RESUMEN
Earthquakes threaten humanity globally in complex ways that mainly include various socioeconomic consequences of life and property losses. Resilience against seismic risks is of high importance in the modern world and needs to be sustainable. Sustainable earthquake resilience (SER) from the perspective of structural engineering means equipping the built environment with appropriate aseismic systems. Shape memory alloys (SMAs) are a class of advanced materials well suited for fulfilling the SER demand of the built environment. This article explores how this capability can be realized by the innovative SMA-based superelasticity-assisted slider (SSS), recently proposed for next-generation seismic protection of structures. The versatility of SSS is first discussed as a critical advantage for an effective SER. Alternative configurations and implementation styles of the system are presented, and other advantageous features of this high-tech isolation system (IS) are studied. Results of shaking table experiments, focused on investigating the expected usefulness of SSS for seismic protection in hospitals and conducted at the structural earthquake engineering laboratory of the University of Bonab, are then reported. SSS is compared with currently used ISs, and it is shown that SSS provides the required SER for the built environments and outperforms other ISs by benefitting from the pioneered utilization of SMAs in a novel approach.
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Terremotos , Aleaciones con Memoria de FormaRESUMEN
Separase is an endopeptidase that separates sister chromatids by cleaving cohesin Rad21 during the metaphase-to-anaphase transition. Conditional expression of Separase in tetracycline-inducible diploid FSK3 mouse mammary epithelial cells with both p53 WT and mutant (Ser-233-234) alleles of unknown physiological significance develops aneuploidy within 5 days of Separase induction in vitro. Overexpression of Separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges. In an in vivo mouse mammary transplant model, induction of Separase expression in the transplanted FSK3 cells for 3-4 weeks results in the formation of aneuploid tumors in the mammary gland. Xenograft studies combined with histological and cytogenetic analysis reveal that Separase-induced tumors are clonal in their genomic complements and have a mesenchymal phenotype suggestive of an epithelial-mesenchymal transition. Induction of Separase resulted in trisomies for chromosomes 8, 15, and 17; monosomy for chromosome 10; and amplification of the distal region of chromosomes 8 and 11. Separase protein is found to be significantly overexpressed in human breast tumors compared with matched normal tissue. These results collectively suggest that Separase is an oncogene, whose overexpression alone in mammary epithelial cells is sufficient to induce aneuploidy and tumorigenesis in a p53 mutant background.
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Aneuploidia , Neoplasias de la Mama/enzimología , Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/metabolismo , Neoplasias Mamarias Experimentales/enzimología , Anafase , Animales , Western Blotting , Línea Celular Tumoral , Cromátides/enzimología , Inestabilidad Cromosómica , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Humanos , Metafase , Ratones , Hibridación de Ácido Nucleico , Separasa , TetraciclinaRESUMEN
Large/giant congenital nevi (L/GCMN) are benign neoplasms of the melanocytic neural crest lineage covering extensive areas of skin presenting risk for melanoma. Surgical resection often leads to scarring and trauma. Histone deacetylase inhibitors (iHDACs) as topical therapeutic agents may prove beneficial as an alternative/adjunct to surgery in this disease. Here we describe the effect of in vitro treatment of iHDACs drugs on primary nevocytes isolated from L/GCMN patients. Micropthalmia transcription factor (MITF) expression in L/GCMN patients' lesions was detected by immunohistochemistry, in cultured nevocytes by immunofluorescence, immunoblot and quantitative polymerase chain reaction. Cellular senescence was detected by SA-ß galactosidase activity. Markers for melanocytic differentiation were evaluated by immunoblot analysis and extracted melanin content was estimated spectrophotometrically. Cell death was measured by lactate dehydrogenase (LDH) assay and necrosis confirmed by polymerase (PARP) cleavage and acridine orange staining of the nuclei. MITF was expressed ubiquitously in nevocytes and melanocytes in patients' lesions. In culture, iHDAC treatment suppressed MITF protein and mRNA expression resulting in a senescent-like phenotype with positive ß-galactosidase staining, progressing to necrotic cell death as evidenced by increased LDH activity, appearance of cleaved PARP and necrotic nuclei. This is the first report showing evidence of iHDACs-induced MITF suppression in congenital nevocytes in vitro leading to a morphologic change with positive ß-galactosidase staining, followed by necrotic cell death in nevocytes, indicating that iHDAC drugs could be valuable therapeutic agents for treatment of L/GCMN lesions.
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Inhibidores de Histona Desacetilasas/uso terapéutico , Nevo Pigmentado/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Factores de Transcripción/efectos de los fármacos , Vorinostat/uso terapéutico , Muerte Celular , Diferenciación Celular , Preescolar , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Lactante , Vorinostat/farmacologíaRESUMEN
Resumption of meiosis from diplotene arrest during the first meiotic prophase in vertebrate oocytes is universally controlled by MPF, a heterodimer of Cdk1 and cyclin B. Activation of MPF depends on the withdrawal of Cdk1 inhibition by Wee1/Myt1 kinase on the one hand and the activation of Cdk1 by Cdc25 phosphatase on the other. It is relevant to know whether both these pathways are necessary to rescue diplotene arrest or if either one of them is sufficient. In MIH (17alpha, 20beta dihydroxy-4-pregnen-3-one) incubated perch (Anabas testudineus) oocytes we have examined these possibilities. Perch oocyte extract following MIH incubation showed a significant increase in Myt1 phosphorylation from 12 to 16 hr indicating its progressive deactivation. MIH induced Mos expression markedly increased at 16 hr effecting 95% GVBD. Cycloheximide inhibited MIH induced Mos expression and its phosphorylation, which in turn reduced Myt1 phosphorylation and GVBD. Myt1 phosphorylation was blocked in Mos immunodepleted oocytes. All these suggest the involvement of Mos in Myt1 phosphorylation. Oocytes incubated in MIH for 16 hr activated Cdc25, but such activation could not rescue the inhibition of GVBD due to Myt1 in Mos immunodepleted oocytes. Blocking Cdc25 with an antisense oligo significantly inhibited GVBD even though Myt1 remained deactivated during this period. Taken together, our findings indicate that MIH requires both pathways for perch oocyte maturation: the expression and activation of Mos, which is linked to Myt1 deactivation on the one hand, and the activation of Cdc25 on the other, as blocking either pathway compromised G2-M transition in perch oocytes.
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Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiología , Proteínas Oncogénicas v-mos/metabolismo , Oocitos/crecimiento & desarrollo , Percas/fisiología , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Hidroxiprogesteronas/metabolismo , Factor Promotor de Maduración/genética , Factor Promotor de Maduración/metabolismo , Proteínas Oncogénicas v-mos/genética , Oogénesis/efectos de los fármacos , Oogénesis/fisiología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Fosfatasas cdc25/metabolismoRESUMEN
Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.
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Endopeptidasas/análisis , Oligopéptidos/análisis , Espectrometría de Fluorescencia/métodos , Anafase , Animales , Línea Celular , Endopeptidasas/metabolismo , Activación Enzimática , Estabilidad de Enzimas , Femenino , Humanos , Cinética , Leucemia/enzimología , Metafase , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Oligopéptidos/química , Péptidos/química , Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Xenopus laevisRESUMEN
Factors regulating transcription of pluripotency genes in congenital nevo-melanocytes are not known. Nevo-melanocytes belong somewhere in-between the ends of a spectrum where the normal epidermal melanocyte represents one end and a melanoma cell with multiple genetic abnormalities represents the other. Cells from large/giant congenital nevi (L/GCMN), unlike normal melanocytes, grow colonies on soft agar and express pluripotency markers, similar to melanoma cells. In this study normal melanocytes, SKMEL28 melanoma cells and nevo-melanocytes isolated from three L/GCMN patients were exposed to niche factors bFGF and IGF1 in vitro at physiological doses, and expression of a panel of pluripotency markers was determined by RT-PCR. While normal melanocytes did not show any significant transcriptional change in the genes studied, bFGF induced transcription of Sox2 and Bmi1 in melanoma cells. Patients' cells showed differential expression, with Sox10 being common to C76N and PD1N, while only Sox2 and Bmi1 were upregulated in C139N. IGF1 on the other hand induced unique sets of genes in each individual sample. We conclude that expression of pluripotency genes in L/GCMN cells is affected by niche factors bFGF and IGF1; however, each individual growth factor induced a unique set of genes in a patient's cells.
RESUMEN
BACKGROUND: Omipalisib has been found to affect the viability of cancer cells. However, its effect on clonogenicity - a feature of cancer stem cells, is not clear. Cells isolated from neurocutaneous melanocytosis (NCM) patients' lesions grow clonogenically. The aim of this study was to investigate the effect of omipalisib treatment on clonogenic growth of NCM cells in vitro. MATERIALS AND METHODS: Clonogenic growth efficiency was evaluated by colony formation assays with or without specific growth factors. Activation of MEK and Akt was determined by immunoblots. Colony formation and cell viability were assessed upon pharmacological inhibition of MEK, Akt and mToR. RESULTS: Clonogenicity appeared to depend on bFGF and IGF1signaling through ERK and Akt. Omipalisib treatment prevented colony formation and induced autophagic cell death. CONCLUSION: Signaling through Akt is important for survival of clonogenic cells in NCM, and omipalisib treatment as a monotherapy or in combination with MEK162 could be an effective therapeutic strategy to inhibit clonogenic growth.
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Transformación Celular Neoplásica/efectos de los fármacos , Melanoma/prevención & control , Melanosis/complicaciones , Síndromes Neurocutáneos/complicaciones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinolinas/farmacología , Neoplasias Cutáneas/prevención & control , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Femenino , Humanos , Lactante , Melanoma/etiología , Melanoma/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridazinas , Transducción de Señal , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Involvement of novel PKCs (nPKCs) in the negative regulation of insulin-signaling pathway is a current interest of many workers investigating the cause for insulin resistance and type 2 diabetes. Free fatty acids (FFAs) are recently shown to be the major players in inducing insulin resistance in insulin target cells. They are also found to be involved in activating nPKCs associated with the impairment of insulin sensitivity. In this overview, we describe PKC delta, theta and epsilon linked to the FFA induced damage of insulin-signaling molecules.
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Ácidos Grasos no Esterificados/farmacología , Insulina/fisiología , Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Ácidos Grasos no Esterificados/sangre , Humanos , Modelos Biológicos , Isoformas de Proteínas/química , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/fisiología , Proteína Quinasa C/química , Proteína Quinasa C/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Neurocutaneous melanocytosis (NCM) is characterized by clonal nevomelanocytic proliferations in the CNS and skin. Given the scarcity of effective therapeutic targets, testing new drugs requires a reliable and reproducible in vitro cellular model of the disease. METHODS: We generated nevomelanocytic spheroids in vitro from lesions of the spinal cord, brain, and skin from 4 NCM patients. Nevomelanocytic cells were grown as monolayers or spheroids and their growth characteristics were evaluated. Cultured cell identity was confirmed by demonstration of the same NRAS mutation found in the original lesions and by immunophenotyping. Nevomelanocytic spheroids were treated with inhibitors of specific mediators of the NRAS signaling pathway (vemurafenib, MEK162, GDC0941, and GSK2126458). Drug sensitivity and cell viability were assessed. RESULTS: Cultured cells were growth-factor dependent, grew as spheroids on Geltrex matrix, and maintained their clonogenicity in vitro over passages. Skin-derived cells formed more colonies than CNS-derived cells. Inhibitors of specific mediators of the NRAS signaling pathway reduced viability of NRAS mutated cells. The highest effect was obtained with GSK2126458, showing a viability reduction below 50%. CONCLUSIONS: NRAS mutated cells derived from clinical NCM samples are capable of continuous growth as spheroid colonies in vitro and retain their genetic identity. Drugs targeting the NRAS signaling pathway reduce in vitro viability of NCM cells. NCM lesional spheroids represent a new and reliable experimental model of NCM for use in drug testing and mechanistic studies.
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Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , Neoplasias Encefálicas/patología , GTP Fosfohidrolasas/antagonistas & inhibidores , Melanoma/patología , Proteínas de la Membrana/antagonistas & inhibidores , Neoplasias Cutáneas/patología , Esferoides Celulares/patología , Western Blotting , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , Técnica del Anticuerpo Fluorescente , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Técnicas para Inmunoenzimas , Lactante , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación/genética , Estudios Prospectivos , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Esferoides Celulares/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The mechanisms behind malignant progression in patients with giant nevi are largely unknown. Here, we aim to describe novel genetic findings and explain possible mechanisms resulting in the most severe form of neurocutaneous melanocytosis. Detailed histological (biopsy and post-mortem) studies, tissue culture, and high-resolution cytogenetic analysis, including chromosome and array comparative genomic hybridization, Ion AmpliSeq Cancer Panel, and Sanger sequencing, were performed on tissues from a white male who succumbed at 17 months of age to congenital melanoma associated with a bathing-trunk nevus. We also used quantitative PCR to quantitatively assess the expression of NRAS among normal cells, including fibroblast and melanocytes, as well as melanoma cells from our patient. Full autopsy documented tumors in the brain, spinal cord, lung, liver, testis, bone marrow, and, retrospectively, in the placenta. Next-generation sequencing and chromosome microarray in our patient revealed novel findings, including duplication of a mutated NRAS gene, leading to an aggressive clinical course and disseminated disease. Quantitative PCR showed a five-fold increase in NRAS expression in the melanoma cell line when compared with normal melanocytes. Finally, three amino acid-changing germline variants were detected: homozygous TP53 p.P72R, heterozygous KIT p.M541L, and homozygous KDR (VEGFR2) p.Q472H. These genes are involved in malignancy and other potentially relevant pathways, such as mast cell and melanocytic signaling, as well as angiogenesis. These findings provide novel insights into the biology of congenital melanocytic proliferations, showing that amplification of mutated NRAS seems to represent a new genetic mechanism leading to melanoma in the context of neurocutaneous melanocytosis.
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GTP Fosfohidrolasas/genética , Melanoma/congénito , Melanoma/patología , Melanosis/patología , Proteínas de la Membrana/genética , Síndromes Neurocutáneos/patología , Nevo Pigmentado/patología , Neoplasias Cutáneas/patología , Autopsia , Biopsia , Hibridación Genómica Comparativa , Resultado Fatal , Amplificación de Genes , Humanos , Lactante , Masculino , Melanocitos/patología , Melanoma/genética , Melanosis/genética , Mutación , Síndromes Neurocutáneos/genética , Nevo Pigmentado/genética , Neoplasias Cutáneas/genéticaRESUMEN
NRAS and BRAF mutations occur in congenital melanocytic nevi (CMN), but results are contradictory. Sixty-six prospectively collected CMN patients were analyzed for NRAS Q61 mutations using Sanger sequencing. Negative cases were evaluated for BRAF V600E mutation. NRAS Q61 mutations affected 51 patients (77.3%), and BRAF V600E was found in 5 (7.6%). NRAS Q61 mutation affected 29 (80.6%) of 36 giant, 16 (80.0%) of 20 large, and 5 (62.5%) of 8 medium-size CMN; BRAF mutation affected 1 (5%) of 20 large and 4 (11.4%) of 36 giant CMN. Compared to NRAS, BRAF-mutated nevi show scattered/extensive dermal and subcutaneous nodules (100% BRAF+ vs 34.8% NRAS+) (P=0.002). Neurocutaneous melanocytosis (NCM) affected 16 (24.2%) of 66 patients, with NRAS Q61 mutation in 12 (75.0%), and BRAF V600E in 2 (12.5%), P=0.009. Two patients were negative for both mutations (12.5%). In conclusion, although NRAS Q61 mutations predominate, BRAF V600E mutation also affects patients with large/giant CMN (L/GCMN), and with NCM, a novel finding. BRAF V600E is also associated with increased dermal/subcutaneous nodules. These findings open the possibility of BRAF-targeted therapy in some L/GCMN and NCM cases.
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Mutación , Nevo Pigmentado/congénito , Nevo Pigmentado/genética , Proteínas Proto-Oncogénicas B-raf/genética , Adolescente , Proliferación Celular , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , GTP Fosfohidrolasas/metabolismo , Genotipo , Humanos , Lactante , Masculino , Melanocitos/metabolismo , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Síndromes Neurocutáneos , Nevo Pigmentado/patología , Fenotipo , Estudios Prospectivos , Neoplasias Cutáneas/congénito , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Resultado del TratamientoRESUMEN
Embryonic signaling pathways, in particular those mediated by Wnt and TGF-ß, are known to play key roles in tumor progression through the induction of epithelial-mesenchymal transition (EMT). Their simultaneous targeting could therefore represent a desirable anticancer strategy. On the basis of recent findings that both Wnt and TGF-ß-associated pathways are regulated by Hippo signaling in mammalian cells, we reasoned that targeting the latter would be more effective in inhibiting EMT. In a search for such inhibitors, we identified a small molecule (C19) with remarkable inhibitory activity not only against Hippo, but also against Wnt and TGF-ß pathways. C19 inhibited cancer cell migration, proliferation, and resistance to doxorubicin in vitro, and exerted strong antitumor activity in a mouse tumor model. Mechanistically, C19 induced GSK3-ß-mediated degradation of the Hippo transducer TAZ, through activation of the Hippo kinases Mst/Lats and the tumor suppressor kinase AMPK upstream of the degradation complex. Overall, this study identified C19 as a multi-EMT pathway inhibitor with a unique mechanism of action. The findings that both AMPK and Mst/Lats mediate the antitumor activity of C19 shed light on a potential cross-talk between metabolic and organ size control pathways in regulating cancer progression. By simultaneously targeting these two pathways, C19 may represent a new type of agents to suppress cancer progression and/or its recurrence.
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Antineoplásicos/administración & dosificación , Neoplasias/tratamiento farmacológico , Propanoles/administración & dosificación , Proteínas Serina-Treonina Quinasas/metabolismo , Tiadiazoles/administración & dosificación , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Aciltransferasas , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Vía de Señalización Hippo , Humanos , Ratones , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Nevocytes (NC) and mastocytes (MC) have different progenitors but share stem cell factor as regulator/activator of NC and for differentiation/proliferation of MC. Both cell types express stem cell factor receptor CD117. We hypothesize that large/giant congenital melanocytic nevi (L/GCMN) may associate with MC hyperplasia. Forty-nine L/GCMN were examined, 12 samples from uninvolved skin of L/GCMN patients and 6 control skin samples studied with Giemsa and immunohistochemistry for CD117 and MC-tryptase. Picrosirius red (PR) was used to assess fibrosis. Digital images were used to count MC/mm(2) using ImageJ software. Western blot (WB) for MC-tryptase in 12 GCMN and 12 non-nevus samples was performed. Analysis of variance (Tukey) and Pearson statistical tests were applied. Increased MCs were observed in nevus tissue (75.1 ± 35.3 MCs/mm(2)) and in uninvolved skin (53.74 ± 27.7 MC/ mm(2)). P â=â 0.109 from patients with L/GCMN, compared with controls from individuals without L/GCMN (28.74 ± 8.4 MC/mm(2)); P â=â 0.001 supported by results of WB analysis for tryptase. A positive trend toward correlation of MC numbers with fibrosis, assessed by PR staining fell short of statistical significance (r â=â 0.245; P â=â 0.086); no difference in fibrosis was found between nevus and non-nevus skin from patients with L/GCMN (P â=â 0.136). We found a higher density of MC, both in normal-appearing skin and nevus areas of L/GCMN patients, compared with control skin samples from individuals without nevi. Given the abnormal wound healing and allergic reactions described in L/GCMN patients, these findings suggest a potential role for MC in the biology of L/GCMN, making them a potential target for therapeutic intervention.
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Mastocitos/patología , Nevo Pigmentado/patología , Neoplasias Cutáneas/patología , Adolescente , Western Blotting , Proliferación Celular , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Lactante , Masculino , Mastocitos/inmunología , Nevo Pigmentado/inmunología , Neoplasias Cutáneas/inmunologíaRESUMEN
The Hippo pathway is a signaling cascade recently found to play a key role in tumorigenesis therefore understanding the mechanisms that regulate it should open new opportunities for cancer treatment. Available data indicate that this pathway is controlled by signals from cell-cell junctions however the potential role of nuclear regulation has not yet been described. Here we set out to verify this possibility and define putative mechanism(s) by which it might occur. By using a luciferase reporter of the Hippo pathway, we measured the effects of different nuclear targeting drugs and found that chromatin-modifying agents, and to a lesser extent certain DNA damaging drugs, strongly induced activity of the reporter. This effect was not mediated by upstream core components (i.e. Mst, Lats) of the Hippo pathway, but through enhanced levels of the Hippo transducer TAZ. Investigation of the underlying mechanism led to the finding that cancer cell exposure to histone deacetylase inhibitors induced secretion of growth factors and cytokines, which in turn activate Akt and inhibit the GSK3 beta associated protein degradation complex in drug-affected as well as in their neighboring cells. Consequently, expression of EMT genes, cell migration and resistance to therapy were induced. These processes were suppressed by using pyrvinium, a recently described small molecule activator of the GSK 3 beta associated degradation complex. Overall, these findings shed light on a previously unrecognized phenomenon by which certain anti-cancer agents may paradoxically promote tumor progression by facilitating stabilization of the Hippo transducer TAZ and inducing cancer cell migration and resistance to therapy. Pharmacological targeting of the GSK3 beta associated degradation complex may thus represent a unique approach to treat cancer.
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Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Acetilación , Aciltransferasas , Antineoplásicos/farmacología , Movimiento Celular , Ensamble y Desensamble de Cromatina , Citocinas/metabolismo , Daño del ADN , Resistencia a Antineoplásicos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Estabilidad Proteica , Receptores Acoplados a Proteínas G/metabolismo , Sulfonamidas/farmacología , Factores de Transcripción/metabolismoRESUMEN
Cellular senescence is considered as a tumor suppressive mechanism. Recent evidence indicates however that senescent cells secrete various growth factors and cytokines, some of which may paradoxically promote cancer progression. This phenomenon termed senescence-associated secretory phenotype (SASP) must be inhibited in order for anti-proliferative agents to be effective. The present study was designed to determine whether the ß-catenin destruction complex (BCDC), known to integrate the action of various growth factors and cytokines, would represent a suitable target to inhibit the activity of SASP components. For this, we carried out experiments to determine the effect of drug-induced senescence on secretion of SASP, ß-catenin transactivation, and the relationship between these processes. Moreover, genetic and pharmacological approaches were used to define the implication of BCDC in mediating the effects of SASP components on cell migration and resistance to drugs. The findings indicate that drug-induced senescence was associated with expression of various Wnt ligands in addition to previously known SASP components. Beta catenin transactivation and expression of genes implicated in epithelial-mesenchymal transition (EMT) also increased in response to drug-induced SASP. These effects were prevented by Pyrvinium, a recently described activator of BCDC. Pyrvinium also suppressed the effects of SASP on cell migration and resistance to doxorubicin. Together, these findings provide insights on the potential role of BCDC in mediating the effects of drug-induced SASP on cancer cell invasion and resistance to therapy, and suggest that targeting this pathway may represent an effective approach to enhance the activity of current and prospective anti-cancer therapeutics.
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Antineoplásicos/farmacología , Complejo de Señalización de la Axina/metabolismo , Senescencia Celular/efectos de los fármacos , Fenotipo , Complejo de Señalización de la Axina/antagonistas & inhibidores , Biomarcadores , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Humanos , Ligandos , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/metabolismoRESUMEN
Maturation of vertebrate oocytes is regulated by maturation inducing hormone (MIH), which is progesterone in all vertebrates except in fish, where it is 17alpha, 20beta dihydroxy progesterone. Once the full growth of the oocytes is achieved, they arrest at prophase of meiosis I. MIH releases oocytes from this arrest. MIH promotes the formation of a dimeric protein kinase complex known as maturation promoting factor (MPF), the regulatory component of which is cyclin B and the catalytic component is cell division cycle (Cdc2) kinase. This complex is activated by phosphorylation at Thrl61 but remains inactive due to the inhibitory phosphorylation at Thrl4 and Tyrl5. MIH stimulates Cdc25, a dual specific phosphatase, that dephosphorylates both Thrl4 and Tyrl5 and converts pre- or inactive MPF to active MPF. Germinal vesicle break down (GVBD) is the marker of oocyte maturation. In an Indian freshwater perch, Anabas testudineus, MIH induced GVBD between 18-20 h. MIH induced oocytes extract in SDS-PAGE showed over-expression of a 30 kDa protein, which is confirmed to be cyclin B by using both monoclonal and polyclonal anti-cyclin B antibodies from various sources. The size of cyclin B in other vertebrates including mammals lies between 46-55 kDa. We have cloned cyclin B gene from perch oocyte and found it to contain the domains required for its function and immunological recognition. We also cloned Cdkl gene, which is very similar to other vertebrates Cdkl. Perch oocyte Cdc25 is overexpressed prior to GVBD converting inactive MPF to active MPF that affect GVBD. The objective of this overview is to deal with the molecular regulation of MPF activation which causes final maturation of oocytes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor Promotor de Maduración/genética , Oocitos/metabolismo , Oogénesis/genética , Transducción de Señal/fisiología , Vertebrados/metabolismo , Animales , Ciclinas/metabolismo , Retroalimentación Fisiológica , Humanos , Factor Promotor de Maduración/metabolismo , Profase Meiótica I , Oocitos/citologíaRESUMEN
The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1 microg/ml), (b) only melatonin (at concentrations of 50, 100 or 500 pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4 h before addition of MIH, (ii) 2 h before addition of MIH, (iii) co-administered with MIH (0 h interval) or (iv) 2 h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16 h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16 h. Administration of melatonin along with MIH (at 0 h interval) or 2 h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50 pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100 pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, Cdk1. Densitometric analysis of the immunoblot data collected from the melatonin pre-treated MIH incubated oocytes showed that cyclin B level continued to increase even after 4 h of incubation, and reached the peak after 12 h. Moreover, determination of H1 kinase activity as an indicator of MPF activity in oocytes revealed that melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Thus, the present study demonstrates for the first time that prior incubation with melatonin accelerates the action of MIH on carp oocyte maturation.