Selective vitamin D receptor modulators and their effects on colorectal tumor growth

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is an endocrine hormone whose classic role is the maintenance of calcium homeostasis. It is well documented that 1,25(OH)(2)D(3) also has anti-tumor effects on a number of cancers and cancer cell lines including breast, colorectal, gastric, liver, ovarian, prostate, and non-melanoma skin cancers. Included in the anti-tumor activities of 1,25(OH)(2)D(3) are its ability to cause antiproliferation, prodifferentation and decrease angiogenesis. Furthermore, through regulation of the plaminogen activator (PA) system and a class of proteolytic enzymes called matrix metalloproteinases (MMPs), 1,25(OH)(2)D(3) reduces the invasive spread of tumor cells. Because of the calcemic limitations of using 1,25(OH)(2)D(3) as a therapy, we have tested the effects of a novel Gemini vitamin D analogue, Deuterated Gemini (DG), on mouse colorectal cancer. We demonstrated that DG is more potent in reducing tumor volume and mass, compared to control and 1,25(OH)(2)D(3). DG significantly prevented (100% reduction, p<0.05) the invasive spread of colorectal tumor cells into the surrounding muscle, and had no effect on serum calcium levels. Thus, DG acts as a selective vitamin D receptor modulator (SVDRM) by enhancing select anti-tumor characteristic 1,25(OH)(2)D(3) activities, without inducing hypercalcemia. Thus, DG shows promise in the development of colorectal cancer therapies.     

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-β-glucuronide [β-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or β-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)(2)D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.     

Nuclear lipid microdomains regulate nuclear vitamin D3 uptake and influence embryonic hippocampal cell differentiation

Despite recent advances in the understanding of the role of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in the CNS, the mechanism of action remains obscure. We demonstrate that some 1,25-(OH)(2)D(3) receptor (VDR) is localized in the cell nucleus in specialized microdomains enriched in sphingomyelin and cholesterol; the integrity of these microdomains is necessary for embryonic hippocampal cell differentiation. Sphingomyelinase (SMase) treatment reduces both VDR and labeled 1,25-(OH)(2)D(3) content in nuclear microdomains. We have previously shown that HN9.10e embryonic hippocampal cells differentiate when incubated with 100 nM 1,25-(OH)(2)D(3) in the presence of 10% fetal calf serum, while serum deprivation induces cell death. In this study, we have investigated whether conditions that alter lipid content of nuclear microdomains modify 1,25-(OH)(2)D(3)-induced differentiation. Serum deprivation activates SMase and modifies the composition of nuclear microdomains, which lose the 1,25-(OH)(2) vitamin D(3) receptor. The incubation of serum-deprived cells with 100 nM 1,25-(OH)(2)D(3) prevents differentiation. However, treatment with 400 nM 1,25-(OH)(2)D(3) during serum withdrawal increases the lipid content of the nuclear microdomains, allows the interaction of 1,25-(OH)(2)D(3) with its receptor, and results in differentiation. These results suggest the presence of VDR in nuclear microdomains is necessary for 1,25-(OH)(2)D(3)-induced differentiation in embryonic hippocampal cells.     

Microarray analysis of 1alpha,25-dihydroxyvitamin D3-treated MC3T3-E1 cells

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH)(2)D(3) in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH)(2)D(3) treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12h after treatment with 1,25-(OH)(2)D(3) (10(-8)M), preceding (6h) or coinciding with (12h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12h incubation period with 1,25-(OH)(2)D(3) were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH)(2)D(3) was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH)(2)D(3) resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest.     

Vitamin D analogs: therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.     

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).     

Synergistic anticancer activity of 1,25-dihydroxyvitamin D(3) and immune cytokines: the involvement of reactive oxygen species

It was previously shown that 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) enhances the cytotoxic activity of tumor necrosis factor alpha (TNFalpha), doxorubicin and menadione. A feature shared by these anticancer agents is the involvement of reactive oxygen species (ROS) in their action. In this work we found that 1, 25(OH)(2)D(3) acted synergistically with interleukin 1 beta (IL-1beta) or interleukin 6 (IL-6) to inhibit the proliferation of MCF-7 breast cancer cells. The extent of the synergism was maximal at 1 nM, a concentration at which 1,25(OH)(2)D(3), acting singly, only marginally reduced the cell number. The thiol antioxidant, N-acetylcysteine (NAC) abolished the synergism between IL-1beta or IL-6 and 1,25(OH)(2)D(3), but had only a small protective effect when the cytokines acted alone. NAC and reduced glutathione (GSH) protected MCF-7 cells from cytotoxicity induced both by TNFalpha alone and by TNFalpha and 1,25(OH)(2)D(3). A two-day exposure to TNFalpha caused a 27.7+/-3.1% (mean +/- SEM) reduction in GSH content. This effect increased to 46.4+/-5.5% by co-treatment with 1, 25(OH)(2)D(3) which did not affect GSH levels on it own. We conclude that 1,25(OH)(2)D(3) can act synergistically with anticancer cytokines present in the tumor milieu and that ROS plays a mediatory role in this interaction.     

A local effect of CYP24 inhibition on lung tumor xenograft exposure to 1,25-dihydroxyvitamin D(3) is revealed using a novel LC-MS/MS assay

The vitamin D(3) catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)(2)D(3)in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC-MS/MS assay that permits absolute quantitation of 1,25(OH)(2)D(3) in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vitro, and 1,25(OH)(2)D(3) rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)(2)D(3) reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20μg/kg 1,25(OH)(2)D(3)). When co-administered with the CYP24 selective inhibitor CTA091 (250μg/kg), 1,25(OH)(2)D(3) plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)(2)D(3) AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)(2)D(3) independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)(2)D(3) concentration versus time data confirmed that: 1,25(OH)(2)D(3) was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)(2)D(3).     

Immunological modulation by 1α,25-dihydroxyvitamin D3 in patients with squamous cell carcinoma of the head and neck

Prior studies showing that treatment of head and neck squamous cell carcinoma (HNSCC) patients with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] stimulated intratumoral immune infiltration were extended to analysis of cytokine profiles in the periphery and in oral tissues. Most prominent was the disparity between cytokine levels in plasma and in either pathologically normal oral tissue or HNSCC tissue from patients that were untreated or treated with 1,25(OH)(2)D(3). Levels of IL-6 and IL-10, but not IL-2, IFN-γ or TNF-α, tended to be increased in the plasma of HNSCC patients and 1,25(OH)(2)D(3) further increased plasma levels of all of these cytokines. While these cytokines tended to be increased in HNSCC tissue, 1,25(OH)(2)D(3) resulted in variable cytokine responses that showed a general tendency toward further increased levels. Levels of IL-8 and VEGF were increased in plasma and tissue of untreated HNSCC patients, and were further increased in plasma, but not in tissues, of patients treated with 1,25(OH)(2)D(3). Levels of IL-1α and IL-1β were similar in plasma of controls and HNSCC patients, but were increased in HNSCC tissues. In contrast to that seen in plasma where 1,25(OH)(2)D(3) increased levels of IL-1α and IL-1β, this was not seen in tissue following 1,25(OH)(2)D(3) treatment. These results show a discordant relationship between systemic and intratumoral cytokine profiles and suggest a tendency of 1,25(OH)(2)D(3)to increase a multitude of cytokines within tumor tissue.     

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis,Jun kinase activation,and interleukin-6 production in primary human keratinocytes

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.     

Hypercalcemia produced by parathyroid hormone suppresses experimental autoimmune encephalomyelitis in female but not male mice

Besides its role in regulating serum levels of calcium and phosphorus, 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) has potent effects on the immune system and suppresses disease in several animal models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While the amount of 1,25-(OH)2D3 needed to prevent EAE is dependent on the gender of the mouse and amount of calcium available in the diet, the minimum levels of 1,25-(OH)2D3 sufficient to prevent disease cause hypercalcemia. To test if hypercalcemia independent of high levels of 1,25-(OH)2D3 can suppress EAE, we used a 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase) knockout mouse strain. Because these 1alpha-hydroxylase knockout mice lack the parathyroid hormone (PTH)-regulated enzyme that synthesizes 1,25-(OH)2D3, hypercalcemia from increased bone turnover was created by continuous administration of PTH without changing the circulating levels of 1,25-(OH)2D3. This PTH-mediated hypercalcemia generated after EAE induction prevented disease in female mice but not male mice. When hypercalcemia was prevented by diet manipulation, PTH administration no longer prevented EAE. We conclude that hypercalcemia is able to prevent EAE after disease induction in female mice.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

1,25(OH)2D3 and calcipotriol (MC903) have similar effects on the induction of osteoclast-like cell formation in human bone marrow cultures

MC903 is a novel analogue of 1,25(OH)2D3 which exhibits similar inhibitory effects on cell proliferation and like, 1,25(OH)2D3, stimulates synthesis of osteoblast specific proteins by osteoblast-like cells in vitro. It is less active than 1,25(OH)2D3 in causing hypercalcemia in vivo. Since 1,25(OH)2D3 is known to stimulate bone resorption and increase the number of osteoclasts in several systems (in vivo and in vitro) we examined the effects of MC903 on the formation of osteoclast-like cells in vitro. As reported previously 1,25(OH)2D3 promoted the formation of multinucleated cells with phenotypic and functional characteristics of osteoclasts from adult human bone-marrow cultures at concentrations between 10(-8)M to 10(-12)M. Higher doses consistently suppressed multinucleated cell formation to values seen in the absence of 1,25(OH)2D3. Cells cultured in the presence of MC903 or for three weeks consistently induced the formation of multinucleated cells at concentrations 10(-8)M to 10(-12)M. As seen with 1,25(OH)2D3, MC903 also inhibited multinucleated cell formation at very high concentrations (10(-6)M). In two separate experiments MC903 appeared to be more potent than 1,25(OH)2D3 at lower concentrations (10(-10)M - 10(-12)M). From this study we conclude that MC903 is at least as potent as 1,25(OH)2D3 in inducing the formation human osteoclast-like cells in vitro. The decreased ability of MC903 to induce hypercalcemia in vivo is not therefore a result of a less marked effect than 1,25(OH)2D3 on the regulation of osteoclast formation.     

PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3)

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the anti-proliferative effects of 1,25(OH)(2)D(3). Since PTHrP increases the expression of the pro-invasive integrin alpha6beta4, we also asked whether 1,25(OH)(2)D(3) decreases integrin alpha6beta4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the effects of 1,25(OH)(2)D(3) on integrin alpha6beta4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (-36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin alpha6beta4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH)(2)D(3) decreased cell proliferation and integrin alpha6 and beta4 expression. These 1,25(OH)(2)D(3) effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH)(2)D(3) regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH)(2)D(3) on cell proliferation and integrin alpha6beta4 expression were significantly attenuated in cells overexpressing PTHrP (-36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH)(2)D(3) exerts its anti-proliferative effects is through down-regulation of PTHrP expression.     

1 alpha,25-Dihydroxyvitamin D(3) inhibits rat liver ultrastructural changes in diethylnitrosamine-initiated and phenobarbital promoted rat hepatocarcinogenesis

The active metabolite of vitamin D, 1 alpha,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] has been receiving increasing attention and has come to the forefront of cancer chemoprevention research as being a regulator of cellular growth, differentiation and death. In the present study, attempts have been made to investigate the in vivo chemopreventive effect of 1,25(OH)(2)D(3) in two-stage rat liver carcinogenesis. Hepatocarcinogenesis was initiated with a single intraperitoneal injection of diethylnitrosamine [DEN] (200 mg/kg b. wt.) at week 4. After a brief recovery period of 2 weeks, all the DEN-treated rats were given phenobarbital (0.05%) in the basal diet and continued thereafter till the completion of the experiment. The results of our experiment showed that the rats which received 1,25(OH)(2)D(3) for 14 weeks (0.3 microg/100 microL propylene glycol, per os, twice a week), starting the treatment 4 weeks prior to DEN injection, exhibited maximum protective effect in maintaining the normal cellular architecture of the hepatocytes than the group of rats which received this micronutrient for only 9 weeks. Moreover, continuous supplementation of 1,25(OH)(2)D(3) maintains the concentration of hepatic microsomal cytochrome P-450 like that of normal vehicle control. Thus, long-term supplementation of 1,25(OH)(2)D(3) significantly (P < 0.001) inhibits hepatic cytosolic lipid peroxidation, thereby protecting the cell membranes from free-radical mediated damage. These results suggest that 1,25(OH)(2)D(3) is useful in the inhibition of rat liver carcinogenesis.     

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.     

Enhancing effects of ceramide derivatives on 1,25-dihydroxyvitamin D(3)-induced differentiation of human HL-60 leukemia cells

Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] represents a useful approach for the treatment for cancer, including acute myeloid leukemia (AML). Recent studies demonstrated that the combined administration of 1,25-(OH)(2)D(3) and differentiation-enhancing agents could alleviate the side effects of 1,25-(OH)(2)D(3) and improve the rate of long term survival. In this study, we determined the enhancing activities of ceramide derivatives on 1,25-(OH)(2)D(3)-induced differentiation of human myeloid leukemia HL-60 cells. Importantly, some of these derivatives -- namely, A2, B3, and H9 -- enhanced the 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells in a concentration-dependent manner. In addition, the morphologic studies using Giemsa staining and flow cytometric analysis demonstrated that the combined treatment of 1,25-(OH)(2)D(3) with one of the three analogues, A2, B3, and H9, directed the HL-60 cells into monocytic lineage, but not into granulocytic lineage. The inhibition studies demonstrated that A2, B3, and H9, enhanced 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells via the PI3-K/PKC/JNK/ERK pathways. The ability of ceramide derivatives to enhance the differentiation-inducing potential of 1,25-(OH)(2)D(3) may contribute to an effective therapy for AML.     

p27Kip1 is essential for the antiproliferative action of 1,25-dihydroxyvitamin D3 in primary,but not immortalized,mouse embryonic fibroblasts

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of numerous cancer cell types. The intracellular proteins that mediate 1,25(OH)(2)D(3)-induced growth inhibition are poorly defined, although it is speculated that p21 and p27 are involved. We tested the requirement of p21 and p27 by treating primary wild-type, p21(-/-), and p27(-/-) mouse embryonic fibroblasts (MEFs) with 100 nm 1,25(OH)(2)D(3). In response to treatment, the wild-type and p21(-/-) MEFs exhibited 54 and 60% growth inhibition (p < 0.05), respectively, whereas the growth of p27(-/-) MEFs was unaffected. Western analyses indicated that p27 expression is induced by 1,25(OH)(2)D(3) treatment in wild-type and p21(-/-) MEFs. p21 expression is also induced by 1,25(OH)(2)D(3) treatment in wild-type and p27(-/-) MEFs, although the effect is less robust than for p27. Next, we spontaneously immortalized each MEF strain, which resulted in a gain of responsiveness to 1,25(OH)(2)D(3) by the p27(-/-) MEFs, as exhibited by 87% growth inhibition (p < 0.05). Both wild-type and p21(-/-) MEFs retained responsiveness (43 and 72% growth inhibition (p < 0.05), respectively). These data from primary and immortalized MEFs demonstrate that there are both p27-dependent and -independent pathways that mediate the antiproliferative action of 1,25(OH)(2)D(3).