1α,25(OH)2D3 regulates pro-angiogenic factors in endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein coupled receptor.

When tumoral cell expansion exceeds the vascular supply, regions of hypoxia or low oxygen concentration are generated promoting the formation of new vessels through cell proliferation and migration. Viral G protein-coupled receptor (vGPCR) is associated to Kaposi's sarcoma pathology and induces a paracrine transformation when is stably expressed in murine endothelial cells activating hypoxia-induced transcription factors. Previously, we reported the antiproliferative actions of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in endothelial cells transformed by the vGPCR (SVEC-vGPCR). Herein, we further investigated if pro-angiogenic factors as AP-1, HIF-1α and VEGF are modulated by 1α,25(OH)2D3. We found by qRT-PCR analysis that the mRNA level of JunB, a negative regulator of cell proliferation, was similarly increased at all-time points tested after 1α,25(OH)2D3 treatment in SVEC-vGPCR cells. Also, mRNA levels of the pro-angiogenic factor c-Fos, which induces tumor invasion, were only decreased during one short period treatment. In addition, Hif-1α mRNA and protein levels were significantly reduced after 1α,25(OH)2D3 treatment in a VDR dependent fashion. However, mRNA levels of the angiogenic activator Vegf, promoted in turn by Hif-1α expression, were surprisingly high depending on VDR expression as well. Moreover, Egr-1, which has been reported to induce VEGF expression independently of HIF-1α, diminished its expression with 1α,25(OH)2D3 treatment, fact that was related to the decline of p-ERK1/2. Altogether, these results suggest a negative modulation of some pro-angiogenic factors like AP-1 and HIF-1α, as part of the antiproliferative mechanism of 1α,25(OH)2D3 in SVEC-vGPCR endothelial cells.

In Vitro Studies of Pegylated Magnetite Nanoparticles in a Cellular Model of Viral Oncogenesis: Initial Studies to Evaluate Their Potential as a Future Theranostic Tool.

Magnetic nanosystems represent promising alternatives to the traditional diagnostic and treatment procedures available for different pathologies. In this work, a series of biological tests are proposed, aiming to validate a magnetic nanoplatform for Kaposi's sarcoma treatment. The selected nanosystems were polyethylene glycol-coated iron oxide nanoparticles (MAG.PEG), which were prepared by the hydrothermal method. Physicochemical characterization was performed to verify their suitable physicochemical properties to be administered in vivo. Exhaustive biological assays were conducted, aiming to validate this platform in a specific biomedical field related to viral oncogenesis diseases. As a first step, the MAG.PEG cytotoxicity was evaluated in a cellular model of Kaposi's sarcoma. By phase contrast microscopy, it was found that cell morphology remained unchanged regardless of the nanoparticles' concentration (1-150 µg mL-1). The results, arising from the crystal violet technique, revealed that the proliferation was also unaffected. In addition, cell viability analysis by MTS and neutral red assays revealed a significant increase in metabolic and lysosomal activity at high concentrations of MAG.PEG (100-150 µg mL-1). Moreover, an increase in ROS levels was observed at the highest concentration of MAG.PEG. Second, the iron quantification assays performed by Prussian blue staining showed that MAG.PEG cellular accumulation is dose dependent. Furthermore, the presence of vesicles containing MAG.PEG inside the cells was confirmed by TEM. Finally, the MAG.PEG steering was achieved using a static magnetic field generated by a moderate power magnet. In conclusion, MAG.PEG at a moderate concentration would be a suitable drug carrier for Kaposi's sarcoma treatment, avoiding adverse effects on normal tissues. The data included in this contribution appear as the first stage in proposing this platform as a suitable future theranostic to improve Kaposi's sarcoma therapy.

Molecular and cellular outcomes of quercetin actions on healthy and tumor osteoblasts.

There is a global trend in the use of natural bioactive compounds to complement conventional therapies in bone diseases. In this work, we studied the effects of the phytoestrogen quercetin (QUE) in healthy and tumor osteoblasts. We found that QUE (1 µM, 48 h) significantly increased the cell number and the viability of healthy human osteoblasts (hFOB cells) determined by a trypan blue and a MTS assay, respectively, among other concentrations tested. In addition, wound healing and cellular adhesion assays also demonstrated that 1 µM of QUE significantly stimulated both parameters in osteoblasts. Moreover, osteoblast differentiation was also triggered by QUE in an osteogenic medium by measuring alkaline phosphatase activity, calcium deposition, and collagen levels. Herein, a concentration of 0.01 µM of QUE showed an increment in these differentiation markers and an activation of AKT/GSK3ß/ß-catenin pathway, determined by a Western blot analysis. In addition, immunocytochemistry and subcellular fraction studies indicated an increase of ß-catenin localization in the plasma membrane after QUE treatment. Otherwise, QUE (20-100 µM) decreased the cell number and the viability in tumor osteoblasts (ROS 17/2.8 cells) after 48 h. Furthermore, QUE (100 µM) decreased AKT(Ser473) and the pro-apoptotic protein BAD(Ser136) phosphorylation. In addition, the ERK1/2 phosphorylation increased leading to osteosarcoma cell death since pre-treatment with the MEK inhibitor PD98059 had reverted QUE effect. Altogether, these results indicate that low concentrations of QUE stimulate osteoblastogenesis but have no effect on the growth of tumor osteoblast cells, for which only high concentrations are efficient.

Down-regulation of COX-2 activity by 1α,25(OH)2D3 is VDR dependent in endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

Our previous reports showed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) has antiproliferative actions in endothelial cells stably expressing viral G protein-coupled receptor (vGPCR) associated with the pathogenesis of Kaposi's sarcoma. It has been reported that COX-2 enzyme, involved in the tumorigenesis of many types of cancers, is induced by vGPCR. Therefore, we investigated whether COX-2 down-regulation is part of the growth inhibitory effects of 1α,25(OH)2D3. Proliferation was measured in presence of COX-2 inhibitor Celecoxib (10-20 µM) revealing a decreased in vGPCR cell number, displaying typically apoptotic features in a dose dependent manner similarly to 1α,25(OH)2D3. In addition, the reduced cell viability observed with 20 µM Celecoxib was enhanced in presence of 1α,25(OH)2D3. Remarkably, although COX-2 mRNA and protein levels were up-regulated after 1α,25(OH)2D3 treatment, COX-2 enzymatic activity was reduced in a VDR-dependent manner. Furthermore, an interaction between COX-2 and VDR was revealed through GST pull-down and computational analysis. Additionally, high-affinity prostanoid receptors (EP3 and EP4) were found down-regulated by 1α,25(OH)2D3. Altogether, these results suggest a down-regulation of COX-2 activity and of prostanoid receptors as part of the antineoplastic mechanism of 1α,25(OH)2D3 in endothelial cells transformed by vGPCR.

In vitro studies revealed a downregulation of Wnt/ß-catenin cascade by active vitamin D and TX 527 analog in a Kaposi's sarcoma cellular model.

The Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously demonstrated that 1α,25(OH)2D3 or its less calcemic analog TX 527 exerts antiproliferative effects in endothelial cells stable expressing vGPCR. Since it is well documented that vGPCR activates the canonical Wnt/ß-catenin signaling pathway, the aim of this study was to evaluate if Wnt/ß-catenin cascade is target of 1α,25(OH)2D3 or TX 527 as part of their antineoplastic mechanism. Firstly, Western blot studies showed an increase in ß-catenin protein levels in a dose and time dependent manner; and when VDR was knockdown, ß-catenin protein levels were significantly decreased. Secondly, ß-catenin localization, investigated by immunofluorescence and subcellular fractionation techniques, was found increased in the nucleus and plasma membrane after 1α,25(OH)2D3 treatment. VE-cadherin protein levels were also increased in the plasma membrane fraction. Furthermore, ß-catenin interaction with VDR was observed by co-immunoprecipitation and mRNA expression of ß-catenin target genes was found decreased. Finally, DKK-1, the extracellular inhibitor of Wnt/ß-catenin pathway, showed an initial upregulation of mRNA expression. Altogether, the results obtained by different techniques revealed a downregulation of Wnt/ß-catenin cascade after 1α,25(OH)2D3 or TX 527 treatment, showing the foundation for a potential chemotherapeutic agent.

VDR agonists down regulate PI3K/Akt/mTOR axis and trigger autophagy in Kaposi's sarcoma cells.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously shown that 1α,25(OH)2D3 or its less-calcemic analog TX 527 inhibits the proliferation of endothelial cells expressing vGPCR, NF-κB activity and induces apoptosis in a VDR dependent manner. In this work, we further explored whether 1α,25(OH)2D3 or TX 527 regulates PI3K/Akt/mTOR axis and induces autophagy as part of its antineoplastic mechanism of action. Proliferation assays indicated that vGPCR cell number decreased in presence of LY294002 (PI3K/Akt inhibitor) likewise 1α,25(OH)2D3 or TX 527 (10 nM, 48 h). Also, Akt phosphorylation was found decreased in dose (0.1-100 nM) and time response studies (12-72 h) after both compounds treatments. In addition, decreased phosphorylated Akt was significantly observed in the nucleus. Moreover, regulation of Akt phosphorylation was NF-κB and VDR dependent. TNFAIP3/A20, an ubiquitin-editing enzyme, a direct NF-κB target gene and a negative regulator of Beclin-1, was down-regulated whereas Beclin-1 was up-regulated after 10 nM of 1α,25(OH)2D3 or TX 527 treatment. Decrement in Akt phosphorylation was accompanied by a reduced mTOR phosphorylation and an increase in the autophagy marker LC3-II. Since increment in autophagosomes not always indicates increment in autophagy activity, we used Chloroquine (CQ, 1 µM), an inhibitor of autophagy flow, to confirm autophagy after both VDR agonists treatment. In conclusion, VDR agonists, 1α,25(OH)2D3 or TX 527, inhibited PI3K/Akt/mTOR axis and induced autophagy in endothelial cells expressing vGPCR by a VDR-dependent mechanism.

In vitro 6-hydroxydopamine-induced neurotoxicity: New insights on NFκB modulation.

Neuronal exposure to 6-hydroxydopamine (6-OHDA), a hydroxylated analog of dopamine, constitutes a very useful strategy for studying the molecular events associated with neuronal death in Parkinson's disease. 6-OHDA increases oxidant levels and impairs mitochondrial respiratory chain, thus promoting neuronal injury and death. Despite the extensive use of 6-OHDA in animal models, the exact molecular events triggered by this neurotoxicant at the neuronal level have not been yet fully understood. Human IMR-32 neuroblastoma cells exposed to increasing concentrations of 6-OHDA displayed high levels of reactive oxygen species and increased plasma membrane permeability with concomitant cell viability diminution. As part of the neuronal response to 6-OHDA exposure, the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) p65 subunit was observed. NFκB nuclear localization was also accompanied by an increase of IκB phosphorylation as well as a rise in cyclooxygenase-2 (COX-2) and the prostaglandin receptor, EP4, mRNA levels. Even though the canonical pathways participating in the modulation of NFκB have been extensively described, here we tested the hypothesis that 6-OHDA-induced injury can activate lipid signaling and, in turn, modulate the transcriptional response. 6-OHDA challenge triggered the activation of lipid signaling pathways and increased phosphatidic acid (PA), diacylglycerol and free fatty acid levels in human neuroblastoma cells. The inhibition of PA production was able to prevent the decrease in cell viability triggered by 6-OHDA, the nuclear translocation of NFκB p65 subunit and the rise in COX-2 mRNA expression. Our results indicate that the onset of the inflammatory process triggered by 6-OHDA involves the activation of PA signaling that, in turn, governs NFκB subcellular localization and COX-2 expression.

Antineoplastic effect of 1α,25(OH)2D3 in spheroids from endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein coupled receptor.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. In endothelial cells, tumor maintenance and NF-κB activation depends on vGPCR constitutive expression and activity. We have previously demonstrated that 1α,25(OH)2D3 induces apoptosis in a VDR dependent manner, inhibits vGPCR cell growth and NF-κB activity. In this study, we developed a method to obtain multicellular spheroids (MCS) from endothelial cells expressing vGPCR in order to test whether MCS have a similar response to 2D-cultures after 1α,25(OH)2D3 treatment. Firstly, we found that vGPCR MCS started to form at 2nd day-growth, reaching a diameter up to 300 µm at 7th day-growth, whereas cells without vGPCR expression (SVEC) developed spheroids earlier and remained smaller throughout the period monitored. Secondly, vGPCR MCS size and architecture were analyzed during 1α,25(OH)2D3 (0.1-100 nM, 48 h) treatment. We found that once treated with 10 nM of 1α,25(OH)2D3 the initials MCS began a slight disaggregation with no changes in size; whereas at the higher dose (100 nM) the architecture of MCS was found completely broken. Furthermore, VDR mRNA expression increased significantly and this change was accompanied by a reduction of HIF-1α, an increase of VEGF, p21 and Bim mRNA expression. Finally, results from Western blot analysis showed that 1α,25(OH)2D3 decreased Akt and ERK1/2 protein phosphorylation. In conclusion, these data have revealed that 1α,25(OH)2D3 inhibits vGPCR MCS proliferation and induces apoptosis similar to vGPCR cells growing in 2D-cultures.

1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation.

We have previously shown that Solanum glaucophyllum leaf extract (SGE) increases VDR protein levels and promotes myoblast differentiation. Here, we investigated whether p38 MAPK and AKT are involved in SGE actions. Cell-cycle studies showed that SGE prompted a peak of S-phase followed by an arrest in the G0/G1-phase through p38 MAPK. Time course studies showed that p38 MAPK and AKT phosphorylation were statistically increased by SGE (10 nM) or synthetic 1α,25(OH)2D3 (1 nM) treatment. Furthermore, p38 MAPK and AKT inhibitors, SB203580 and LY294002 respectively, suppressed myoblasts fusion induced by SGE or synthetic 1α,25(OH)2D3 We have also studied differentiation genes by qRT-PCR. myoD1 mRNA increased significantly by SGE (24-72 h) or 1α,25(OH)2D3 (24 h) treatment. mRNA expression of myogenin also increased upon SGE or 1α,25(OH)2D3 treatment. Finally, MHC2b mRNA expression, a late differentiation marker, was increased significantly by both compounds at 72 h compared to control. Taken together, these results suggest that SGE, as synthetic 1α,25(OH)2D3, promotes myotube formation through p38 MAPK and AKT activation.

The proapoptotic protein Bim is up regulated by 1α,25-dihydroxyvitamin D3 and its receptor agonist in endothelial cells and transformed by viral GPCR associated to Kaposi sarcoma.

We have previously shown that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 induce apoptosis via caspase-3 activation in endothelial cells (SVEC) and endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we studied whether intrinsic apoptotic pathway could be activated by changing the balance between anti and pro-apoptotic proteins. Time response qRT-PCR analysis demonstrated that the mRNA level of anti-apoptotic gene Bcl-2 decreased after 12h and increased after 48h treatment with 1α,25(OH)2D3 or TX 527 in SVEC and vGPCR cells, whereas its protein level remained unchanged through time. mRNA levels of pro-apoptotic gene Bax significantly increased only in SVEC after 24 and 48h treatment with 1α,25(OH)2D3 and TX 527 although its protein levels remained unchanged in both cell lines. Bim mRNA and protein levels increased in SVEC and vGPCR cells. Bim protein increase by 1α,25(OH)2D3 and TX 527 was abolished when the expression of vitamin D receptor (VDR) was suppressed. On the other hand, Bortezomib (0.25-1nM), an inhibitor of NF-κB pathway highly activated in vGPCR cells, increased Bim protein levels and induced caspase-3 cleavage. Altogether, these results indicate that 1α,25(OH)2D3 and TX 527 trigger apoptosis by Bim protein increase which turns into the activation of caspase-3 in SVEC and vGPCR cells. Moreover, this effect is mediated by VDR and involves NF-κB pathway inhibition in vGPCR.

Cell cycle arrest and apoptosis induced by 1α,25(OH)2D3 and TX 527 in Kaposi sarcoma is VDR dependent.

We have previously shown that 1α,25(OH)2-Vitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 inhibit the proliferation of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR) and this could be partially explained by the inhibition of the NF-κB pathway. In this work, we further explored the mechanism of action of both vitamin D compounds in Kaposi sarcoma. We investigated whether the cell cycle arrest and subsequent apoptosis of endothelial cells (SVEC) and SVEC transformed by vGPCR (SVEC-vGPCR) elicited by 1α,25(OH)2D3 and TX 527 were mediated by the vitamin D receptor (VDR). Cell cycle analysis of SVEC and SVEC-vGPCR treated with 1α,25(OH)2D3 (10nM, 48h) revealed that 1α,25(OH)2D3 increased the percentage of cells in the G0/G1 phase and diminished the percentage of cells in the S phase of the cell cycle. Moreover, the number of cells in the S phase was higher in SVEC-vGPCR than in SVEC due to vGPCR expression. TX 527 exerted similar effects on growth arrest in SVEC-vGPCR cells. The cell cycle changes were suppressed when the expression of the VDR was blocked by a stable transfection of shRNA against VDR. Annexin V-PI staining demonstrated apoptosis in both SVEC and SVEC-vGPCR after 1α,25(OH)2D3 and TX 527 treatment (10nM, 24h). Cleavage of caspase-3 detected by Western blot analysis was increased to a greater extent in SVEC than in SVEC-vGPCR cells, and this effect was also blocked in VDR knockdown cells. Altogether, these results suggest that 1α,25(OH)2D3 and TX 527 inhibit the proliferation of SVEC and SVEC-vGPCR and induce apoptosis by a mechanism that involves the VDR.

Age-related changes in the response of intestinal cells to 1α,25(OH)2-vitamin D3.

The hormonally active form of vitamin D(3), 1α,25(OH)(2)-vitamin D(3), acts in intestine, its major target tissue, where its actions are of regulatory and developmental importance: regulation of intracellular calcium through modulation of second messengers and activation of mitogenic cascades leading to cell proliferation. Several causes have been postulated to modify the hormone response in intestinal cells with ageing, among them, alterations of vitamin D receptor (VDR) levels and binding sites, reduced expression of G-proteins and hormone signal transduction changes. The current review summarizes the actual knowledge regarding the molecular and biochemical basis of age-impaired 1α,25(OH)(2)-vitamin D(3) receptor-mediated signaling in intestinal cells. A fundamental understanding why the hormone functions are impaired with age will enhance our knowledge of its importance in intestinal cell physiology.

NFκB pathway is down-regulated by 1α,25(OH)(2)-vitamin D(3) in endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein coupled receptor.

We have previously demonstrated that 1α,25 dihydroxy-vitamin D(3) (1α,25(OH)(2)D(3)) has antiproliferative effects on the growth of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we have investigated whether 1α,25(OH)(2)D(3) exerts its growth inhibitory effects by inhibiting the Nuclear Factor κ B (NFκB) pathway which is highly activated by vGPCR. Cell proliferation studies demonstrated that 1α,25(OH)(2)D(3), similarly to bortezomib, a proteosome inhibitor that suppresses the activation of NFκB, reduced the proliferation of endothelial cells transformed by vGPCR (SVEC-vGPCR). The activity of NFκB in these cells decreased by 70% upon 1α,25(OH)(2)D(3) treatment. Furthermore, time and dose response studies showed that the hormone significantly decreased NFκB and increased IκBα mRNA and protein levels in SVEC-vGPCR cells, whereas in SVEC only IκBα increased significantly. Moreover, NFκB translocation to the nucleus was inhibited and occurred by a mechanism independent of NFκB association with vitamin D(3) receptor (VDR). 1α,25(OH)(2)D(3)-induced increase in IκBα required de novo protein synthesis, and was independent of MAPK and PI3K/Akt pathways. Altogether, these results suggest that down-regulation of the NFκB pathway is part of the mechanism involved in the antiproliferative effects of 1α,25(OH)(2)D(3) on endothelial cells transformed by vGPCR.

[Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3]. / Vitamina D y cáncer: acción antineoplásica de la 1α,25(OH)2-vitamina D3.

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.

Vitamina D y cáncer: acción antineoplásica de la 1α, 25(OH)2 -vitamina D3 / Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3

La forma hormonalmente activa de la vitamina D, 1α,25(OH)2-vitamina D3 (1α,25(OH)2D3), además de desempeñar un rol crucial en el mantenimiento de la homeostasis de calcio en el cuerpo, también regula el crecimiento y la diferenciación de diferentes tipos celulares, incluyendo células cancerosas. Actualmente hay numerosos estudios que investigan los efectos de la hormona en estas células, debido al interés en el uso terapéutico del 1α,25(OH)2D3 y de análogos con menor actividad calcémica para el tratamiento o prevención del cáncer. En este trabajo de revisión se describe el sistema endocrino de la vitamina D, su mecanismo de acción, su acción antineoplásica y se provee información sobre los últimos avances en el estudio de nuevos análogos de la hormona con menos actividad calcémica para el tratamiento del cáncer.

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.

Vitamina D y cáncer: acción antineoplásica de la 1α, 25(OH)2 -vitamina D3 / Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3

La forma hormonalmente activa de la vitamina D, 1α,25(OH)2-vitamina D3 (1α,25(OH)2D3), además de desempeñar un rol crucial en el mantenimiento de la homeostasis de calcio en el cuerpo, también regula el crecimiento y la diferenciación de diferentes tipos celulares, incluyendo células cancerosas. Actualmente hay numerosos estudios que investigan los efectos de la hormona en estas células, debido al interés en el uso terapéutico del 1α,25(OH)2D3 y de análogos con menor actividad calcémica para el tratamiento o prevención del cáncer. En este trabajo de revisión se describe el sistema endocrino de la vitamina D, su mecanismo de acción, su acción antineoplásica y se provee información sobre los últimos avances en el estudio de nuevos análogos de la hormona con menos actividad calcémica para el tratamiento del cáncer.(AU)

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.(AU)

Vitamina D y cáncer: acción antineoplásica de la 1α, 25(OH)2 -vitamina D3 / Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3

La forma hormonalmente activa de la vitamina D, 1α,25(OH)2-vitamina D3 (1α,25(OH)2D3), además de desempeñar un rol crucial en el mantenimiento de la homeostasis de calcio en el cuerpo, también regula el crecimiento y la diferenciación de diferentes tipos celulares, incluyendo células cancerosas. Actualmente hay numerosos estudios que investigan los efectos de la hormona en estas células, debido al interés en el uso terapéutico del 1α,25(OH)2D3 y de análogos con menor actividad calcémica para el tratamiento o prevención del cáncer. En este trabajo de revisión se describe el sistema endocrino de la vitamina D, su mecanismo de acción, su acción antineoplásica y se provee información sobre los últimos avances en el estudio de nuevos análogos de la hormona con menos actividad calcémica para el tratamiento del cáncer.(AU)

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.(AU)

1 Alpha,25-dihydroxyvitamin D3 and its TX527 analog inhibit the growth of endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein-coupled receptor in vitro and in vivo.

The Kaposi sarcoma-associated herpes virus-G protein-coupled receptor is a key molecule in the pathogenesis of Kaposi sarcoma, playing a central role in promoting vascular endothelial growth factor-driven angiogenesis and spindle cell proliferation. We studied the effects of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] and the analog TX527 on the proliferation of endothelial cells (SVECs) and SVECs transformed by the viral G protein-coupled receptor (SVEC-vGPCR). 1 alpha,25(OH)(2)D(3) and TX527 decreased SVEC-vGPCR and SVEC numbers, the response being time dependent and similar in both cell lines. Vitamin D receptor (VDR) levels increased on treatment with 10 nm 1 alpha,25(OH)(2)D(3) or 1 nm TX527 in a time-dependent manner (1.5-24 h) in SVECs and SVEC-vGPCR. Basal VDR levels were increased in SVEC-vGPCR. The antiproliferative effects were accompanied by reduction in cyclin D1 and accumulation of p27 in SVECs but not SVEC-vGPCR. Induction of VDR was blocked by transfection of short hairpin RNA against VDR in SVEC-vGPCR and the antiproliferative effects of 1 alpha,25(OH)(2)D(3) and TX527 were decreased, involving the VDR genomic pathway in the hormone and analog mechanism of action. In vivo experiments showed that 1 alpha,25(OH)(2)D(3) and TX527 decreased SVEC-vGPCR tumor progression when the tumor cells were implanted in nude mice. In conclusion, we have demonstrated that 1 alpha,25(OH)(2)D(3) and its TX527 analog have antiproliferative effects on the growth of endothelial cells transformed by the vGPCR in vitro and in vivo, the vitamin D receptor being part of the inhibitory mechanism of action.

Vitamin D receptor levels and binding are reduced in aged rat intestinal subcellular fractions.

The hormonal form of vitamin D, 1alpha,25(OH)(2)-vitaminD(3) [1alpha,25(OH)(2)D(3)], stimulates signal transduction pathways in intestinal cells. To gain insight into the relative importance of the vitamin D receptor (VDR) in the rapid hormone responses, the amounts and localization of the VDR were evaluated in young (3 months) and aged (24 months) rat intestinal cells. Immune-fluorescence and Western blot studies showed that VDR levels are diminished in aged enterocytes. Confocal microscopy assays revealed that the VDR and other immune-reactive proteins have mitochondrial, membrane, cytosol and perinuclear localization. Western blot analysis using specific antibodies detected the 60 and 50 kDa bands expected for the VDR in the cytosol and microsomes and, to a lesser extent, in the nucleus and mitochondria. Low molecular weight immune-reactive proteins were also detected in young enterocytes subcellular fractions. Since changes in hormone receptor levels appear to constitute a common manifestation of the ageing process, we also analyzed 1alpha,25(OH)(2)D(3) binding properties and VDR levels in subcellular fractions from young and aged rats. In competition binding assays, employing [(3)H]-1alpha,25(OH)(2)D(3) and 1alpha,25(OH)(2)D(3), we have detected specific binding in all subcellular fractions, with maximum binding in mitochondrial and nuclear fractions. Both, VDR protein levels and 1alpha,25(OH)(2)D(3) binding, were diminished with ageing. Age-related declines in VDR may have important consequences for correct receptor/effector coupling in the duodenal tissues and may explain age-related declines in the hormonal regulation of signal transduction pathways that we previously reported.

Nongenomic action of 1 alpha,25(OH)(2)-vitamin D3. Activation of muscle cell PLC gamma through the tyrosine kinase c-Src and PtdIns 3-kinase.

We have previously demonstrated that the steroid hormone 1 alpha,25(OH)(2)-vitamin D(3)[1 alpha,25(OH)(2)D(3)] stimulates the production of inositol trisphosphate (InsP(3)), the breakdown product of phosphatidylinositol 4,5-biphosphate (PtdInsP(2)) by phospholipase C (PtdIns-PLC), and activates the cytosolic tyrosine kinase c-Src in skeletal muscle cells. In the present study we examined whether 1 alpha,25(OH)(2)D(3) induces the phosphorylation and membrane translocation of PLC gamma and the mechanism involved in this isozyme activation. We found that the steroid hormone triggers a significant phosphorylation on tyrosine residues of PLC gamma and induces a rapid increase in membrane-associated PLC gamma immunoreactivity with a time course that correlates with that of phosphorylation in muscle cells. Genistein, a tyrosine kinase inhibitor, blocked the phosphorylation of PLC gamma. Inhibition of 1 alpha,25(OH)(2)D(3)-induced c-Src activity by its specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA, prevented hormone stimulation of PLC gamma tyrosine phosphorylation. The isozyme phosphorylation is also blocked by both wortmannin and LY294002, two structurally different inhibitors of phosphatidyl inositol 3-kinase (PtdIns3K), the enzyme that produces PtdInsP(3) known to activate PLC gamma isozymes specifically by interacting with their SH2 and pleckstrin homology domains. The hormone also increases the physical association of c-Src and PtdIns3K with PLC gamma and induces a c-Src-dependent tyrosine phosphorylation of the p85 regulatory subunit of PtdIns3K. The time course of hormone-dependent PLC gamma phosphorylation closely correlates with the time course of its redistribution to the membrane, suggesting that phosphorylation and redistribution to the membrane of PLC gamma are two interdependent events. 1 alpha,25(OH)(2)D(3)-induced membrane translocation of PLC gamma was prevented to a great extent by c-Src and PtdIns3K inhibitors, PP1 and LY294002. Taken together, the present data indicates that the cytosolic tyrosine kinase c-Src and PtdIns 3-kinase play indispensable roles in 1 alpha,25(OH)(2)D(3) signal transduction cascades leading to PLC gamma activation.