RESUMEN
Thyroid hormones (THs) T3 and T4, play an essential role in the development and metabolism of many tissues and organs, and have profound metabolic effects in adult life. THs action is mediated mainly by the thyroid hormone receptor (TRs) which seem to be ubiquitous. To-date thyroid-associated disease are not thought to be related in tendinopathies and tendons tears. Recent study demonstrated the presence of TRs in tendons and their possible role in the proliferation and apoptosis of human tenocyte isolated from tendon. We review new discovery that revisit our current thinking on the tendon biology focusing on thyroid hormones (THs) T3 and T4, and their possible role on human tenocyte.
RESUMEN
Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.(wt)p53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.
Asunto(s)
Carcinoma Corticosuprarrenal/patología , Carcinoma Corticosuprarrenal/radioterapia , Factor II del Crecimiento Similar a la Insulina/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Carcinoma Corticosuprarrenal/genética , Carcinoma Corticosuprarrenal/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Activación Enzimática/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Etiquetado Corte-Fin in Situ , Factor II del Crecimiento Similar a la Insulina/genética , Datos de Secuencia Molecular , Estabilidad Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiación Ionizante , Análisis de Secuencia de ADN , Transducción de Señal/efectos de la radiación , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Infertility is a dramatic and frequent side effect in women who are undergoing chemotherapy. Actual strategies are mainly focused on oocyte cryopreservation, but this is not always a suitable option. Considering the key role that granulosa cells play in follicle life, we studied whether thyroid hormone 3,5,3'-triiodothyronine (T(3)) protects rat ovarian granulosa cells from chemotherapy-induced apoptosis. To this aim, a cell line was established from fresh isolated rat granulosa cells and named rGROV. Cells were exposed to paclitaxel (PTX) and T(3), and apoptosis, cell viability, and cell cycle distribution were analyzed under different conditions. First, the integrity of the steroidogenic pathway was demonstrated, and the presence of thyroid receptors, transporters, and deiodinases was confirmed by quantitative PCR. Cells were then exposed to PTX alone or contemporary to T(3). MTT and TUNEL assays revealed that while there was a relevant percentage of dying cells when exposed to PTX (40-60%), the percentage was sensibly reduced (20-30%) in favor of living cells if T(3) was present. Cell cycle analysis showed that cells exposed to PTX alone were first collected in G2 and then died by apoptosis; on the other hand, the T(3) granted the cells to cycle regularly and survive PTX insult. In addition, western blot and FCM analyses confirmed that caspases activation, casp 3 and Bax, were downregulated by T(3) and that Bcl2 and cyclins A and B together with cdk1 were upregulated by T(3). In conclusion, we demonstrated that thyroid hormone T(3) can counteract the lethal effect of taxol on granulosa cells.
Asunto(s)
Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Paclitaxel/efectos adversos , Triyodotironina/metabolismo , Animales , Proteínas Portadoras , Caspasas/metabolismo , Ciclo Celular , Línea Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , ARN/genética , ARN/metabolismo , Ratas , Ratas Wistar , Receptores de Hormona Tiroidea , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrificed. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Triyodotironina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Experimental/inducido químicamente , Activación Enzimática/efectos de los fármacos , Glucosa/farmacología , Transportador de Glucosa de Tipo 2/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Estreptozocina/antagonistas & inhibidores , Triyodotironina/administración & dosificación , Triyodotironina/farmacologíaRESUMEN
Thiazolidinediones, specific peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.
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Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Neoplasias de la Corteza Suprarrenal/patología , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Hipoglucemiantes/farmacología , PPAR gamma/antagonistas & inhibidores , Tiazolidinedionas/farmacología , Neoplasias de la Corteza Suprarrenal/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina E/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , RosiglitazonaRESUMEN
Several findings suggest that the patient's hormonal context plays a crucial role in determining cancer outcome. The exact nature of thyroid hormone action on tumour growth has not been established yet, in fact contrasting data show thyroid hormones have a promotory or an inhibitory action on cancer cell proliferation depending on the case. We hypothesized that not only tissue specificity, but also specific mutations occurring during tumoral development in different thyroid hormone cellular targets are responsible for this dual effect. To test our hypothesis we analysed, by time-course and bromodeoxyuridine assay, thyroid hormone effects on the proliferation of six cancer cell lines originating from the same tissue or organ but carrying different mutations (in phospho-inositide 3 kinase or ß-catenin genes). The data obtained in this study show how mutations that affect the balance between degradation and stabilization of ß-catenin assume a remarkable importance in determining the cell-specific thyroid hormone effect on cell growth.
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Proliferación Celular/efectos de los fármacos , Neoplasias/patología , Tiroxina/farmacología , Triyodotironina/farmacología , Western Blotting , Expresión Génica/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Ex vivo islet cell culture in the presence of stimulating factors prior to transplantation is considered a good strategy in contrast to the short conclusion of islets transplantation. Previously, we demonstrated how T3 can increase b-cell function via specific activation of Akt; therefore we hypothesized that thyroid hormone T3 can be considered a promising candidate for the in vitro expansion of islet cell mass. Rat pancreatic islets have been isolated by the collagenase digestion and cultured in the presence or not of the thyroid hormone T3 10â»7 M. Islets viability has been evaluated by the use of two different dyes, one cell-permeable green fluorescent dye and propidium iodide, and by the analysis of core cell damage upcoming. Moreover, islets function has been evaluated by insulin secretion. The ability of b-cells to counteract apoptosis induced by streptozotocin has been analyzed by TUNEL assay. We demonstrated that treatment of primary cultures of rat pancreatic islets with T3 results in augmented ß-cell vitality with an increase of their functional properties. In addition, a sensible reduction of the core cell damage has been observed in the T3 treated islets, suggesting the preservation of the ß-cells integrity during the culture period. Nonetheless, the insulin secretion is sensibly augmented after T3 stimulation. The strong increment shown in Akt activation suggests the involvement of this pathway in the observed phenomena. In conclusion we indicate T3 as a good factor to improve ex vivo islets cell culture.
Asunto(s)
Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Triyodotironina/farmacología , Animales , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Citoprotección/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Masculino , Ratas , Ratas Wistar , Estreptozocina/toxicidad , Receptores beta de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea/metabolismoRESUMEN
Mitotane inhibits steroid synthesis by an action on steroidogenic enzymes, as 11beta-hydroxylase and cholesterol side chain cleavage. It also has a cytotoxic effect on the adrenocortical cells and represents a primary drug used in the adrenocortical carcinoma (ACC). H295R and SW13 cell lines were treated with mitotane 10(-5) M and ionizing radiations (IR) in combination therapy, inducing an irreversible inhibition of cell growth in both adrenocortical cancer cells. As shown in a previous report, mitotane/IR combination treatment induced a cell accumulation in the G2 phase. Here, we report the radiosensitizing properties of mitotane in two different ACC cell lines. The drug reveals the effectiveness to enhance the cytotoxic effects of IR by attenuating DNA repair and interfering on the activation of mitosis promoting factor (MPF), mainly regulated by the degradation of cyclin B1 in the mitotic process. These events may explain the inappropriate activation of cdc2, implicated in G2/M phase arrest and probably induced by the mitotane and IR in the combined treatment. Indeed, treatment with purvalanol, a cdc2-inhibitor prevents cell cycle arrest, triggering the G2/M transition. The observation that mitotane and IR in combination treatment amplifies the activation level of cyclin B/cdc2 complexes contributing to cell cycle arrest, suggests that the MPF could function as a master signal for controlling the temporal order of different mitotic events. Moreover, we report that mitotane interferes in modulation of mismatch repair (MMR) enzymes, revealing radiosensitizing drug ability.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/radioterapia , Carcinoma Corticosuprarrenal/radioterapia , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Mitotano/farmacología , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Reparación de la Incompatibilidad de ADN/fisiología , Evaluación Preclínica de Medicamentos , Fase G2/efectos de los fármacos , Fase G2/fisiología , Humanos , Complejos Multiproteicos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/farmacología , Células Tumorales CultivadasRESUMEN
Numerous evidences indicate that thyroid hormones exert an important role in the regulation of the reproductive system in the adult female. Although a clear demonstration of the thyroid-ovarian interaction is still lacking, it is conceivable that thyroid hormones might have a direct role in ovarian physiology via receptors in granulosa cells. In this study we analyzed if thyroid hormone treatment could affect cell proliferation and survival of COV434 cells. To this aim cell growth experiments and cell cycle analyses by flow cytometry were performed. Secondly the T(3) survival action was tested by TUNEL assay and MD30 cleavage analysis. We showed that T(3), and not T(4), can protect ovarian granulosa cells COV434 from apoptosis, regulating cell cycle and growth in the same cells. The increase in cell growth resulted in an augmented percentage of the cells in the S phase and, in a reduction of the doubling time (18%). Subsequently apoptotic pathway induced by serum deprivation has been evaluated in the cells exposed or not to thyroid hormone treatment. The T(3) treatment was able to remarkably counteract the apoptotic process. Even at the ultrastructural level there was an evident protective effect of T(3) in the cells that, besides the maintenance of the original morphology and, the absence of basophilic cytoplasm, conserved normal junctional areas. Furthermore, the protective T(3) effect evaluated by FACS analysis in the presence of a PI3K inhibitor revealed, as also confirmed by Western Blot on pAkt, that the PI3K pathway is crucial in T(3) survival action.
Asunto(s)
Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Tiroxina/farmacología , Triyodotironina/farmacología , Apoptosis/efectos de los fármacos , Caspasa 9/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Femenino , Células de la Granulosa/enzimología , Células de la Granulosa/ultraestructura , Humanos , Queratina-18/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor beta1 has been hypothesized. The serine-threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic beta-cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non-genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRbeta1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor beta1. We showed that the thyroid receptor beta1 mediates the T3 regulation of the cdk4.cyc D1.p21(CIP1).p27(KIP1) complex formation and activity. In addition TRbeta1 is essential for the T3 upregulation of the Akt targets beta-catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the beta1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway.
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Insulinoma/patología , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores beta de Hormona Tiroidea/fisiología , Triyodotironina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Tamaño de la Célula , Supervivencia Celular , Humanos , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Biosíntesis de ProteínasRESUMEN
The pancreatic adenocarcinoma is an aggressive and devastating disease, which is characterized by invasiveness, rapid progression, and profound resistance to actual treatments, including chemotherapy and radiotherapy. At the moment, surgical resection provides the best possibility for long-term survival, but is feasible only in the minority of patients, when advanced disease chemotherapy is considered, although the effects are modest. Several studies have shown that thyroid hormone, 3,3',5-triiodo-l-thyronine (T(3)) is able to promote or inhibit cell proliferation in a cell type-dependent manner. The aim of the present study is to investigate the ability of T(3) to reduce the cell growth of the human pancreatic duct cell lines chosen, and to increase the effect of chemotherapeutic drugs at conventional concentrations. Three human cell lines hPANC-1, Capan1, and HPAC have been used as experimental models to investigate the T(3) effects on pancreatic adenocarcinoma cell proliferation. The hPANC-1 and Capan1 cell proliferation was significantly reduced, while the hormone treatment was ineffective for HPAC cells. The T(3)-dependent cell growth inhibition was also confirmed by fluorescent activated cell sorting analysis and by cell cycle-related molecule analysis. A synergic effect of T(3) and chemotherapy was demonstrated by cell kinetic experiments performed at different times and by the traditional isobologram method. We have showed that thyroid hormone T(3) and its combination with low doses of gemcitabine (dFdCyd) and cisplatin (DDP) is able to potentiate the cytotoxic action of these chemotherapic drugs. Treatment with 5-fluorouracil was, instead, largely ineffective. In conclusion, our data support the hypothesis that T(3) and its combination with dFdCyd and DDP may act in a synergic way on adenopancreatic ductal cells.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Triyodotironina/uso terapéutico , Antimetabolitos/uso terapéutico , Western Blotting , Carcinoma Ductal Pancreático/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/uso terapéutico , Ciclina D1/análisis , Ciclina D2 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Ciclinas/análisis , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Fluorouracilo/uso terapéutico , Humanos , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/análisis , Receptores de Hormona Tiroidea/análisis , Quinasas p21 Activadas , GemcitabinaRESUMEN
It has recently been recognized that thyroid hormones may rapidly generate biological responses by non-genomic mechanisms that are unaffected by inhibitors of transcription and translation. The signal transduction pathways underlying these effects are just beginning to be defined. We demonstrated that thyroid hormone T3 rapidly induces Akt activation in pancreatic beta cells rRINm5F and hCM via thyroid hormone receptor (TR) beta1. The phosphorylation of Akt was T3 specific and dependent. Coimmunoprecipitation and colocalization experiments revealed that the phosphatidylinositol 3 kinase (PI3K) p85alpha subunit and the thyroid receptor beta1 were able to form a complex at the cytoplasmic level in both the cell lines, suggesting that a 'cytoplasmic TRbeta1' was implicated. Moreover, we evidenced that T3 treatment was able to induce kinase activity of the TRbeta1-associated PI3K. The silencing of TRbeta1 expression through RNAi confirmed this receptor to be crucial for the T3-induced activation of Akt. This action involved a T3-induced nuclear translocation of activated Akt, as demonstrated by confocal immunofluorescence. In summary, T3 is able to specifically activate Akt in the islet beta cells rRINm5F and hCM through the interaction between TRbeta1 and PI3K p85alpha, demonstrating the involvement of TRbeta1 in this novel T3 non-genomic action in islet beta cells.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores beta de Hormona Tiroidea/fisiología , Triyodotironina/fisiología , Línea Celular Tumoral , HumanosRESUMEN
BACKGROUND: Radiation therapy (RT) is a well established therapeutic modality for the treatment of solid tumors. In particular, post-operative RT is considered the standard treatment adjuvant to surgery since its ability to prolong median survival of patients with malignant astrocytoma has been shown; nevertheless the ionizing radiation (IR) treatment fails in a considerable number of astrocytoma patients. MATERIALS AND METHODS: Using an ADF human astrocytoma cell line the molecular mechanisms involved in the DNA damage induced by fractionated irradiation (FIR) and single IR treatment have been investigated. RESULTS: FIR and single IR treatment inhibited the growth of the ADF human astrocytoma cell line. FACS analysis revealed that FIR treatment, but not single IR treatment, induced growth inhibition associated with the induction of apoptosis. Apoptosis was related to caspase-3 activation and reactive oxygen species (ROS) generation. ROS formation depends on the up-regulation of the cytochrome P450 enzyme gene. On the contrary, 12.5 Gy induced necrotic cell death up-regulating the HSPD1, HSPCB, HSPCA and HSPB1 genes. CONCLUSION: FIR treatment induced cell death through caspase-3 and ROS-mediated apoptosis.
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Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Caspasa 3/metabolismo , Radiación Ionizante , Especies Reactivas de Oxígeno/metabolismo , Apoptosis , Astrocitoma/enzimología , Astrocitoma/genética , Astrocitoma/patología , Western Blotting , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Fraccionamiento de la Dosis de Radiación , Activación Enzimática , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
3,5,3'-triiodothyronine (T3) is essential for the growth and the regulation of metabolic functions, moreover, the growth-stimulatory effect of T3 has largely been demonstrated and the pathways via which T3 promotes cell growth have been recently investigated. Type 1 diabetes (T1D) is due to the destruction of beta-cells, which occurs even through apoptosis. Aim of our study was to analyze whether T3 could have an antiapoptotic effect on cultured beta-cells undergoing apoptosis. We have demonstrated that T3 promotes cell proliferation in islet beta-cell lines (rRINm5F and hCM) provoking an increment in cell number (up to 55%: rRINm5F and 45%: hCM), cell viability, and BrdU incorporation, and regulating the cell cycle-related molecules (cyc A, D1, E, and p27(kip1)). T3 inhibited the apoptotic process induced by streptozocin, S-Nitroso-N-Acetylpenicylamine (SNAP), and H2O2 via regulation of the pro- and anti-apoptotic factors Bcl-2, Bcl-XL, Bad, Bax, and Caspase 3. The T3 protective effect was PI-3 K-, but not MAPK- or PKA-mediated, involving pAktThr308. Thus, T3 could be considered a survival factor protecting islet beta-cells from apoptosis.
Asunto(s)
Apoptosis , Células Secretoras de Insulina/efectos de los fármacos , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Humanos , Células Secretoras de Insulina/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Receptores de Hormona Tiroidea/fisiología , Transducción de Señal , Triyodotironina/farmacologíaRESUMEN
The thyroid hormone, 3,5,3'-Triiodo-L-thyronine (T3), is essential for growth, differentiation, and regulation of metabolic functions in multicellular organisms, although the specific mechanisms of this control are still unknown. In this study, treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype, we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion, our results suggest, for the first time, that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Islotes Pancreáticos/citología , Neoplasias Pancreáticas/patología , Triyodotironina/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 2 , Humanos , Insulina/genética , Islotes Pancreáticos/fisiología , Proteínas de Transporte de Monosacáridos/genética , Fenotipo , Receptores de Hormona Tiroidea/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The insulin receptor susbtrate-3 (IRS-3) is a member of a family of intermediate adapter proteins that function as major intracellular targets for phosphorylation by the activated insulin and IGF-I receptors. Among the four IRS proteins identified so far, IRS-3 exhibits a rather peculiar expression pattern during both the embryonic development and adult life, suggesting a different mechanism of regulation of its expression. In this study, we cloned the 5' flanking region of the mIRS-3 gene and analyzed its promoter activity. The mIRS-3 promoter is inhibited by wild-type p53, and this effect is completely abolished by cotransfection of a dominant negative p53. Tumor-derived p53 mutants show variable, but lower suppressing capability than wt p53. In addition, treatment with doxorubicin inhibits endogenous expression of mIRS-3 mRNA in C2C12 and 3T3-L1 cells. The DNA region spanning from nucleotides -287 and -178 in the mIRS-3 promoter is responsible for a 32.2% reduction of the mouse double minute 2 (MDM2) promoter activity, suggesting its involvement in the p53-mediated inhibitory effect. In conclusion, our study demonstrates that the mIRS-3 promoter is regulated by p53 at the transcriptional level. The inhibition of mIRS-3 promoter by wild-type p53, and its de-repression by tumor-derived p53 mutants, appears to be similar to that previously reported for the IGF-I receptor promoter, suggesting a common role of these two genes in p53-mediated cell growth and differentiation.