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 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.     

Antiproliferative Activity and in Vivo Toxicity of Double-Point Modified Analogs of 1,25-Dihydroxyergocalciferol

Analogs of 1,25-dihydroxyergocalciferol, modified in the side-chain and in the A-ring, were tested for their antiproliferative activity against a series of human cancer cell lines in vitro and in vivo toxicity. The proliferation inhibition caused by the analogs was higher than that of the parent compounds, while the toxicity, measured as the serum calcium level, was lower. All analogs were able to induce, in HL-60 and MV4-11 leukemic cells, G₀/G₁ cell cycle arrest and differentiation expressed as morphological signs typical for monocytes. The analogs also induced the expression of CD11b and/or CD14 cell-differentiation markers. The most potent analogs, PRI-5105, PRI-5106, PRI-5201 and PRI-5202, were also able to induce vitamin D receptor (VDR) protein expression, mainly in the cytoplasmic fraction of HL-60 or MV4-11 cells. The most active analogs were the 19-nor ones with an extended and rigidified side-chain (PRI-5201 and PRI-5202), as in the former analogs PRI-1906 and PRI-1907. Epimerization at C-24 (PRI-5101) or introduction of an additional hydroxyl at C-23 (PRI-5104) reduced the toxicity of the analog with retained antiproliferative activity.     

Chemical Synthesis of Side-Chain-Hydroxylated Vitamin D3 Derivatives and Their Metabolism by CYP27B1

1α,25-Dihydroxyvitamin D₃ (abbreviated here as 1,25D₃) is a hormonally active form of vitamin D₃ (D₃), and is produced from D₃ by CYP27 A1-mediated hydroxylation at C25, followed by CYP27B1-mediated hydroxylation at C1. Further hydroxylation of 25D₃ and 1,25D₃ occurs at C23, C24 and C26 to generate corresponding metabolites, except for 1,25R,26D₃. Since the capability of CYP27B1 to hydroxylate C1 of side-chain-hydroxylated metabolites other than 23S,25D₃ and 24R,25D₃ has not been examined, we have here explored the role of CYP27B1 in the C1 hydroxylation of a series of side-chain-hydroxylated D₃ derivatives. We found that CYP27B1 hydroxylates the R diastereomers of 24,25D₃ and 25,26D₃ more effectively than the S diastereomers, but shows almost no activity towards either diastereomer of 23,25D₃. This is the first report to show that CYP27B1 metabolizes 25,26D₃ to the corresponding 1α-hydroxylated derivative, 1,25,26D₃. It will be interesting to examine the physiological relevance of this finding.     

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.     

Micro-RNAs in Response to Active Forms of Vitamin D3 in Human Leukemia and Lymphoma Cells

Non-coding micro-RNA (miRNAs) regulate the protein expression responsible for cell growth and proliferation. miRNAs also play a role in a cancer cells’ response to drug treatment. Knowing that leukemia and lymphoma cells show different responses to active forms of vitamin D₃, we decided to investigate the role of selected miRNA molecules and regulated proteins, analyzing if there is a correlation between the selected miRNAs and regulated proteins in response to two active forms of vitamin D₃, calcitriol and tacalcitol. A total of nine human cell lines were analyzed: five leukemias: MV-4-1, Thp-1, HL-60, K562, and KG-1; and four lymphomas: Raji, Daudi, Jurkat, and U2932. We selected five miRNA molecules—miR-27b, miR-32, miR-125b, miR-181a, and miR-181b—and the proteins regulated by these molecules, namely, CYP24A1, Bak1, Bim, p21, p27, p53, and NF-kB. The results showed that the level of selected miRNAs correlates with the level of proteins, especially p27, Bak1, NFκB, and CYP24A1, and miR-27b and miR-125b could be responsible for the anticancer activity of active forms of vitamin D₃ in human leukemia and lymphoma.     

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.     

Differential Response of Lung Cancer Cells,with Various Driver Mutations,to Plant Polyphenol Resveratrol and Vitamin D Active Metabolite PRI-2191

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.     

Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida

The active vitamin D metabolites 25-OH−D and 1α,25-(OH)₂−D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D₂ (VD₂) and vitamin D₃ (VD₃) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH−D₂ and 25-OH−D₃ was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH−D₃ and tenfold higher concentrations of 25-OH−D₂ after addition of 5 % (w/v) 2-hydroxypropyl β-cyclodextrin (2-HPβCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)₂−D₂ and 1,25-(OH)₂−D₃ and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD₂ and VD₃ metabolites.     

Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism

Two 24-fluoro-25-hydroxyvitamin D₃ analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D₃ side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D₃ revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D₃ (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.     

CYP24A1 Expression Inversely Correlates with Melanoma Progression: Clinic-Pathological Studies

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3) homeostasis. Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities. Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas. Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases. In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis. The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas. With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases. Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity. Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes. Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival. In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.     

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.     

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.     

The Effects of Vitamin D on the Expression of IL-33 and Its Receptor ST2 in Skin Cells; Potential Implication for Psoriasis

Interleukin 33 (IL-33) belongs to the IL-1 family and is produced constitutively by epithelial and endothelial cells of various organs, such as the skin. It takes part in the maintenance of tissue homeostasis, repair, and immune response, including activation of Th2 lymphocytes. Its involvement in pathogenesis of several inflammatory diseases including psoriasis was also suggested, but this is not fully understood. The aim of the study was to investigate expression of IL-33 and its receptor, ST2, in psoriasis, and the effects of the active form of vitamin D (1,25(OH)₂D₃) on their expression in skin cells. Here we examined mRNA and protein profiles of IL-33 and ST2 in 18 psoriatic patients and healthy volunteers by qPCR and immunostaining techniques. Potential effects of 1,25(OH)₂D₃ and its receptor (VDR) on the expression of IL-33 and ST2 were tested in cultured keratinocytes, melanocytes, fibroblasts, and basal cell carcinoma cells. It was shown that 1,25(OH)₂D₃ effectively stimulated expression of IL-33 and its receptor ST2’s mRNAs in a time-dependent manner, in keratinocytes and to the lesser extends in melanocytes, but not in fibroblasts. Furthermore, the effect of vitamin D on expression of IL-33 and ST2 was VDR-dependent. Finally, we demonstrated that the expression of mRNA for IL-33 was mainly elevated in the psoriatic skin but not in its margin. Interestingly, ST2 mRNA was downregulated in psoriatic lesion compared to both marginal tissue as well as healthy skin. Our data indicated that vitamin D can modulate IL-33 signaling, opening up new perspectives for our understanding of the mechanism of vitamin D action in psoriasis therapy.     

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.     

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.