in the expression of proteins involved in cell cycle regulation, angiogenesis and the action of vitamin D were analyzed. Moreover, the effects of 1,24(OH)2D3 on the anticancer activity of sunitinib and sunitinib in combination with docetaxel were examined in an A549 lung cancer model in vivo. Experiments aiming at evaluating the cytotoxicity of the combinations of the test agents revealed that imatinib and sunitinib together with cisplatin or docetaxel exerted potent anti-proliferative effects in vitro on A549 lung cancer cells and in HLMECs; however, 1,24(OH)2D3 and 1,25(OH)2D3 enhanced the cytotoxic effects only in the endothelial cells. Among the test agents, sunitinib and cisplatin decreased the secretion of vascular endothelial growth factor (VEGF)‑A from the A549 lung cancer cells. The decrease in the VEGF‑A level following incubation with cisplatin correlated with a higher p53 protein expression, while no such correlation was observed following treatment of the A549 cells with sunitinib. Sunitinib together with docetaxel and 1,24(OH)2D3 exhibited a more potent anticancer activity in the A549 lung cancer model compared to double combinations and to treatment with the compounds alone. The observed anticancer activity may be the result of the influence of the test agents on the process of tumor angiogenesis, for example, through the downregulation of VEGF‑A expression in tumor and also on the induction of cell death inside the tumor.

Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI‑2191 and PRI‑2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, real‑time PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17β-estradiol and transforming growth factor β (TGFβ) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFβ levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFβ level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFβ and 17β-estradiol levels contributed to the facilitation of metastatic process.

Background: Oxidative damage may lead to the dysfunction of melanocytes (MCs) and is one of the causative mechanisms involved in the pathogenesis of vitiligo., Objectives: This study was designed to investigate the protective effects of the vitamin D3 analog tacalcitol on oxidative damage induced by hydrogen peroxide (H 2 O 2 ) in human epidermal MCs., Methods: Human epidermal MCs were cultured and identified by l-DOPA staining and HMB-45 immunohistochemical staining. The model of oxidative damage induced by H 2 O 2 was established, and the cells were treated with tacalcitol. The viability of MCs was determined using an MTS assay. Morphological changes in cell dendrites were observed by microscopy, and the rate of change of dendrites was calculated. The reactive oxygen species (ROS) level in MCs was determined using immunofluorescence microscopy. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in MCs were determined using the WST-1 and TBA methods, respectively., Results: In comparison with the control group, the viability of MCs and SOD activity were significantly decreased in the H 2 O 2 group (P < 0.05) and significantly increased in the tacalcitol group (P < 0.05). In comparison with the control group, the rate of change of cell dendrites and levels of ROS and MDA were significantly increased in the H 2 O 2 group (P < 0.05) and significantly decreased in the tacalcitol group (P < 0.05)., Conclusions: Tacalcitol can reduce oxidative damage induced by H 2 O 2 in MCs by inhibiting intracellular ROS overproduction, increasing SOD activity, and decreasing the level of MDA, thereby reducing cell apoptosis., (© 2017 The International Society of Dermatology.)

The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory, and antiproliferative activities.

This study was aimed to determine whether hypocalcemic analogs of active forms of vitamins D modulate expression of genes related to stem-like phenotype in colon cancer cell lines HT-29 and HCT-116 undergoing renewal after the treatment with 5-fluorouracil (5-FU). Both lines express vitamin D receptor, but differ in differentiation stage and vitamin D sensitivity. Cells that resisted the 5-FU exposure were treated with synthetic analog of 1,25-dihydroxyvitamin D2 (PRI-1906) and analogs of 1,25-dihydroxyvitamin D3 (PRI-2191 and PRI-2205). Proliferative activity was more profoundly affected by vitamin D analogs in HT-29/5-FU than in HCT-116/5-FU cells. In HT-29/5-FU cells, analogs PRI-1906 and PRI-2191 downregulated the expression of genes related to survival, re-growth, and invasiveness during renewal, while PRI-2205 increased expression of genes related to differentiation only. In HCT-116/5-FU cells, PRI-2191 decreased the expression of stemness- and angiogenesis-related genes, whereas PRI-1906 augmented their expression. The effects in HCT-116/5-FU cells were observed at higher concentrations of the analogs than those used for HT-29/5-FU cells. Out of the series of analogs studied, PRI-2191 might be used to counteract the renewal of both moderately and poorly differentiated cancer cells following conventional treatment.

In an attempt to prepare vitamin D analogues that are superagonists, (20R)- and (20S)-isomers of 1α-hydroxy-2-methylenevitamin D3 and 1α,25-dihydroxy-2-methylenevitamin D3 have been synthesized. To prepare the desired A-ring dienyne fragment, two different approaches were used, both starting from the (-)-quinic acid. The obtained derivative was subsequently coupled with the C,D-ring enol triflates derived from the corresponding Grundmann ketones, using the Sonogashira reaction. Moreover, (20R)- and (20S)-1α,25-dihydroxy-2-methylenevitamin D3 compounds with an (5E)-configuration were prepared by iodine catalyzed isomerization. All four 2-methylene analogues of the native hormone were characterized by high in vitro activity. As expected, the 25-desoxy analogues were much less potent. Among the synthesized compounds, two of them, 1α,25-dihydroxy-2-methylenevitamin D3 and its C-20 epimer, were found to be almost as active as 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) on bone but more active in intestine.

Vitamin D and its derivatives have modulatory effects in immunological and inflammatory responses. Such properties suggest that they might have an impact on chronic inflammatory airway diseases, including nasal polyposis. The aim of this study was to understand the role of vitamin D in chronic rhinosinusitis with nasal polyps (CRSwNP) by investigating its effect on the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9 in nasal polyp-derived fibroblasts. Two primary fibroblast cultures were established from nasal polyp tissues obtained during surgery. The nasal polyp-derived fibroblasts were stimulated with tumor necrosis factor-α (TNF-α; 10 ng/mL) for 24 hours, followed by replacement with media alone or with vitamin D derivatives (calcitriol or tacalcitol; 10μM) and incubated for another 24 hours. After the treatments, the levels of MMP-2 and MMP-9 secreted were evaluated by both enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. ELISA results revealed that TNF-α could substantially stimulate the secretion of MMP-2 (p < 0.01) and MMP-9 (p < 0.001) in nasal polyp-derived fibroblasts. More importantly, such stimulatory effect was significantly suppressed by adding calcitriol (p ≤ 0.01 for MMP-2 and p < 0.001 for MMP-9) or tacalcitol (p < 0.005 for both MMP-2 and MMP-9). The ELISA results were also confirmed by Western blot analysis. The inhibitory effect of vitamin D derivatives on MMP-2 and MMP-9 secretion could potentiate their application in pharmacotherapy of Taiwanese CRSwNP patients., (Copyright © 2015. Published by Elsevier Taiwan.)

Cross-complementation studies offer the possibility to overcome limitations imposed by the inherent complexity of multicellular organisms in the study of human diseases, by taking advantage of simpler model organisms like the budding yeast Saccharomyces cerevisiae. This review deals with, (1) the use of S. cerevisiae as a model organism to study human diseases, (2) yeast-based screening systems for the detection of disease modifiers, (3) Hailey-Hailey as an example of a calcium-related disease, and (4) the presentation of a yeast-based model to search for chemical modifiers of Hailey-Hailey disease. The preliminary experimental data presented and discussed here show that it is possible to use yeast as a model system for Hailey-Hailey disease and suggest that in all likelihood, yeast has the potential to reveal candidate drugs for the treatment of this disorder. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the present study, we evaluated the antitumor effect of two synthetic analogs of vitamin D, namely PRI-2191 [(24R)-1,24-dihydroxyvitamin D3] and PRI-2205 (5,6-trans calcipotriol), in combined human colon HT-29 cancer treatment with 5-fluorouracil (5-FU). Mice bearing HT-29 tumors transplanted subcutaneously or orthotopically were injected with vitamin D analogs and 5-FU in various schedules. A statistically significant inhibition of subcutaneous or orthotopic tumor growth was observed as a result of combined therapy. In HT-29 tumors and in cells from in vitro culture, we observed increased vitamin D receptor (VDR) expression after treatment with either PRI-2205 or 5-FU alone, or in combination. Moreover, PRI-2205 decreased the percentage of cells from intestinal tumors in G2/M and S stages and increased sub-G1. Increased VDR expression was also observed after combined treatment of mice with 5-FU and PRI-2191. Moreover, our docking studies showed that PRI-2205 has stronger affinity for VDR, DBP and CAR/RXR ligand binding domains than PRI-2191. PRI-2191 analog, used with 5-FU, increased the percentage of subcutaneous tumor cells in G0/G1 and decreased the percentage in G2/M, S and sub-G1 populations as compared to 5-FU alone. In in vitro studies, we observed increased expression of p21 and p-ERK1/2 diminution via use of both analogs as compared to use of 5-FU alone. Simultaneously, PRI-2191 antagonizes some pro-apoptotic activities of 5-FU in vitro. However, in spite of these disadvantageous effects in terms of apoptosis, the therapeutic effect expressed as tumor growth retardation by PRI-2191 is significant. Our results suggest that the mechanism of potentiation of 5-FU antitumor action by both analogs is realized via increased p21 expression and decreased p-ERK1/2 level which may lead to diminution of thymidylate synthase expression. Higher binding affinity for VDR, DBP, but also for CAR\RXR ligand binding domain of PRI-2205 may, in part, explain its very low toxicity with sustained anticancer activity.

The active metabolite of vitamin D, 1α, 25 dihydroxyvitamin D3 (calcitriol) is classically known to regulate calcium and phosphate homeostasis and bone mineralization. In addition, calcitriol has also been documented to act as a potent anticancer agent in multiple cell culture and animal models of cancer. However, major side effects, such as hypercalcemia, hinder broad-spectrum therapeutic uses of calcitriol in cancer chemotherapy. Synthesis of calcitriol analogues with the same or increased antiproliferative and pro-differentiating activities, and with reduced undesired effects on calcium and bone metabolism, is getting significant attention towards rational therapeutics to treat cancer. In this regard, phosphonate analogues have been shown to display a certain degree of dissociation between the vitamin D activity in vitro and undesired hypercalcemia in vivo. However, few phosphonates have been described in the literature and fewer of them tested for antitumoral effects. Our group has synthesized a novel vitamin D analogue (EM1) bearing an alkynylphosphonate moiety that combines the low calcemic properties of phosphonates with the decreased metabolic inactivation due to the presence of a triple bond between C-23 and C-24. Biological assays demonstrated that this analogue has potent antiproliferative effects in a wide panel of tumour cell lines, even in those resistant to calcitriol treatment. Importantly, EM1 does not show toxic effects in animals, even administered at high doses and for extended periods of time. In the current review we discuss the effects and the potential application in cancer of vitamin D and its derivatives, with an emphasis on phosphonate analogues.

The hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1a), has a wide variety of biological activities and its major molecular target is considered to be the vitamin D receptor (VDR). The A-ring stereoisomers of 1a as well as its C2-modified analogues, which have different stereochemistry at the C1 and/or C3 hydroxy groups, are of interest since recent metabolic studies have shown that catabolism could occur through A-ring modification. In this review, a practical and versatile synthesis of the A-ring enyne precursors by the convergent method of Trost and coworkers, which is needed to construct all possible A-ring stereoisomers of 1,25-dihydroxyvitamin D3 (1a-d), and the C2-modified analogues (4a-d, 5a-d, 6a-d and 7a-d) is described. A strategy for the synthesis and evaluation of all possible A-ring stereoisomers of 1a and their A-ring modified analogues is important, and this will stimulate synthesis and biological studies into vitamin D.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor which mediates a variety of functions in the skin including cutaneous inflammation. SLIGKV-NH(2) , an agonist peptide for PAR2, enhanced the interleukin (IL)-17-induced production of two CXC chemokines, CXCL1 (GRO-α) and CXCL8 (IL-8), in normal human epidermal keratinocytes (NHEK) in a concentration-dependent manner. The enhanced production of those chemokines was suppressed by a PAR2-specific siRNA. The SLIGKV-NH(2) -induced production of both CXCL1 and CXCL8 was markedly reduced by cyclosporine A. The enhanced production of CXCL1 was suppressed by 1α, 24R-dihydroxyvitamin D(3) , an active form of vitamin D(3) , and weakly by glucocorticoids, dexamethasone and clobetasol propionate, whereas production of CXCL8 was not altered by any of those receptor agonists. In psoriatic skin, the thickened upper spinous layer of the epidermis was positive for PAR2 protein and the expression of the IL17A mRNA was increased. These results suggest that the IL-17-induced pro-inflammatory reaction is enhanced by the activation of PAR2 in keratinocytes, and that the effect of PAR2 is differentially modulated by cyclosporine A, the active form of vitamin D(3) and glucocorticoids., (© 2011 Japanese Dermatological Association.)

Imatinib mesylate (Gleevec, STI571) is a specific inhibitor of the Bcr/Abl fusion tyrosine kinase that exhibits potent antileukemic effects in chronic myelogenous leukemia. Bcr/Abl-positive K562 and Bcr/Abl-negative HL-60 human leukemia cells were used to investigate the effect of PRI-2191, a calcitriol analog, on the biological effects of imatinib combined with other anticancer drugs. The results show that PRI-2191 enhances the antiproliferative effect of imatinib on HL-60 cells. When these two agents together are applied with either docetaxel or cisplatin, but not with idarubicin, the antiproliferative effect could still be enhanced. Moreover, when the interaction between the chemotherapy agents was antagonistic or additive, PRI-2191 could even shift it to synergism. This effect correlated with an accumulation of HL-60 cells in the G0/G1 phase of the cell cycle and a decrease in the percentage of cells in the G2/M and S stage in the ternary combinations used. PRI-2191 did not influence apoptosis induced by imatinib alone or in ternary combinations with all the chemotherapy agents used. These results may suggest that the stronger antiproliferative effect of the combined treatment with PRI-2191 on HL-60 cells is related to cell cycle arrest rather than to the induction of apoptosis.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

Background: Vitamin D and its metabolites play an important role in calcium homeostasis, bone remodelling, hormone secretion, cell proliferation and differentiation. Recent studies also suggest a beneficial role of vitamin D in slowing the progression of tissue fibrosis. However, their effects on dermal fibrosis and keloids are unknown. Objectives  To investigate the effect of 1,25-dihydroxyvitamin D3 (1,25D) in the pathogenesis of tissue fibrosis by keloid fibroblasts (KFs)., Methods: KFs were cultured and exposed to different concentrations of 1,25D in the presence or absence of transforming growth factor (TGF)-β1. KF phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, immunofluorescence and multiplex enzyme-linked immunosorbent assay techniques. Collagen synthesis was evaluated by measuring (3) H-proline incorporation. The effect of 1,25D on cell proliferation and viability was evaluated by Formazan assay, proliferating cell nuclear antigen expression and the colorimetric conversion of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide., Results: We confirmed the presence of vitamin D receptors (VDRs) in cultured keloid fibroblasts. Fibroblasts transfected with a vitamin D response element reporter construct and exposed to the active vitamin D metabolite 1,25D showed increased promoter activity indicating VDR functionality in these cells. Incubation of KFs with 1,25D suppressed TGF-β1-induced collagen type I, fibronectin and α-smooth muscle actin expression. 1,25D also modulated plasminogen activator inhibitor-1 and matrix metalloproteinase-9 expression induced by TGF-β1. Interestingly, 1,25D induced hepatocyte growth factor mRNA expression and protein secretion in keloid fibroblasts., Conclusions: This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for keloid and related fibrotic disorders., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

Background: Recently, 1α,25-dihydroxycholecalciferol (vitD) has received increasing interest for its effects on many tissues and organs other than bone. A number of experimental studies have shown that vitD may have an important role in modifying risk for cardiovascular disease., Aims: This study was planned to test the effects of vitD on endothelial nitric oxide (NO) production and to study the intracellular pathways leading to NO release., Methods: In human umbilical vein endothelial cells (HUVEC) cultures the effects of vitD on NO production and p38, Akt, ERK and eNOS phosphorylations were examined in absence or in presence of the NO synthase inhibitor L-NAME and protein kinases specific inhibitors SB203580, wortmannin and UO126., Results: VitD caused a concentration-dependent increase in NO production. The maximum effect was observed at a concentration of 1 nM and the optimal time of stimulation was 1 min. Effects induced by vitD were abolished by L-NAME and by pre-treatment with protein kinases inhibitors. To verify the effective involvement of vitD receptor (VDR) in the action mechanism of vitD, experiments were repeated in presence of the specific VDR ligands ZK159222 and ZK191784., Conclusions: The results of this study demonstrate that vitD can induce a significant increase in endothelial NO production. VitD interaction with VDR caused the phosphorylation of p38, AKT and ERK leading to eNOS activation., (Copyright © 2011 S. Karger AG, Basel.)

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and a series of 2-methylene-19-nor analogs of 1,25(OH)(2)D(3) were evaluated for their ability to reduce the size of utricles (comedolytic activity) in a rhino mouse model of acne. All analogs tested, as well as the native hormone, increased the skin epidermal thickness. In contrast, only a subset of analogs that lacked a full side chain and 25-hydroxyl group were found to possess comedolytic activity. A reduction in comedone area could be achieved without adversely affecting serum calcium levels. Although all compounds that contained a side chain ranging from 2 to 5 carbons in length had similar potency as comedolytic agents, increasing the length of the side chain resulted in a progressive increase in calcemic liability. Dose-response studies of the comedolytic analogs showed that an increase in epidermal thickness was achieved at a lower dose than that needed to induce comedolysis. Thus, we have identified a unique subset of vitamin D analogs that produce comedolysis in the absence of hypercalcemia. Further, the activity of vitamin D analogs in causing epidermal hyperproliferation has been distinguished from that resulting in a reduction in utricle size.

Background: 1,24-Dihydroxyvitamin D₃ (tacalcitol), a vitamin D(3) compound, has been used to treat T cell-mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best-known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T-cell recruitment have not yet been evaluated. Cutaneous lymphocyte-associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T-cell infiltration. We recently reported that 1,25-dihydroxyvitamin D₃ inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells., Objectives: In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells., Methods: We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin-homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen-dependent delayed-type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin-infiltrating CD4+ T cells., Results: Tacalcitol downregulated the expression of CLA and, in parallel, the E- and P-selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model., Conclusions: These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels., (© 2010 The Authors. Journal Compilation © 2010 British Association of Dermatologists.)

We have examined effects of the 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), on differentiation and proliferation of human keratinocytes and the anti-inflammatory potential of 20,23(OH)2D3 from its action on nuclear factor-kappaB (NF-kappaB). 20,23(OH)2D3 inhibited growth of keratinocytes with a potency comparable to that for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell cycle analysis showed that this inhibition was associated with G1/G0 and G2/M arrests. 20,23(OH)2D3 stimulated production of involucrin mRNA and inhibited production of cytokeratin 14 mRNA in a manner similar to that seen for 1,25(OH)2D3. Flow cytometry showed that these effects were accompanied by increased involucrin protein expression, and an increase in the cell size and granularity. Silencing of the vitamin D receptor (VDR) by corresponding siRNA abolished the stimulatory effect on involucrin gene expression demonstrating an involvement of VDR in 20,23(OH)2D3 action. This mode of action was further substantiated by stimulation of CYP24 gene expression and stimulation of the CYP24 promoter-driven reporter gene activity. 20,23(OH)2D3 displayed several fold lower potency for induction of CYP24 gene expression than 1,25(OH)2D3. Finally, 20,23(OH)2D3 inhibited the transcriptional activity of NF-kappaB in keratinocytes as demonstrated by EMSA, NF-kappaB-driven reporter gene activity assays and measurements of translocation of p65 from the cytoplasm to the nucleus. These inhibitory effects were connected with stimulation of the expression of IkappaBalpha with subsequent sequestration of NF-kappaB in the cytoplasm and consequent attenuation of transcriptional activity. In summary, we have characterized 20,23(OH)2D3 as a novel secosteroidal regulator of keratinocytes proliferation and differentiation and a modifier of their immune activity., (J. Cell. Physiol. 223: 36-48, 2010. (c) 2009 Wiley-Liss, Inc.)

Purpose: Biologically active vitamin D3 (VD) derivatives possess modulatory activities on immunological and inflammatory responses which can be reflected by altered levels of pro-inflammatory chemokines. Nasal polyposis (NP), defined as a chronic inflammatory process of upper respiratory system, could be influenced by VD derivatives. The purpose of this study was to investigate the influence of 1alpha,25-dihydroxyvitamin D3 (calcitriol) and 1alpha,24(R)-dihydroxyvitamin D3 (tacalcitol) on the secretion of IL-6 and IL-8 by fibroblasts derived from NP., Material and Methods: The study involved 12 fibroblast cultures derived from NP samples obtained from surgically treated patients. Measurements were performed on the polyp cells after the 6-9 passages. Culture stimulation involved treatment with tacalcitol and calcitriol at a defined strength (from 10(-7)M to 10(-4)M). IL-6 and IL-8 concentrations were estimated with ELISA., Results: Treatment with calcitriol or tacalcitol inhibits the synthesis of both IL-6 and IL-8 compared to the control group. The dose dependence of this effect has been confirmed. VD derivatives influence was marked at higher concentrations. Significant interleukin decrease was observed at 10(-5) and 10(-4) for calcitriol and 10-4 in the case of tacalcitol., Conclusions: The present study demonstrates that calcitriol and tacalcitol are capable of affecting pro-inflammatory cytokine (IL-6 and IL-8) levels in NP cultures. Our data imply a potential therapeutical application of topical VD derivates in NP and warrant further investigation.

Unlabelled: Vitamin D (VD) and its different analogues, besides their classic role as regulators of calcium and phosphor homeostasis, have emerged as a large family of antiproliferative agents. Such properties suggested VD potential as a therapy for chronic inflammatory diseases, including nasal polyposis (NP). NP growth involves both an inflammatory process and the proliferation of fibroblast as an important factor inducing aberrations in the phenotype of the epithelium. The aim of this study was to investigate the possible influence of 1alpha,25-dihydroxyvitamin D(3) (calcitriol) and 1alpha,24(R)-dihydroxyvitamin D(3) (tacalcitol) in monotherapy and in combination with budesonid R (BR) on NP fibroblast proliferation., Material and Methods: The study involved 26 samples of NP. NP cells were cultured on 96-well plates beginning with a concentration of 5 x 10(3) cells per well with RPMI 1640 medium supplemented with antibiotics and 10% foetal bovine serum. After the fourth to sixth passage the medium was replaced with a nutrient medium with calcitriol or tacalcitol in a defined concentration (from 10(-9) M to 10(-3) M) alone or in combination with BR in 1:1, 1:3 or 3:1 ratios, each at concentrations from 10(-5) M to 10(-3) M., Results: Growth inhibition of nasal fibroblasts exposed to calcitriol or tacalcitol was noted. Significant antiproliferating activity was observed at calcitriol concentrations of 10(-4) M and 10(-3) M after 48 h, and at a concentration of 10(-3) M after 72 h with the percentage of proliferating cells reduced to 30% compared to the control samples (P < 0.05). In cells treated with tacalcitol the maximal effect was seen at 10(-4) M after 48 h and at 10(-3)M after 72 h with a 60% inhibition with respect to the control (P < 0.05). The inhibition of fibroblast proliferation reached the maximal level when they were exposed to calcitriol: BR (1 : 1) or tacalcitol: BR (1 : 1), each at a concentration of 10(-4) M, after 72 h (82% and 69%, respectively)., Conclusions: The antiproliferative activity of calcitriol and tacalcitol in NP cultures was confirmed. Because of its lower toxicity and higher activity tacalcitol seems to be the more promising agent in NP therapy, both as a single medication and in treatment protocols with BR.

We have shown that activin A, a cytokine implicated in regulating B-cell proliferation, is severely deficient in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP), an autoimmune disorder characterized by surfactant accumulation and neutralizing autoantibodies to granulocyte-macrophage colony stimulating factor. Mechanisms of activin regulation in alveolar macrophages are not well understood. Based on previous gene array results from PAP bronchoalveolar lavage cells suggesting deficiencies in vitamin D target genes, and on recent evidence of vitamin D receptor elements (VDREs) in the human activin A gene promoter, we investigated the effects of 1,25-dihydroxyvitamin D (vitamin D(3)) on activin A expression in alveolar macrophages from healthy individuals and PAP patients. Activin A expression was stimulated by LPS in cultures of either healthy control or PAP alveolar macrophages; in contrast, vitamin D(3) increased activin A only in healthy controls but not in PAP. Compared to healthy controls, freshly obtained (uncultured) PAP alveolar macrophages displayed healthy intrinsic vitamin D receptor expression but deficient expression of vitamin D target genes, cathelicidin and thioredoxin interacting protein. PAP patients also demonstrated a relative insufficiency of circulating vitamin D. Investigation of activin A in murine alveolar macrophages confirmed a lack of functional response to vitamin D as anticipated since murine activin A does not contain VDREs. Results suggest that mechanisms of activin A deficiency in PAP alveolar macrophages may involve dysregulation of a novel species-specific vitamin D-activin A pathway.

Chronic kidney disease results in a reduction in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) synthesis and an accumulation of phosphorus in the blood, leading to secondary hyperparathyroidism and renal osteodystrophy. Vitamin D analogs that retain the ability to suppress PTH but that are less calcemic and phosphatemic than the native hormone are preferred therapies for secondary hyperparathyroidism. However, even the most favored analog currently approved for the treatment of chronic kidney disease patients, i.e. 1,25-dihydroxy-19-nor-vitamin D2 (19-nor-D2, Zemplar), still retains some ability to stimulate intestinal absorption of calcium and phosphate. A recently described analog of vitamin D3, 2-methylene-19-nor-20(S)-1alpha-hydroxy-bishomopregnacalciferol [20(S)-2MbisP], suppresses PTH levels, but is unable to stimulate intestinal calcium absorption or bone resorption in rats. The present study shows that 20(S)-2MbisP is unable to stimulate intestinal phosphate absorption at levels known to suppress PTH secretion. Further, 19-nor-vitamin D2 under the same circumstances does stimulate phosphate absorption. Thus, 2MbisP has significant potential in the management of secondary hyperparathyroidism of renal failure.

Integrins are cell-surface receptors engaged in important cancer invasion processes, such as adhesion, migration, proliferation and differentiation. The aim of this study was to evaluate the effect of 1,25-dihydroxyvitamin D3 (calcitriol) and its metabolite 1,24-dihydroxyvitamin D3 (PRI-2191) on alphavbeta3 integrin expression in various cancer cell lines. The expression levels of the beta3 and alphav integrins were reduced only in the WEHI-3 and LLC cell lines by the two compounds. Calcitriol or PRI-2191 treatment caused differentiation of WEHI-3 mouse leukemia cells, but apoptosis of LLC cells. WEHI-3 and LLC cells exposed to calcitriol or PRI-2191 lost their migratory and adhesive potentials. The inhibition of migratory potential was higher in the LLC cells than in the WEHI-3 cells and appeared to correlate with the increased down-regulation of alphavbeta3 integrin by calcitriol or PRI-2191. The observed in vivo effects (antitumor and antimetastatic) in mice bearing subcutaneously transplanted LLC cancer are possibly associated with inhibited migratory potential as a consequence of the lowered integrin expression caused by calcitriol or PRI-2191.

The active form of vitamin D3 and some of its related compounds show neuroprotective effects in various models of neuronal damage, however, mechanism of their anti-apoptotic action has not been elucidated. Therefore, the present study was designed to investigate the effects of 1,25-dihydroxyvitamin D3 and its low-calcemic analogues, PRI-2191, PRI-1890 and PRI-1901 on staurosporine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Twenty-four hour incubation with staurosporine (1 microM) enhanced the caspase-3 activity, decreased mitochondrial membrane potential and increased the number of apoptotic cells as visualized by Hoechst staining. 1,25-Dihydroxyvitamin D3 and PRI-2191 attenuated the staurosporine-induced caspase-3 activity at 5, 50 and 500 nM, whereas PRI-1890 and PRI-1901 were active only at higher concentrations. Furthermore, 1,25-dihydroxyvitamin D3 (50 and 500 nM) and PRI-2191 (500 but not 50 nM) reversed the staurosporine-evoked decrease in mitochondrial membrane potential. Hoechst and calcein staining confirmed the neuroprotective effects of the secosteroids under study. Further study revealed that a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, at concentration of 100 nM antagonized the effect of 1,25-dihydroxyvitamin D3 and PRI-2191 on staurosporine-induced caspase-3 activation. These data indicate that 1,25-dihydroxyvitamin D3 and its low-calcemic analogues at nanomolar concentrations inhibited mitochondrial pathway of apoptosis in SH-SY5Y neuronal cells, though with different potency. Moreover, the activation of PI3-K/Akt signaling pathway appears to play a role in anti-apoptotic effects of the secosteroids.

Unlabelled: Vitamin D analogs are being developed that retain therapeutic effects but are less calcemic and phosphatemic, a concern in CKD patients who are prone to vascular calcification. We tested a new analog of vitamin D, 2MbisP, and found that it suppresses PTH at doses that do not affect serum Ca or P., Introduction: Calcitriol is used for the treatment of secondary hyperparathyroidism. However, its use is often limited by the development of hypercalcemia and hyperphosphatemia, an important consideration in patients with chronic kidney disease (CKD) because they are prone to vascular calcification. To minimize this toxicity, structural modifications in the vitamin D molecule have led to the development of calcitriol analogs with selective actions., Materials and Methods: In this study, we compared the effects of 1,25(OH)(2)D(3) and a new analog, 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), on the development of secondary hyperparathyroidism and established secondary hyperparathyroidism in uremic rats and on mobilization of calcium and phosphorus from bone in parathyroidectomized rats. The clearance from circulation, half-life, and binding affinities to the vitamin D-binding protein and vitamin D receptor of this compound were also evaluated., Results: Uremia produced a marked rise in plasma PTH, but treatment every other day for 2 wk with either 1,25(OH)(2)D(3) (4 ng) or 2MbisP (250, 750, 1500, or 3000 ng) suppressed this increase by >50%. The suppression by 1,25(OH)(2)D(3), however, was accompanied by increases in ionized calcium, phosphorus, and the calcium x phosphorus product, whereas these three parameters were unchanged by 2MbisP. The binding affinity of 2MbisP was 10-20 times less for the vitamin D receptor and 1000 times less for the serum vitamin D-binding protein compared with 1,25(OH)(2)D(3). Also, 2MbisP was cleared more rapidly from the circulation (t1/2 = 10 min) than 1,25-(OH)(2)D(3) (t1/2=7-9 h). In parathyroidectomized rats fed calcium-or phosphorus-deficient diets, daily injections of 2MbisP (1500 or 3000 ng), unlike 1,25(OH)(2)D(3) (50 ng), had no effect on calcium or phosphorus mobilization from bone., Conclusions: In uremic rats, 2MbisP can suppress PTH at doses that do not affect plasma calcium, phosphorus, and calcium x phosphorus product. This new vitamin D analog may represent an important tool in the treatment of secondary hyperparathyroidism in patients with CKD.

Introduction: Recurrent polyposis in the same patient resulting in the necessity of repeated surgeries forced to search for new pharmacological therapeutic methods. At present, locally acting glycocorticosteroids have the greatest value in the treatment of nasal polyposis. Polyps grow is connected with inflammation process and proliferation of fibroblasts., Objective: An evaluation of calcitriol and tacalcitol influence on proliferation of fibroblasts extracted from nasal polyps., Material: consisted of 9 tissue samples coming from nasal polyps sampled during polypectomies. The testing was performed on the polyps fibroblasts after the sixth passage after the primary culture was established. Three days after the culture was started the cells were poured with nutrient medium without serum added and after further 24 hours was replaced by nutrient medium with takalcitol and calcitriol in the defined concentrations. The expression of the genes coding histone H3 was evaluated with the use of RT-PCR technique., Results: Tacalcitiol and calcitriol in vitro decrease proliferation of fibroblasts sampled from nasal polyps. Inhibition is most effective for the concentration of 10-4M. Tacalcitiol and calcitriol also inhibit level of histone H3 gene expression., Conclusion: Experimental data suggest tacalcitiol to be more effective in the same concentration. Present studies may indicate the direction of further investigation in the potential pharmacological treatment on nasal polyps.

New vitamin D analogs are interesting candidates for anticancer treatment, including squamous cell carcinomas (SCCs), especially in combination with cytostatics. In order to evaluate the effect of combined application of cisplatin, imatinib, or docetaxel and new vitamin D analogs [PRI-1906: (24E)-24a-homo-(1S)-1,25-dihydroxyergocalciferol, and PRI-2191: (24R)-1,24-dihydroxyvitamin D3], against the cells of two human SCCs lines (SCC-25 and FaDu), cytotoxic activity and the effect on cell cycle and apoptosis were determined. The synergistic or additive antiproliferative effect was observed for all cytostatics used after treatment of FaDu cell line with calcitriol or its analogs. Antagonism caused by combination of calcitriol and docetaxel was shown only in the lowest dose. FaDu cells treated with cytostatics and vitamin D analogs cumulated in G0/G1 stage. A statistically significant decrease (2x) in the percentage of apoptotic cells was observed only in combination of imatinib and calcitriol or PRI-1906. On the other hand, when the SCC-25 cell line incubated with cisplatin and imatinib in combination with calcitriol or PRI-2191 (100 nM) was used, the quantitative method of Chou and Talalay indicated antagonism. In the lower doses of calcitriol or PRI-2191 combined with imatinib, the synergistic effect was observed, but in the case of combination with cisplatin or docetaxel only weak additivity was detected. Moreover, a significant decrease (2x) of the percentage of SCC-25 cells undergoing apoptosis induced by docetaxel, cisplatin, and imatinib was observed. The combination of all cytostatic drugs applied with PRI-1906 in all doses caused synergism or additivity. These results might indicate that PRI-1906 is more effective than calcitriol or PRI-2191 as a potential anticancer agent, when used in combination therapy with cytostatic agents. To our knowledge, this is the first observation of interaction with calcitriol or its analogs and imatinib.

The active form of 1,25-dihydroxyvitamin D(3) prevents neuronal damage in vitro and in vivo , however, it induces also hypercalcemia and hyperphosphatemia. Side-chain-modified analogues of 1,25-dihydroxyvitamin D(3), which show low calcemic activity, may be potentially useful in the treatment of some neurodegenerative diseases. Previously, we have found that PRI-2191 more potently than 1,25-dihydroxyvitamin D(3) protects human neuroblastoma (SH-SY5Y) cells against hydrogen-peroxide-induced toxicity. In the present study, we evaluated effects of two other 1,25-dihydroxyvitamin D(3) analogues - PRI-1890 and PRI-1901 on the neuronal degeneration in the same cell model. In line with the previous study, 24-h incubation with hydrogen peroxide (0.5 mM) was toxic to cells, as evidenced by an enhanced efflux of lactate dehydrogenase into the culture medium, and these effects were prevented by PRI-1890 and PRI-1901 at concentration of 5, 50 and 500 nM. Comparing the neuroprotective effects of secosteroids, we found that all three analogues were efficient at lower concentration than 1,25-dihydroxyvitamin D(3) and among them the PRI-2191 showed the most evident concentration-dependent effect. In the second part of this study, an involvement of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K), kinases which play a crucial role in neurodegenerative processes, in neuroprotective action of 1,25 dihydroxyvitamin D(3) and its the most potent analogue PRI-2191 has been investigated. The inhibitor of c-Jun N-terminal kinase (JNK)-MAPK (SP600125, 1 microM), inhibitor of p38-MAPK (SB-203580, 1 and 10 microM) and inhibitor of extracellular signal-regulated kinase (ERK)-MAPK (PD-98059, 15 and 30 microM) attenuated the hydrogen peroxide-induced toxicity. Moreover, PD-98059 (30 microM) enhanced neuroprotective effects of 1,25 dihydroxyvitamin D(3) but not that of PRI-2191. In contrast, the inhibitor of PI3-K (wortmannin, 10, 100 nM) did not affect hydrogen peroxide-induced cell damage itself, however, it significantly antagonized the effect of PRI-2191. On the other hand, wortmannin did not affect the neuroprotective effects of 1,25 dihydroxyvitamin D(3) This suggests that the activation of PI3-K/Akt signaling pathway plays an important role in the mechanism of inhibitory action of PRI-2191 on hydrogen peroxide-evoked toxicity in SH-SY5Y cells. Furthermore, these data point to differential involvement of ERK-MAPK and PI3-K in neuroprotective effects of 1,25 dihydroxyvitamin D(3) and its low-calcemic analogue - PRI-2191.

Monocytic differentiation is coordinated through the ordered activation of multiple signalling pathways, controlling transcription of specific subsets of genes that regulate the development of the mature phenotype. To identify key transcription factors involved in this process, we used the human monoblastic U-937 cell line as a model of monocytic differentiation. U-937 cells can be differentiated by treatment with all-trans retinoic acid (ATRA) and 1,25alpha-dihydroxycholecalciferol (VitD3), resulting in G(0)/G(1)-arrested cells expressing monocytic surface markers. We have previously shown that ATRA-induced differentiation and cell cycle arrest specifically requires Stat1 activation, through phosphorylation of tyrosine 701 and serine 727. In this report, we used U-937 cells expressing phosphorylation-deficient mutants of Stat1 (Stat1Y701F and Stat1S727A) to determine myeloid-specific transcription factors that are activated downstream of Stat1 during induced monocytic differentiation. We demonstrate that ATRA-induced upregulation of Stat2, ICSBP/IRF8 and C/EBPepsilon, key transcription factors linked to myelomonocytic differentiation, is selectively impaired in cells expressing mutant Stat1. In contrast, ATRA-induced expression of PU.1, C/EBPalpha, C/EBPbeta and IRF-1 was unaffected. Taken together, our data suggest that ATRA-induced regulation of Stat2, ICSBP and C/EBPepsilon is dependent on active Stat1, and that a failure to correctly regulate these transcription factors is associated with the inhibition of monocytic differentiation.

Calcitriol is a potent antiproliferative agent against various tumour cells in vitro. Here, the results of a study on vitamin D compounds (calcitriol's analogues PRI-1906 and PRI-2191) as potential agents in combined antitumour therapy in vitro are presented. Applying antiproliferative SRB and MTT assays, the growth inhibitory effects of the vitamin D compounds, applied alone or in combination with either cisplatin or doxorubicin, were measured. The following cancer cell lines were employed: A549 (human non-small cell lung carcinoma), B16 (murine melanoma), CCRF, HL-60 (human leukaemia), SW707 (human colon cancer), MCF-7, T47D (human breast cancer), WEHI-3 (mouse leukaemia) and normal cells: BALB 3T3 (normal murine fibroblast cell line). It was shown that the treatment of tumour cells, which are sensitive to vitamin D compounds, with the combination of vitamin D compounds and cytostatics decreased the inhibitory concentration 50% (IC50) values compared with the effects of the cytostatics applied alone.

Calcitriol is a potent antiproliferative agent against various tumour cells in vitro. Its biological activity is mediated by the vitamin D receptors (VDRs). Here, we present the results of a study on vitamin D3 compounds (calcitriol and its analogue PRI-2191) as potential agents in combined antitumour therapy in vitro. Applying antiproliferative SRB and MTT assays, we measured the growth inhibitory effects of vitamin D compounds applied alone or in combination with either cisplatin or doxorubicin. Next, we examined the correlation of this effect with the presence of nVDR (nuclear VDR). The following cancer cell lines were applied: HL-60 (human leukaemia), SW707 (human colon cancer), A549 (human lung cancer), WEHI-3 (mouse leukaemia). The treatment of tumour cells with the combination of vitamin D compounds and cytostatics decreased the inhibitory concentration 50% (IC50) values compared with the effects of cytostatics applied alone. The synergistic effect was positively correlated with nVDR expression.

Several topical treatments such as ointments, keratolytics, dithranol, tar, corticosteroids and Vitamin D3 analogues are commonly used in the treatment of mild and/or moderate psoriasis. These treatments can be associated with a variety of local and systemic side effects, as well as to very often unsatisfactory results. The purpose of this critical review of the literature is to evaluate the efficacy and tolerability of the synthesis of new analogues of the Vitamin D3 Tacalcitol, which is formulated in ointment form at a concentration of 4 microg/g, for the treatment of mild and/or moderate psoriasis (involvement of <20% of the surface of the skin) and to evaluate whether this drug can be used in the treatment of other skin conditions. Based on existing data in the literature, Tacalcitol is an effective drug for the topical treatment of psoriasis and is also able to ensure that the effects last over time, even after treatment has stopped. Tacalcitol is also well tolerated because the onset of side effects, such as local irritation, pruriginous or burning sensations, were reported in only a small percentage of the subjects who were treated. Lastly, the marked regulatory effects it has on the proliferation and differentiation of keratinocytes, as well as on the immunocompetent cells, has led to suggestions that Tacalcitol may be used in other keratinisation disorders and in some hyperproliferative skin diseases. Evaluation of the effective indications to use in these conditions still requires further data confirming its effectiveness, opening the way to wider use of this molecule in dermatology.

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.

The active form of Vitamin D(3) has been reported to prevent neuronal damage caused by a variety of insults, however, it may also induce undesirable hypercalcemic effects. In the present study, we evaluated effects of (24R)-1,24-dihydroxycholecalciferol (PRI-2191) on hydrogen peroxide- and excitatory amino acid-induced neuronal damage in human neuroblastoma (SH-SY5Y) cell line. Exposure of SH-SY5Y cells to N-methyl-d-aspartate (NMDA; 5mM), kainate (0.2mM) and hydrogen peroxide (0.1-1mM) significantly enhanced lactate dehydrogenase release. Furthermore, the neurotoxic effects of hydrogen peroxide was dependent on c-Jun N-terminal kinase (JNK)- and p38- mitogen-activated protein kinase (MAPK) activity. Both secosteroids at nanomolar concentrations inhibited neuronal damage, but their efficacy varied depending on the toxic agent. PRI-2191 was equipotent as 1alpha,25-dihydroxyVitamin D(3) in protecting SH-SY5Ycells against NMDA toxicity, and had stronger effect against hydrogen peroxide-induced damage, but was less efficient against kainate-induced injury. The obtained results suggest potential usefulness of PRI 2191 in the treatment of neurodegenerative diseases.

Tacalcitol (1,24(R)(OH)2D3, TV-02) inhibited the TPA-induced inflammatory cell infiltration (largely neutrophils) histopathologically and myeloperoxidase (MPO) activity dose-dependently. Tacalcitol inhibited the mRNA expression and protein production of TPA-induced macrophage inflammatory protein-2 (MIP-2) and KC, the functional analogue of human interleukin (IL)-8, in the skin. Immunohistochemical staining of the TPA-applied skin revealed that mast cells expressed MIP-2, whereas KC was observed in keratinocytes, fibroblasts and outer root sheath of hair follicles. Furthermore, tacalcitol inhibited TPA-induced mast cell degranulation 24 hr after application without influence on the total number of mast cells. In this study, tacalcitol was found to have an inhibitory effect on cutaneous inflammation such as inhibition of neutrophil infiltration, MIP-2 and KC production, and mast cell degranulation in TPA-treated hairless mice. These results suggest that tacalcitol modulates cutaneous inflammation as well as keratinocyte proliferation and differentiation, and the inhibitory effect of tacalcitol on cutaneous inflammation may contribute to clinical the effectiveness in the treatment of psoriasis.

Steroids are implicated in the regulation of neurodegenerative processes including oxidative stress. The aim of this study was to compare the effects of 1alpha,25-dihydroxyvitamin D3 (calcitriol) and its low-calcemic analogue (24R)-1,24-dihydroxycholecalciferol (tacalcitol, PRI-2191) and some putative neuroprotective steroids belonging to various pharmacological groups on the hydrogen peroxide-induced cytotoxicity in NG108-15 cell line. Both secosteroids prevented in a concentration-dependent manner (50-500 nM) the hydrogen peroxide-induced lactate dehydrogenase release in the NG108-15 cells. Estrogens (17beta-estradiol and estriol, 100 and 1000 nM) also protected these cells. Of the two neurosteroids tested at micromolar concentrations, dehydroepiandrosterone sulphate markedly reduced cell damage, whereas the allopregnanolone showed a weak neuroprotective effect. These data support some previous observations on the antineurotoxic action of neurosteroids and indicate that calcitriol and its low-calcemic analogue PRI-2191 have neuroprotective properties comparable to those of estrogens. Considering the undesired effects of estrogens, it seems that some calcitriol analogues may be alternative drugs in the treatment of some oxidative stress-related neurodegenerative disorders.

Vitiligo vulgaris is a common skin disease, however some cases show poor clinical responses to topical steroid ointment or PUVA therapy. Such regimens are generally avoided in the treatment of facial lesions or in pediatric cases because of the undesirable side effects. To confirm the excellent response to combination therapy with topical vitamin D3 ointment and solar irradiation for vitiligo achieved in the initial patients, we conducted an open trial on other patients, most of whom had poor clinical responses to the prior therapies. Fifteen patients (9 men and 6 women) with vitiligo vulgaris were enrolled in this study. Each patient was instructed to sunbathe for 30 minutes within 1 hour after topical application of the tacalcitol [1 alpha 24(OH)(2)D(3)] ointment or cream to the skin lesions every day. Six of 15 patients showed a fair and excellent clinical response to the combination therapy (more than 30% clearance of the vitiligo). The clinical effect was more apparent in patients with a history of less than 5 years of vitiligo (4 of 6 cases) in contrast to those with a history of more than 5 years (2 of 9 cases). In vitro experiments revealed that tacalcitol upregulated the expression of c-Kit mRNA by melanocytes irradiated with linear polarized infrared, UVA or short period solar irradiation. These results suggest that combination therapy with topical vitamin D(3) ointment and solar irradiation can be used as an alternate therapy for vitiligo vulgaris.

This study focused on the effects of tacalcitol (1,24 (R) (OH)2D3, TV-02) ointment (20 micro g/g) on cutaneous inflammation, epidermal proliferation, and differentiation and compared them with tacalcitol ointment (2 micro g/g) and other anti-psoriatic ointments using hairless mice. Tacalcitol ointment (0, 2 and 20 micro g/g) significantly inhibited 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cutaneous inflammation, histopathologically. The effect of tacalcitol ointment (20 micro g/g) on cutaneous inflammation was much stronger than that of tacalcitol ointment (0, 2 micro g/g), and as effective as calcipotriol ointment (50 micro g/g) or betamethasone valerate ointment (1.2 mg/g). Tacalcitol ointment (20 micro g/g) also significantly inhibited TPA-induced myeloperoxidase (MPO) activity, as effectively as calcipotriol ointment (50 micro g/g) or betamethasone valerate ointment (1.2 mg/g). The effect of tacalcitol ointment on epidermal proliferation [ornithine decarboxylase (ODC) activity] and differentiation [transglutaminase (TGase) activity] was dose-dependent from 0 micro g/g to 20 micro g/g. The effect of tacalcitol ointments on epidermal proliferation was significant at the doses of 2 micro g/g and 20 micro g/g, and that on epidermal differentiation was significant at the doses of 0.2 micro g/g or more. The effect of tacalcitol ointment (20 micro g/g) on epidermal differentiation was significantly stronger than tacalcitol ointment (2 micro g/g). In this study, tacalcitol ointment (20 micro g/g) was found to have a marked effect on cutaneous inflammation and improved effect on epidermal differentiation, although tacalcitol ointment (2 micro g/g) also had significant effects on epidermal proliferation and differentiation. These findings support the clinical effectiveness of tacalcitol ointment (20 micro g/g) against psoriasis.

Background: The active vitamin D3 regulates proliferation and differentiation of epidermal keratinocytes. Recently topical vitamin D3, tacalcitol, calcipotriol, and maxacalcitol are widely used for psoriasis., Objective: To examine the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cultured normal keratinocytes (NHK) and compared its effect with those of various vitamin D3 analogues., Methods: Cell proliferation of NHK cells was analyzed by MTS, BrdU and 3H-thymidine incorporation. The expression of involucrin, transglutaminase 1, keratin 5 and keratin 1 was investigated by western blot and PCR amplification and quantitative assay. Furthermore, we performed cornified cell envelope (CE) formation assay., Results: 1,25(OH)2D3, tacalcitol, calcipotriol, and maxacalcitol decreased NHK cell proliferation in a concentration-dependent manner and the maximal effect was observed at 10(-7) M. There was no significant difference in the anti-proliferative effect among the active vitamin D3 analogues. The expression of involucrin and transglutaminase 1 were induced by 1,25(OH)2D3 and its analogues in mRNA and protein levels. CE formation was also induced by 1,25(OH)2D3 and its analogues. There was no significant difference in the potency among these chemicals. Keratin 5 and 1 expression was not altered by these active vitamin D3 analogues., Conclusions: The present study demonstrated that active vitamin D3 analogues, tacalcitol, calcipotriol, and maxacalcitol, suppress keratinocyte proliferation and induce differentiation with similar potency.