Publications by authors named "Verónica González-Pardo"

20 Publications

  • Page 1 of 1

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

Heliyon 2020 Oct 2;6(10):e05149. Epub 2020 Oct 2.

Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Bahía Blanca, Argentina.

Our previous reports showed that 1α,25-dihydroxyvitamin D (1α,25(OH)D) 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)D. 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)D. In addition, the reduced cell viability observed with 20 μM Celecoxib was enhanced in presence of 1α,25(OH)D. Remarkably, although COX-2 mRNA and protein levels were up-regulated after 1α,25(OH)D 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)D 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)D in endothelial cells transformed by vGPCR.
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http://dx.doi.org/10.1016/j.heliyon.2020.e05149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549067PMC
October 2020

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

Toxicol In Vitro 2020 Mar 12;63:104748. Epub 2019 Dec 12.

Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)-CONICET, San Juan 670, 8000 Bahía Blanca, Argentina. Electronic address:

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)D 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/β-catenin signaling pathway, the aim of this study was to evaluate if Wnt/β-catenin cascade is target of 1α,25(OH)D or TX 527 as part of their antineoplastic mechanism. Firstly, Western blot studies showed an increase in β-catenin protein levels in a dose and time dependent manner; and when VDR was knockdown, β-catenin protein levels were significantly decreased. Secondly, β-catenin localization, investigated by immunofluorescence and subcellular fractionation techniques, was found increased in the nucleus and plasma membrane after 1α,25(OH)D treatment. VE-cadherin protein levels were also increased in the plasma membrane fraction. Furthermore, β-catenin interaction with VDR was observed by co-immunoprecipitation and mRNA expression of β-catenin target genes was found decreased. Finally, DKK-1, the extracellular inhibitor of Wnt/β-catenin pathway, showed an initial upregulation of mRNA expression. Altogether, the results obtained by different techniques revealed a downregulation of Wnt/β-catenin cascade after 1α,25(OH)D or TX 527 treatment, showing the foundation for a potential chemotherapeutic agent.
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http://dx.doi.org/10.1016/j.tiv.2019.104748DOI Listing
March 2020

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

Heliyon 2019 Aug 27;5(8):e02367. Epub 2019 Aug 27.

Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Departamento de Biología Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)-CONICET, San Juan 670, 8000, Bahía Blanca, Argentina.

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)D 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)D 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)D 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)D 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)D or TX 527, inhibited PI3K/Akt/mTOR axis and induced autophagy in endothelial cells expressing vGPCR by a VDR-dependent mechanism.
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http://dx.doi.org/10.1016/j.heliyon.2019.e02367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722267PMC
August 2019

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

Toxicol In Vitro 2019 Oct 25;60:400-411. Epub 2019 Jun 25.

Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Argentina; Departamento de Biología, Bioquímica y Farmacia-Universidad Nacional del Sur (UNS), Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Bahía Blanca, Argentina. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tiv.2019.06.019DOI Listing
October 2019

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

J Steroid Biochem Mol Biol 2019 02 9;186:122-129. Epub 2018 Oct 9.

Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Departamento de Biología Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)-CONICET, San Juan 670, 8000 Bahía Blanca, Argentina. Electronic address:

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)D 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)D treatment. Firstly, we found that vGPCR MCS started to form at 2 day-growth, reaching a diameter up to 300 μm at 7 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)D (0.1-100 nM, 48 h) treatment. We found that once treated with 10 nM of 1α,25(OH)D 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)D decreased Akt and ERK1/2 protein phosphorylation. In conclusion, these data have revealed that 1α,25(OH)D inhibits vGPCR MCS proliferation and induces apoptosis similar to vGPCR cells growing in 2D-cultures.
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http://dx.doi.org/10.1016/j.jsbmb.2018.10.004DOI Listing
February 2019

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

Biol Open 2018 May 14;7(5). Epub 2018 May 14.

Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Universidad Nacional del Sur-CONICET, 8000 Bahía Blanca, Argentina

We have previously shown that 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)D (1 nM) treatment. Furthermore, p38 MAPK and AKT inhibitors, SB203580 and LY294002 respectively, suppressed myoblasts fusion induced by SGE or synthetic 1α,25(OH)D We have also studied differentiation genes by qRT-PCR. mRNA increased significantly by SGE (24-72 h) or 1α,25(OH)D (24 h) treatment. mRNA expression of also increased upon SGE or 1α,25(OH)D treatment. Finally, 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)D, promotes myotube formation through p38 MAPK and AKT activation.
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http://dx.doi.org/10.1242/bio.033670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992525PMC
May 2018

In vitro effects of 1α,25(OH)₂D₃-glycosides from Solbone A (Solanum glaucophyllum leaves extract; Herbonis AG) compared to synthetic 1α,25(OH)₂D₃ on myogenesis.

Steroids 2016 May 8;109:7-15. Epub 2016 Mar 8.

INBIOSUR-CONICET-Depto. Biología Bioquímica & Farmacia, Universidad Nacional del Sur, Bahía Blanca 8000, Argentina. Electronic address:

The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts. For these, differentiation markers and myogenic parameters were studied in C2C12 myoblasts. Results showed that Solbone A, likewise the synthetic hormone, increased creatine kinase activity at day 2 after differentiation induction (60%, p<0.05). Solbone A and synthetic 1α,25(OH)2D3 increased vitamin D3 receptor protein expression at 10nM (50% and 30%, respectively) and the transcription factor myogenin (80%, p<0.05). However, tropomyosin expression was not affected by both compounds. In addition, myosin heavy chain (MHC) protein expression was increased 30% at day 2 of differentiation. Solbone A or synthetic 1α,25(OH)2D3 had no effects on myogenin nor MHC cell localization. Cellular mass increased with myogenesis progression, being Solbone A more effective than synthetic 1α,25(OH)2D3. Finally, Solbone A, as well as synthetic 1α,25(OH)2D3, augmented the index fusion of cultured muscle fibers. In conclusion, these results demonstrated that Solbone A exhibit at least equal or greater effects on early myoblast differentiation as synthetic hormone, suggesting that plant glycosides could be an effective, accessible and cheaper substitute for synthetic 1α,25(OH)2D3 to promote muscle growth.
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http://dx.doi.org/10.1016/j.steroids.2016.03.002DOI Listing
May 2016

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.

Steroids 2015 Oct 6;102:85-91. Epub 2015 Aug 6.

INBIOSUR (CONICET-UNS), 8000 Bahía Blanca, Argentina. Electronic address:

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.
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http://dx.doi.org/10.1016/j.steroids.2015.08.005DOI Listing
October 2015

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

J Steroid Biochem Mol Biol 2014 Oct 5;144 Pt A:197-200. Epub 2013 Dec 5.

Departamento de Biología, Bioquímica & Farmacia, Universidad Nacional del Sur-CONICET, 8000 Bahía Blanca, Argentina.

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.
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http://dx.doi.org/10.1016/j.jsbmb.2013.11.014DOI Listing
October 2014

Role of VDR in 1α,25-dihydroxyvitamin D3-dependent non-genomic activation of MAPKs, Src and Akt in skeletal muscle cells.

J Steroid Biochem Mol Biol 2013 Jul 5;136:125-30. Epub 2013 Mar 5.

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, 8000 Bahía Blanca, Argentina.

1α,25-dihydroxyvitamin D3 [1,25D] is recognized as a steroid hormone that rapidly elicits intracellular signals in various tissues. In skeletal myoblasts, we have previously demonstrated that one of the 1,25D-induced non-genomic effects is the upstream stimulation of MAPKs through Src activation. In this work, the data obtained suggest that the classical receptor of vitamin D (VDR) participates in non-transcriptional actions of 1,25D. We significantly reduced VDR expression by infection of C2C12 murine myoblasts with lentiviral particles containing the pLKO.1 plasmid with information to express a shRNA against mouse VDR. In these cells (C2C12-shVDR), Western blot analyses show that 1,25D-induced p38 MAPK activation and Src tyr416 phosphorylation were abolished. In addition, 1,25D-dependent activity of ERK1/2 was diminished in cells lacking VDR but to a lesser extent (∼-60%). Phosphorylation of Akt by 1,25D, recently demonstrated in C2C12 cells, in the present work also appeared to be partially dependent on VDR expression (∼50% in C2C12-shVDR cells). Our results indicate that VDR is involved in 1,25D-induced rapid events related to survival/proliferation responses in skeletal muscle cells, providing relevant information on the mechanism of initiation of the non-genomic hormone signal. The participation of a VDR-independent non-genomic mechanism of action should also be taken into consideration. This article is part of a Special Issue entitled 'Vitamin D Workshop'.
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http://dx.doi.org/10.1016/j.jsbmb.2013.02.013DOI Listing
July 2013

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

Ageing Res Rev 2013 Jan 15;12(1):76-89. Epub 2012 Jun 15.

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Consejo Nacional de Investigaciones Científicas y Técnicas, Bahía Blanca, Argentina.

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.
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http://dx.doi.org/10.1016/j.arr.2012.06.001DOI Listing
January 2013

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.

Steroids 2012 Sep 7;77(11):1025-32. Epub 2012 Jun 7.

Departamento de Biología, Bioquímica & Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.

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.
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http://dx.doi.org/10.1016/j.steroids.2012.05.006DOI Listing
September 2012

[Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3].

Medicina (B Aires) 2012 ;72(2):143-9

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

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.
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November 2014

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.

Endocrinology 2010 Jan 13;151(1):23-31. Epub 2009 Nov 13.

Departamento de Biología Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.

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.
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http://dx.doi.org/10.1210/en.2009-0650DOI Listing
January 2010

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

Biogerontology 2008 Apr 1;9(2):109-18. Epub 2007 Dec 1.

Departamento de Biología, Bioquímica & Farmacia, Universidad Nacional del Sur, San Juan 670, 8000, Bahia Blanca, Argentina.

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.
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http://dx.doi.org/10.1007/s10522-007-9118-2DOI Listing
April 2008

Age-related alteration of 1alpha,25(OH)2-vitamin D3-dependent activation of p38 MAPK in rat intestinal cells.

Biogerontology 2007 Feb 20;8(1):13-24. Epub 2006 Jul 20.

Departamento de Biología, Bioquímica & Farmacia, Universidad Nacional del Sur, San Juan 670, Bahia Blanca 8000, Argentina.

In intestinal cells, 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) regulates gene expression via the specific intracellular vitamin D receptor and induces fast non-transcriptional responses involving stimulation of transmembrane signal transduction pathways. In the present study, we analyzed, for the first time, alterations in p38 MAPK response to 1alpha,25(OH)(2)D(3) in rat enterocytes with ageing. In enterocytes from young rats, the hormone increased, in a time- and dose-dependent fashion, the phosphorylation of p38 MAPK, peaking at 3 min (+2-fold). Basal levels of p38 MAPK phosphorylation were lower in enterocytes from old rats and the hormone response was greatly diminished (+0.5-fold at 3 min). p38 MAPK phosphorylation impairment in old animals was not related to significant changes of the kinase protein expression and do not explain the decreased response to 1alpha,25(OH)(2)D(3). Extracellular and intracellular Ca(2+) chelation or c-Src pharmacological inhibition suppressed hormone activation of p38 MAPK in both, young and aged rats, demonstrating that Ca(2+) and the non-receptor tyrosine kinase c-Src are required for full activation of p38 MAPK in cells stimulated with 1alpha,25(OH)(2)D(3). Two other vitamin D(3) metabolites, 25(OH)D(3) and 24,25(OH)(2)D(3, )also enhanced p38 phosphorylation, and to a similar extent than 1alpha,25(OH)(2)D(3), an ability that is lost with ageing. Enterocyte exposure to the hormone also resulted in the rapid induction of c-fos protein (peaking at 5 min, +3-fold) and to a greater extent than that of mRNA induction. With ageing, 1alpha,25(OH)(2)D(3)-dependent increase of c-fos protein level was diminished, but c-fos mRNA expression was not different from young animals. Impairment of 1alpha,25(OH)(2)D(3) activation of p38 MAPK upon ageing and abnormal hormone regulation of the c-fos oncoprotein synthesis may affect intestinal cell function.
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http://dx.doi.org/10.1007/s10522-006-9031-0DOI Listing
February 2007

1alpha,25(OH)(2)-Vitamin D(3) stimulates intestinal cell p38 MAPK activity and increases c-Fos expression.

Int J Biochem Cell Biol 2006 24;38(7):1181-90. Epub 2006 Jan 24.

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, 8000 Bahía Blanca, Argentina.

In intestinal cells, as in other target cells, the steroid hormone 1alpha,25(OH)(2)-Vitamin D(3) (1alpha,25(OH)(2)D(3)) regulates gene expression via the specific intracellular Vitamin D receptor and induces fast non-transcriptional responses involving stimulation of transmembrane signal transduction pathways. We have previously shown that the hormone activates the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2 in rat intestinal cells. In the present study, we have demonstrated that 1alpha,25(OH)(2)D(3) also induces the phosphorylation and activation of p38 MAPK in these cells. The hormone effects were time and dose-dependent, with maximal stimulation at 2min (+3-fold) and 1nM. 1alpha,25(OH)(2)D(3)-dependent p38 phosphorylation was suppressed by SB 203580, a selective inhibitor of p38 MAPK. Ca(2+) chelation with EGTA, inhibition of the c-Src-tyrosine kinase family with PP1 or protein kinase A (PKA) with Rp-cAMP, attenuated hormone activation of p38 MAPK. The physiological significance of 1alpha,25(OH)(2)D(3)-dependent activation of ERK1/2 and p38 MAP kinases was addressed by monitoring c-Fos expression. Incubation of intestinal cells with the hormone was followed by a rapid induction of c-Fos expression which was blocked by SB 203580 and partially suppressed by the ERK1/2 inhibitor PD 98059. Our results suggest that 1alpha,25(OH)(2)D(3) activates p38 MAPK, involving Ca(2+), c-Src and PKA as upstream regulators, and that p38 MAPK has a central role in hormone-induction of the oncoprotein c-Fos in rat intestinal cells.
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http://dx.doi.org/10.1016/j.biocel.2005.12.018DOI Listing
August 2006

Tyrosine phosphorylation signalling dependent on 1alpha,25(OH)2-vitamin D3 in rat intestinal cells: effect of ageing.

Int J Biochem Cell Biol 2004 Mar;36(3):489-504

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur., 8000 Bahía Blanca, Argentina.

In intestinal cells, as in other target cells, 1alpha,25(OH)(2)D(3) elicits long-term and short-term responses which involve genomic and non-genomic mode of actions, respectively. There is evidence indicating that activation of tyrosine phosphorylation pathways may participate in the responses induced by 1alpha,25(OH)(2)D(3) through its non-genomic mechanism. In this study we have evaluated the involvement of 1alpha,25(OH)(2)D(3) in the tyrosine phosphorylation of PLCgamma and MAPK (ERK1/2) in enterocytes from young (3 months) and aged (24 months) rats. Immunochemical analysis revealed that the hormone stimulates PLCgamma tyrosine phosphorylation in young rat enterocytes. Hormone effect on PLCgamma is rapid, peaking at 2 min (+100%), is dose-dependent (10(-10) to 10(-8)M) and decreases with ageing. 1alpha,25(OH)(2)D(3) also induces the phosphorylation and activation of the mitogen-activated-protein kinases ERK1 and ERK2, effect which was evident at 1 min (three-fold) and reached a maximum at 2 min (six-fold). Hormone-dependent ERK1 and ERK2 phosphorylation and activity is greatly reduced in enterocytes from old rats. In both, young and aged animals, 1alpha,25(OH)(2)D(3)-induced PLCgamma and ERK1/2 phosphorylation was effectively suppressed by the tyrosine kinase inhibitor genistein (100 uM) and suppressed to a great extent by PP1, an inhibitor of c-Src kinases. LY294002, a specific inhibitor of PI3 kinase (PI3K), enzyme with an important role in mitogenesis, did not affect hormone-dependent ERK1/2 phosphorylation, indicating that PI3K is not involved in 1alpha,25(OH)(2)D(3)-induced MAPK activation. In agreement with this data, enzyme activity assays and tyrosine phosphorylation of the regulatory subunit (p85) of PI3K showed that the hormone has no effect on the enzyme activity in rat enterocytes. Taken together, the present study suggest that in intestinal cells, tyrosine phosphorylation is an important mechanism of 1alpha,25(OH)(2)D(3) involved in PLCgamma and MAPK regulation and that this mechanism is impair with ageing.
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http://dx.doi.org/10.1016/j.biocel.2003.08.005DOI Listing
March 2004

Activation of RAF-1 through Ras and protein kinase Calpha mediates 1alpha,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells.

J Biol Chem 2003 Jan 1;278(4):2199-205. Epub 2002 Nov 1.

Departamento de Biología, Bioquímica and Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca, Argentina.

We have previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) is mediated by activation of the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how 1alpha,25(OH)(2)D(3) up-regulates the MAPK cascade, we have investigated whether the hormone acts upstream through stimulation of Raf-1 and the signaling mechanism by which this effect might take place. Treatment of chick myoblasts with 1alpha,25(OH)(2)D(3) (1 nm) caused a fast increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a specific Ras inhibitor peptide that involves Ras in 1alpha,25(OH)(2)D(3) stimulation of Raf-1. 1alpha,25(OH)(2)D(3) rapidly induced tyrosine de-phosphorylation of Ras-GTPase-activating protein, suggesting that inhibition of Ras-GTP hydrolysis is part of the mechanism by which 1alpha,25(OH)(2)D(3) activates Ras in myoblasts. The protein kinase C (PKC) inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked 1alpha,25(OH)(2)D(3)-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of Raf-1 also involves PKC. In addition, transfection of muscle cells with an antisense oligodeoxynucleotide against PKCalpha mRNA suppressed serine phosphorylation by 1alpha,25(OH)(2)D(3). The increase in MAPK activity and tyrosine phosphorylation caused by 1alpha,25(OH)(2)D(3) could be abolished by Ras inhibitor peptide, compound PD 98059, which prevents the activation of MEK by Raf-1, or incubation of cell lysates before 1alpha,25(OH)(2)D(3) exposure with an anti-Raf-1 antibody. In conclusion, these results demonstrate for the first time in a 1alpha,25(OH)(2)D(3) target cell that activation of Raf-1 via Ras and PKCalpha-dependent serine phosphorylation plays a central role in hormone stimulation of the MAPK-signaling pathway leading to muscle cell proliferation.
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http://dx.doi.org/10.1074/jbc.M205732200DOI Listing
January 2003

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.

Eur J Biochem 2002 May;269(10):2506-15

Department Biología, Bioquímica & Farmacia. Universidad Nacional del Sur, San Juan Bahia Blanca, Argentina.

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.
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http://dx.doi.org/10.1046/j.1432-1033.2002.02915.xDOI Listing
May 2002