Vitamin D compounds in leukemia

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3,] possess in vitro multiple anti-cancer activities including growth arrest, induction of apoptosis and differentiation of a variety of different types of malignant cells. However, its use as a therapeutic agent is hindered by its calcemic effects. Analogs of 1,25(OH)(2)D3 have enhanced anti-tumor activity, with reduced calcemic effects. However, limited clinical studies using vitamin D compounds have not yet achieved major clinical success. Nevertheless, pre-clinical studies suggest that the combination of either 1,25(OH)(2)D3 or its analogs with other agents can have additive or synergistic anti-cancer activities, suggesting future clinical studies.     

Induction of tissue plasminogen activator secretion from rat heart microvascular cells by fM 1,25(OH)(2)D(3)

We investigated the effects of 1,25-dihydroxyvitamin D(3) [25(OH)(2)D(3)] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)(2)D(3) and several of its analogs. The results showed that 1,25(OH)(2)D(3) induced tPA secretion at 10(-10) to 10(-16) M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca(2+) channels, Ro-24-5531, were approximately 10% as active as 1,25(OH)(2)D(3). An analog that binds the vitamin D receptor poorly but is an effective Ca(2+) channel agonist, Ro-24-2287, required approximately 10(-13) M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)(2)D(3). Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca(2+)]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)(2)D(3) was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)(2)D(3) on two different induction pathways of tPA release.     

A vitamin D analog regulates mesangial cell smooth muscle phenotypes in a transforming growth factor-beta type II receptor-mediated manner.

Mesangial cells share features with contractile smooth muscle cells and mechanically support the capillary wall. The role of vitamin D compounds and the transforming growth factor-beta (TGF-beta) type II receptor in modulating the smooth muscle phenotype of cultured mesangial cells was examined. Cell proliferation was significantly inhibited by the vitamin D analog 22-oxa-1,25-dihydroxyvitamin D(3) (22-oxacalcitriol; OCT) rather than by 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) in a dose-dependent manner. OCT-treated early passage mesangial cells (MC-E cells) had increased expression levels of type IV collagen and smooth muscle alpha actin mRNA, but 1, 25(OH)(2)D(3)-treated MC-E cells did not. The addition of a TGF-beta(1)-neutralizing antibody to the OCT-treated MC-E cells blocked this inhibitory effect for cell proliferation and attenuated the up-regulated mRNA levels. However, after exposure to 1, 25(OH)(2)D(3) or OCT, there was no significant difference in the secretion of active TGF-beta. We next investigated whether TGF-beta type II receptor (RII) was involved in this regulation. OCT treatment significantly increased the expression of the RII mRNA in MC-E cells. These results suggest that the vitamin D analog OCT induces smooth muscle phenotypic alterations and that this phenomenon was mediated through the induction of RII in cultured mesangial cells.     

1,25(OH)2D3 alters growth plate maturation and bone architecture in young rats with normal renal function

Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)(2)D(3), the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)(2)D(3) is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)(2)D(3) treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 μg/kg 1,25(OH)(2)D(3) for one week, or intermittent 3 μg/kg 1,25(OH)(2)D(3) for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)(2)D(3)-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)(2)D(3) increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)(2)D(3) lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)(2)D(3)-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)(2)D(3) on intracortical bone formation. This study shows negative effects of 1,25(OH)(2)D(3) on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.     

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.     

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.     

1,25-(OH)2-16ene-23yne-D3 reduces secondary hyperparathyroidism in uremic rats with little calcemic effect

OBJECTIVES: To compare the effects of vitamin D analogs versus calcitriol on serum levels of Ca, P and parathyroid hormone (PTH). A compound better than calcitriol should increase the Ca x P product less than calcitriol for an equivalent decrease in PTH levels. METHODS: Biological activity of 4 vitamin D analogs, 1,25-(OH)(2)-16ene- D(3) (RO(1)), 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)), 1,25-(OH)(2)-26,27-hexafluoro-16ene-23yne-D(3) (RO(3)) and 1,25-(OH)(2)-16ene-23yne-26,27-hexafluoro-19nor-D(3) (RO(4)) was tested vs. calcitriol in parathyroidectomized rats. In a second set of experiments, the effects of RO(2), RO(4) and calcitriol were studied in 5/6 nephrectomized rats with secondary hyperparathyroidism. RESULTS: In parathyroidectomized rats, all analogs (250 pmol/day) led calcemia to rise after 7 days. In uremic rats, all treatments reduced PTH levels. RO(4) revealed toxicity. RO(2) was as effective as calcitriol in suppressing PTH in a dose dependent manner. Mean plasma ionized calcium did not change from baseline to day 14 and day 28 on RO(2) (250 or 500 pmol/day) whereas it increased significantly on RO(2) (1,000 pmol/day) and calcitriol (125 or 250 pmol/day). Increasing the dose of calcitriol led Ca x P to rise more dramatically than increasing the dose of RO(2), which appears to have a wider therapeutic window than calcitriol. CONCLUSION: 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)) may represent a novel candidate for the treatment of renal osteodystrophy in humans.     

Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)(2)D(3) levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3), although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1 alpha-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly, 1,25(OH)(2)D(3) up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.     

1alpha,25(OH)2-vitamin D3 inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells

Several mesenchymally derived cells, including osteoblasts, secrete hepatocyte growth factor (HGF). 1alpha,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of MG-63 osteoblastic cells. Here we show that MG-63 cells secrete copious amounts of HGF and that 1,25(OH)(2)D(3) inhibits HGF production. MG-63 cells also express HGF receptor (c-Met) mRNA, suggesting an autocrine action of HGF. Indeed, although exogenous HGF failed to stimulate cellular proliferation, neutralizing endogenous HGF with a neutralizing antibody inhibited MG-63 cell proliferation; moreover, inhibiting HGF synthesis with 1,25(OH)(2)D(3) followed by addition of HGF rescued hormone-induced inhibition of proliferation. Nonneutralized cells displayed constitutive phosphorylation of c-Met and the mitogen-activated protein kinases mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2, which were inhibited by anti-HGF antibody. Constitutive phosphorylation of Erk1/2 was also abolished by 1,25(OH)(2)D(3). Addition of HGF to MG-63 cells treated with neutralizing HGF antibody induced rapid phosphorylation of c-Met, MEK1, and Erk1/2. Thus endogenous HGF induces a constitutively active, autocrine mitogenic loop in MG-63 cells. The known antiproliferative effect of 1,25(OH)(2)D(3) on MG-63 cells can be accounted for by the concomitant 1,25(OH)(2)D(3)-induced inhibition of HGF production.     

Toxicity and antineoplastic effect of (24R)-1,24-dihydroxyvitamin D3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.     

20-Hydroxyvitamin D2 is a noncalcemic analog of vitamin D with potent antiproliferative and prodifferentiation activities in normal and malignant cells

20-hydroxyvitamin D(2) [20(OH)D(2)] inhibits DNA synthesis in epidermal keratinocytes, melanocytes, and melanoma cells in a dose- and time-dependent manner. This inhibition is dependent on cell type, with keratinocytes and melanoma cells being more sensitive than normal melanocytes. The antiproliferative activity of 20(OH)D(2) is similar to that of 1,25(OH)(2)D(3) and of newly synthesized 1,20(OH)(2)D(2) but significantly higher than that of 25(OH)D(3). 20(OH)D(2) also displays tumorostatic effects. In keratinocytes 20(OH)D(2) inhibits expression of cyclins and stimulates involucrin expression. It also stimulates CYP24 expression, however, to a significantly lower degree than that by 1,25(OH)(2)D(3) or 25(OH)D(3). 20(OH)D(2) is a poor substrate for CYP27B1 with overall catalytic efficiency being 24- and 41-fold lower than for 25(OH)D(3) with the mouse and human enzymes, respectively. No conversion of 20(OH)D(2) to 1,20(OH)(2)D(2) was detected in intact HaCaT keratinocytes. 20(OH)D(2) also demonstrates anti-leukemic activity but with lower potency than 1,25(OH)(2)D(3). The phenotypic effects of 20(OH)D(2) are mediated through interaction with the vitamin D receptor (VDR) as documented by attenuation of cell proliferation after silencing of VDR, by enhancement of the inhibitory effect through stable overexpression of VDR and by the demonstration that 20(OH)D(2) induces time-dependent translocation of VDR from the cytoplasm to the nucleus at a comparable rate to that for 1,25(OH)(2)D(3). In vivo tests show that while 1,25(OH)(2)D(3) at doses as low as 0.8 μg/kg induces calcium deposits in the kidney and heart, 20(OH)D(2) is devoid of such activity even at doses as high as 4 μg/kg. Silencing of CY27B1 in human keratinocytes showed that 20(OH)D(2) does not require its transformation to 1,20(OH)(2)D(2) for its biological activity. Thus 20(OH)D(2) shows cell-type dependent antiproliferative and prodifferentiation activities through activation of VDR, while having no detectable toxic calcemic activity, and is a poor substrate for CYP27B1.     

Vitamin D(3) and analogues modulate the expression of CSF-1 and its receptor in human dendritic cells

The active vitamin D(3)-metabolite 1,25(OH)(2)D(3) inhibits the interleukin 4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF)-induced differentiation of human monocytes into dendritic cells without altering survival. Colony-stimulating factor 1 (CSF-1) is an important survival factor for cells of the monocytic lineage. We therefore investigated whether the inhibitory activity of 1,25(OH)(2)D(3) is paralleled by a regulation of CSF-1 and its receptor. Purified human monocytes were cultured together with IL-4/GM-CSF in the presence of 1,25(OH)(2)D(3), its analogue tacalcitol, the low-affinity vitamin D receptor ligand 24,25(OH)(2)D(3), or the solvent ethanol for up to 5 days. Expression of CSF-1, CSF-1R, and GM-CSF mRNA was measured by RT-PCR. Protein secretion for CSF-1 was measured by ELISA, expression of CSF-1R by flow cytometry. The results showed that 1,25(OH)(2)D(3) and tacalcitol significantly up-regulated CSF-1 mRNA-expression and protein secretion in a dose-dependent manner. The effect of 1,25(OH)(2)D(3) occurred already after 1h of pre-treatment. In contrast, CSF-1R mRNA- and cell surface-expression was down-regulated simultaneously. The solvent ethanol and 24,25(OH)(2)D(3) were without effect. GM-CSF mRNA expression was not modulated in 1,25(OH)(2)D(3)-treated cells. These data point towards a distinct and specific regulation of CSF-1 and its receptor by 1,25(OH)(2)D(3) and its analogue tacalcitol in human monocytes which parallels the inhibition of differentiation into dendritic cells without altering survival.     

1,25-Dihydroxyvitamin D and three low-calcemic analogs decrease UV-induced DNA damage via the rapid response pathway

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is anti-apoptotic in human keratinocytes, melanocytes and fibroblasts after ultraviolet (UV)-exposure. To date, there is no published data on the effects of 1,25(OH)(2)D(3) or its analogs on DNA damage in irradiated skin cells. In these skin cells, 24h pre-treatment with 1,25(OH)(2)D(3) dose-dependently (10(-12) to 10(-8)M) decreased CPD damage by up to 60%. This photoprotective effect was also seen if the 1,25(OH)(2)D(3) was added immediately after irradiation and was mimicked by QW-1624F2-2 (QW), a low-calcemic 1beta-hydroxymethyl-3-epi-16-ene-24,24-difluoro-26,27-bis homo hybrid analog. The well-studied low calcemic, rapid acting agonist analogs 1alpha,25(OH)(2)lumisterol(3) (JN) and 1alpha,25(OH)(2)-7-dehydrocholesterol (JM) also protected skin cells from UV-induced cell loss and CPD damage to an extent comparable with that of 1,25(OH)(2)D(3). In contrast, the rapid response antagonist analog 1beta,25(OH)(2)D(3) (HL) completely abolished the photoprotective effects (reduced cell loss and reduced CPD damage) produced by treatment with 1,25(OH)(2)D(3), JN, JM and QW. Evidence for involvement of the nitric oxide pathway in the protection from CPD damage by 1,25(OH)(2)D(3) was obtained. These data provide further evidence for a role of the vitamin D pathway in the intrinsic skin defenses against UV damage. The data also support the hypothesis that the photoprotective effects of 1,25(OH)(2)D(3) are mediated via the rapid response pathway(s).     

1,25-Dihydroxyvitamin D3 inhibits tumor necrosis factor-alpha-induced adhesion molecule expression in endothelial cells

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.     

Inhibitory effect of 22-oxa-1,25-dihydroxyvitamin D3, maxacalcitol, on the proliferation of pancreatic cancer cell lines

Effective chemotherapy for pancreatic cancer is urgently needed. The aim of this study was to compare the anti-proliferative activity on pancreatic cancer cell lines of the vitamin D(3) analog, 22-oxa-1,25-dihydroxyvitamin D(3), maxacalcitol, with that of 1,25-dihydroxyvitamin D(3), calcitriol, with analysis of vitamin D receptor status and the G(1)-phase cell cycle-regulating factors. Antiproliferative effects of both agents were compared using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and by measuring the tumor size of xenografts inoculated into athymic mice. Scatchard analysis of vitamin D receptor contents, and mutational analysis of receptor complementary DNA were performed. Levels of expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, p21 and p27, were analysed by western blotting. In vitro, maxacalcitol and calcitriol markedly inhibited the proliferation and caused a G(1) phase cell cycle arrest with the appearance of numerous domes. In vivo, maxacalcitol inhibited the growth of BxPC-3 xenografts more significantly than calcitriol, without inducing hypercalcemia. Responsive cells had abundant functional vitamin D receptors. However, Hs 766T, showing no response to either agent, had the second highest receptor contents with no abnormalities in its primary structure deduced by receptor complementary DNA. In the responsive cells, p21 and p27 were markedly up-regulated after 24h of treatment with both agents. In non-responsive cells, no such changes were observed. In conclusion, maxacalcitol and calcitriol up-regulate p21 and p27 as an early event, which in turn could block the G(1)/S transition and induce growth inhibition in responsive cells, and maxacalcitol may provide a more useful tool for the chemotherapy of pancreatic cancer than calcitriol because of its low toxicity.