Biomedical subjects

Milan Uskokovic

Publications and source records attributed to Milan Uskokovic.

14 recordsLinked to original sources

PubMed2006

Cooperative antitumor effects of vitamin D3 derivatives and rosemary preparations in a mouse model of myeloid leukemia.

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) is a powerful differentiation agent, which has potential for treatment of myeloid leukemias and other types of cancer, but the calcemia produced by pharmacologically active doses precludes the use of this agent in the clinic. We have shown that carnosic acid, the major rosemary polyphenol, enhances the differentiating and antiproliferative effects of low concentrations of 1,25D(3) in human myeloid leukemia cell lines (HL60, U937). Here we translated these findings to in vivo conditions using a syngeneic mouse leukemia tumor model. To this end, we first demonstrated that as in HL60 cells, differentiation of WEHI-3B D(-) murine myelomonocytic leukemia cells induced by 1 nM 1,25D(3) or its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol (Ro25-4020), can be synergistically potentiated by carnosic acid (10 microM) or the carnosic acid-rich ethanolic extract of rosemary leaves. This effect was accompanied by cell cycle arrest in G0 + G1 phase and a marked inhibition of cell growth. In the in vivo studies, i.p. injections of 2 microg Ro25-4020 in Balb/c mice bearing WEHI-3B D(-) tumors produced a significant delay in tumor appearance and reduction in tumor size, without significant toxicity. Another analog, 1,25-dihydroxy-16,23Z-diene-20-epi-26,27-hexafluoro-19-nor-cholecalciferol (Ro26-3884) administered at the same dose was less effective than Ro25-4020 and profoundly toxic. Importantly, combined treatment with 1% dry rosemary extract (mixed with food) and 1 microg Ro25-4020 resulted in a strong cooperative antitumor effect, without inducing hypercalcemia. These results indicate for the first time that a plant polyphenolic preparation and a vitamin D derivative can cooperate not only in inducing leukemia cell differentiation in vitro, but also in the antileukemic activity in vivo. These data may suggest novel protocols for chemoprevention or differentiation therapy of myeloid leukemia.

Abietanes↗

PubMed2006

Novel Gemini-vitamin D3 analog inhibits tumor cell growth and modulates the Akt/mTOR signaling pathway.

We have shown previously that 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3 (Gemini) compounds, which have two side chains attached to carbon-20, had increased anti-tumor activities against breast, prostate and leukemia cell lines in comparison to 1,25(OH)2 vitamin D3. This prompted us to synthesize additional Gemini compounds with further modifications and evaluate their anticancer effects. Most effective in this series was 1,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-vitamin D3 [Gemini-23-yne-26,27-hexafluoro-D3]. This analog was approximately 10-fold more potent than previously characterized Gemini compounds in inhibiting the clonal growth of HL-60, MCF-7 and LNCaP cell lines. Also in MCF-7 cells, Gemini-23-yne-26,27-hexafluoro-D3 caused dephosphorylation of the oncogenic kinase, Akt, resulting in dephosphorylation of the Akt target proteins, Forkhead transcription factor and mammalian target of rapamycin (mTOR). Downstream effectors of mTOR were also inhibited by the analog as demonstrated by decreased phosphorylation of both S6 kinase, and the translation inhibitor, 4E-BP1. The mTOR pathway regulates mRNA translation; exposure of MCF-7 cells to Gemini-23-yne-26,27-hexafluoro-D3 decreased their rate of protein synthesis and increased the association of 4EBP-1 with the translation initiation factor, eIF4E. Inhibition of the Akt-mTOR pathway represents a novel mechanism by which vitamin D3 analogs may modulate the expression and activity of proteins involved in cancer cell proliferation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Gene expression profiling changes induced by a novel Gemini Vitamin D derivative during the progression of breast cancer.

We investigated gene expression changes induced by a novel Gemini Vitamin D(3) analog, RO-438-3582 (1alpha,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-cholecalciferol, Ro3582), in a unique human breast MCF10 model. We used two breast epithelial cell lines from this model, namely MCF10AT1 (Ha-ras oncogene transfected MCF10A, early premalignant) and MCF10CA1a (fully malignant and metastatic derived from the MCF10AT1 line). We analyzed gene expression changes induced by Ro3582 using GeneChip technology, quantitative RT-PCR, Western blot analysis, or a gene transcription assay. Interestingly, we found distinct gene expression profile differences between Ro3582-induced response of the early premalignant MCF10AT1 and the malignant and metastatic MCF10CA1a cell lines. Moreover, while the Gemini Vitamin D(3) analog Ro3582 modulated the expression of several Vitamin D target genes such as the 24-hydroxylase, CD14, osteocalcin, and osteopontin in both cell lines, Ro3582 regulated many genes involved in cell proliferation and apoptosis, cell adhesion, invasion, angiogenesis as well as cell signaling pathways, such as the BMP and TGF-beta systems, differently in the two cell lines. The Gemini Vitamin D(3) analog Ro3582 induced more significant gene changes in the early premalignant MCF10AT1 cells than in the malignant metastatic MCF10CA1a cells, suggesting that Gemini Vitamin D(3) analogs may be more effective in preventing the progression of an early stage of breast carcinogenesis than in treating late stage breast cancer.

Apoptosis↗

PubMed2006

A novel vitamin D derivative activates bone morphogenetic protein signaling in MCF10 breast epithelial cells.

We investigated the action of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], a novel Gemini vitamin D(3) analog Ro-438-3582 [1alpha,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluorocholecalciferol (Ro3582)], and a classic vitamin D(3) analog Ro-26-2198 [1alpha,25-dihydroxy-16,23(Z)-diene-26,27-hexafluoro-19-nor-cholecalciferol (Ro2198)] in modulating the transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP) system in MCF10 immortalized breast epithelial cells. We found that 1alpha,25(OH)(2)D(3), Ro3582, and Ro2198 all enhanced BMP/Smad signaling by increasing the phosphorylation of receptor-regulated Smads. Ro3582 was more active than Ro2198, but both were considerably more active than 1alpha,25(OH)(2)D(3.) Ro3582 enhanced BMP/Smad signaling by 1) inducing the phosphorylation of receptor-regulated Smads (Smad1/5), 2) increasing the accumulation of phosphorylated Smad1/5 in the nucleus, and 3) activating BMP-mediated transcription in MCF10 breast epithelial cells. Furthermore, Ro3582 induced the synthesis of BMP-2 and BMP-6 mRNA and protein, and the expression of Smad6 mRNA in MCF10 breast epithelial cells was inhibited by Ro3582. The induction of phospho-Smad1/5 by Ro3582 was inhibited by treatment with the BMP antagonist Noggin, whereas neutralizing antibody to TGF-beta did not block the induction of phospho-Smad1/5 by Ro3582. Treatment with Noggin also blocked the effect of Ro3582 on nuclear accumulation of phospho-Smad1/5 and the induction of BMP-2 and BMP-6 mRNA synthesis. These results indicate that the activation of BMP/Smad signaling by the Gemini vitamin D(3) analog Ro3582 may be through the production of BMP ligands, including BMP-2 and BMP-6, and/or down-regulation of the inhibitory Smad6. This is the first report to show that 1alpha,25(OH)(2)D(3) and its derivatives activate BMP/Smad-specific signaling in human breast epithelial cells.

Antibodies↗

PubMed2006

The synthetic triterpenoid CDDO-imidazolide induces monocytic differentiation by activating the Smad and ERK signaling pathways in HL60 leukemia cells.

Synthetic triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) or CDDO-imidazolide [2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid imidazolide (CDDO-Im)], induce cell differentiation in myeloid leukemia cells but their mechanism of action is not known. CDDO-Im induces monocytic differentiation markers, CD14, and nonspecific esterase in HL60 leukemia cells. We show that CDDO-Im activates the extracellular signal-regulated kinase (ERK) signaling pathway and up-regulates CCAAT/enhancer-binding protein beta, a transcription factor critical for monocytic differentiation. The monocytic differentiation induced by CDDO-Im was partially blocked by the mitogen-activated protein kinase/ERK kinase 1 inhibitor PD98059, suggesting that the mitogen-activated protein kinase-ERK1/2 pathway plays a role in the differentiation induced by CDDO-Im. Furthermore, CDDO-Im activates the transforming growth factor beta (TGF-beta)/Smad signaling pathway. CDDO-Im enhanced the phosphorylation of the receptor-regulated Smads, phospho-Smad3, and phospho-Smad1/5, but not phospho-Smad2, and induced the expression of Smad4. Monocytic differentiation induced by CDDO-Im was blocked by both TGF-beta antibody and the bone morphogenetic protein (BMP) antagonist Noggin. This indicates that activation of the Smad signaling pathway by triterpenoids is an important mechanism of monocytic differentiation. CDDO-Im induced the synthesis of mRNA for TGF-beta2, BMP6, TGF-beta type II receptor, and BMP type II receptor. CDDO-Im synergized with members of the TGF-beta superfamily or with 1alpha,25(OH)2vitamin D3 (D3) in monocytic differentiation, and the synergistic effect was particularly striking in combination with D3. The combination of triterpenoids and D3 may have a practical use in differentiation therapy of myeloid leukemia as well as for promoting the formation of bone and cartilage.

Bone Morphogenetic Protein 6↗

PubMed2005

Analogs of 1alpha,25-dihydroxyvitamin D(3) as novel inhibitors of renin biosynthesis.

The renin-angiotensin system (RAS) plays a central role in the pathogenesis of hypertension. Recently, we discovered that 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] functions as a negative endocrine regulator of renin biosynthesis, which provides a molecular basis to explore the potential of Vitamin D analogs as renin inhibitors to control the RAS. To search for renin-inhibiting Vitamin D analogs, we screened 20 Vitamin D analog compounds using As4.1-hVDR cell (a juxtaglomerular cell line) culture by Northern blot and luciferase reporter assays. We found that the Gemini compounds, which have two side-chains at carbon-20 position, were particularly active in suppressing renin expression. Eight Gemini compounds were identified that were equally or 10- to 100-times more potent than 1,25(OH)(2)D(3) in renin inhibition. These Gemini compounds also potently stimulate 25-hydroxyvitamin D 24-hydroxylase expression in As4.1-hVDR cells. Administration of compound RO-27-5646 [1,25-dihydroxy-21-(3-methyl-3-hydroxy-butyl)-19-nor-cholecalciferol] in mice caused a marked reduction in renal renin mRNA expression without invoking severe hypercalcemia as seen in 1,25(OH)(2)D(3) treatment. These data establish in principle that Vitamin D analogs can indeed inhibit renin expression in vitro and in vivo, and support the notion that low calcemic Vitamin D analogs can potentially be used as therapeutic agents to control the RAS.

Animals↗

PubMed2004

Vitamin D: a negative endocrine regulator of the renin-angiotensin system and blood pressure.

The renin-angiotensin system (RAS) plays a central role in the regulation of blood pressure, volume and electrolyte homeostasis. Inappropriate activation of the RAS may lead to hypertension. Clinical and epidemiological studies have suggested a correlation between Vitamin D-deficiency and high blood pressure. Our recent studies demonstrate that Vitamin D is a potent endocrine suppressor of renin biosynthesis to regulate the RAS. Mice lacking the Vitamin D receptor (VDR) have elevated production of renin and angiotensin (Ang) II, leading to hypertension, cardiac hypertrophy and increased water intake. These abnormalities can be prevented by treatment with an ACE inhibitor or AT(1) receptor antagonist. Vitamin D repression of renin expression is independent of calcium metabolism, the volume- and salt-sensing mechanisms and the Ang II feedback regulation. In normal mice, Vitamin D-deficiency stimulates renin expression, whereas injection of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] reduces renin synthesis. In cell cultures, 1,25(OH)(2)D(3) directly suppresses renin gene transcription by a VDR-dependent mechanism. Furthermore, we have found that Gemini compounds have more potent renin-suppressing activity than 1,25(OH)(2)D(3). Collectively, our studies reveal a critical role of the Vitamin D endocrine system in the regulation of blood pressure and volume homeostasis, and suggest that low calcemic Vitamin D analogs may potentially be developed into a new class of anti-hypertensive agents to control renin production and blood pressure.

Blood Pressure↗

PubMed2004

Dendritic cells as key targets for immunomodulation by Vitamin D receptor ligands.

Vitamin D receptor (VDR) ligands, in addition to controlling calcium metabolism, exert important effects on the growth and differentiation of many cell types and possess pronounced pro-tolerogenic immunoregulatory activities. VDR ligands can act directly on T cells, but antigen-presenting cells (APCs), and in particular dendritic cells (DCs), appear to be primary targets for their tolerogenic properties. The capacity of VDR ligands to target APCs and T cells is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor NF-kB that is down-regulated in APCs and in T cells. VDR ligands can induce in vitro and in vivo tolerogenic DCs able to enhance CD4(+)CD25(+) suppressor T cells that, in turn, inhibit Th1 cell responses. These mechanisms of action can explain some of the immunoregulatory properties of VDR ligands, and are potentially relevant for the treatment of Th1-mediated autoimmune diseases and allograft rejection.

Dendritic Cells↗

PubMed2003

Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases.

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL-12 and enhanced IL-10 secretion. We have found that a short treatment with 1,25(OH)(2)D(3) induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. This effect is enhanced by co-administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)(2)D(3) on DCs. Graft acceptance is associated with an increased percentage of CD4(+)CD25(+) regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4(+)CD25(+) cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non-hypercalcemic doses. The enhancement of CD4(+)CD25(+) regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach.

Animals↗

PubMed2003

A low-calcemic vitamin D analog (Ro 25-4020) inhibits the growth of LNCaP human prostate cancer cells with increased potency by producing an active 24-oxo metabolite (Ro 29-9970).

In this study, we have characterized a novel less-calcemic vitamin D analog Ro 25-4020 (1alpha, 25 dihydroxy-16-ene-5,6-trans-vitamin D3) and investigated the mechanisms underlying its enhanced growth inhibitory properties. We found that Ro 25-4020 (IC50 = 0.3 nM) exhibited greater inhibitory activity than 1,25(OH)2D3 (IC50 = 1 nM) on LNCaP human prostate cancer cell growth. However, Ro 25-4020 was tenfold less active than 1,25(OH)2D3 in receptor-binding assays, ligand-induced heterodimerization and transactivation assays using VDR. HPLC and GC-MS analyses revealed that 1,25(OH)2D3 is converted to a 24-hydroxy metabolite, which has been shown to be less potent than 1,25(OH)2D3. In contrast, Ro 25-4020 was converted to a major 24-oxo metabolite that was more stable. Ligand-binding assays reveal that both Ro 25-4020 and its 24-oxo metabolite have similar affinity for VDR. Synthetic 24-oxo-Ro 25-4020, however, inhibited LNCaP cell proliferation as potently as 1,25(OH)2D3 and was more potent in transactivation of two out of three vitamin D target genes tested. These results suggest that conversion of Ro 25-4020 into an active and more stable 24-oxo metabolite with longer half-life contributes significantly to its potent antiproliferative actions on the LNCaP cells.

Animals↗

PubMed2002

Vitamin D3 analogs stimulate hair growth in nude mice.

The active form of vitamin D3 can regulate epidermal keratinization by inducing terminal differentiation; and mice lacking the vitamin D receptor display defects leading to postnatal alopecia. These observations implicate the vitamin D3 pathway in regulation of hair growth. We tested the ability of 1,25 dihydroxyvitamin D3 and its synthetic analogs to stimulate hair growth in biege/nude/xid (BNX) nu/nu (nude) mice exhibiting congenital alopecia. Nude mice were treated with different vitamin D3 analogs at doses that we had previously found to be the highest dose without inducing toxicity (hypercalcemia). The mice were monitored for hair growth and were scored according to a defined scale. Skin samples were taken for histological observation of hair follicles and for extraction of RNA and protein. Vitamin D3 analogs dramatically stimulated the hair growth of nude mice, although parental 1,25 dihydroxyvitamin D3 had no effect. Hair growth occurred in a cyclical pattern, accompanied by formation of normal hair follicles and increased expression of certain keratins (Ha7, Ha8, and Hb3). Vitamin D3 analogs seem to act on keratinocytes to initiate hair follicle cycling and stimulate hair growth in mice that otherwise do not grow hair.

Alopecia↗

PubMed2002

Cellular and molecular events associated with the bone-protecting activity of the noncalcemic vitamin D analog Ro-26-9228 in osteopenic rats.

We have examined several analogs of 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in an animal model of osteoporosis (ovariectomized rats) to identify a compound with a greater therapeutic range than 1,25-(OH)(2)D(3) for treatment of this bone disease. Here, we report that one analog, Ro-26-9228, had a bone-protecting effect but did not induce hypercalcemia at a wide concentration range. Analysis of biochemical markers and the bone histomorphometry of analog-treated rats suggested that Ro-26-9228 acted by inhibiting bone resorption and increasing the number of differentiated osteoblasts. To determine the basis for the segregation between hypercalcemia and bone-protecting action, we examined gene expression in tissues that regulate calcium homeostasis. We found that 1,25-(OH)(2)D(3) induced 24-hydroxylase mRNA expression in the duodena of ovariectomized rats, but Ro-26-9228 did not. Furthermore, in the duodena of intact animals, 1,25-(OH)(2)D(3) induced a significant increase in calbindin D 9K and plasma membrane calcium pump 1 mRNAs, but Ro-26-9228 had no effect on these mRNAs. On the other hand, the osteoblast-specific gene products osteocalcin and osteopontin were significantly up-regulated in trabecular bone by both the natural hormone and Ro-26-9228. Further investigation of gene-regulatory events in trabecular bone revealed that both 1,25-(OH)(2)D(3) and Ro-26-9228 up-regulated TGF beta1 and beta2 mRNAs. We concluded that the unique properties of Ro-26-9228 include preferential gene regulation in osteoblasts over duodenum and effective induction of growth factors in bone.

Animals↗

PubMed2002

A 1alpha,25-dihydroxyvitamin D(3) analog enhances regulatory T-cells and arrests autoimmune diabetes in NOD mice.

Type 1 diabetes is a chronic progressive autoimmune disease characterized by mononuclear cell infiltration, dominated by interleukin-12 (IL-12)-dependent Th1 cells, of the pancreatic islets, with subsequent destruction of insulin-producing beta-cells. Here, we demonstrate that treatment of adult nonobese diabetic (NOD) mice with an analog of 1alpha,25-dihydroxyvitamin D(3), an immunomodulatory agent preventing dendritic cell maturation, decreases lipopolysaccharide-induced IL-12 and gamma-interferon production, arrests Th1 cell infiltration and progression of insulitis, and inhibits diabetes development at nonhypercalcemic doses. Arrest of disease progression is accompanied by an enhanced frequency in the pancreatic lymph nodes of CD4(+)CD25(+) regulatory T-cells that are able to inhibit the T-cell response to the pancreatic autoantigen insulinoma-associated protein 2 and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells. Thus, a short treatment of adult NOD mice with an analog of 1,25-dihydroxyvitamin D(3) inhibits IL-12 production, blocks pancreatic infiltration of Th1 cells, enhances CD4(+)CD25(+) regulatory cells, and arrests the progression of type 1 diabetes, suggesting its possible application in the treatment of human autoimmune diabetes.

Animals↗

PubMedDate not supplied

Ability of potent vitamin D3 analogs to inhibit growth of prostate cancer cells in vivo.

BACKGROUND: Studies have identified analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which in vitro are 10- to 3,000-fold more active than 1,25(OH)2D3. We compared in vivo the anti-cancer activity of three potent vitamin D3 analogs and 1,25(OH)2D3 at near to each of their maximal tolerated dose (MTD). MATERIALS AND METHODS: Human LNCaP prostate cancer xenografts were grown in nude mice and the animals were treated with intraperitoneal injections of either diluant; 1,25(OH)2D3; 1,25-Dihydroxy-20epi-22-oxa-24,26,27-trisho-mocholecalciferol (KH 1060); 1,25-Dihydroxy-22E,24E-diene-24,26,27-trishomocholecalciferol (EB 1039); and 1,25-Dihydroxy-16-ene-24-oxo-19-norcholecalciferol (RO 26-9114). Tumor sizes were measured weekly and tumor weights were measured at autopsy on the 12th week. RESULTS: Each of the analogs equally and markedly inhibited growth of the prostate cancer xenografts. The 1,25(OH)2D3 initially inhibited growth but, by the time of sacrifice, the tumors were nearly the same size as diluant controls. The histological examination of the tumors showed that the analogs produced tumor necrosis and microcalcification. None of the mice developed hypercalcemia, which is the major toxicity of vitamin D3 compounds. CONCLUSION: The MTD of the analogs varied by 400-fold but each had similar efficacy suggesting that, when choosing a vitamin D analog for clinical study, overall efficacy without toxicity is more important than the total amount of the compound that can be administered. In summary, we have identified three vitamin D analogs that show marked potency in vivo to inhibit growth of human prostate cancer xenografts; each had no detectable toxicity. This study should help lay the foundation for clinical studies.

Animals↗