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Bimetallic gold based catalysts have been prepared using a sol immobilisation technique. Despite a very similar metal dispersion, different structures are revealed depending on the second metal, with alloyed systems being preferred in the case of Pd, Pt and Cu, and core-shell in the case of Ru. A positive synergistic effect between the metals has been revealed only in the cases of Pd and Cu in the oxidation of benzyl alcohol. AuPd/C has been also studied in the hydrogenation of benzaldehyde where the bimetallic catalyst revealed a different selectivity compared to the monometallic counterpart.
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BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.
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Acalasia del Esófago/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Regulación hacia Abajo , Femenino , Humanos , Células Intersticiales de Cajal/metabolismo , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Transcriptoma , Adulto JovenRESUMEN
A class of heterogeneous catalysts based on commercial bentonite from natural origin, containing at least 80 wt% of montmorillonite clay, was designed to transform selectively and under mild conditions toxic organosulfur and organophosphorus chemical warfare agents into non-noxious products with a reduced impact on health and environment. The bentonite from the natural origin was modified by introducing iron species and acid sites in the interlayer space, aiming to obtain a sorbent with strong catalytic oxidising and hydrolytic properties. The catalytic performance of these materials was evaluated in the oxidative abatement of (2-chloroethyl)ethyl sulfide (CEES), a simulant of sulfur mustard, in the presence of aqueous hydrogen peroxide as an oxidant. A new decontamination formulation was, moreover, proposed and obtained by mixing sodium perborate, as a solid oxidant, to iron-bentonite catalysts. Solid-phase decontamination tests, performed on a cotton textile support contaminated with organosulfide and organophosphonate simulant agents revealed the good activity of the solid formulation, especially in the in situ detoxification of blistering agents. Tests carried out on the real blistering warfare agent, sulfur mustard (HD agent), showed that, thanks to the co-presence of the iron-based clay together with the solid oxidant component, a good decontamination of the test surface from the real warfare agent could be achieved (80% contaminant degradation, under ambient conditions, in 24 h).
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CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.
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Quinasa de la Caseína II/metabolismo , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
Sphingolipids, such as ceramide, sphingosine and sphingosine 1-phosphate (S1P) are bioactive molecules that have important functions in a variety of cellular processes, which include proliferation, survival, differentiation and cellular responses to stress. Sphingolipids have a major impact on the determination of cell fate by contributing to either cell survival or death. Although ceramide and sphingosine are usually considered to induce cell death, S1P promotes survival of cells. Sphingosine kinases (SPHKs) are the enzymes that catalyze the conversion of sphingosine to S1P. There are two isoforms, SPHK1 and SPHK2, which are encoded by different genes. SPHK1 has recently been implicated in contributing to cell transformation, tumor angiogenesis and metastatic spread, as well as cancer cell multidrug-resistance. More recent findings suggest that SPHK2 also has a role in cancer progression. This review is an overview of our understanding of the role of SPHKs and S1P in hematopoietic malignancies and provides information on the current status of SPHK inhibitors with respect to their therapeutic potential in the treatment of hematological cancers.
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Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/enzimología , Terapia Molecular Dirigida/métodos , Progresión de la Enfermedad , Humanos , Lisofosfolípidos/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Esfingosina/análogos & derivados , Esfingosina/antagonistas & inhibidoresRESUMEN
Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.
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Antineoplásicos/uso terapéutico , Quinasa de la Caseína II/antagonistas & inhibidores , Naftiridinas/uso terapéutico , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Transducción de Señal , Respuesta de Proteína Desplegada , Animales , División Celular , Chaperón BiP del Retículo Endoplásmico , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/química , Fenazinas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologíaRESUMEN
The phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) are two major signaling molecules in the PI3K/Akt/mTOR signal transduction cascade. This pathway is a key regulator of a wide range of physiological cell processes which include proliferation, differentiation, survival, metabolism, exocytosis, motility, and autophagy. However, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences response to therapeutic treatments. Therefore, targeting PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome. The PI3K/Akt/mTOR signaling network is activated in acute leukemias of both myelogenous and lymphoid lineage, where it correlates with poor prognosis and enhanced drug-resistance. The catalytic sites of PI3K and mTOR share a high degree of sequence homology. This feature has allowed the synthesis of ATP-competitive compounds that targeted the catalytic site of both PI3K and mTOR (e.g. PI-103, NVP-BEZ235). In preclinical settings, dual PI3K/mTOR inhibitors displayed a much stronger cytotoxicity against leukemic cells than either PI3K inhibitors or allosteric mTOR inhibitors, such as rapamycin and its derivatives (rapalogs). At variance with rapamycin/rapalogs, dual PI3K/mTOR inhibitors targeted both mTOR complex 1 and mTOR complex 2, and inhibited the rapamycin-resistant phosphorylation of eukaryotic initiation factor 4E-binding protein 1, resulting in a marked inhibition of oncogenetic protein translation in leukemic cells. Hence, they strongly reduced the proliferation rate and induced an important apoptotic response. Here, we reviewed the evidence documenting that dual PI3K/mTOR inhibitors represent a promising option for future targeted therapies of leukemic patients.
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BACKGROUND: GDNF/RET and Endothelin-3 (ET-3)/EDNRB regulate survival, differentiation, migration, and proliferation of neural crest-derived cells. Although several RET and EDNRB signalling mediators have been characterized, most of the genes targeted by these two pathways are still largely unknown. We focused our study on apolipoprotein B (APOB) as a novel target gene of the RET and EDNRB pathways, based on previous data obtained using a Caenorhabditis elegans strain mutant for the homologue of mammalian ECE1. METHODS: Molecular and cellular studies of Apob were performed in the murine Neuro2a cells, an in vitro model for studying neural crest-derived cell development, along with a mouse knock-in for the Hirschsprung-associated mutation Ret(C620R). Silencing for Apob and Ret has been performed via shRNA. KEY RESULTS: GDNF/RET and ET-3/EDNRB cooperated in inducing neuronal differentiation resulting in Apob activation in Neuro2a cell line. Apob expression was downregulated in mouse embryos homozygous for the Ret(C620R) mutation and presenting a severe Hirschsprung phenotype. Ret silencing prevented Apob expression increase. MAPK P38 kinase activation evoked Apob expression via GDNF/RET signalling in Neuro2a cells. A p53-dependent repressor element in Apob promoter resulted in a reduced Apob expression. Silencing of Apob reduced HuD protein expression. CONCLUSIONS & INFERENCES: Apob is a novel downstream target of the RET/EDNRB pathways with a role in neuronal survival and maintenance, as indicated by its effect on HuD expression. Our data provide a conceptual framework to investigate and establish the role of APOB gene in severe gut dysmotility.
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Apolipoproteínas B/metabolismo , Endotelina-3/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , Apolipoproteínas B/genética , Western Blotting , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Endotelina-3/genética , Técnicas de Sustitución del Gen , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Enfermedad de Hirschsprung/genética , Enfermedad de Hirschsprung/metabolismo , Humanos , Inmunohistoquímica , Ratones , Regiones Promotoras Genéticas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/ß and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.
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Compuestos Heterocíclicos con 3 Anillos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Doxorrubicina/farmacología , Sinergismo Farmacológico , Humanos , Fosforilación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Transducción de SeñalRESUMEN
Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.
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Mamíferos/metabolismo , Células Madre Neoplásicas/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Humanos , Células Madre Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway. Recent works have demonstrated a crucial role for IDO in the induction of immune tolerance during infections, pregnancy, transplantation, autoimmunity, and neoplasias. IDO is widely expressed in human tissues and cell subsets, including dendritic cells, where it modulates their function by increasing tolerogenic capacities. The aim of the present paper is to highlight the most recent data about IDO expression in dendritic cells and its role as a potent inducer of T regulatory cells.
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Células Dendríticas/inmunología , Tolerancia Inmunológica , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Linfocitos T Reguladores/inmunología , Autoinmunidad , Enfermedades Transmisibles/enzimología , Enfermedades Transmisibles/inmunología , Células Dendríticas/enzimología , Femenino , Humanos , Neoplasias/enzimología , Neoplasias/inmunología , Embarazo , Linfocitos T Reguladores/enzimología , Inmunología del TrasplanteRESUMEN
The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health.
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Antineoplásicos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Leucemia/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Terapia Molecular Dirigida , Proteínas de Neoplasias/fisiología , Quinasas raf/fisiología , Proteínas ras/fisiología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular/efectos de los fármacos , División Celular/genética , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Modelos Biológicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinasas raf/antagonistas & inhibidores , Quinasas raf/genética , Proteínas ras/antagonistas & inhibidores , Proteínas ras/genéticaRESUMEN
It has become apparent that regulation of protein translation is an important determinant in controlling cell growth and leukemic transformation. The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway is often implicated in sensitivity and resistance to therapy. Dysregulated signaling through the PI3K/PTEN/Akt/mTOR pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Furthermore, this pathway is activated by autocrine transformation mechanisms. PTEN is a critical tumor suppressor gene and its dysregulation results in the activation of Akt. PTEN is often mutated, silenced and is often haploinsufficient. The mTOR complex1 (mTORC1) regulates the assembly of the eukaryotic initiation factor4F complex, which is critical for the translation of mRNAs that are important for cell growth, prevention of apoptosis and transformation. These mRNAs have long 5'-untranslated regions that are G+C rich, rendering them difficult to translate. Elevated mTORC1 activity promotes the translation of these mRNAs via the phosphorylation of 4E-BP1. mTORC1 is a target of rapamycin and novel active-site inhibitors that directly target the TOR kinase activity. Although rapamycin and novel rapalogs are usually cytostatic and not cytotoxic for leukemic cells, novel inhibitors that target the kinase activities of PI3K and mTOR may prove more effective for leukemia therapy.
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Antineoplásicos/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia/tratamiento farmacológico , Terapia Molecular Dirigida , Proteínas de Neoplasias/fisiología , Fosfohidrolasa PTEN/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/fisiología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica/genética , Humanos , Leucemia/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , MicroARNs/genética , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/fisiología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas/antagonistas & inhibidores , Proteínas/efectos de los fármacos , Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Seudogenes , ARN Mensajero/genética , ARN Neoplásico/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/fisiologíaRESUMEN
The mammalian Target Of Rapamycin (mTOR) serine/threonine kinase belongs to two multi-protein complexes, referred to as mTORC1 and mTORC2. mTOR-generated signals have critical roles in leukemic cell biology by controlling mRNA translation of genes that promote proliferation and survival. However, allosteric inhibition of mTORC1 by rapamycin has only modest effects in T-cell acute lymphoblastic leukemia (T-ALL). Recently, ATP-competitive inhibitors specific for the mTOR kinase active site have been developed. In this study, we have explored the therapeutic potential of active-site mTOR inhibitors against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. The inhibitors affected T-ALL cell viability by inducing cell-cycle arrest in G(0)/G(1) phase, apoptosis and autophagy. Western blot analysis demonstrated a Ser 473 Akt dephosphorylation (indicative of mTORC2 inhibition) and a dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors. The inhibitors strongly synergized with both vincristine and the Bcl-2 inhibitor, ABT-263. Remarkably, the drugs targeted a putative leukemia-initiating cell sub-population (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, the inhibitors displayed remarkable anti-leukemic activity, which emphasizes their future development as clinical candidates for therapy in T-ALL.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Western Blotting , Dominio Catalítico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Inmunosupresores/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismoRESUMEN
Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6-12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34(+) leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34(+) CD38(Low/Neg) CD123(+)) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Erucicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , MAP Quinasa Quinasa 4/metabolismo , Fosforilcolina/análogos & derivados , Células Precursoras de Linfocitos B/efectos de los fármacos , Proteína Fosfatasa 2/metabolismo , Animales , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilcolina/farmacología , Células Precursoras de Linfocitos B/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.
Asunto(s)
Apoptosis/fisiología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Animales , Western Blotting , Caspasas/metabolismo , Línea Celular , ADN/análisis , ADN/biosíntesis , ADN/genética , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Etiquetado Corte-Fin in Situ , Potenciales de la Membrana/fisiología , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Membranas Mitocondriales/fisiología , Fibras Musculares Esqueléticas/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Mioblastos/fisiología , Mioblastos/ultraestructura , Rayos UltravioletaRESUMEN
We examined the effect of arousals (shifts from sleep to wakefulness) on breathing during sleep using a mathematical model. The model consisted of a description of the fluid dynamics and mechanical properties of the upper airways and lungs, as well as a controller sensitive to arterial and brain changes in CO(2), changes in arterial oxygen, and a neural input, alertness. The body was divided into multiple gas store compartments connected by the circulation. Cardiac output was constant, and cerebral blood flows were sensitive to changes in O(2) and CO(2) levels. Arousal was considered to occur instantaneously when afferent respiratory chemical and neural stimulation reached a threshold value, while sleep occurred when stimulation fell below that value. In the case of rigid and nearly incompressible upper airways, lowering arousal threshold decreased the stability of breathing and led to the occurrence of repeated apnoeas. In more compressible upper airways, to maintain stability, increasing arousal thresholds and decreasing elasticity were linked approximately linearly, until at low elastances arousal thresholds had no effect on stability. Increased controller gain promoted instability. The architecture of apnoeas during unstable sleep changed with the arousal threshold and decreases in elasticity. With rigid airways, apnoeas were central. With lower elastances, apnoeas were mixed even with higher arousal thresholds. With very low elastances and still higher arousal thresholds, sleep consisted totally of obstructed apnoeas. Cycle lengths shortened as the sleep architecture changed from mixed apnoeas to total obstruction. Deeper sleep also tended to promote instability by increasing plant gain. These instabilities could be countered by arousal threshold increases which were tied to deeper sleep or accumulated aroused time, or by decreased controller gains.
Asunto(s)
Nivel de Alerta/fisiología , Fenómenos Fisiológicos Respiratorios , Sueño/fisiología , Retroalimentación , Humanos , Masculino , Modelos Neurológicos , Síndromes de la Apnea del Sueño/fisiopatologíaRESUMEN
A cytokine-dependent (FL5.12), drug-sensitive, p53 wild type (WT) and a doxorubicin-resistant derivative line (FL/Doxo) were used to determine the mechanisms that could result in drug resistance of early hematopoietic precursor cells. Drug resistance was associated with decreased p53 induction after doxorubicin treatment, which was due to a higher level of proteasomal degradation of p53. Dominant-negative (DN) p53 genes increased the resistance to chemotherapeutic drugs, MDM-2 and MEK inhibitors, further substantiating the role of p53 in therapeutic sensitivity. The involvement of signal transduction and apoptotic pathways was examined, as drug resistance did not appear to be due to increased drug efflux. Drug-resistant FL/Doxo cells had higher levels of activated Raf/MEK/ERK signaling and decreased induction of apoptosis when cultured in the presence of doxorubicin than drug-sensitive FL5.12 cells. Introduction of DN MEK1 increased drug sensitivity, whereas constitutively active (CA) MEK1 or conditionally active BRAF augmented resistance, documenting the importance of the Raf/MEK/ERK pathway in drug resistance. MEK inhibitors synergized with chemotherapeutic drugs to reduce the IC(50). Thus the p53 and Raf/MEK/ERK pathways play key roles in drug sensitivity. Targeting these pathways may be effective in certain drug-resistant leukemias that are WT at p53.
Asunto(s)
Resistencia a Medicamentos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Quinasas raf/metabolismo , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Caspasas/metabolismo , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Doxorrubicina/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Genes Dominantes , Células Madre Hematopoyéticas/metabolismo , Imidazoles/farmacología , Leupeptinas/farmacología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Quinasas raf/antagonistas & inhibidoresRESUMEN
Increased loop gain (a function of both controller gain and plant gain), which results in instability in feedback control, is of major importance in producing recurrent central apnoeas during sleep but its role in causing obstructive apnoeas is not clear. The purpose of this study was to investigate the role of loop gain in producing obstructive sleep apnoeas. Owing to the complexity of factors that may operate to produce obstruction during sleep, we used a mathematical model to sort them out. The model used was based on our previous model of neurochemical control of breathing, which included the effects of chemical stimuli and changes in alertness on respiratory pattern generator activity. To this we added a model of the upper airways that contained a narrowed section which behaved as a compressible elastic tube and was tethered during inspiration by the contraction of the upper airway dilator muscles. These muscles in the model, as in life, responded to changes in hypoxia, hypercapnia and alertness in a manner similar to the action of the chest wall muscles, opposing the compressive action caused by the negative intraluminal pressure generated during inspiration which was magnified by the Bernoulli Effect. As the velocity of inspiratory airflow increased, with sufficiently large increase in airflow velocity, obstruction occurred. Changes in breathing after sleep onset were simulated. The simulations showed that increases in controller gain caused the more rapid onset of obstructive apnoeas. Apnoea episodes were terminated by arousal. With a constant controller gain, as stiffness decreased, obstructed breaths appeared and periods of obstruction recurred longer after sleep onset before disappearing. Decreased controller gain produced, for example, by breathing oxygen eliminated the obstructive apnoeas resulting from moderate reductions in constricted segment stiffness. This became less effective as stiffness was reduced more. Contraction of the upper airway muscles with hypercapnia and hypoxia could prevent obstructed apnoeas with moderate but not with severe reductions in stiffness. Increases in controller gain, as might occur with hypoxia, converted obstructive to central apnoeas. Breathing CO2 eliminated apnoeas when the activity of the upper airway muscles was considered to change as a function of CO2 to some exponent. Low arousal thresholds and increased upper airway resistance are two factors that promoted the occurrence and persistence of obstructive sleep apnoeas.
Asunto(s)
Química Encefálica/fisiología , Mecánica Respiratoria/fisiología , Apnea Obstructiva del Sueño/fisiopatología , Presión del Aire , Algoritmos , Nivel de Alerta/fisiología , Dióxido de Carbono/sangre , Retroalimentación/fisiología , Humanos , Pulmón/fisiología , Modelos Anatómicos , Oxígeno/sangre , Faringe/fisiología , Nervio Frénico/fisiología , Recurrencia , Músculos Respiratorios/fisiologíaRESUMEN
The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4-11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34(+) cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K-Akt survival pathway.