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4. Morin loaded mesoporous molecular sieves as novel devices to the potential treatment of tumor pathologies.

Author

Montiel Schneider, María Gabriela, Martín, María Julia, Cuello, Natalia, Favatela, María Florencia, Gentili, Claudia, Elias, Verónica, Eimer, Griselda, and Lassalle, Verónica

Subjects
MOLECULAR sieves, IRON oxide nanoparticles, MESOPOROUS materials, MORIN, TUMOR treatment, IRON oxides, MESOPOROUS silica
Abstract

Morin is an antioxidant and anticancer flavonoid, extracted from natural sources, that may exert beneficial effects for several pathologies. Despite this, the administration of morin represents a challenge due to its low aqueous solubility. Mesoporous silica materials have emerged as biocompatible tools for drug delivery, as their pore size can be modulated for maximum surface area to volume ratio. In this contribution, we evaluate the ability of iron-modified mesoporous materials, for morin loading and controlled delivery. The SBA-15 and MCM-41 sieves were synthesized and modified with iron (metal content 4.02 and 6.27 % wt, respectivily). Characterization by transmission electron microscopy, XRD and UV-Vis revealed adequate pore size and agglomerates of very small metallic nanospecies (nanoclusters), without larger iron oxide nanoparticles. FT-IR spectra confirmed the presence of silanol groups in the solid hosts, which can interact with different groups present in morin molecules. SBA-15 materials were more efficient in terms of morin loading capacity (LC) due to their larger pore diameter. LC was more than 35% for SBA-15 materials when adsorptions studies were carried out with 9 mg of drug. Antioxidant activity were assayed by using DPPH test. Free iron materials presented a significate improvement as antioxidants after morin incorporation, reaching a scavenging activity of almost a 90%. On the other hand, in iron modified mesoporous materials, the presence of morin did not affect the scavenging activity. The results could be related with the formation of a complex between the flavonoid and the iron. Finally, biosafety studies using normal epithelial cells revealed that neither the loaded nor the unloaded materials exerted toxicity, even at doses of 1 mg/ml. These findings expand knowledge about mesoporous materials as suitable carriers of flavonoids with the aim of improving therapies for a wide range of pathologies. [ABSTRACT FROM AUTHOR]

Published
2024
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9. Chitosan‐Based Formulations Intended as Protective Spray for Mask Surfaces in Prevention of Coronavirus Dissemination

Author

Ayala‐Peña, Victoria Belen, primary, Martin, María Julia, additional, Favatela, Florencia, additional, Otarola, Jessica, additional, Morán, Pedro, additional, Ventura, María, additional, Gentili, Claudia, additional, Salcedo, María Florencia, additional, Mansilla, Andrea, additional, Pérez, Sandra, additional, Dolcini, Guillermina, additional, Alvarez, Vera, additional, and Lassalle, Verónica, additional

Published
2022
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14. Involvement of ERK1/2, p38 MAPK, and PI3K/AKT signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells

Author

Calvo, Natalia, Martin, Maria Julia, de Boland, Ana Russo, and Gentili, Claudia

Subjects
Oncology, Experimental, Colon cancer -- Physiological aspects, Adenocarcinoma -- Physiological aspects, Cell research, Cell cycle -- Research, Phosphotransferases -- Health aspects, Cellular signal transduction -- Research, Cancer cells -- Health aspects, Cancer -- Research, Biological sciences
Abstract

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells. Key words: PTHrP, Caco-2 cells, cell cycle, MAPKs, PI3K/AKT. Le peptide apparente a l'hormone parathyroidienne (PTHrP, acronyme de Parathyroid Hormone-related Peptide) est distribue dans la plupart des tissus fetaux et adultes, et son expression est correlee avec la severite du carcinome du colon. Recemment, les auteurs ont obtenu des indications que chez les cellules Caco-2, une lignee cellulaire humaine d'adenocarcinome colorectal, le PTHrP exogene accroit le nombre de cellules vivantes par l'intermediaire de ERK1/2, de la p38 MAPK et de la PI3-kinase, et induit l'expression de la cycline D1, une proteine regulatrice du cycle cellulaire. Dans cette etude, ils ont examine de maniere plus approfondie le role du PTHrP dans la regulation de la progression du cycle cellulaire chez ces cellules intestinales. L'analyse en cytometrie de flux a revele que le traitement au PTHrP diminuait le nombre de cellules en G0/G1 et accroissait le nombre de cellules en S et en G2/M. L'hormone accroissait l'expression de CDK6 et diminuait la quantite de regulateurs negatifs du cycle cellulaire, p27Kip1, p15INK4B et p53. Cependant, le PTHrP ne modifiait pas l'expression de la cycline D3, de CDK4 et de p16INK4B. De plus, les inhibiteurs de ERK1/2 (PD98059), de la p38 MAPK (SB203580) et de la PI3-kinase (LY294002) renversaient la reponse des cellules Caco-2 au PTHrP. Dans l'ensemble, ces resultats suggerent que le PTHrP module positivement la progression du cycle cellulaire et modifie l'expression de proteines impliquees dans la regulation du cycle cellulaire par l'intermediaire des voies de signalisation de ERK1/2, de la p38 MAPK et de la PI3-kinase chez les cellules Caco-2. [Traduit par la Redaction] Mots-cles: PTHrP, cellules Caco-2, cycle cellulaire, MAPKs, PI3K/AKT., Introduction Parathyroid hormone-related protein (PTHrP) was originally identified as the causative agent of humoral hypercalcemia of malignancy, one of the most frequent paraneoplastic syndromes (Strewler 2000). The PTHrP molecule is [...]

Published
2014
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18. Pro-apoptotic effects of parathyroid hormone in intestinal cells

Author

Calvo, Natalia, German, Olga, de Boland, Ana Russo, and Gentili, Claudia

Subjects
Parathyroid hormone -- Physiological aspects -- Health aspects -- Research, Homeostasis -- Research -- Health aspects -- Physiological aspects, Apoptosis -- Causes of -- Physiological aspects -- Research -- Health aspects, Biological sciences, Physiological aspects, Research, Causes of, Health aspects
Abstract

Apoptosis, a form of programmed cell death, is a process fundamental to normal growth and development, immune response, tissue remodeling after injury or insult, and homeostasis of the intestinal epithelium. Recently, it has become apparent that apoptosis is a crucial process in skeletal development and homeostasis, and that signaling by the parathyroid hormone (PTH) receptor can either promote or suppress apoptosis depending on the cellular context. In this study, we evaluated the role of PTH in intestinal apoptosis using human colonic Caco-2 cells. To that end, Caco-2 cells were exposed to PTH ([10.sup.-8] mol/L) for 1-5 days. Evaluation of cell survival by use of resazurin staining, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS) staining, and crystal violet staining revealed that PTH treatment diminishes the number of viable cells. Assessment of cells after PTH treatment by use of propidium iodide showed that the hormone increased the number of red-stained (dead) cells (178%, 5 days). Moreover, we found that the hormone induced disruption of actin filaments with changes to cellular shape, alteration of cell-to-cell junctions, externalization of membrane phosphatidylserine, chromatin condensation, and DNA fragmentation of the nucleus, which are morphological features consistent with apoptosis. In addition, PTH treatment revealed a cytosolic staining pattern of 14-3-3. However, the significance of such differential localization for 14-3-3 function remains unknown. Taken together, the present study suggests that PTH promotes apoptosis in Caco-2 intestinal cells. Key words: parathyroid hormone, Caco-2 cells, apoptosis. L'apoptose, une forme de mort cellulaire programmee, est un processus fondamental de la croissance et du developpement normaux, de la reponse immunitaire, du remodelage tissulaire apres un dommage ou une agression, et elle joue un role critique dans le maintien de l'homeostasie de l'epithelium intestinal. Recemment, il est devenu clair que l'apoptose constituait un processus crucial du developpement et de l'homeostasie du squelette et que la signalisation issue du recepteur de la PTH pouvait soit promouvoir, soit supprimer l'apoptose selon le contexte cellulaire. Dans cette etude, nous avons evalue le role de la PTH dans l'apoptose intestinale a l'aide des cellules de colon humain Caco-2. Pour ce faire, les cellules Caco-2 ont ete exposees pendant 1 a 5 jours a la PTH ([10.sup.-8] M). L'evaluation de la survie cellulaire par des marquages a la resazurine, au 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS) ou au Crystal violet, a revele que le traitement a la PTH diminue le nombre de cellules viables. L'evaluation de la mortalite apres le traitement a la PTH avec l'iodure de propidium a montre que l'hormone augmente le nombre de cellules colorees en rouge (178 %, 5 jours). De plus, nous avons trouve que l'hormone induit une dissociation des filaments d'actine et des changements dans la forme des cellules, une alteration des jonctions cellule-cellule, une externalisation de la phosphatidylserine membranaire, ainsi qu'une condensation de la chromatine et une fragmentation de l'ADN, des caracteristiques morphologiques correspondant a l'apoptose. Un patron de coloration cytosolique de la 14-3-3 σ aussi ete observe suite au traitement a la PTH. Cependant, la signification de cette localisation differentielle de la fonction de la 14-3-3 reste inconnue. En somme, cette etude suggere que la PTH est un promoteur de l'apoptose des cellules intestinales Caco-2. Mots-cles : PTH, cellules Caco-2, apoptose. [Traduit par la Redaction], Introduction Apoptosis is recognized as an important process responsible for maintaining the cellular balance between proliferation and death. This form of cell death can be induced by a wide range [...]

Published
2009

19. Severe hypercalcaemia in an infant

Author

Martín, María Julia, primary, Maury Fuentes, Kelly, additional, Novoa Díaz, María Belén, additional, Cao, Gabriel, additional, Gigola, Graciela, additional, Brunetto, Oscar, additional, and Gentili, Claudia, additional

Published
2021
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30. Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells

Author

Martín, María Julia, Calvo, Natalia Graciela, Russo, Ana Josefa, and Gentili, Claudia Rosana

Subjects
Activating Transcription Factor 1, Cell Nucleus, PTHrP, SIGNAL TRANSDUCTION, PROLIFERATION, Parathyroid Hormone-Related Protein, Bioquímica y Biología Molecular, Ciencias Biológicas, Proto-Oncogene Proteins c-myc, Protein Transport, Colonic Neoplasms, CACO-2 CELLS, Humans, Cyclin D1, Caco-2 Cells, Mitogen-Activated Protein Kinases, Phosphorylation, Cyclic AMP Response Element-Binding Protein, Protein Processing, Post-Translational, Proto-Oncogene Proteins c-akt, CIENCIAS NATURALES Y EXACTAS, beta Catenin, Cell Proliferation, Signal Transduction
Abstract

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco-2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP-dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF-1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of β-catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c-Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco-2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco-2 cells Fil: Martín, María Julia. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina Fil: Calvo, Natalia Graciela. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina Fil: Russo, Ana Josefa. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina Fil: Gentili, Claudia Rosana. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina

Published
2013

31. Cell cycle modulation by parathyroid hormone in human colon adenocarcinoma cells

Author

Calvo, Natalia Graciela, Russo, Ana Josefa, and Gentili, Claudia Rosana

Subjects
Ciencias Biológicas, CÉLULAS CACO-2, purl.org/becyt/ford/1 [https], Bioquímica y Biología Molecular, purl.org/becyt/ford/1.6 [https], CICLO CELULAR, CIENCIAS NATURALES Y EXACTAS, PTH
Abstract

La hormona paratiroidea (PTH) es un importante mediador de la remodelación ósea y actúa como regulador esencial de la homeostasis del calcio. Dependiendo del contexto celular, PTH también puede estimular o inhibir la apoptosis y provocar alteraciones en la regulación del ciclo celular. En trabajos previos demostramos que, en las células Caco-2 derivadas de adenocarcinoma de colon humano, la hormona (10-8 M) en ausencia de suero tiene efectos proapoptoticos y, con baja dosis de suero, efectos anti-proliferativos: aumenta la expresión de la proteína inhibitoria del ciclo celular p27Kip1 y disminuye la expresión de las ciclinas D1 y D3 y de la quinasa dependiente de ciclina CDK6. En este trabajo profundizamos el estudio de los mecanismos moleculares que median la respuesta a PTH de las células Caco-2. Se evidencio que, en ausencia de suero, el receptor de PTH tipo 1 (RPTH1) se localiza exclusivamente en el núcleo. Además, el tratamiento con la hormona solo en la dosis de 10-8 M disminuye el número de células vivas, pero no se observan cambios en concentraciones menores. Mas aun, en ausencia de suero, solamente la expresión de la proteína inhibitoria del ciclo celular p15ink4b es modificada por el tratamiento hormonal. Estos resultados sugieren que PTH en una dosis de 10-8 M arresta la progresión del ciclo celular en las células Caco-2; sin embargo, según las condiciones experimentales, este efecto seria mediado por distintas proteínas asociadas al ciclo celular. Fil: Calvo, Natalia Graciela. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahia Blanca; Argentina Fil: Russo, Ana Josefa. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahia Blanca; Argentina Fil: Gentili, Claudia Rosana. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahia Blanca; Argentina

Published
2013

32. Pro-apoptotic effects of parathyroid hormone in intestinal cells

Author

Calvo, Natalia Graciela, Russo, Ana Josefa, and Gentili, Claudia Rosana

Subjects
Ciencias Biológicas, PARATHYROID HORMONE, Otras Ciencias Biológicas, CACO-2 CELLS, CIENCIAS NATURALES Y EXACTAS, APOPTOSIS
Abstract

Apoptosis, a form of programmed cell death, is a process fundamental to normal growth and development, immune response, tissue remodeling after injury or insult, and homeostasis of the intestinal epithelium. Recently, it has become apparent that apoptosis is a crucial process in skeletal development and homeostasis, and that signaling by the parathyroid hormone (PTH) receptor can either promote or suppress apoptosis depending on the cellular context. In this study, we evaluate the role of PTH in the intestinal apoptosis using the human colonic Caco-2 cells. To that end, Caco-2 cells were exposed to PTH (10-8 mol/L) for 1-5 days. Evaluation of the cell survival by use of resazurin staining, 3-(4,5-dimethylthiazol-2-yl) -5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS) staining, and crystal violet staining revealed that PTH treatment diminishes the number of viable cells. Assessment of cells after PTH treatment by used of propidium iodide shows that the hormone increases the number of red-stained (dead) cells (+ 178%, 5 days). Moreover, we found that the hormone induces disruption of actin filaments with changes to cellular shape, alteration of cell-to-cell junctions, externalization of membrane phosphatidylserine, chromatin condensation, and DNA fragmentation of the nucleus, which morphological features consistent with apoptosis. In addition, PTH treatment revealed a cytosolic staining pattern of 14-3-3. However, the significance of such differential localization for 14-3-3 function remains unknown. Taken together, the present study suggests that PTH promotes apoptosis in Caco-2 intestinal cells. Fil: Calvo, Natalia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Russo, Ana Josefa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Gentili, Claudia Rosana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina

Published
2009
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41. MODULACIÓN POR LA HORMONA PARATIROIDEA DEL CICLO CELULAR EN CÉLULAS DE ADENOCARCINOMA DE COLON HUMANO.

Author

Calvo, Natalia, de Boland, Ana Russo, and Gentili, Claudia

Abstract

Copyright of Actualizaciones en Osteología is the property of Asociacion Argentina de Osteologia y Metabolismo Mineral and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Published
2013

42. MECANISMO DE ACCIÓN DE PTH EN CÉLULAS DE ADENOCARCINOMA DE COLON HUMANO.

Author

Gentili, Claudia, Calvo, Natalia, and de Boland, Ana Russo

Abstract

Copyright of Actualizaciones en Osteología is the property of Asociacion Argentina de Osteologia y Metabolismo Mineral and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Published
2010

43. 1α,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

Author

Gentili, Claudia, Morelli, Susana, and Boland, Ana Russo de

Subjects
*PARATHYROID hormone, *CYTOSOL, *CYTOPLASM, *PHOSPHOLIPASES
Abstract

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1α,25,dihydroxy-vitamin D3 [1α,25(OH)2D3] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) [3H-AA] one–two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca2+ chelator BAPTA-AM (5 μM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca2+-dependent mechanism involving activation of Ca2+-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 μM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1α,25(OH)2D3 (1 nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1α,25(OH)2D3 activate in duodenal cells, a Ca2+-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1α,25(OH)2D3 and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function. [Copyright &y& Elsevier]

Published
2004
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44. Implication of Gβγ proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes

Author

Gentili, Claudia, Boland, Ricardo, and de Boland, Ana Russo

Abstract

Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca2+(EGTA, 0.5 mM) and chelation of intracellular Ca2+with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid acetoxymethyl ester (BAPTA) (5 μM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca2+is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Gαs and Gβ, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gβ subunits by preincubation of cells with a Gβ antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gβγ–c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.

Published
2005
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46. Mecanismos de acción del análogo tumoral de la hormona paratiroidea (PTHrP) en células de cáncer de colon humano

Author

Martín, María Julia and Gentili, Claudia Rosana

Subjects
purl.org/becyt/ford/1 [https], Ciencias Biológicas, PTHrP, Bioquímica y Biología Molecular, purl.org/becyt/ford/1.6 [https], CANCER COLORRECTAL, PROLIFERACIÓN, hormones, hormone substitutes, and hormone antagonists, SEÑALIZACIÓN CELULAR, CIENCIAS NATURALES Y EXACTAS
Abstract

La hormona paratiroidea (PTH) es un importante mediador de la remodelación ósea y actúa junto al calcitriol (1α,25(OH)2vitamina D3), como regulador esencial de la homeostasis del calcio y fósforo. El péptido relacionado con la hormona paratiroidea (PTHrP) es una poli hormona que tiene un papel importante en el desarrollo del feto y en la homeostasis de tejidos adultos actuando principalmente en forma parácrina. Puede ser expresada y secretada por varios tumores y es responsable de la hipercalcemia humoral maligna, siendo éste un signo de mal pronóstico en las neoplasias malignas. Al momento de comenzar este Trabajo de Tesis había poca información acerca del rol de PTHrP actuando como factor parácrino sobre células derivadas del cáncer colorrectal (CCR). En el presente Trabajo se evidencia que PTHrP es capaz de inducir un aumento en la proliferación celular de dos líneas celulares derivadas de CCR, las células Caco-2 y las células HCT116. Además, este efecto inducido por la hormona está modulado por las cascadas de señalización PKC, Src, Akt, ERK MAPK y β-catenina. Se observó que en las células tumorales intestinales la hormona mediante ERK MAPK desencadena el aumento de la expresión y/o activación por fosforilación de CREB/ATF-1, c-myc y ciclina D1, que son moléculas relevantes en la proliferación celular. Acorde con lo observado in vitro, los experimentos desarrollados en el modelo murino revelan que la administración continua de la hormona en forma intra-tumoral modula la expresión de los marcadores claves en la progresión del CCR ERK, ciclina D1 y CREB/ATF-1. Tanto las células tumorales intestinales normales como aquellas que sobre-expresan PTHrP responden a la acción de las hormonas calciotrópicas PTH y calcitriol, al menos a nivel transcripcional, disminuyendo la expresión de PTHrP. PTHrP no sólo favorece la activación de vías mitogénicas y la proliferación de las células Caco-2 y HCT116 sino que también es capaz de disminuir la sensibilidad de estas células al Irinotecán (que es una droga comúnmente utilizada para la quimioterapia del CCR) mediante las vías de ERK MAPK y β-catenina.Este trabajo aporta información original respecto al rol del análogo tumoral de PTH en células tumorales intestinales y los mecanismos moleculares involucrados en la respuesta hormonal de estas células tanto in vitro como in vivo. Además, profundiza el conocimiento respecto de los mecanismos moleculares modulados por las hormonas calciotrópicas PTH y calcitriol en células tumorales intestinales. Un aporte de este Trabajo de Tesis es que al elucidar las vías de señalización que regulan los procesos inducidos por PTHrP y que favorecen la proliferación y/o quimiorresistencia, se podrían generar estrategias terapéuticas alternativas en el tratamiento del CCR cuya finalidad es inhibir la expresión de PTHrP o su mecanismo de acción en estas células tumorales. Parathyroid hormone (PTH) is an important mediator of bone remodeling and acts together with calcitriol (1α, 25 (OH)2vitamin D3), as an essential regulator of calcium and phosphorus homeostasis. The parathyroid hormone related peptide (PTHrP) is a poly hormone that has an important role in the development of the fetus and in the homeostasis of adult tissues acting mainly through paracrine pathways. It can be expressed and secreted by several tumors and is responsible for malignant humoral hypercalcemia, which is a sign of poor prognosis in malignant neoplasms. When this Thesis Work started, there was little information about the role of PTHrP acting as a paracrine factor on cells derived from colorectal cancer (CRC). In the present work it is demonstrated that PTHrP is able to induce an increase in the cellular proliferation of two cell lines derived from CRC, Caco-2 cells and HCT116 cells. In addition, this effect is modulated by PKC, Src, Akt, ERK MAPK and β-catenin. It was observed that in the intestinal tumor cells, the hormone triggers the increase of the expression and/or activation by phosphorylation of CREB/ATF-1, c-myc and cyclin D1, which are relevant molecules in the cell proliferation, through ERK MAPK. In agreement with the observed in vitro, the experiments developed in the murine model revealed that the continuous administration of the hormone in an intra-tumoral manner modulates the expression of ERK, cyclin D1 and CREB / ATF-1, which are key markers in the progression of CRC. Both, normal intestinal tumor cells and those that over-express PTHrP respond to the action of the calcitropic hormones PTH and calcitriol, at least at the transcriptional level, by decreasing the expression of PTHrP. PTHrP not only activates mitogenic pathways and induces the proliferation of Caco-2 and HCT116 cells, but it is also capable of decreasing the sensitivity of these cells to Irinotecan (which is a drug commonly used for CRC chemotherapy) through ERK MAPK and β-catenin pathways. This work provides original information about the role of the tumor analog of PTH in intestinal tumor cells and the molecular mechanisms involved in the hormonal response of these cells, both in vitro and in vivo. In addition, this work expands the knowledge regarding the molecular mechanisms modulated by the calcitropic hormones PTH and calcitriol in intestinal tumor cells. The contribution of this Thesis work is that by elucidating the signaling pathways that regulate the processes induced by PTHrP and that contribute to the proliferation and/or chemoresistance, alternative therapeutic strategies could be generated in the treatment of CRC whose purpose is to inhibit the expression of PTHrP or its mechanism of action in these tumor cells. Fil: Martín, María Julia.

Published
2018

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