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.

Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization.

Protein prenylation is a post-translational modification controlling the localization, activity, and protein-protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.

Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase.

Rab geranylgeranyltransferase (GGTase-II, RGGT) catalyses the post-translational modification of eukaryotic Rab GTPases, proteins implicated in several pathologies, including cancer, diabetes, neurodegenerative, and infectious diseases. Thus, RGGT inhibitors are believed to be a potential platform for the development of drugs and tools for studying processes related to the abnormal activity of Rab GTPases. Here, a series of new α-phosphonocarboxylates have been prepared in the first attempt of rational design of covalent inhibitors of RGGT derived from non-covalent inhibitors. These compounds were equipped with electrophilic groups capable of binding cysteines, which are present in the catalytic cavity of RGGT. A few of these analogues have shown micromolar activity against RGGT, which correlated with their ability to inhibit the proliferation of the HeLa cancer cell line. The proposed mechanism of this inhibitory activity was rationalised by molecular docking and mass spectrometric measurements, supported by stability and reactivity studies.

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.

Synthesis of the 6-Substituted Imidazo[1,2-a]Pyridine-3-yl-2- Phosphonopropionic Acids as Potential Inhibitors of Rab Geranylgeranyl Transferase.

Twelve phosphonopropionates derived from 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC) were synthesized and evaluated for their activity as inhibitors of protein geranylgeranylation. The nature of the substituent in the C6 position of imidazo[1,2-a]pyridine ring was responsible for the compound's activity against Rab geranylgeranyl transferase (RGGT). The most active inhibitors disrupted Rab11A prenylation in the human cervical carcinoma HeLa cell line. The esterification of carboxylic acid in the phosphonopropionate moiety turned the inhibitor into an inactive analog.

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 and Biological Evaluation of Imidazole-Bearing α-Phosphonocarboxylates as Inhibitors of Rab Geranylgeranyl Transferase (RGGT).

Rab geranylgeranyl transferase (RGGT) is an interesting therapeutic target, as it ensures proper functioning of Rab GTPases, a class of enzymes responsible for the regulation of vesicle trafficking. Relying on our previous studies, we synthesized a set of new α-phosphonocarboxylic acids as potential RGGT inhibitors, with emphasis on the elaboration of imidazole-containing analogues. We identified two compounds with activity similar to that of previously reported RGGT inhibitors, showing structural similarity to imidazo[1,2-a]pyridine-containing analogues in terms of their substitution pattern. Interestingly, analogues of the N-series, derived from another phosphonocarboxylate RGGT inhibitor, 2-fluoro-3-(1H-imidazol-1-yl)-2-phosphonopropanoic acid, turned out to be inactive in our model, indicating that an additional substituent localized at positions C2 or C4 of the imidazole ring, may adversely affect the potency against the targeted enzyme.

Diverse Regulation of Vitamin D Receptor Gene Expression by 1,25-Dihydroxyvitamin D and ATRA in Murine and Human Blood Cells at Early Stages of Their Differentiation.

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.

Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells.

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D3 to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARß are activated by sub-nM all-trans retinoic acid (EC50-0.3 nM): ~50-fold more is required for activation of RARα (EC50-16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

Therapeutic use of selective synthetic ligands for retinoic acid receptors: a patent review.

INTRODUCTION: Differentiation therapy using all-trans retinoic acid (ATRA) revolutionised the treatment of acute promyelocytic leukaemia to such an extent that it is now one of the most curable types of leukaemia, with ATRA and anthracycline-based chemotherapy providing cure rates above 80%. Isotretinoin is used to treat chronic acne. Here, we examine the information described in recent patents and the extent to which new findings are influencing extending retinoid-based differentiation therapy to other cancers, as well as the development of new therapies for other disorders. AREAS COVERED: A search has been performed on the literature and worldwide patents filed during 2014 to the present time, focusing on synthetic agonists and antagonists of retinoic acid receptors and novel compositions for the delivery of these agents. EXPERT OPINION: New potential therapeutic applications have been described, including lung, breast and head and neck cancers, T cell lymphoma and neurodegenerative, metabolic, ophthalmic, muscle, and inflammatory disorders. Recent patents have described the means to maximise retinoid activity. Two decades of efforts to extend retinoid-based therapies have been disappointing and new synthetic retinoids, target diseases and modes of delivery may well resolve this long standing issue.

Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D.

Restored expression of vitamin D receptor and sensitivity to 1,25-dihydroxyvitamin D3 in response to disrupted fusion FOP2-FGFR1 gene in acute myeloid leukemia cells.

BACKGROUND: Acute myeloid leukemia (AML) cells can be induced to undergo terminal differentiation with subsequent loss of tumorigenicity using 1,25-dihydroxyvitamin D3 (1,25D) alone or in combination with hematopoietic cytokines. KG1 cells are resistant to 1,25D-induced cell differentiation. These cells have the aberrant signal transduction resulting from a constitutively active fusion protein FOP2-FGFR1, a constitutively active STAT1 and a high level of interferon (IFN) stimulated genes (ISGs). METHODS: In this paper we report that in KG1 cells with constitutively activated protein FOP2-FGFR1 delivery of plasmid DNA disrupted FOP2-FGFR1 fusion gene. RESULTS: As a consequence, STAT1 signal transduction pathway became switched off, the expression of vitamin D receptor (VDR) gene was increased and sensitivity to 1,25D-induced differentiation was restored. The activation of ISGs in KG1 cells resulted in resistance to externally added IFNs, and also this effect was reversed in cells with disrupted FOP2-FGFR1 fusion gene. DISCUSSION: In this paper we have documented for the first time a link between constitutively active STAT1 signal transduction pathway, high level of ISGs and low expression of VDR gene. CONCLUSIONS: We show in this paper that delivery of plasmid DNA to the cells may disrupt fusion gene FOP2-FGFR1 which occurs in a disease entity called 8p11 myeloproliferative syndrome. Inhibition of the FOP2-FGFR1 signal transduction pathway restored sensitivity of the cells to 1,25D-induced cell differentiation.

Perspectives of differentiation therapies of acute myeloid leukemia: the search for the molecular basis of patients' variable responses to 1,25-dihydroxyvitamin d and vitamin d analogs.

The concept of differentiation therapy of cancer is ~40 years old. Despite many encouraging results obtained in laboratories, both in vitro and in vivo studies, the only really successful clinical application of differentiation therapy was all-trans-retinoic acid (ATRA)-based therapy of acute promyelocytic leukemia (APL). ATRA, which induces granulocytic differentiation of APL leukemic blasts, has revolutionized the therapy of this disease by converting it from a fatal to a curable one. However, ATRA does not work for other acute myeloid leukemias (AMLs). Since 1,25-dihydroxyvitamin D3 (1,25D) is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs (VDAs) was widely accepted. Also, some types of solid cancers responded to in vitro applied VDAs, and hence it was postulated that VDAs can be used in many clinical applications. However, early clinical trials in which cancer patients were treated either with 1,25D or with VDAs, did not lead to conclusive results. In order to search for a molecular basis of such unpredictable responses of AML patients toward VDAs, we performed ex vivo experiments using patient's blast cells. Experiments were also performed using 1,25D-responsive and 1,25D-non-responsive cell lines, to study their mechanisms of resistance toward 1,25D-induced differentiation. We found that one of the possible reasons might be due to a very low expression level of vitamin D receptor (VDR) mRNA in resistant cells, which can be increased by exposing the cells to ATRA. Our considerations concerning the molecular mechanism behind the low VDR expression and its regulation by ATRA are reported in this paper.

NADPH-cytochrome P450 reductase is regulated by all-trans retinoic acid and by 1,25-dihydroxyvitamin D3 in human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) cell lines can be driven to differentiate to monocyte-like cells by 1,25- dihydroxyvitamin D3 (1,25D) and to granulocyte-like cells by all-trans-retinoic acid (ATRA). Both compounds activate their specific intracellular receptors, vitamin D receptor (VDR) and retinoic acid receptors (RARs) respectively. Inside the cells 1,25D is degraded to calcitrioic acid by a mitochondrial enzyme CYP24A1, while ATRA is degraded to several polar metabolites by CYP26. NADPH-cytochrome P450 oxidoreductase (POR) is a membrane-bound enzyme required for electron transfer to cytochrome P450 (CYP), vital in the processes of the metabolism of drugs and steroid production in humans. In this paper we report that POR in AML cells, from both cell lines and patients, is upregulated by ATRA and by 1,25D at the level of mRNA and protein. Partial silencing of POR in HL60 cells resulted in augmented differentiation response to 1,25D.

Regulation of Leukemic Cell Differentiation through the Vitamin D Receptor at the Levels of Intracellular Signal Transduction, Gene Transcription, and Protein Trafficking and Stability.

1α,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D) exerts its biological activities through vitamin D receptor (VDR), which is a member of the superfamily of steroid receptors, that act as ligand-dependent transcription factors. Ligated VDR in complex with retinoid X receptor (RXR) binds to regulatory regions of 1,25(OH)(2)D-target genes. 1,25(OH)(2)D is able to induce differentiation of leukemic blasts towards macrophage-like cells. Many different acute myeloid leukemia (AML) cell lines respond to 1,25(OH)(2)D by increasing CD14 cell surface receptor, some additionally upregulate CD11b and CD11c integrins. In untreated AML cells VDR protein is present in cytosol at a very low level, even though its mRNA is continuously expressed. Ligation of VDR causes protein stabilization and translocation to the cell nuclei, where it regulates transcription of target genes. Several important groups of genes are regulated by 1,25(OH)(2)D in HL60 cells. These genes include differentiation-related genes involved in macrophage function, as well as a gene regulating degradation of 1,25(OH)(2)D, namely CYP24A1. We summarize here the data which demonstrate that though some cellular responses to 1,25(OH)(2)D in AML cells are transcription-dependent, there are many others which depend on intracellular signal transduction, protein trafficking and stabilization. The final effect of 1,25(OH)(2)D action in leukemic cells requires all these acting together.

Studies on the mechanisms of superagonistic pro-differentiating activities of side-chain modified analogs of vitamin D2.

1,25-Dihydroxyvitamin D3 (1,25D) is implicated in many cellular functions including cell proliferation and differentiation, thus, exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D are its potent calcemic and phosphatemic activities. Therefore, synthetic analogs of 1,25D for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. Previously, we described pro-differentiating effects of vitamin D2 analogs with extended and branched side-chains. Analogs with side-chains extended by a pair of one (PRI-1906) or two carbon units (PRI-1907) displayed elevated cell-differentiating activity towards some acute leukemia cell lines (AML) in comparison to 1,25D. In this study, the potential mechanism of this superagonistic activity of the analogs was addressed. At first, possible differences in the expression of CYP24A1, a major catabolizing enzyme for vitamin D compounds and resulting differences in the degradation of analogs were investigated. In addition, interactions of the analogs with vitamin D receptor (VDR) and resulting activation of CCAAT-enhancer-binding protein ß (C/EBPß) were studied. The results obtained show that superagonistic pro-differentiating activities of analogs PRI-1906 and PRI-1907 do not seem to be caused by their altered catabolism, but most probably by altered interactions with VDR and resulting downstream proteins.

Opposite regulation of vitamin D receptor by ATRA in AML cells susceptible and resistant to vitamin D-induced differentiation.

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D(3) (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.