Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells

Calcitriol or 1,25(OH)₂D₃ is a negative growth regulator of MCF-7 breast cancer cells. The growth arrest is due to apoptosis activation, which involves mitochondrial disruption. This effect is blunted in vitamin D resistant cells (MCF-7^DRes cells). Menadione (MEN), a glutathione (GSH)-depleting compound, may potentiate antitumoral effects of anticancer drugs. The aim of this study was to investigate whether MEN enhances cellular responsiveness of MCF-7 cells to 1,25(OH)₂D₃. Cells were cultured and treated with different concentrations of 1,25(OH)₂D₃ ± MEN or vehicle for 96 h. GSH levels and the activity of antioxidant enzymes were determined by spectrophotometry and ROS production by flow cytometry. Both drugs decreased growth and enhanced ROS in MCF-7 cells, obtaining the maximal effects when 1,25(OH)₂D₃ was combined with MEN (P < 0.01 vs. Control and vs. each compound alone). MCF-7^DRes cells were not responsive to 1,25(OH)₂D₃, but the cell proliferation was slightly inhibited by the combined treatment. Calcitriol and MEN separately enhanced antioxidant enzyme activities, but when they were used in combination, the effect was more pronounced (P < 0.05 vs. Control and vs. each compound alone). MEN, calcitriol and the combined treatment decreased GSH levels (P < 0.05 vs. Control). The data indicate that MEN potentiates the effect of 1,25(OH)₂D₃ on growth arrest in MCF-7 cells by oxidative stress and increases the activities of antioxidant enzymes, probably as a compensatory mechanism.     

The cellular and molecular regulation of 1,25(OH)2D3 production.

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues

The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH)₂D₃ provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH)₂D₃, we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH)₂D₃ to look for possible cooperative activation of the gene. We identified 1,25(OH)₂D₃-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH)₂D₃ is potentially important in a wide range of tissues.     

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1α,25dihydroxycholecalciferol (1,25D₃ = calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1α,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.     

Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

1,25(OH)2D3 attenuates pulmonary arterial hypertension via microRNA-204 mediated Tgfbr2/Smad signaling

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high pulmonary artery pressure. It is reported that microRNA-204 (miR-204) plays an important role in the development of PAH. Calcitriol [1,25-dihydroxyvitamin D3, 1,25(OH)₂D₃] mediates multiple pathophysiological processes. The aim of the current study was to explore the role of 1,25(OH)₂D₃ in PAH. PAH was induced in rats and rat pulmonary arterial endothelial cells (PAECs) were isolated as in vitro PAH model. The mean pulmonary artery pressure, morphologic changes, and expressions of transforming growth factor-beta1 (Tgfbr2), Smad2/7, alpha smooth muscle actin (α-SMA), and p21 were then measured. Furthermore, the effect of 1,25(OH)₂D₃ on rat PAECs with or without hypoxia treatment was also assessed by measuring the proliferation, migration, and cell cycle distribution of PAECs. The potential targets of miR-204 were also predicted and validated with a dual-luciferase reporter system. Then the role of miR-204 and Tgfbr2 in the anti-PAH effect of 1,25(OH)₂D₃ was further explored by modulating the expression of the two genes. The overall pulmonary hypertension and hypoxia-induced proliferation and migration of PAECs were attenuated by administration of 1,25(OH)₂D₃, which was associated with the suppressed expressions of Tgfbr2, α-SMA, and Smad7 and induced expressions of miR-204, p21 and Smad2 both in vitro and in vivo. Moreover, the luciferase reporter assay identified Tgfbr2 as a novel direct target of miR-204. Both overexpression of miR-204 and inhibition of Tgfbr2 would strengthen the effect of 1,25(OH)₂D₃ administration. Findings outlined in the current study demonstrated that 1,25(OH)₂D₃ was a promising therapeutic modality for treatment of PAH, function of which was exerted through miR-204 mediated Tgfbr2 signaling.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

Intestinal Na/Ca exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)₂D₃ on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)₂D₃ were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)₂D₃ simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)₂D₃. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)₂D₃. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)₂D₃ treatment. Rapid effects of 1,25(OH)₂D₃ on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)₂D₃ on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)₂D₃ enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Effect of dietary calcium and 1,25-(OH)2D3 on the expression of calcium transport genes in calbindin-D9k and -D28k double knockout mice

The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (KO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we generated CaBP-9k/CaBP-28k double knockout (DKO) mice in order to investigate the importance of CaBP-9k and CaBP-28k in active calcium processing. Under normal dietary conditions, DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to WT or CaBP-28k KO mice. Under calcium-deficient dietary conditions, the phenotype and expression of calcium transport genes in CaBP-28k KO mice were similar to WT, whereas in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of transient receptor potential vanilloid member 6 (TRPV6) mRNA was significantly decreased in DKO mice fed a calcium-deficient diet as compared to CaBP-28k KO or WT mice, and DKO mice died after 4 weeks on a calcium-deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25-(OH)₂D₃-deficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. These results indicate that deletion of CaBP-28k alone does not affect body calcium homeostasis, but that deletion of CaBP-9k and CaBP-28k has a significant effect on calcium processing under calcium-deficient conditions, confirming the importance of dietary calcium and 1,25-(OH)₂D₃ during growth and development.     

A 2-D polymer assembled by cubane-like clusters [Tb4(OH)4(phen)3(H2O)3] and 3-sulfobenzoate

A coordination polymer {[Tb₄(3-SBA)₄(OH)₄(phen)₃(H₂O)₃] · 7H₂O}_n (3-SBA = 3-sulfobenzoate, phen = 1,10-phenanthroline) was synthesized and structurally characterized. The complex contains cubane-like clusters, [Tb₄(μ₃-OH)₄(phen)₃(H₂O)₃]⁸⁺, which are further linked through 3-SBA ligands to form a 2-D grid-like network structure with topology of (3³, 4⁴, 5³). The complex exhibits strong photoluminescence of the Tb³⁺ ion.     

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48 h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.     

Synthesis and characterisation of a trinuclear uranyl complex: Crystal structure of (CN3H6)5[(UO2)3O(OH)2(CH3COO)(C2O4)3]

The reaction of uranyl oxalate trihydrate with guanidinium acetate at room temperature in water yields known uranyl complex with composition (CN₃H₆)₂[UO₂(C₂O₄)₂(H₂O)]·H₂O as a first phase and a novel complex (CN₃H₆)₅[(UO₂)₃O(OH)₂(CH₃COO)(C₂O₄)₃] as a second. The second phase was investigated by means of IR spectroscopy and X-ray diffraction. The trinuclear discrete complex contains two symmetrically independent uranyl ions with a pentagonal bipyramid structure and has a nonplanar geometry. The distortion of its equatorial plane is caused by substitution of a monodentate bridge hydroxide anion by a bidentate bridge acetate-anion. The acidic ligands found in the complex are usually in competition for a place in coordination sphere of an uranyl ion, thus peculiarities of the complex formation are discussed in terms of ‘crystallochemical analysis’.     

Syntheses and characterization of the W(II) and W(III) N2 complexes, [WCl2(PMe3)3]2N2 and [WCl3(PMe3)2]2N2

Refluxing WCl₄(PMe₃)₃ under a nitrogen atmosphere in the presence of two equivalents of sodium amalgam leads to a reduction to the W(II) complex [cis,mer-WCl₂(PMe₃)₃]₂N₂ (1), which can be converted to [mer,trans-WCl₃(PMe₃)₂]₂N₂ (2) via appropriate oxidation/chlorination. Structural data have been obtained for both complexes, and demonstrate significantly increased steric crowding in 1 due to PMe₃/PMe₃ interactions. The N-N bond distances in the two compounds are similar, at 1.279(4) and 1.243(18) Å, respectively.     

不明原因复发性流产再次妊娠患者血清1,25(OH)2D3、sTim-3与Th17/Treg免疫失衡和妊娠结局的关系

摘要 目的：探讨不明原因复发性流产（URSA）再次妊娠患者血清1,25-二羟维生素D3[1,25（OH)）₂D₃]、可溶性T细胞免疫球蛋白黏蛋白分子3（sTim-3）与辅助性T细胞17（Th17）/调节性T细胞（Treg）免疫失衡和妊娠结局的关系。方法：选择于湖南省妇幼保健院2020年1月~2022年1月就诊的62例URSA再次妊娠患者作为研究组,另选择同期进行孕检的正常早孕妇女30例作为对照组。比较两组孕早期血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平、Th17/Treg比值。Pearson法分析URSA再次妊娠患者血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值平的相关性。根据URSA再次妊娠患者妊娠结局的不同分为妊娠成功分娩组和妊娠再次流产组,比较两组孕早期血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值。受试者工作特征（ROC）曲线分析血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值对妊娠结局的预测价值。结果：研究组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于对照组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于对照组（P<0.05）。Pearson相关分析显示,URSA再次妊娠患者血清1,25(OH) ₂D₃与血清sTim-3、外周血Th17细胞水平、Th17/Treg比值呈负相关,与Treg细胞水平呈正相关（P<0.05）;血清sTim-3与外周血Treg细胞水平呈负相关,与Th17细胞水平、Th17/Treg比值呈正相关（P<0.05）。妊娠再次流产组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于妊娠成功分娩组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于妊娠成功分娩组（P<0.05）。ROC曲线分析显示,血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平及Th17/Treg比值均可预测URSA再次妊娠患者妊娠再次流产的发生风险,且上述指标联合检测的预测效能更高。结论：血清1,25(OH) ₂D₃水平异常降低、sTim-3水平异常升高可导致Th17/Treg免疫失衡,导致URSA再次妊娠患者再次发生流产。上述指标联合检测对URSA再次妊娠患者妊娠再次流产的预测效能更高。     

Synthesis and characterization of SrCu2(O2CR)3(bdmap)3 and Bi2(O2CCH3)4(bdmap)2(H2O) (R = CH3, CF3; bdmap = 1,3-bis(dimethylamino)-2-propanolato)

New mixed metal complexes SrCu₂(O₂CR)₃(bdmap)₃ (R = CF₃ (1a), CH₃ (1b)) and a new dinuclear bismuth complex Bi₂(O₂CCH₃)₄(bdmap)₂(H₂O) (2) have been synthesized. Their crystal structures have been determined by single-crystal X-ray diffraction analyses. Thermal decomposition behaviors of these complexes have been examined by TGA and X-ray powder diffraction analyses. While compound 1a decomposes to SrF₂ and CuO at about 380°C, compound 1b decomposes to the corresponding oxides above 800°C. Compound 2 decomposes cleanly to Bi₂O₃ at 330°C. The magnetism of 1a was examined by the measurement of susceptibility from 5–300 K. Theoretical fitting for the susceptibility data revealed that 1a is an antiferromagnetically coupled system with g = 2.012(7), −2J = 34.0(8) cm⁻¹. Crystal data for 1a: C₂₇H₅₁N₆O₉F₉Cu₂Sr/THF, monoclinic space group P2₁/m, A = 10.708(6), B = 15.20(1), C = 15.404(7) Å, β = 107.94(4)°, V = 2386(2) ų, Z = 2; for 1b: C₂₇H₆₀N₆O₉Cu₂Sr/THF, orthorhombic space group Pbcn, A = 19.164(9), B = 26.829(8), C = 17.240(9) Å, V = 8864(5) ų, Z = 8; for 2: C₂₂H₄₈O₁₁N₄Bi₂, monoclinic space group P2₁/c, A = 17.614(9), B = 10.741(3), C = 18.910(7) Å, β = 109.99(3)°, V = 3362(2) ų, Z = 4.