Combination Of D3 Research Articles

Combinations of vitamin A and D induced are synergistic in breast cancer cells and alter gene expression in the endoplasmic reticulum stress, unfolded protein and estrogen signaling canonical pathways

Introduction: Numerous studies over the past 30 years have shown that bioactive compounds present in functional foods, including vitamins, minerals and phytochemicals reduce cancer risk. For example, vitamins A and D derivatives found in fruits, vegetables, and dairy products, reduce the growth of breast, bladder, head, neck, lung, prostate, and skin cancers. However, the effects of these combined vitamins have not been previously reported for breast cancer.Aims: To investigate the activities of vitamin A (all-trans-retinoic acid; ATRA), as well as vitamins D2 and D3 in combination in the breast epithelial cancer cell lines T47D:A18, MCF-7, and SK-BR-3 and perform whole genome analysis in MCF-7 cells using RNA-seq.Methods: Breast cancer cells were cultured in appropriate media and treated with ATRA, D2 or D3 alone in concentrations from 1-10 μg/ml, or in combination at 1, 5, and 10 μg/ml. The CellTiter-Glo® 2.0 assay, Caspase-Glo®3/7, Caspase®8, and ApoTox-Glo™ Triplex assays measured cell viability and apoptosis. The effect of treatment on autophagy in MCF-7 cells was measured with a CYTO-ID® Autophagy Detection Kit 2.0. The whole transcriptome analysis was assessed using mRNA-seq and qPCR.Results: Separately, ATRA, D2, and D3 all reduced the viability of all breast cancer cell lines tested, with median inhibitory concentrations (IC50) between 2.1 to 31.7 μg/ml. However, when breast cancer cells were treated with combinations of ATRA+D2+D3, the IC50 was reduced indicating synergism. In MCF-7 cells, 5-flurouracil (5-FLU) had an IC50 of 1.37 µg/ml, while the vitamin A and D combination had an IC50 of 1.5 µg/ml, indicating the combination was ~90% as effective as 5-FLU. Treatment of MCF-7 cells with ATRA+D2+D3 enhanced caspase 3/7 activity, as well as the expression of Bax, BAD, PTEN and p53 (apoptosis canonical pathway), as well as induced autophagy. Whole genome analysis of treated MCF-7 cells showed a significant upregulation in gene expression in the autophagy, endoplasmic reticulum stress and the unfolded protein response apoptosis canonical pathways. Furthermore, MCF-7 cells treated with ATRA+D2+D3 demonstrated significant downregulation of gene expression in estrogen-mediated S phase entry and estrogen signaling canonical pathways suggesting antiestrogenic effects. Conclusions: Vitamins A and D combinations had synergistic effects in breast cancer cells and induced both apoptosis and autophagy. Transcriptional profiling showed significant alterations in gene expression patterns and upregulation of multiple cancer signaling pathways supporting the hypothesis that combining vitamins A and D is a more effective treatment than either vitamin alone. Keywords: all-trans-retinoic acid (ATRA), Vitamin D2, Vitamin D3, breast cancer cells, apoptosis, estrogen receptors

The effects of 1a(OH)D3 individually or in combination with phytase, and different levels of cholecalciferol on performance, tibia criteria, and plasma minerals of Japanese quails

The aim of studywas to compare efficacy of 1-α(OH)D3 alone or in combination with phytase and 1-α(OH)D3 in combination of phytase and different concentration of cholecalciferol on performance, tibia parameters, andplasma minerals of quails fed Ca-P deficient diet. A total of 280 mixed sex 5-d-old quails were allocated to 7 treatments with 5 replicates. The vitamin supplement which incorporated to basal diet did not contain cholecalciferol. The dietary treatments were as follows: Ca-P deficient diet (basal diet); basal diet + 500 FTU phytase/kg of diet; basal diet + phytase + 5 μg of 1-α(OH)D3 kg-1 of diet;basal diet + phytase + 5 μg of 1-α(OH)D3 and 250, 500, 750 and 1,000 IU of cholecalciferol kg-1of diet. The highest final body weight and the best feed conversion ratioobtained in the group supplemented with 1,000 IU cholecalciferol kg-1 of diet (p < 0.05). Supplementation of 1-α(OH)D3 alone or in combination with phytase and phytase and different concentration of cholecalciferol could improve tibia parameters (p < 0.05). In conclusion, supplementation of 1-α(OH)D3 alone to Ca-P deficient diet could maximize tibia mineralization, whereas it couldn't maximize performance, performance criteria were maximized by supplementation of 1,000 IU cholecalciferol kg-1 of diet.

Vitamin D3 and zinc synergistically induce regulatory T cells and suppress interferon-γ production in mixed lymphocyte culture

Although vitamin D3 (cholecalciferol) and zinc are known to individually shift the immune response towards tolerance, little is known about the effect of their combined administration. This work contributes to understanding the combined action of zinc and vitamin D3 in different in vitro models for immune reactions. Zinc and vitamin D3 in combination boosted the differentiation into Foxp3+CD4+ T cells (Treg). Vitamin D3 alone reduced the percentage of CD4+T-bet+ T cells (TH1). In mixed lymphocyte culture (MLC), therapeutic concentrations of vitamin D3 and zinc in combination suppressed interferon-γ (IFN-γ) secretion more strongly than the single agent treatment. This effect was also detected for a combination of subtherapeutic concentrations of both vitamin D3 and zinc. Vitamin D3, even at nanomolar concentrations, increased intracellular zinc levels. PBMC (peripheral blood mononuclear cells) of individuals at risk of zinc deficiency responded to vitamin D3 treatment with a higher mRNA expression of Zip13. In PBMC, both agents reduced activation-induced IL-17 secretion. In summary, this study shows, for the first time, a vitamin D3-induced upregulation of CD4+Foxp3+ T cells in MLC. The data propose a model where zinc augments the effect of vitamin D3 in certain therapeutic and subtherapeutic concentrations. Lower concentrations of both vitamin D3 and zinc could be used for effective treatment, thus reducing possible side effects from vitamin D3 and zinc. Vitamin D3 and zinc in combination may be a promising and cheap option to treat dysregulated immune response in various conditions.

Vitamin D3/VDR inhibits inflammation through NF-κB pathway accompanied by resisting apoptosis and inducing autophagy in abalone Haliotis discus hannai.

Vitamin D3 is believed to be a contributing factor to innate immunity. Vitamin D receptor (VDR) has a positive effect on inhibiting nuclear factor κB (NF-κB)-mediated inflammation. The underlying molecular mechanisms remain unclear, particularly in mollusks. Consequently, this study will investigate the process of vitamin D3/VDR regulating NF-κB pathway and further explore their functions on inflammation, autophagy, and apoptosis in abalone Haliotis discus hannai. Results showed that knockdown of VDR by using siRNA and dsRNA of VDR in vitro and in vivo led to more intense response of NF-κB signaling to lipopolysaccharide and higher level of apoptosis and autophagy. In addition, 1,25(OH)2D3 stimulation after VDR silencing could partially alleviate apoptosis and induce autophagy. Overexpression of VDR restricted the K48-polyubiquitin chain-dependent inhibitor of κB (IκB) ubiquitination and apoptosis-associated speck-like protein containing CARD (ASC) oligomerization. Besides, VDR silencing resulted in increase of ASC speck formation. In further mechanistic studies, we showed that VDR can directly bind to IκB and IKK1 in vitro and in vivo. In the feeding trial, H&E staining, TUNEL, and electron microscope results showed that vitamin D3 deficiency (0IU/kg) could recruit more basophilic cells and increase more TUNEL-positive apoptotic cells and lipid droplets (LDs) than vitamin D3 supplement (1000IU/kg and 5000IU/kg). In summary, abalone VDR plays a negative regulator role in NF-κB-mediated inflammation via interacting with IκB and inhibiting ubiquitin-dependent degradation of IκB. Vitamin D3 in combination with VDR is essential to establish a delicate balance between autophagy and apoptosis in response to inflammation.

Prophylactic and Therapeutic Role of Vitamin D3 in Combination with Fluconazole Against Vaginal Candidiasis in a Murine Model.

In the present study, we assessed the adjunct effect of vitamin D3 in combination with Fluconazole (FLZ) against Vulvovaginal Candidiasis (VVC) in mice. Prophylactic effect was assessed by pretreating mice with vitamin D3 before exposure of mice with 2 X 106 CFUs of Candida albicans followed by treatment with FLZ. To determine the combined therapeutic efficacy, C. albicans infected mice were treated with a combination of vitamin D3 (10 μg/kg) and FLZ (10 and 20 mg/kg). The efficacy of the treatment was assessed by analyzing the fungal load and blood cell count. Moreover, the levels of inflammatory cytokines, including IL- 1β, IL-17 and TNF-α, were analyzed in the vaginal tissues. The histological analysis of the vaginal tissue from the untreated and treated mice was performed to assess the efficacy of the treatment. Prophylactic treatment with vitamin D3 (10 and 20 μg/kg) significantly increased the therapeutic effect of FLZ against VVC. In a therapeutic experiment, mice in the infected control group showed the highest vaginal fungal load of 83627 ± 10058 CFUs. Treatment with FLZ at a dose of 10 mg/kg reduced fungal load to 55523 ± 14823 CFUs, whereas the mice treated with a combination of vitamin D3 and FLZ (10 mg/kg) had the fungal burden of 12156 ± 3219. Similarly, treatment with FLZ (20 mg/kg) reduced fungal load to 36394 ± 5648 CFUs, whereas the addition of vitamin D3 to FLZ (20 mg/kg) further reduced the fungal burden to 2179 ± 1188. The leukocyte numbers in the infected mice increased to 9802 ± 505 as compared to 5152 ± 778 in normal control mice. Whereas, a combination of vitamin D3 with FLZ (10 and 20 mg/kg) reduced leukocyte numbers to 7284 ± 607 and 5739 ± 1126. The histological analysis data revealed epithelial necrosis, shedding and ulceration in the vaginal wall. Treatment with FLZ or a combination of FLZ and vitamin D3 brought regenerative changes in the vaginal epithelium and lamina proparia. The results of the present work recommend that the addition of vitamin D3 may be considered to increase the efficacy of FLZ against VVC.

Effect of Vitamin D3 in combination with Omega-3 Polyunsaturated Fatty Acids on NETosis in Type 2 Diabetes Mellitus Patients.

An understanding of the consequences of oxidative/halogenative stress triggered by neutrophil activation is impossible without considering NETosis. NETosis, formation of neutrophil extracellular traps (NETs), is known to promote microthrombus formation and impair wound healing in type 2 diabetes mellitus (T2DM) patients. Therefore, there is a need to search for drugs and treatment approaches that could prevent excessive NET formation. We aimed to evaluate the effect of vitamin D3 in combination with omega-3 polyunsaturated fatty acids (vitamin D3/omega-3 PUFAs) on NETosis in T2DM patients with purulent necrotizing lesions of the lower extremities. Patients and healthy subjects had vitamin D3 deficiency. Patients received, beyond standard treatment, 6000 IU of vitamin D3 and 480 mg of omega-3 PUFAs, and healthy subjects 1000 IU of vitamin D3 and 240 mg of omega-3 PUFAs daily for seven days. Neutrophil activation in ex vivo blood by phorbol-12-myristate-13-acetate (PMA) was used as a NETosis model. The percentage of blood NETs relative to leukocytes (NETbackground) before vitamin D3/omega-3 PUFA supplementation was 3.2%-4.9% in healthy subjects and 1.7%-10.8% in patients. These values rose, respectively, to 7.7%-9.1% and 4.0%-17.9% upon PMA-induced NETosis. In addition, the leukocyte count decreased by 700-1300 per 1 μL in healthy subjects and 700-4000 per 1 μL in patients. For both patients and healthy subjects, taking vitamin D3/omega-3 PUFAs had no effect on NETbackground but completely inhibited PMA-induced NET formation, though neutrophils exhibited morphological features of activation. Also, leukocyte loss was reduced (to 500 per 1 μL). For patients on standard treatment alone, changes occurred neither in background NETs and leukocytes nor in their amount after PMA stimulation. The decreased ability of neutrophils to generate NETs, which can be achieved by vitamin D3/omega-3 PUFA supplementation, could have a positive effect on wound healing in T2DM patients and reduce the incidence and severity of complications.

Investigation of different levels of cholecalciferol and its metabolite in calcium and phosphorus deficient diets on growth performance, tibia bone ash and development of tibial dyschondroplasia in broilers

This experiment was conducted to examine the effects of 1-α(OH)D3 alone or in combination with different levels of cholecalciferol on performance, and tibia parameters of one-d–old male broilers fed a tibial dyschondroplasia (TD)-inducing diet. A total of three hundred male broilers were randomly allocated to 5 treatment groups with 4 replicates. The dietary treatments consisted of TD inducing diet, TD inducing diet supplemented with 5 μg per kg of 1-α(OH)D3; TD inducing diet supplemented with 5 μg per kg of 1-α(OH)D3 and 1,500; 3,000 or 5,000 IU cholecalciferol kg-1 of diet. At 42 d of age, broiler chickens fed diets containing 1-α(OH)D3 and 1,500 IU cholecalciferol kg-1 of diet had higher body weight (p < 0.05). In the complete experimental period the best FCR and the highest daily weight gain were obtained in broilers supplemented with 1-α(OH)D3 and 1,500 IU cholecalciferol kg-1 of diet. Broilers supplemented with 1-α(OH)D3 and 1,500 IU cholecalciferol kg-1 of diet had significantly lower incidence and severity of TD in comparison with other groups. In conclusion, the results indicated that the supplementation of 1-α(OH)D3 in combination of 1,500 IU cholecalciferol kg-1 of diet could maximize tibia bone ash, performance and prevent TD in broilers fed TD inducing diet.

Vitamin D increases killing of intracellular Leishmania amazonensis in vitro independently of macrophage oxidative mechanisms.

Vitamin D has been reported to activate macrophage microbicidal mechanisms by inducing the production of antimicrobial peptides and nitric oxide (NO), but conversely has been shown to contribute to a greater susceptibility to Leishmania amazonensis infection in mice. Thus, this study aimed to evaluate the role of vitamin D during intracellular infection with L. amazonensis by examining its effect on macrophage oxidative mechanisms and parasite survival in vitro. Vitamins D2 and D3 significantly inhibited promastigote and amastigote growth in vitro. Vitamin D3 was not able to induce NO and reactive oxygen species (ROS) production in uninfected macrophages or macrophages infected with L. amazonensis. In addition, vitamin D3 in combination with interferon (IFN)-γ did not enhance amastigote killing and in fact, significantly reduced NO and ROS production when compared with the effect of IFN-γ alone. In this study, we demonstrated that vitamin D directly reduces parasite growth in infected macrophages (approximately 50-60% at 50 μm) but this effect is independent of the activation of macrophage oxidative mechanisms. These findings will contribute to a better understanding of the role of vitamin D in cutaneous leishmaniasis.

Vitamin D, magnesium, calcium, and their interaction in relation to colorectal cancer recurrence and all-cause mortality

ABSTRACTBackgroundHigher concentrations of 25-hydroxyvitamin D3 [25(OH)D3] at diagnosis are associated with a lower mortality risk in colorectal cancer (CRC) patients. However, magnesium and calcium are important in vitamin D metabolism.ObjectivesWe aimed to investigate 25(OH)D3, magnesium, or calcium and their interaction among patients with CRC in relation to recurrence and all-cause mortality.MethodsThe study population included 1169 newly diagnosed stage I–III CRC patients from 2 prospective cohorts. Associations between 25(OH)D3 concentrations, magnesium or calcium intake through diet and/or supplements at diagnosis, and recurrence and all-cause mortality were evaluated using multivariable Cox proportional hazard models. The interaction between 25(OH)D3 and magnesium or calcium was assessed by investigating 1) joint compared with separate effects, using a single reference category; and 2) the effect estimates of 1 factor across strata of another.ResultsSerum 25(OH)D3, calcium, and magnesium, alone and their interactions, were not associated with recurrence. Serum 25(OH)D3 concentrations seemed to be associated with all-cause mortality. An inverse association between magnesium intake (HRQ3 vs. Q1: 0.55; 95% CI: 0.32, 0.95 and HRQ4 vs. Q1: 0.65; 95% CI: 0.35, 1.21), but not calcium intake, and all-cause mortality was observed. When investigating the interaction between 25(OH)D3 and magnesium, we observed the lowest risk of all-cause mortality in patients with sufficient vitamin D concentrations (≥50 nmol/L) and a high magnesium intake (median split) (HR: 0.53; 95% CI: 0.31, 0.89) compared with patients who were vitamin D deficient (<50 nmol/L) and had a low magnesium intake. No interactions between calcium and vitamin D in relation to all-cause mortality were observed.ConclusionsOur findings suggest that the presence of an adequate status of 25(OH)D3 in combination with an adequate magnesium intake is essential in lowering the risk of mortality in CRC patients, yet the underlying mechanism should be studied. In addition, diet and lifestyle intervention studies are needed to confirm our findings. The COLON study was registered at clinicaltrials.gov as NCT03191110. The EnCoRe study was registered at trialregister.nl as NTR7099.

Does Vitamin D3 Prevent the Inhibitory Effect of Vancomycin on Osteoblasts?

BackgroundThe utility of vancomycin powder to prevent surgical site infection, mainly in spinal surgery, has been widely examined, and the local administration of vancomycin powder to wounds has been reported to be effective in preventing surgical site infections after spine surgery. However, in vitro studies have shown that high local concentrations of vancomycin may inhibit osteogenesis, although it remains unclear how these high concentrations influence osteoblasts. No candidate drug has been reported to recover cytotoxicity with high concentrations of vancomycin, but we suggest that vitamin D3, which induces osteoblast proliferation, may be administrated concomitantly with vancomycin in these situations.Questions/purposes(1) Does a high concentration of vancomycin reduce viable osteoblast numbers in cell culture compared with controls? (2) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with continuous vancomycin? (3) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with pulsed vancomycin (24 hours of administration)? (4) Does vitamin D3 administration confer alkaline phosphatase, mineralization, and gene expression when administered with pulsed vancomycin?MethodsMC3T3-E1 cells were cultured at 37° C in an α-minimum essential medium supplemented with 10% fetal bovine serum in a humidified incubator containing 5% CO2. The experimental concentrations of vancomycin (2500 μg/mL, 5000 μg/mL, and 7500 μg/mL) were determined based on previous reports and preliminary experiments. We concomitantly administered vitamin D3 (0.01 nM) to prevent cytotoxicity in osteoblasts, using two different treatments: continuous vancomycin administration (measured at 6 hours, 12 hours, 24 hours, and 72 hours) and pulsed vancomycin for 24 hours (measured at 1 days, 3 days, and 7 days). We analyzed cell numbers and morphologic changes in cells treated with vancomycin or vancomycin plus 0.01 nM vitamin D3. Osteoblast differentiation was assessed with alkaline phosphatase staining, alkaline phosphatase activity, and Alizarin red S staining.ResultsThe number of cells was reduced at 6 hours, 24 hours, 48 hours, and 72 hours in response to continuous vancomycin administration at 7500 µg/mL (at 72 hours, control 14.6 × 104 cells/mL ± 0.260 × 104 cells/mL, vancomycin at 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, mean difference -13.7 × 104 cells/mL ± 0.388 × 104 cells/mL [95% CI -14.5 to -12.9]; p < 0.001). Vitamin D3 did not have a protective effect when vancomycin was administered continuously at 7500 µg/mL (at 72 hours, vancomycin alone 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, vancomycin + vitamin D3 1.67 × 104 cells/mL ± 0.310 × 104 cells/mL, mean difference 0.75 × 104 cells/mL ± 0.423 × 104 cells/mL [95% CI -0.127 to 1.63]; p = 0.09).With pulsed administration for only the first 24 hours, the number of cells was reduced at 1 day, 3 days, and 7 days at 7500 μg/mL (at 7 days, control 18.6 × 104 cells/mL ± 1.29 × 104 cells/mL, vancomycin at 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, mean difference -15.1 × 104 cells/mL ±1.33 × 104 cells/mL [95% CI -17.9 to -12.4]; p < 0.001 for all). However, vitamin D3 had a recovery effect when vancomycin was administered only for 24 hours (cell number with 7500 μg/mL, day 7: vancomycin alone 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, vancomycin +vitamin D3 10.6 × 104 cells/mL ± 0.900 × 104 cells/mL, mean difference 7.13 × 104 cells/mL ± 0.946 × 104 cells/mL [95% CI 5.16 to 9.09]; p < 0.001).With the addition of vitamin D3, we observed recovery of alkaline phosphatase staining and Alizarin red staining (evidence of calcification) but no difference in the gene expression of Type I collagen (vancomycin alone 0.319 ± 0.0730, vancomycin + vitamin D3 0.511 ± 0.139, mean difference 0.192 ± 0.157 [95% CI -0.483 to 0.867]; p = 0.345), alkaline phosphatase (vancomycin alone 0.532 ± 0.0210, vancomycin + vitamin D3 0.785 ± 0.0590, mean difference 0.253 ± 0.0620 [95% CI -0.0150 to 0.521]; p = 0.0550), and cathelicidin antimicrobial peptide (vancomycin alone 0.885 ± 0.0520, vancomycin + vitamin D3 1.24 ± 0.125, mean difference 0.355 ± 0.135 [95% CI -0.0200 to 0.730]; p = 0.0580).ConclusionWe found that 7500 µg/mL of vancomycin is cytotoxic to osteoblasts. Cytotoxicity could be prevented by administering vitamin D3 in combination with vancomycin.Clinical RelevanceThe high concentrations of vancomycin routinely used clinically raises concerns related to osteoblast cytotoxicity, which may contribute to pseudoarthrosis after spinal surgery. Thus, vitamin D3, which is frequently used to treat osteoporosis, may have efficacy as a concomitantly administered drug by inducing the proliferation of osteoblasts. These results indicate that a combination therapy of vancomycin and vitamin D3 may prevent adverse events such as osteoblast cytotoxicity.

The effects of dietary 1-alpha-hydroxycholecalciferol individually or in combination with different levels of cholecalciferol on growth performance and tibia criteria in broiler chickens

This study was conducted to evaluate the effects of 1 alpha-hydroxycholecalciferol (1-α(OH)D3) individually or in combination with different concentrations of cholecalciferol on growth performance and tibia criteria of 3-day-old broiler chickens fed diets deficient in Ca and phosphorus P. A total of 300 hatched broiler chickens were weighed and randomly allotted to 5 treatments with 4 pens and 15 broiler chickens/pen. The vitamin premix that included in the diets did not contain cholecalciferol. The dietary treatments were as follows: Ca–P deficient diet + phytase (basal diet); basal diet + 5 μg of 1-α(OH)D3/kg of diet; basal diet + 5 μg of 1-α(OH)D3 and 1500 IU of cholecalciferol/kg of diet; basal diet + 5 μg of 1-α(OH)D3 and 3000 IU of cholecalciferol/kg of diet; basal diet + 5 μg of 1-α(OH)D3 and 5000 IU of cholecalciferol/kg of diet. Final body weight (BW) of broiler chickens supplemented with 1-α(OH)D3 and 1500 IU of cholecalciferol/kg of diet and 1-α(OH)D3 and 3000 IU of cholecalciferol/kg of diet was greater (P < 0.05) than those fed basal diet, basal diet supplemented with 1-α(OH)D3 alone and basal diet supplemented with 1-α(OH)D3 and 5000 IU of cholecalciferol/kg of diet. In the whole experiment, feed intake of broiler chickens fed basal diet was greater (P < 0.05) than those fed basal diet supplemented with 1-α(OH)D3 alone, basal diet supplemented with 1-α(OH)D3 and 1500, 3000, and 5000 IU of cholecalciferol/kg of diet. Feed conversion ratio of broiler chickens fed basal diet supplemented with 1-α(OH)D3 and 1500 or 3000 IU of cholecalciferol/kg of diet were significantly better than those fed basal diet, basal diet supplemented with 1-α(OH)D3 alone or basal diet supplemented with 1-α(OH)D3 and 5000 IU of cholecalciferol/kg of diet. Broiler chickens fed basal diet had lower tibia length and diameter than those in other groups (P < 0.05). Broiler chickens fed basal diet supplemented with 1-α(OH)D3 and 3000 and 5000 IU of cholecalciferol/kg of diet had greater (P < 0.05) tibia weight than those fed basal diet or basal diet supplemented with 1-α(OH)D3 alone, but did not differ from broiler chickens fed basal diet supplemented with 1-α(OH)D3 and 1500 IU of cholecalciferol/kg of diet that were intermediate. Broiler chickens fed basal diet supplemented with 1-α(OH)D3 alone had greater (P < 0.05) tibia ash than those in other groups. In conclusion, addition of 1-α(OH)D3 alone to the Ca-and P-deficient diet could maximize bone mineralization, but it couldn't maximize growth performance and growth performance criteria including BW and feed efficiency were maximized in broiler chickens fed dietary containing 1-α(OH)D3 in combination with 1500 IU cholecalciferol/kg of diet.

1α,25‐dihydroxyvitamin D3 in combination with transforming growth factor‐β increases the frequency of Foxp3+ regulatory T cells through preferential expansion and usage of interleukin‐2

A high prevalence of vitamin D insufficiency and deficiency exists worldwide, which is associated with an increased incidence and severity of a range of immune-mediated diseases. This has resulted in considerable interest in the immunodulatory functions of vitamin D. The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to increase the frequency of Foxp3+ CD4+ T regulatory (Treg) cells when present at high concentrations or under strong T-cell stimulation in culture. Supporting evidence exists in vivo for a positive association between serum 25(OH)D and Foxp3+ Treg cell numbers in humans. The aim of this work was to identify the cytokine milieu required in vitro to promote Foxp3+ Treg cells in cultures containing 1,25(OH)2D3 at more moderate concentrations (10−7 m). Stimulation of human CD4+ T cells with a combination of 1,25(OH)2D3 and transforming growth factor-β (TGF-β) greatly increased the frequency of Foxp3+ Treg cells, which is proposed to result from the preferential expansion of Foxp3+ Treg cells, as compared with the Foxp3− effector T cells, in culture. The differential effect on proliferation may result from enhanced availability and usage of interleukin-2 by the Foxp3+ Treg cells compared with Foxp3− effector T cells. In summary, modulation of the cytokine environment to one high in TGF-β in the presence of 1,25(OH)2D3 (10−7 m) significantly increased Foxp3+ Treg cell frequency. These data provide additional evidence for the important immunomodulatory properties of 1,25(OH)2D3 that exist and may help to control inflammatory diseases.

Vitamin D fortification of eggs for human health

Vitamin D is an essential component of vertebrate nutrition, and epidemiological surveys confirm a chronic vitamin D insufficiency in the human population. Eggs are one of the few natural sources rich in vitamin D, containing both vitamin D₃ (D₃) and 25-hydroxyvitamin D₃ (25(OH)D₃). 25-Hydroxyvitamin D₃ is especially useful because it provides five times the relative biological activity of vitamin D. In order to establish the potential for enrichment of eggs with D₃ and 25(OH)D₃, a total of 162 hens were fed three levels of D₃ in combination with three levels of 25(OH)D₃. Egg yolks were analysed for their D₃ and 25(OH)D₃ contents, and egg production, egg weights and feed efficiencies were recorded. The contents of D₃ and 25(OH)D₃ in egg yolk increased significantly with increasing dietary concentrations. There were no significant differences in egg mass, egg weight or feed efficiency. Depending on the dietary concentrations used, it was possible to produce eggs with between 100 and 500 IU vitamin D, providing scope to meet the recommended daily requirement of vitamin D for children or adults. The addition of higher levels of D₃ and 25(OH)D₃ produced eggs with sufficient vitamin D to meet the recommended daily requirements of adults and children without any detrimental effect on production parameters.

Inhibition of Proliferation and Induction of Apoptosis by the Combination of β-carotene and 1,25-dihydroxyvitamin D3 in Human Esophageal Cancer EC9706 Cells

Esophageal cancer is a common malignant tumor occurring in human esophageal epithelial tissue. The primary purpose of this paper was to define the effects of β-carotene and 1,25-dihydroxyvitamin D3, alone and in combination, on cell proliferation, cell cycle and apoptosis of human esophageal cancer EC9706 cells. Treatment with different concentrations of β-carotene and/or 1,25-dihydroxyvitamin D3. MTT assay showed that β-carotene and 1,25-dihydroxyvitamin D3 significantly inhibited proliferation of EC9706 cells in a dose- and time-dependent manner. Further studies also demonstrated that β-carotene alone or 1,25-dihydroxyvitamin D3 alone caused a marked increase on the induction of apoptosis in EC9706 cells. The percentage of G0/G1-phase cells significantly increased on addition of 1,25-dihydroxyvitamin D3 alone, but there were no significant changes with β-carotene alone. These two agents in combination synergistically inhibited cell growth and induced apoptosis. Therefore, our results indicate that β-carotene and 1,25-dihydroxyvitamin D3 in combination may provide a novel strategy for preventing and treating esophageal cancer.

Thyroid hormone transporters and deiodinases in the developing human hypothalamus

Thyroid hormone (TH) signaling in brain cells is dependent on transport of TH across the plasma membrane followed by intracellular deiodination and binding to the nuclear TH receptors. The aim of this study is to investigate the expression of the specific TH transporters monocarboxylate transporter 8 (MCT8 (SLC16A2)), MCT10, organic anion transporting polypeptide 1C1 (OATP1C1 (SLCO1C1)), and the types 2 and 3 deiodinases (D2 and D3) in the developing human hypothalamus. Fifteen postmortem brain samples of fetuses and young children ranging between 17 weeks of gestation and 29 months of postnatal age including one child (28 months) with central congenital hypothyroidism were studied. Sections of the different hypothalami were stained with polyclonal rabbit antisera against MCT8, MCT10, OATP1C1, D2, and D3. We found MCT8 and D3 but not D2 protein expression to be present in our earliest sample of 17 weeks of gestation, indicating triiodothyronine degradation, but not production at this time of development. At term, expression of TH transporters and D2 decreased and D3 expression increased, suggesting decreased TH signaling just before birth. The child with central congenital hypothyroidism showed higher MCT8 and D2 expression compared with the other children of similar age. This study reports the developmental timing of expression of components crucial for central TH signaling in the human hypothalamus. In general, during fetal hypothalamic development, the coordinated expression of D2 and D3 in combination with the different TH transporters suggests that proper TH concentrations are regulated to prevent untimely maturation of brain cells.

A phase I/II clinical trial of intravenous (I.V.) calcitriol with fixed doses of cisplatin and docetaxel in advanced non-small cell lung cancer.

e18118 Background: In vitro and in vivo studies have demonstrated the antiproliferative effects of 1, 25 (OH)2D3 (calcitriol) as single agent and antitumor synergy with cisplatin. The goals of this Phase I/II study were to determine the maximum tolerated dose (MTD) of 1, 25 (OH)2 D3 in combination with cisplatin and docetaxel, and to evaluate the efficacy in patients (pts) with metastatic NSCLC.Methods: The study was a multicenter, open-label study in pts with metastatic NSCLC. Pts were adults 18 yrs., PS 0-1 with normal liver/kidney function. For the phase I study, pts (3–6 per cohort) received 1, 25 (OH)2 D3 I.V. every 21 days prior to docetaxel and cisplatin. The starting dose of 1,25 (OH)2D3 was 15 mcg/m2 at sequential ascending dose levels (DL) (15, 30, 60 and 80 mcg/m2) using a 3+3 design targeting a dose-limiting toxicity (DLT) rate of &lt;33%. Docetaxel was administered at 75 mg/m2 and cisplatin 75mg/m2 following 1, 25 (OH)2 D3 for 4 cycles. We analyzed SNPs in the CYP24A1 gene.Results: 37 pts were enrolled (16 in phase I and 21 in phase II) with a median age of 54 (range 34–79) yrs.; M: F, 12:17. At the 80 mcg/m2 dose level, 2/4 pts had DLT of grade 4 neutropenia. There were no cases of hypercalcemia or azotemia. The MTD and recommended Phase II dose was 60 mcg/m2. Among 6 response-evaluable Phase I pts, and 21 phase II pts, there were: 2 confirmed partial responses (PR), 6 unconfirmed PRs and 10 pts with stable disease. The median time to progression was 6.9 months (95% CI 4.4, 12.9) and the median overall survival was 8.3 months (95% CI 5.8, 14.9). Of the CYP24A1 SNPs, the IVS4-308C&gt;G was associated with progressive disease (Chi-Square=0.0062)Conclusions: The MTD of 1,25 (OH)2D3 in combination with docetaxel and cisplatin was 60 mcg/m2 IV every 21 days. Pre-specified endpoint of a 50% response rate was not met in the phase II study. However, disease control in 66% of patients argues for further study of 1,25 (OH)2D3 as maintenance therapy. The CYP24A1 polymorphism IVS4-308C&gt;G may be associated with resistance to a 1,25 (OH)2D3 based therapeutic regimen

Abstract 3846: Vitamin D3 potentiates the antitumor effects of arsenic trioxide in human leukemia (HL-60) cells

Abstract Arsenic trioxide (ATO) is an innovative form of therapy that has recently been found to benefit acute promyelocytic leukemia (APL) patients. Our laboratory has demonstrated that ATO stimulates oxidative stress therefore causing ATO-induced cytotoxicity in human leukemia (HL-60) cells. Pro-oxidants have been known to play a role in free radical-mediated oxidative stress. Vitamin D3, an active metabolite of vitamin D, inhibits the growth of a number of cancer types such as prostate, breast, colorectal, leukemia, and skin cancers. The specific aim of this research was to determine whether co-exposure to vitamin D3 increases the efficacy of ATO through cytotoxicity, oxidative stress and apoptosis associated with increased ATO toxicity in human leukemia (HL-60) cells. In our current investigation, we performed the MTT assay and trypan blue exclusion test to measure cell viability. We also performed the lipid peroxidation assay to determine the levels of malondialdehyde (MDA) production in HL-60 cells co-exposed to ATO and vitamin D3 in combination. The results of trypan blue exclusion and MTT assay indicated that vitamin D3 exposure potentiates the toxic activity of ATO in HL-60 cells in a dose dependent manner. Co-administration of vitamin D3 and ATO resulted in a significant (P&amp;lt;0.05) increase in MDA level compared to ATO alone. A statistically significant and dose-dependent increase (p &amp;lt;0.05) was recorded in annexin V positive cells (apoptotic cells) with increasing doses of vitamin D3 in ATO-treated cells. This finding was confirmed by the result of DNA laddering assay showing clear evidence of DNA fragmentation in ATO and vitamin D3 treated HL-60 cells. Overall, the present study indicates that vitamin D3 potentiates the antitumor effects of ATO at least part, via cytotoxicity, oxidative stress, nucleosomal fragmentation and phosphatidylserine externalization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3846. doi:1538-7445.AM2012-3846

Abstract 5418: The immunomodulatory drug lenalidomide restores a Vitamin D sensitive phenotype to the Vitamin D resistant breast cancer cell line MDA-MB-231: Potential for breast cancer therapeutics

Abstract 1α,25-Dihydroxyvitamin D3, (1,25 D3) the biologically active form of vitamin D, is well established as an inhibitor of cancer cell growth in addition to its primary role in maintaining bone mineralization. In breast cancer cell lines, inhibitory effects on cell cycle arrest, angiogenesis, invasion and metastasis have been observed in addition to pro-apoptotic effects. 1,25 D3 also inhibits and prevents breast cancer growth in several mouse models, and a correlation between vitamin D receptor expression on breast cancer cells and disease free survival of breast cancer patients has also been observed. However, resistance to vitamin D and hypercalcaemia at higher doses are key limiting factors in clinical use. The drug Revlimid ® (lenalidomide) which has shown great promise in multiple myeloma, can also modulate signalling in apoptotic and cell growth pathways leading to inhibition of cell growth, inhibition of metastasis and invasion. Our study aimed to determine whether lenalidomide treatment of breast cancer cells resistant to vitamin D would result in an acquisition of sensitivity to vitamin D and a resultant inhibition of cell growth. The cell lines MCF-12A, MCF-7 and MDA-MB-231,representing non-tumorigenic, tumorigenic and metastatic breast lines respectively were used. The latter line was also vitamin D resistant. Cells were treated with lenalidomide and/or 1,25 D3 using a dose of 100 nM 1,25 D3 (a clinically tolerable dose giving IC50 inhibition of MCF-7 and MCF-12A cells). Results showed that whereas lenalidomide had no effect on the growth of the vitamin D sensitive lines, and gave only 20% inhibition of growth of MDA-MB-231 at 10 µM, a 50% inhibition of cell growth by 1,25 D3 (equivalent to that seen with the sensitive lines) was achieved in the presence of lenalidomide at a concentration of 1 µM. Further investigation revealed that the mechanism of this effect was an increase in apoptosis of the cell line, shown by an increase in parp cleavage and annexin V expression. An array measuring proteins associated with several signalling cascades showed that the combination of 1,25 D3 and lenalidomide resulted in an increase in proapoptotic proteins (phosphorylated P53, P21 and claspin, in addition to a decrease in BCL-2. Although both drugs had individual effects on pro-apototic proteins, and anti-apoptotic proteins, the combination resulted in an overall increase of pro-apoptotic protein expression leading to the observed inhibition of cell viability and growth. These results demonstrate the potential for the use of lenalidomide and 1,25 D3 in combination for tumours that are refractory to vitamin D. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5418.

High-dose oral vitamin D3 supplementation in rheumatology patients with severe vitamin D3 deficiency

Objectives Recent large trials indicate that adherence associated with a daily regimen of vitamin D is low and limits anti-fracture efficacy with vitamin D supplementation. The aim of this report is to describe changes of 25-hydroxyvitamin D (25(OH)D) serum concentrations achieved with a single oral dose of 300 000 IU vitamin D3. Methods Over a course of 4 months, we identified 33 elderly with severe vitamin D deficiency (25(OH)D < 25 nmol/l) on admission to acute care. Patients were admitted for musculoskeletal pain, bone disease, or gait abnormalities. The mean age was 80.5 years (SD ± 6.1). All patients were treated with a single oral dose of 300 000 IU D3 in combination with 500–1000 mg calcium supplements per day depending on their dietary calcium intake. Results Baseline mean 25(OH)D serum concentrations were 15 nmol/l (SD ± 5.5). Mean 25(OH)D serum concentrations increased to 81.4 nmol/l (SD ± 29.7) at 3 months (29 patients) and were still 69.0 nmol/l (SD ± 17.9) at 6 months (26 patients). Mean serum calcium levels were 2.24 mmol/l (SD ± 0.11) at baseline, 2.28 mmol/l (SD ± 0.18) at 3 months, and 2.28 mmol/l (SD ± 0.13) at 6 months. Two patients with mild hypercalcemia (2.69 mmol/l) at 3 months had normal values at 6 months. Conclusion Based on our observations, a single oral dose of 300 000 IU vitamin D3 raises mean 25(OH)D serum concentrations to the target mean of above 75 nmol/l at 3 months and a mean level of 69 nmol/l at 6 months. As calcium absorption is enhanced with higher 25(OH)D serum concentrations, calcium supplementation may need downward adjustment with this regimen to avoid mild hypercalcemia.

Overexpression of cyclin D1, D3, and p21 in an infantile renal carcinoma with Xp11.2 TFE3-gene fusion

Renal carcinomas harboring the TFE-3 translocation are rare and occur predominately in children and adolescents. Here, we report a case of infantile renal carcinoma with TFE3 translocation and show that the cell cycle is deregulated in this type of carcinoma. It is characterized by nuclear accumulation of cyclin D1 and D3 in combination with high levels of cyclin-dependent kinase inhibitor p21 Cip1/Waf1 but without accumulation of p53, p16 INK4a, or mdm2. The combined overexpression of p21, cyclin D1, and cyclin D3 was found exclusively in this type but not in other, more common types of renal carcinoma/oncocytoma ( n=27). These results further underscore that renal carcinomas with Xp11. 2 translocations/TFE3-gene fusion represent a special type of renal neoplasm showing deregulation of specific cell cycle components. The analysis of further cases has to prove whether the derangement of the cell cycle is uniform and correlates with the specific type of molecular genetic derangement.