Les macrophages (Mɸ) infiltrés dans les tumeurs orchestrent les différentes étapes du développement tumoral. De par leur capacité à internaliser les nanoparticules (NPs) et leur plasticité phénotypique, ils sont impliqués dans l’efficacité thérapeutique des actifs vectorisés par un rôle de réservoir de NPs ou une modulation de leur réponse envers les cellules néoplasiques. Le calcitriol, métabolite actif de la vitamine D, possède des activités à la fois anticancéreuse et immunomodulatrice. Sa vectorisation via des NPs est une approche thérapeutique intéressante pour potentialiser ses activités tout en limitant les effets secondaires s’opposant à son utilisation clinique dans le cadre de la chimiothérapie. Une étude de formulation a permis de développer des NPs à base d’acide poly(D,L)lactique et de triglycérides (ratio 1:2) d’une taille de 200 nm et présentant une libération prolongée du calcitriol. Des études in vitro menées sur les cellules de cancer du sein MCF-7 ont permis de mettre en évidence l’avantage d’une libération prolongée du calcitriol vis-à-vis de son activité antiproliférative aboutissant à une réduction de 65% de la viabilité cellulaire après 10 jours par rapport au contrôle, non observable avec le calcitriol libre. La participation active des M? à l’activité cytotoxique du calcitriol sur les lignées cellulaires de cancer du sein MCF-7 et de leucémie MV4-11 a aussi été mise en évidence par un modèle de co-culture in vitro. En effet, les NPs de calcitriol, après internalisation par les Mɸ, provoquent une action cytotoxique prolongée contre les cellules MCF-7 en co-culture au bout de 10 jours avec seulement 20% de cellules viables vs 70% en l’absence de Mɸ / Tumor associated macrophages (Mɸ) orchestrate the different stages of tumor development. They are able to internalize nanoparticles (NPs) and are known for their phenotypic plasticity, which make them interesting targets for cancer treatment through the storage of NPs or a modulation of their activity towards the neoplastic cells. Calcitriol, the active metabolite of vitamin D, exerts both anticancer and immunomodulatory activities. Its vectorization via NPs is an interesting therapeutic approach to potentiate its activities while limiting its side effects, which hamper its current clinical use in chemotherapy. We developed poly (D, L) lactic acid and triglyceride-based NPs (1:2 ratio) measuring 200 nm and exhibiting a sustained release of calcitriol. In vitro studies, performed on breast cancer cells (MCF-7), showed the advantages of a sustained release of calcitriol regarding its antiproliferative activity with a 65%-decrease in cell viability after 10 days compared to unexposed cells, while it was unobservable for free calcitriol. The implication of Mɸ in the cytotoxic activity of calcitriol towards MCF-7 cells and MV4-11 cells (leukemia) cells has been demonstrated using an in vitro co-culture model. Calcitriol-NPs showed a sustained cytotoxic activity towards MCF-7 cells in co-cultures after 10 days, through their uptake by Mɸ, with a decrease in cell viability of 80% vs 30% in mono-cultures

Orientador: Lilia F. R. de Souza Li / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T11:10:27Z (GMT). No. of bitstreams: 1 Macedo_LuciaCosentinode_M.pdf: 2481805 bytes, checksum: 85ab094926dbe8ad65a47ec2c1c6fcf5 (MD5) Previous issue date: 2006 / Resumo: A 1,25-dihidroxivitamina D é de fundamental importância na homeostase do cálcio. A vitamina D exerce suas ações através da interação com o seu receptor. O receptor da vitamina D (VDR) é membro da superfamília de receptores nucleares. O raquitismo é uma doença causada por mineralização deficiente na matriz óssea ou osteóide, afetando o desenvolvimento e a formação do osso na fase de crescimento. Mutações no receptor da vitamina D causam raquitismo dependente de vitamina D tipo II. Neste tipo de raquitismo ocorre hipocalcemia e as concentrações séricas de 1,25-dihidroxivitamina D3 são elevadas e, na maioria das vezes não há resposta ao tratamento com doses altas de 1,25-dihidroxivitamina D. Pacientes apresentam raquitismo grave de inicio precoce, nos primeiros meses de vida e a maior parte dos indivíduos apresenta pouco ou nenhum pêlo corporal (alopecia). O objetivo principal deste projeto foi a análise molecular do receptor de vitamina D (VDR) em quatro pacientes com raquitismo dependente de vitamina D tipo n que manifestaram raquitismo e alopecia. Amostras de DNA destes pacientes foram usadas para sequenciar o gene do VDR. Os cromatogramas gerados foram analisados em programas específicos, visando a busca de mutações. A análise mostrou 2 novas mutações pontuais que resultam em mudança de aminoácido (Q259E e G319V) e uma mutação que introduz um codon terminal (R73X). A cultura primária de fibroblasto proveniente de biópsia de pele dos pacientes foi usada para análise funcional do receptor. Extrato proteico nuclear mostrou redução de expressão de todos os receptores mutados. O receptor truncado contendo apenas parte do domínio de ligação com o DNA perdeu o epítopo de interação com o anticorpo usado, não sendo identificado no Western blot pelo anticorpo utilizado, impossibilitando a avaliação de sua expressão. Tratamento da cultura primária de fibroblasto com doses crescentes de l,25(OH)2 vitamina D demonstrou que os VDRs mutados foram incapazes de ser ativado e aumentar a expressão de 24-hidroxilase. Este trabalho identificou mutações em quatro pacientes com raquitismo dependente de vitamina D tipo II e estas mutações resultaram em comprometimento funcional do VDR / Abstract: The 1,25-dihydroxyvitamin D plays a fundamental role in the calcium homeostasis. The vitamin D exerts its actions through the interaction with its receptor. The vitamin D receptor (VDR) is a member of the superfamily of nuclear receptors. Rickets is a disease caused by deficient mineralization in the bone matrix or osteoids, affecting the development and formation of the bone during the growth stage. Mutations in the vitamin D receptor are associated to the vitamin D-dependent rickets type II. The biochemical characteristics is hypocalcemia and increase levels of 1,25-dihydroxyvitamin D3, and most of time there is no answer to the treatment with high doses of 1,25-dihydroxyvitamin D. Patients present rickets and or osteomalacia of varying severity beginning in the first months of life and most of the individuals presents little or any body hair (alopecia). The main objective of this project was the molecular analysis of the vitamin D receptor (VDR) in four patients with vitamin D-dependent rickets type II that manifested with rickets and alopecia. DNA samples of these patients were used to sequence the VDR gene. The generated chromatograms were analyzed in specific programs, aiming the search of mutations. The analysis showed two novel missense mutation that result in amino acid change (Q259E e G319V) and one nonsense mutations (R73X). Fibroblast primary culture derived from the patients' skin biopsy was carried out for functional analysis of the receptor. Nuclear protein extract showed reduction of expression of all mutant receptors. The truncated VDR receptor containing only part of the DNA binding domain lost the region containing the epitopo which the antibody was raised against and it was not identified by the Western blot analysis. Treatment of the fibroblast primary culture treatment with increasing doses of l,25(OH)2 vitamin D showed that the mutant VDR were unable to be activated and to increase the expression of the 24-hydroxylase. It was found mutations in the four patients with vitamin D-dependent rickets type II studied. Functional analysis confirmed that the mutations impaired the VDR activation / Mestrado / Mestre em Farmacologia

No description available.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Tese (doutorado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2010 / Hipovitaminose D (25(OH)D) é fator de risco para doença cardiovascular (DCV) na população em hemodiálise (HD). Pelos efeitos desta vitamina na ativação de receptores sistêmicos no sistema imune e miocárdico, pacientes com hipovitaminose D apresentariam in / Background: Hypovitaminosis D (25(OH) D3 deficiency) is a cardiovascular risk factor in chronic kidney disease hemodialysis (HD) population, for its potential actions on systemic vitamin D receptors present in immune system and myocardium. We hypothesize

Vitamin D regulation is associated with several human disorders and contributes to various cellular mechanisms. Calcitriol (commercially available as Rocaltrol), an active Vitamin D metabolite, is known as a neuro-protective and anti-cancer drug but most importantly helps maintaining the calcium homeostasis inside the human body. The effectiveness of calcitriol to perform as an effective therapeutic agent is counteracted by its calcemic effects. In order to obtain better therapeutic results, synthetic calcitriol analogs without these calcemic effects have been recently developed but they are not yet cost-effective and their production is time-consuming. In order to determine the best active form of calcitriol that could provide higher chemotherapeutic activity without these calcemic effects, calcitriol mode of action was studied using yeast as a model system. In order to achieve this, we analyzed the calcitriol effects on yeast cellular growth based on calcium intake levels. In this work, we also assessed yeast strains with gene deletions of selected calcium transporter genes to understand the calcitriol metabolism. For the aim of understanding hypercalcemic effects of calcitriol, we developed a hypothesis based on calcitriol interactions with oxygen. Interestingly, use of an anaerobic model validated the oxygen interactions with calcitriol that might possibly cause calcemic effects on patients. Anaerobically grown yeast treated with calcitriol showed significantly less intracellular calcium levels when imaged under indo-1 calcium binding fluorescence dye as compared to calcitriol treated yeast grown under aerobic conditions. Finally, we predict that calcitriol might control free radical generation within the yeast system based on experiments with AAPH and UV- irradiation.

No description available.

Cinco décadas de descubrimientos de agentes antineoplásicos han generado una interesante batería de agentes quimioterapéuticos; sin embargo, aunque resultan útiles, se requieren agentes más efectivos ya que estos compuestos tienen poco impacto en las tasas de supervivencia o en la calidad de vida del paciente, especialmente en algunos tipos de cáncer, tales como el Carcinoma Celular Escamoso de Cabeza y Cuello (CCECC) y el Glioblastoma Multiforme (GBM). El presente trabajo de tesis describe resultados que demuestran la potencialidad terapéutica de dos productos sintéticos (análogos del calcitriol) y un compuesto natural (extracto triterpénico de las esporas de Ganoderma lucidum) como agentes terapéuticos en estos dos tipos de cáncer. El calcitriol (1α, 25(OH)2-vitamina D3) es esencial para el mantenimiento de la salud debido a su participación en un crisol de procesos fisiológicos como el control del metabolismo fosfo-cálcico, el crecimiento celular y el sistema inmune. Este compuesto, además, posee potenciales propiedades preventivas y terapéuticas contra diversas enfermedades hiperproliferativas como el cáncer, pero debido a la hipercalcemia que produce al seguir la posología terapéutica, se ve limitado su empleo como agente antitumoral. Por dichos motivos, se sintetizan análogos del calcitriol con el fin de encontrar alguno/s que presenten efectos antitumorales pero que carezcan de los efectos calcemiantes. Nuestro laboratorio, en conjunto con un grupo de investigación de Química Orgánica de la Universidad de Vigo, diseñamos y sintetizamos análogos del calcitriol que contienen un grupo oxolano o un grupo amida en la cadena lateral con el fin de estudiar su potencialidad terapéutica en cáncer. Como parte de este trabajo de tesis evaluamos si estos análogos podían ser efectivos como agentes antitumorales en CCECC y GBM. Por otra parte, la metodología más empleada en la búsqueda de nuevos principios activos naturales es la bioprospección, que incrementa en diez veces la probabilidad de éxito. En la actualidad, el Instituto Nacional del Cáncer (NCI) de USA está intensificando la búsqueda de metabolitos provenientes de la naturaleza para el descubrimiento de nuevas drogas debido a la gran variedad de compuestos que se encuentran en los productos naturales. Ganoderma lucidum es un reconocido hongo comestible y medicinal, empleado por más de dos mil años por la medicina tradicional China, India y Japonesa. Dentro de los efectos farmacológicos se documentaron sus propiedades antitumorales y contiene más de 400 compuestos bioactivos, entre ellos los triterpenoides (la actividad antitumoral se le atribuye a ellos), polisacáridos, nucleótidos y esteroles. También, como parte de este trabajo de tesis comenzamos a evaluar la actividad antitumoral, para el CCECC y los GBM, del extracto triterpénico de las esporas de este hongo producidos por el Laboratorio de Hongos Comestibles y Medicinales, CERZOS-CCT-Bahía Blanca-CONICET. / Five decades of research of anticancer agents have generated an interesting battery of chemotherapeutic agents; however, even though it is useful, more effective agents are needed since these have little impact in survival rates or in the patient‟s life quality, particularly in certain kinds of cancer, such as head and neck squamous cell carcinoma (HNSCC) and glioblastoma multiforme (GBM). This thesis describes results that demonstrate the therapeutic feasibility of two synthetic products (analogues of calcitriol), and a natural compound (extract triterpene Ganoderma lucidum spores) as therapeutics agents in these two kind of cancer. Calcitriol (1α, 25(OH)2-vitamin D3) is essential for the maintenance of health due to its participation in a crucible of physiological processes such as the control of phospho-calcic metabolism, cell growth and immune system. Moreover, this compound has potential preventive and therapeutic properties against various hyper proliferative diseases such as cancer, but due to the hypercalcemia that causes the therapeutic dosage, its use as antitumoral agent is limited. For these reasons, analogues of calcitrol were synthesized to evaluate if any of them shows antitumor effects but lacking calcemiantes effects. Our lab together with a research group Organic Chemistry at the University of Vigo designed and synthesized analogues of calcitriol that contain an oxolane or amide group in the side chain with the goal of studying its therapeutic potentiality in cancer. In this thesis we evaluate if these new analogues can be effective as antitumoral agents in CCECC and GBM. Furthermore, the most used methodology in the search of new natural assets is bioprospecting, increasing tenfold the chances of success. At present, the National Cancer Institute (NCI) of USA is putting emphasis on the search of metabolites from nature for drug discovery because of the wide variety of compounds found in nature. Ganoderma lucidum is a recognized edible and medicinal mushroom used for over two thousand years by traditional Chinese, Indian and Japanese medicine. Among the pharmacological effects, its anti-tumor properties were documented and contains over 400 bioactive compounds among them triterpenoids (antitumor activity is ascribed to them), polysaccharides, nucleotides and sterols. In this thesis we also evaluate the antitumoral activity on CCECC and GBM of triterpene extract spores of this fungus produced by the Laboratory of Edible and Medicinal Mushrooms, CERZOS-CCT-CONICET-Bahia Blanca.

El metabolito activo de la vitamina D es el calcitriol o 1α,25(OH)2 vitamina D3 (1,25D). El 1,25D ingresa a la célula difundiendo a través de la membrana plasmática y se une a su receptor, el receptor de la vitamina D (VDR). Este complejo desencadena acciones rápidas, no genómicos (como la activación de vías de señalización MAPKs, y la generación de segundos mensajeros, como el aumento del Ca2+ intracelular) o efectos genómicos que requieren generación de nuevas proteínas. Dado que la mayor parte de los efectos del 1,25D reportados involucran al VDR y éste se expresa en la mayoría de los tejidos, esta hormona esteroide se considera pleiotrópica. En el laboratorio donde trabajo, se ha demostrado que el VDR se expresa en el músculo esquelético y que el tratamiento con 1,25D (10-9 M) es capaz de promover a proliferación y la activación de vías de señalización como las MAPKs, Akt, con la participación de Src, muy rápidamente, tanto en mioblastos proliferativos como en miotubos diferenciados. Por otro lado está ampliamente documentado que el 1,25D tiene efectos anti-proliferativos y pro-apoptóticos en varios tipos de cáncer. En este trabajo de Tesis Doctoral, se evidenció que en la línea de músculo esquelético normal C2C12, durante la fase proliferativa, el tratamiento con 1,25D (10-9 M) promueve la expresión del VDR y la activación de p38 MAPK y ERK 1/2, que promueven la expresión de las CDK 4/6. Más tarde, durante el arresto promotor de diferenciación, iniciado por el 1,25D, la expresión del VDR y la activación de la p38 MAPK son esenciales para inducir el aumento en la expresión de la ciclina D3, de los CKIs p21Cip1/WAF1 y p27Kip1, y del marcador de diferenciación muscular miogenina, el cual también requiere que la ciclina D3 se exprese. En las células C2C12 que están en la etapa de diferenciación temprana, en la fase de alineación (previo a la fusión celular para la formación del miotubo), la hormona esteroide 1,25D unida a su receptor tiene la capacidad de activar la tirosina quinasa no receptora Src y las MAPKs ERK 1/2 y p38 MAPK, a tiempos cortos (hasta de una hora), sin embargo no es capaz de modificar la expresión de proteínas (como las ciclinas D1 y D3 o la caveolin-1). El rabdomiosarcoma (RMS) es el cáncer de tejido blando más común entre los niños y tiene características de músculo esquelético, a pesar de tener múltiples orígenes. La línea RD es una línea celular de rabdomiosarcoma humano, originada de biopsias de una paciente caucásica de siete años de edad. A pesar de la evidencia acumulada de los efectos antineoplásicos de la hormona esteroide 1,25D, en diferentes tipos de cáncer, solo existe un reporte de los efectos de este esteroide en RMS, en líneas diferentes a la línea RD. En esta Tesis Doctoral, se evidenció que las células RD expresan basalmente VDR y que el tratamiento con la hormona durante 24 y 48 horas promueve un incremento de este receptor. Además el 1,25D (10-9 M) promueve la activación de p38 MAPK, vía su quinasa upstream inmediata MKK 3/6 y Src a tiempos cortos en las células RD. De relevancia, se observó que la activación de p38 MAPK es necesaria para la activación de Src, pese a ser esta una quinasa upstream de las MAPKs. El tratamiento con 1,25D durante 72 horas provoca una marcada disminución en la expresión del VDR junto con una inhibición en la proliferación (ya que decrece la cantidad de células vivas y aumenta la expresión de p15INK4). Conjuntamente, la hormona no promueve la diferenciación celular (ya que no se observan cambios morfológicos y disminuye la expresión de marcadores de diferenciación tempranos) y estimula la apoptosis (aumentando la expresión de los mediadores ciclina D1 y CDK4 y causando picnosis mitocondrial y pérdida de la red mitocondrial). En conclusión, el metabolito activo de la vitamina D, el calcitriol tiene efectos opuestos en el músculo esquelético normal y el transformado, ya que en el primero promueve la proliferación seguida de un arresto pro-diferenciativo, mientras que en RMS es capaz de inducir la apoptosis. / The vitamin D active metabolite is the calcitriol or 1α,25(OH)2-vitamin D3 (1,25D). The 1,25D diffuse into the cell through the plasmatic membrane and it binds to its receptor, the vitamin D receptor (VDR). This complex triggers non-genomic, rapid actions (like activation of MAPKs pathways, and second messengers, like intracellular Ca2+ increase) or genomic effects that require protein synthesis. Almost all of the 1,25D actions require the VDR, and this receptor is expressed in almost all tissues, this steroid hormone is pleiotropic. In the laboratory where I investigate, it was demonstrated that the VDR is expressed in skeletal muscle and 1,25D treatment promotes proliferation and activation of MAPK, Akt, and the tyrosine kinase Src, quickly, in both proliferating myoblast and differentiated myotubes. By another hand, it was largely reported that 1,25D exerts antiproliferative and proapoptotic effects in many different cancer types. In this Ph.D. Thesis, it was evidenced that in normal skeletal muscle cell line C2C12, in the proliferative state, 1,25D (10-9 M) treatment promotes VDR expression and p38 MAPK and ERK 1/2 pathways activation, and all these together induce CDK 4/6 expression. Then, during pro-differentiative arrest induced by 1,25D, the VDR expression, and p38 MAPK activation are key events to promote an increase in Cyclin D3, CKIs p21cip1/WAF1 and p27kip1 and myogenin expression. In C2C12 cell, in an early differentiation state, during myoblast alignment, 1,25D through its receptor induce Src, ERK 1/2 and p38 MAPK pathways activation, within a few minutes, but this short treatment cannot modify some proteins expression involved in differentiation process (like Cyclin D3 and D1, or caveolin-1). Rhabdomyosarcoma (RMS) is the soft tissue cancer most common in children with skeletal muscle features, although it has different origins. The RD cell line is a human RMS cell line, originated from biopsies from a seven years old Caucasian patient. There is accumulated evidence about antineoplastic effects of 1,25D, in different cancer types; nevertheless, there is only one report relative to the effects of this steroid hormone treatment developed in RMS cell lines different to RD. In this PhD. Thesis, it was shown that RD cells express basally VDR and hormone treatment during 24 and 48 hours promotes an increase of this receptor. Moreover, 1.25D (10-9 M) promotes p38 MAPK activation via its immediate upstream kinase, MKK 3/6, and Src at short times of steroid exposition. Of relevance, it was observed that p38 MAPK activation is necessary for the activity of Src, despite this being an upstream kinase of MAPKs. Treatment with 1.25D for 72 hours causes a decrease in VDR expression and the inhibition in proliferation (since it decreases the amount of living cells and increases the expression of p15INK4). This steroid treatment does not promote differentiation, since no morphological changes were observed and the expression of early differentiation markers falls. Furthermore, 1,25Dstimulates apoptosis increasing the expression of cyclin D1 and CDK4 mediators and causing mitochondria pyknosis and disruption of mitochondrial network). In conclusion, the active metabolite of vitamin D, calcitriol has opposite effects on normal and transformed skeletal muscle cells, since in the normal cells it promotes proliferation followed by a pro-differentiative arrest, whereas in RMS it is capable of inducing apoptosis.