Expression of Vitamin D Receptor (VDR) Positively Correlates with Survival of Urothelial Bladder Cancer Patients

Vitamin D3 shows tumoristatic and anticancer effects by acting through the vitamin D receptor (VDR), while hydroxylation of 25-hydroxyvitamin D3 at position 1α by CYP27B1 is an essential step in its activation. The expression of both the VDR and CYP27B1 has been found in many normal and cancer tissues, but there is a lack of information about its expression in human bladder cancers. The aim of the present research was to examine whether the expression of the VDR and CYP27B1 in bladder cancer was related to the prognostic markers and disease outcome. We analyzed VDR and CYP27B1 in samples of tumor and normal tissues obtained from 71 urinary bladder cancer patients. The highest VDR immunostaining was found in normal epithelium and was significantly lower in bladder cancer cells (p < 0.001 with Mann–Whitney U test). VDR expression was lowest in more advanced (pT2b–pT4) (p = 0.005 with Mann–Whitney U test) and metastasizing cancers (p < 0.05 and p = 0.004 with Mann–Whitney U test for nuclear and cytoplasmic VDR immunostaining, respectively). The lack of cytoplasmic and nuclear VDR was also related to shorter overall survival (for cytoplasmic VDR immunolocalization 13.3 vs. 55.3 months of survival, HR = 1.92, p = 0.04 and for nuclear VDR immunostaining 13.5 vs. 55.3 months of survival, HR = 2.47, p = 0.002 with Mantel-Cox test). In cases with the lack of high cytoplasmic VDR staining the non-classic differentiations (NDs) was observed in higher percentage of tumor area. CYP27B1 expression was lower in cancer cells than in normal epithelial cells (p = 0.03 with Mann–Whitney U test), but its expression did not correlate with tumor stage (pT), metastasizing, grade, mitotic activity or overall survival. In conclusion, expression of the VDR and CYP27B1 are deregulated in urothelial bladder cancers. Although our results showing a relationship between the decreased VDR expression and prognostic markers and survival time indicate potential usefulness of VDR as a new indicator of a poorer prognosis, further studies are needed in different patient cohorts by independent groups to validate this hypothesis. We also suggest that vitamin D-based therapies may represent an adjuvant strategy in treatment for bladder cancers expressing VDR.     

Association of Vitamin D Metabolism Gene Polymorphisms with Autoimmunity: Evidence in Population Genetic Studies

A high prevalence of vitamin D (calcidiol) serum deficiency has been described in several autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (AR), and systemic lupus erythematosus (SLE). Vitamin D is a potent immunonutrient that through its main metabolite calcitriol, regulates the immunomodulation of macrophages, dendritic cells, T and B lymphocytes, which express the vitamin D receptor (VDR), and they produce and respond to calcitriol. Genetic association studies have shown that up to 65% of vitamin D serum variance may be explained due to genetic background. The 90% of genetic variability takes place in the form of single nucleotide polymorphisms (SNPs), and SNPs in genes related to vitamin D metabolism have been linked to influence the calcidiol serum levels, such as in the vitamin D binding protein (VDBP; rs2282679 GC), 25-hydroxylase (rs10751657 CYP2R1), 1α-hydroxylase (rs10877012, CYP27B1) and the vitamin D receptor (FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232), and TaqI (rs731236) VDR). Therefore, the aim of this comprehensive literature review was to discuss the current findings of functional SNPs in GC, CYP2R1, CYP27B1, and VDR associated to genetic risk, and the most common clinical features of MS, RA, and SLE.     

Development of In Vitro and In Vivo Evaluation Systems for Vitamin D Derivatives and Their Application to Drug Discovery

We have developed an in vitro system to easily examine the affinity for vitamin D receptor (VDR) and CYP24A1-mediated metabolism as two methods of assessing vitamin D derivatives. Vitamin D derivatives with high VDR affinity and resistance to CYP24A1-mediated metabolism could be good therapeutic agents. This system can effectively select vitamin D derivatives with these useful properties. We have also developed an in vivo system including a Cyp27b1-gene-deficient rat (a type I rickets model), a Vdr-gene-deficient rat (a type II rickets model), and a rat with a mutant Vdr (R270L) (another type II rickets model) using a genome editing method. For Cyp27b1-gene-deficient and Vdr mutant (R270L) rats, amelioration of rickets symptoms can be used as an index of the efficacy of vitamin D derivatives. Vdr-gene-deficient rats can be used to assess the activities of vitamin D derivatives specialized for actions not mediated by VDR. One of our original vitamin D derivatives, which displays high affinity VDR binding and resistance to CYP24A1-dependent metabolism, has shown good therapeutic effects in Vdr (R270L) rats, although further analysis is needed.     

Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D₃ 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 (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)₂D₃ 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.     

High-Throughput Gene and Protein Analysis Revealed the Response of Disc Cells to Vitamin D,Depending on the VDR FokI Variants

Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor (VDR), observed as associated with spine pathologies. The present study was aimed at investigating—through high-throughput gene and protein analysis—the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)₂D₃ was administered to the cells with or without interleukin 1 beta (IL-1β). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.     

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.     

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)₂D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)₂D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)₂D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1^−/− deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.     

Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D2 as Potential Anti-Leukemic Agents

Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D₂ (1,25D₂) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D₂. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D₂ and 1,25D₃, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D₂. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist.     

Vitamin D3 Treatment Alters Thyroid Functional Morphology in Orchidectomized Rat Model of Osteoporosis

Vitamin D plays an essential role in prevention and treatment of osteoporosis. Thyroid hormones, in addition to vitamin D, significantly contribute to regulation of bone remodeling cycle and health. There is currently no data about a possible connection between vitamin D treatment and the thyroid in the context of osteoporosis. Middle-aged Wistar rats were divided into: sham operated (SO), orchidectomized (Orx), and cholecalciferol-treated orchidectomized (Orx + Vit. D₃; 5 µg/kg b.m./day during three weeks) groups (n = 6/group). Concentration of 25(OH)D in serum of the Orx + Vit. D₃ group increased 4 and 3.2 times (p < 0.0001) respectively, compared to Orx and SO group. T_4, TSH, and calcitonin in serum remained unaltered. Vit. D₃ treatment induced changes in thyroid functional morphology that indicate increased utilization of stored colloid and release of thyroid hormones in comparison with hormone synthesis, to maintain hormonal balance. Increased expression of nuclear VDR (p < 0.05) points to direct, TSH independent action of Vit. D on thyrocytes. Strong CYP24A1 immunostaining in C cells suggests its prominent expression in response to Vit. D in this cell subpopulation in orchidectomized rat model of osteoporosis. The indirect effect of Vit. D on bone, through fine regulation of thyroid function, is small.     

Vitamin D–VDR Novel Anti-Inflammatory Molecules—New Insights into Their Effects on Liver Diseases

There is consistent evidence that vitamin D deficiency is strongly associated with liver dysfunction, disease severity, and poor prognosis in patients with liver disease. Vitamin D and its receptor (VDR) contribute to the regulation of innate and adaptive immune responses. The presence of genetic variants of vitamin D- and VDR-associated genes has been associated with liver disease progression. In our recent work, we summarized the progress in understanding the molecular mechanisms involved in vitamin D–VDR signaling and discussed the functional significance of VDR signaling in specific cell populations in liver disease. The current review focuses on the complex interaction between immune and liver cells in the maintenance of liver homeostasis and the development of liver injury, the interplay of vitamin D and VDR in the development and outcome of liver disease, the role of vitamin D- and VDR-associated genetic variants in modulating the occurrence and severity of liver disease, and the therapeutic value of vitamin D supplementation in various liver diseases. The association of the vitamin D–VDR complex with liver dysfunction shows great potential for clinical application and supports its use as a prognostic index and diagnostic tool.     

Antitumor Effects of Vitamin D Analogs on Hamster and Mouse Melanoma Cell Lines in Relation to Melanin Pigmentation

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)₂D₃) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)₂D₃ and 25(OH)D₃), and novel relatively non-calcemic ones (20(OH)D₃, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner.     

Expression Pattern of Small Nucleolar RNA Host Genes and Long Non-Coding RNA in X-rays-Treated Lymphoblastoid Cells

A wide variety of biological effects are induced in cells that are exposed to ionizing radiation. The expression changes of coding mRNA and non-coding micro-RNA have been implicated in irradiated cells. The involvement of other classes of non-coding RNAs (ncRNA), such as small nucleolar RNAs (snoRNAs), long ncRNAs (lncRNAs), and PIWI-interacting RNAs (piRNAs) in cells recovering from radiation-induced damage has not been examined. Thus, we investigated whether these ncRNA were undergoing changes in cells exposed to ionizing radiation. The modulation of ncRNAs expression was determined in human TK6 (p53 positive) and WTK1 (p53 negative) cells. The snoRNA host genes SNHG1, SNHG6, and SNHG11 were induced in TK6 cells. In WTK1 cells, SNHG1 was induced but SNHG6, and SNHG11 were repressed. SNHG7 was repressed in TK6 cells and was upregulated in WTK1 cells. The lncRNA MALAT1 and SOX2OT were induced in both TK6 and WTK1 cells and SRA1 was induced in TK6 cells only. Interestingly, the MIAT and PIWIL1 were not expressed in TK6 cells before or after the ionizing radiation treatment. The MIAT and PIWIL1 were upregulated in WTK1 cells. This data provides evidence that altered ncRNA expression is a part of the complex stress response operating in radiation-treated cells and this response depends on functional p53.     

Assessment of the Effects of Dietary Vitamin D Levels on Olanzapine-Induced Metabolic Side Effects: Focus on the Endocannabinoidome-Gut Microbiome Axis

Vitamin D deficiency is associated with poor mental health and dysmetabolism. Several metabolic abnormalities are associated with psychotic diseases, which can be compounded by atypical antipsychotics that induce weight gain and insulin resistance. These side-effects may be affected by vitamin D levels. The gut microbiota and endocannabinoidome (eCBome) are significant regulators of both metabolism and mental health, but their role in the development of atypical antipsychotic drug metabolic side-effects and their interaction with vitamin D status is unknown. We studied the effects of different combinations of vitamin D levels and atypical antipsychotic drug (olanzapine) exposure on whole-body metabolism and the eCBome-gut microbiota axis in female C57BL/6J mice under a high fat/high sucrose (HFHS) diet in an attempt to identify a link between the latter and the different metabolic outputs induced by the treatments. Olanzapine exerted a protective effect against diet-induced obesity and insulin resistance, largely independent of dietary vitamin D status. These changes were concomitant with olanzapine-mediated decreases in Trpv1 expression and increases in the levels of its agonists, including various N-acylethanolamines and 2-monoacylglycerols, which are consistent with the observed improvement in adiposity and metabolic status. Furthermore, while global gut bacteria community architecture was not altered by olanzapine, we identified changes in the relative abundances of various commensal bacterial families. Taken together, changes of eCBome and gut microbiota families under our experimental conditions might contribute to olanzapine and vitamin D-mediated inhibition of weight gain in mice on a HFHS diet.     

LINC01133 Inhibits Invasion and Promotes Proliferation in an Endometriosis Epithelial Cell Line

Endometriosis is a common gynecological disorder characterized by ectopic growth of endometrium outside the uterus and is associated with chronic pain and infertility. We investigated the role of the long intergenic noncoding RNA 01133 (LINC01133) in endometriosis, an lncRNA that has been implicated in several types of cancer. We found that LINC01133 is upregulated in ectopic endometriotic lesions. As expression appeared higher in the epithelial endometrial layer, we performed a siRNA knockdown of LINC01133 in an endometriosis epithelial cell line. Phenotypic assays indicated that LINC01133 may promote proliferation and suppress cellular migration, and affect the cytoskeleton and morphology of the cells. Gene ontology analysis of differentially expressed genes indicated that cell proliferation and migration pathways were affected in line with the observed phenotype. We validated upregulation of p21 and downregulation of Cyclin A at the protein level, which together with the quantification of the DNA content using fluorescence-activated cell sorting (FACS) analysis indicated that the observed effects on cellular proliferation may be due to changes in cell cycle. Further, we found testis-specific protein kinase 1 (TESK1) kinase upregulation corresponding with phosphorylation and inactivation of actin severing protein Cofilin, which could explain changes in the cytoskeleton and cellular migration. These results indicate that endometriosis is associated with LINC01133 upregulation, which may affect pathogenesis via the cellular proliferation and migration pathways.