Vitamin D and immune system.

The active metabolite of vitamin D 1,25(OH)2D is well known for its role in regulating calcium-phosphate homeostasis of the human body. However, the immunomodulating activity of 1,25(OH)2D has been known for many years. There are numerous reports correlating low vitamin D levels in blood serum with the onset of autoimmune diseases and with the severe course of acute infections. In this chapter, we address the role of 1,25(OH)2D in these diseases, and we discuss the possible mechanisms of action of 1,25(OH)2D in immune cells.

Immuno-Stimulating Activity of 1,25-Dihydroxyvitamin D in Blood Cells from Five Healthy People and in Blasts from Five Patients with Leukemias and Pre-Leukemic States.

(1) Hematological malignancies are characterized by an immortalization, uncontrolled proliferation of blood cells and their differentiation block, followed by the loss of function. The primary goal in the treatment of leukemias is the elimination of rapidly proliferating leukemic cells (named blasts). However, chemotherapy, which removes proliferating blasts, also prevents the remaining immune cells from being activated. Acute leukemias affect elderly people, who are often not fit to survive aggressive chemotherapy. Therefore, there is a need of milder treatment, named differentiation therapy, which might simulate the immune system of the patient. 1,25-Dihydroxyvitamin D, or low-calcemic analogs of this compound, were proposed as supporting therapy in acute leukemias. (2) Bone marrow blasts from patients with hematological malignancies, and leukocytes from healthy volunteers were ex vivo exposed to 1,25-dihydroxyvitamin D, and then their genomes and transcriptomes were investigated. (3) Our analysis indicates that 1,25-dihydroxyvitamin D regulates in blood cells predominantly genes involved in immune response, such as CAMP (cathelicidin antimicrobial peptide), CP (ceruloplasmin), CXCL9 (C-X-C motif chemokine ligand 9), CD14 (CD14 molecule) or VMO1 (vitelline membrane outer layer 1 homolog). This concerns blood cells from healthy people, as well as blasts from patients with hematological malignancies. In addition, in one patient, 1,25-dihydroxyvitamin D significantly downregulated transcription of genes responsible for cell division and immortalization. (4) In conclusion, the data presented in this paper suggest that addition of 1,25-dihydroxyvitamin D to the currently available treatments would stimulate immune system, inhibit proliferation and reduce immortal potential of blasts.

Overexpressed fibroblast growth factor receptors increase 1,25-dihydroxyvitamin D-dependent differentiation of acute myeloid leukemia cells.

Many malignancies are driven by mutations within the gene for fibroblast growth factor receptor 1 (FGFR1). Previously, we have shown that signal transduction from the FOP2-FGFR1 fusion protein in acute myeloid leukemia KG1 cells is responsible for a low level of expression of the vitamin D receptor gene. In this paper, we address whether other fibroblast growth factor receptors regulate the vitamin D receptor (VDR) gene. We used the human myeloid leukemia U937 and HL60 cells, the bone cancer cell line U2OS, and cell transfection methods to answer the question. For myeloid leukemia cells, overexpression of FGFRs 1-3 genes caused a shift towards monocytic differentiation; this was extracellular regulated kinase (Erk) 1,2-dependent. Overexpression of FGFRs 1-3 genes also upregulated expression of the VDR gene, further sensitizing these cells to 1,25-dihydroxyvitamin D-induced monocyte differentiation. When we increased expression in bone cells, fibroblast growth factor receptors did not upregulate VDR gene expression, nor influence the activity of VDR. Fibroblast growth factor receptors are overexpressed in many neoplasms. Therefore, it may be reasonable to use vitamin D analogs to treat these cancers, to activate VDR and drive cell differentiation.

Vitamin D Derivatives in Acute Myeloid Leukemia: The Matter of Selecting the Right Targets.

Acute myeloid leukemia (AML) is an aggressive and often fatal hematopoietic malignancy. A very attractive way to treat myeloid leukemia, called "differentiation therapy", was proposed when in vitro studies showed that some compounds are capable of inducing differentiation of AML cell lines. One of the differentiation-inducing agents, all-trans-retinoic acid (ATRA), which can induce granulocytic differentiation in AML cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a chromosomal translocation. ATRA has greatly improved the treatment of APL. Since 1,25-dihydroxyvitamin D (1,25D) is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs was widely accepted. However, early clinical trials in which cancer patients were treated either with 1,25D or with analogs did not lead to conclusive results. Recent results have shown that AML types with certain mutations, such as isocitrate dehydrogenase (IDH) mutations, may be the right targets for differentiation therapy using 1,25D, due to upregulation of vitamin D receptor (VDR) pathway.

Heavy metals in leathers, artificial leathers, and textiles in the context of quality and safety of use.

The article presents research findings on the content of arsenic, cadmium, chromium, copper, lead, and zinc in extracts from leathers, artificial leathers intended for footwear components, and textiles. After extracting the metals using an artificial acidic sweat solution, their contents were quantitatively determined by atomic absorption spectrometry. In the cotton textiles, the metal contents were in accordance with the OEKO-TEX limits, while regarding the artificial leathers, only the acrylic knit fur had a too high chromium content (1.1 mg/kg) as compared with the requirements of the STANDARD 100 by OEKO-TEX for products intended for children (< 1.0 mg/kg). The chromium content in lining and upper leather (> 228.0 mg/kg) exceeds the limits for children's products (< 2.0 mg/kg), but also the less restrictive ones for other products (< 200.0 mg/kg). Regarding the other metals, the leathers met the OEKO-TEX requirements. Approved materials may have elevated heavy metal contents, as demonstrated for chromium. The presence of heavy metals in too large amounts in products is a serious problem due to their allergenic and toxic effect. Therefore, action should be taken aimed at more effective detection and elimination of such products from markets and at reducing the use of chemicals containing harmful metals in manufacturing processes.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists.

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells.

BACKGROUND: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. METHODS: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). RESULTS: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. CONCLUSIONS: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs' structure might lead to new treatment options against ovarian cancer.

Investigating the Role of Methylation in Silencing of VDR Gene Expression in Normal Cells during Hematopoiesis and in Their Leukemic Counterparts.

(1) Background: Vitamin D receptor (VDR) is present in multiple types of blood cells, and its ligand, 1,25-dihydroxyvitamin D (1,25D), is important for the proper functioning of the immune system. Activity of VDR is higher in hematopoietic stem and progenitor cells than in fully differentiated blood cells of mice and humans. In some human acute myeloid leukemia (AML) blasts, the expression of the VDR gene is also high. The mechanism of silencing the VDR gene expression during differentiation of blood cells has been addressed in this work. (2) Methods: The cells have been obtained using fluorescence activated sorting from murine tissues and from human umbilical cord blood (UCB). Then, the expression of the VDR gene and transcriptional activity of the VDR protein has been tested in real-time polymerase chain reaction (PCR). Eventually, the methylation of VDR promoter regions was tested using bisulfite sequencing. (3) Results: The CpG islands in VDR promoters were not methylated in the cells studied both in mice and in humans. The use of hypomethylating agents had no effect toward expression of human VDR transcripts, but it increased expression of the VDR-target gene, CYP24A1. (4) Conclusions: The expression of the VDR gene and transcriptional activity of the VDR protein varies at successive stages of hematopoietic differentiation in humans and mice, and in blasts from AML patients. The experiments presented in this case indicate that methylation of the promoter region of the VDR gene is not the major mechanism responsible for these differences.

Design, synthesis and biological evaluation of novel 2-alkylidene 19-norcalcitriol analogs.

Continuing our studies aimed at A-ring modified vitamin D compounds, we designed novel 19-norcalcitriol derivatives bearing at C-2 pegylated chains of different lengths. The terminal fragments of these substituents contain hydroxyls or moieties possessing nitrogen and/or sulfur atoms capable of transition metal ions complexation. Also, two conjugate-type platinum(II) complexes of 19-norcalcitriol were obtained in which l-methionine served as chelating moiety. The convergent synthesis of the target 19-norcalcitriol analogs involved several steps with the crucial one being condensation of A-ring phosphine oxide and the known Grundmann ketone by Wittig-Horner reaction. Further elaboration of the 2-alkylidene substituent provided all final compounds which were then tested to determine their affinity for the vitamin D receptor and cytotoxic activity.

Synthesis of Gemini analogs of 19-norcalcitriol and their platinum(II) complexes.

Hormonally active vitamin D3 metabolite, calcitriol, plays an important role in calcium-phosphate homeostasis, immune system actions and cell differentiation. Although anticancer activity of calcitriol is well documented and thousands of its analogs have been synthesized, none has been approved as a potential drug against cancer. Therefore, we attempted to introduce the cytotoxic effect to the calcitriol molecule by its linking to cisplatin. Herein, we present the synthesis of vitamin D compounds, designed on the basis of molecular modeling and docking experiments to the vitamin D receptor, and characterized by the presence of significantly different two side chains attached to C-20. In this study, a new synthetic approach to Gemini analogs was developed. Preparation of the target 19-norcalcitriol compounds involved separate syntheses of several building blocks (the A-ring, C/D-rings and side-chain fragments). The convergent synthetic strategy was used to combine these components by the different coupling processes, the crucial one being Wittig-Horner reaction of the Grundmann ketone analog with the known 2-methylene A-ring phosphine oxide. Due to the nature of the constructed steroidal side chains (bidentate ligands), which allowed coordination of metal ions, the first conjugate-type platinum(II) complexes of the vitamin D analogs were also successfully prepared and characterized. The target vitamin D compounds, displaying significant affinity for a vitamin D receptor, were assessed in vitro for their anti-proliferative activities towards several cell lines.

Investigation of finishing of leather for inside parts of the shoes with a natural biocide.

The prevention of decrease of quality caused by microbial activity in footwear materials entails the use of biocides. However, these substances may pose a hazard to humans and to the natural environment. The paper presents the results of antimicrobial effect investigation for cowhide leather treated with oregano oil. In these studies oil was applied by spraying onto the finished leather surface and examined to determine its antimicrobial activity by using the Agar Diffusion Plate Test. These results were compared with those where a cowhide leather was treated with oil at the stage of fatliquoring. In addition, the oregano oil toxicity level was assessed and compared with biocides used in the tanning industry. Introducing oregano oil into the leather at the stage of fatliquoring provides a better antimicrobial effect than by spraying, however hygienic finishing of leather can be obtained by introducing oil into the raw material by these both methods. The oregano oil is characterised by the lowest number of hazards and toxicity as compared with commercial biocides. The use of essential oils as natural biocides in the tanning industry seems to be especially important and suitable solution considering the harmful effects of synthetic biocides to humans and the environment.

Investigating the Role of VDR and Megalin in Semi-Selectivity of Side-Chain Modified 19-nor Analogs of Vitamin D.

1,25-dihydroxyvitamin D3 (1,25D3) is implicated in many cellular functions, including cell proliferation and differentiation, thus exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D3 are potent calcemic activities. Therefore, synthetic analogs of 1,25D3 for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. To obtain this goal, the analogs should effectively activate transcription of genes responsible for cell differentiation, leaving the genes responsible for calcium homeostasis less active. In order to better understand this phenomenon, we selected a series of structurally related 19-nor analogs of 1,25D (PRI-5100, PRI-5101, PRI-5105, and PRI-5106) and tested their activities in blood cells and in cells connected to calcium homeostasis. Affinities of analogs to recombinant vitamin D receptor (VDR) protein were not correlated to their pro-differentiating activities. Moreover, the pattern of transcriptional activities of the analogs was different in cell lines originating from various vitamin D-responsive tissues. We thus hypothesized that receptors which participate in transport of the analogs to the cells might contribute to the observed differences. In order to study this hypothesis, we produced renal cells with knock-out of the megalin gene. Our results indicate that megalin has a minor effect on semi-selective activities of vitamin D analogs.

Regulation of FOXP3 expression in myeloid cells in response to all-trans-retinoic acid, interleukin 2 and transforming growth factor ß.

FoxP3 is a transcription factor essential for differentiation and function of T regulatory cells (Tregs). There are two major subsets of Tregs: natural Tregs (nTregs) generated in thymus and inducible Tregs (iTregs) produced in peripheral immune system. It has been documented that iTreg development is dependent on soluble mediators including interleukin 2 (IL2), transforming growth factor ß (TGFß) and all-trans-retinoic acid (ATRA). In our experiments we performed a gene expression array, followed by Real-time PCR experiments to study expression of genes regulated by ATRA in cells of myeloid origin. Our experiments revealed that ATRA alone, but also a cocktail of mediators consisting of IL2, TGFß and ATRA, upregulate expression of FOXP3 gene in normal and leukemic myeloid cells. Our results indicate that signaling pathways which are used at the late steps of T cell differentiation, are also active in the cells of myeloid lineage.

Synthesis of 19-norcalcitriol analogs with pegylated alkylidene chains at C-2.

The results presented in this paper constitute an extension of our synthetic efforts focused on 19-norvitamin D compounds possessing elongated 2-alkylidene substituents. Based on our previous results, molecular modeling studies, and docking experiments, we selected a novel 19-norcalcitriol analog with long chain at C-2 containing several ether moieties and terminated by 2-(pyridin-2'-yl)ethylamino fragment. It was expected that such structural modification might allow binding of transition metal by the ligand, increase solubility of the formed complexes as well as improve their affinity to the VDR. For comparison, a 19-norcalcitriol analog was also obtained with the terminal hydroxyl group at its pegylated 2-alkylidene substituent. The synthesis of the target vitamin D compounds described in this work was performed using the Wittig-Horner approach. The respective A-ring phosphine oxide was obtained starting from the D-(-)-quinic acid and then coupled with the known Grundmann ketone.

Regulation of Expression of CEBP Genes by Variably Expressed Vitamin D Receptor and Retinoic Acid Receptor α in Human Acute Myeloid Leukemia Cell Lines.

All-trans-retinoic acid (ATRA) and 1α,25-dihydroxyvitamin D (1,25D) are potent inducers of differentiation of myeloid leukemia cells. During myeloid differentiation specific transcription factors are expressed at crucial developmental stages. However, precise mechanism controlling the diversification of myeloid progenitors is largely unknown, CCAAT/enhancer-binding protein (C/EBP) transcription factors have been characterized as key regulators of the development and function of the myeloid system. Past data point at functional redundancy among C/EBP family members during myeloid differentiation. In this study, we show that in acute myeloid leukemia (AML) cells, high expression of vitamin D receptor gene (VDR) is needed for strong and sustained upregulation of CEBPB gene, while the moderate expression of VDR is sufficient for upregulation of CEBPD in response to 1,25D. The high expression level of the gene encoding for retinoic acid receptor α (RARA) allows for high and sustained expression of CEBPB, which becomes decreased along with a decrease of RARA expression. Expression of CEBPB induced by ATRA is accompanied by upregulated expression of CEBPE with similar kinetics. Our results suggest that CEBPB is the major VDR and RARA-responsive gene among the CEBP family, necessary for expression of genes connected with myeloid functions.

Synthesis of 19-norcalcitriol analogs with alkylidene moieties at C-2 based on succinic acid and l-methionine.

On the basis of the literature data, our previous research work and docking experiments, we designed novel 19-norvitamin D compounds having elongated 2-alkylidene substituents. These 19-norcalcitriol derivatives have attached 2-(3'-aminopropylidene) substituent in which the nitrogen atom bears acyl residue derived from succinic acid and l-methionine. Both compounds were obtained by the same synthetic strategy involving Julia coupling of the A-ring ketone with the known C/D-ring sulfone. In the obtained 1α,25-dihydroxy-19-norvitamin D3 derivative, the alkylidene substituent at C-2 was further elaborated to the desired structures.

Synthesis of 19-norcalcitriol analogs with elongated side chain.

Pronounced biological potency of 19-norvitamin D compounds as well as interesting biological action of the vitamin D analogs possessing elongated side chains encouraged us to expand the scope of our structure-activity studies to encompass such modifications of the 1α,25-(OH)2D3 (calcitriol) molecule. The aim of our studies was the synthesis of calcitriol analog, designed on the basis of results of molecular modeling and docking experiments, and characterized by a presence of a long, nitrogen-containing substituent attached to carbon 26, and an exomethylene moiety transferred from C-10 to C-2. The convergent synthesis of such 19-norcalcitriol compound, described in this communication, consisted of the preparation and combining four building blocks. The crucial point of the synthesis, coupling of the known A-ring phosphine oxide and the synthesized Grundmann ketone analog, was achieved using Wittig-Horner protocol. It provided the protected analog of 1α,25-dihydroxy-2-methylene-19-norvitamin D3 which was further transformed into the target compound.

Acute Myeloid Leukaemia: New Targets and Therapies.

The most common acute hematological malignancy in adults is acute myeloid leukaemia (AML), accounting for more than 80% of cases in patients over 60 years of age [...].