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1.
Curr Biol ; 25(19): 2479-92, 2015 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-26387716

RESUMEN

Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis/fisiología , Beclina-1 , Línea Celular Tumoral , Células Cultivadas , Drosophila , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Unión Proteica , Técnicas del Sistema de Dos Híbridos
2.
EMBO J ; 34(8): 1025-41, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25586377

RESUMEN

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1(+/-) mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.


Asunto(s)
Autofagia/efectos de los fármacos , Ácidos Grasos Insaturados/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , Beclina-1 , Caenorhabditis elegans , Células Cultivadas , Femenino , Células HeLa , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ácido Oléico/farmacología , Ácido Palmítico/farmacología , Saccharomyces cerevisiae , Regulación hacia Arriba/efectos de los fármacos
3.
Methods Enzymol ; 543: 73-88, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24924128

RESUMEN

Macroautophagy (hereafter referred to as autophagy), a central mechanism mediating the lysosomal degradation of cytoplasmic components, can be stimulated by a wide panel of adverse stimuli, including a panoply of anticancer agents. The central autophagic organelle is the autophagosome, a double membrane-bound vacuole that sequesters the cytoplasmic material destined to disposal. The ultimate destiny of the autophagosome is to fuse with a lysosome, resulting in the degradation of the autophagic cargo. In this setting, it is important to discriminate whether a particular stimulus actually promotes autophagy or it simply blocks the fusion of autophagosomes with lysosomes. To this aim, the methods that assess autophagy should assess not only the number of autophagosomes but also the so-called autophagic flux, that is, the clearance of the autophagy cargo from the lysosomal compartment. Here, we present a compendium of methods to assess the autophagic flux in cultured malignant cells. This approach should allow for the study of the intimate link between autophagy and oncometabolism in several experimental paradigms.


Asunto(s)
Autofagia , Neoplasias/patología , Línea Celular Tumoral , Humanos , Sondas Moleculares , Neoplasias/inmunología
4.
Autophagy ; 10(7): 1341-2, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24852146

RESUMEN

We recently reported that BAG6/BAT3 (BCL2-associated athanogene 6) is essential for basal and starvation-induced autophagy in E18.5 bag6(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of the EP300/p300-dependent acetylation of TRP53 and autophagy-related (ATG) proteins. We observed that BAG6 increases TRP53 acetylation during starvation and pro-autophagic TRP53-target gene expression. BAG6 also decreases the EP300 dependent-acetylation of ATG5, ATG7, and LC3-I, posttranslational modifications that inhibit autophagy. In addition, in the absence of BAG6 or when using a mutant of BAG6 exclusively located in the cytoplasm, autophagy is inhibited, ATG7 is hyperacetylated, TRP53 acetylation is abrogated, and EP300 accumulates in the cytoplasm indicating that BAG6 is involved in the regulation of the nuclear localization of EP300. We also reported that the interaction between BAG6 and EP300 occurs in the cytoplasm rather than the nucleus. Moreover, during starvation, EP300 is transported to the nucleus in a BAG6-dependent manner. We concluded that BAG6 regulates autophagy by controlling the localization of EP300 and its accessibility to nuclear (TRP53) and cytoplasmic (ATGs) substrates.


Asunto(s)
Autofagia , Proteína p300 Asociada a E1A/metabolismo , Espacio Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Acetilación , Animales , Ratones , Modelos Biológicos , Transporte de Proteínas , Proteína p53 Supresora de Tumor/metabolismo
5.
Proc Natl Acad Sci U S A ; 111(11): 4115-20, 2014 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24591579

RESUMEN

Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B-associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7. Specifically, BAT3 increases p53 acetylation and proautophagic p53 target gene expression, while limiting p300-dependent acetylation of ATG7, a mechanism known to inhibit autophagy. In the absence of BAT3 or when BAT3 is located exclusively in the cytosol, autophagy is abrogated, ATG7 is hyperacetylated, p53 acetylation is abolished, and p300 accumulates in the cytosol, indicating that BAT3 regulates the nuclear localization of p300. In addition, the interaction between BAT3 and p300 is stronger in the cytosol than in the nucleus and, during starvation, the level of p300 decreases in the cytosol but increases in the nucleus only in the presence of BAT3. We conclude that BAT3 tightly controls autophagy by modulating p300 intracellular localization, affecting the accessibility of p300 to its substrates, p53 and ATG7.


Asunto(s)
Autofagia/fisiología , Proteína p300 Asociada a E1A/metabolismo , Embrión de Mamíferos/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Fraccionamiento Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Cartilla de ADN/genética , Embrión de Mamíferos/metabolismo , Inmunoprecipitación , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
6.
Mol Cell ; 53(5): 710-25, 2014 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-24560926

RESUMEN

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.


Asunto(s)
Acetilcoenzima A/química , Autofagia , Citosol/enzimología , Regulación Enzimológica de la Expresión Génica , Adenosina Trifosfato/química , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Proteína p300 Asociada a E1A/química , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Células HeLa , Humanos , Ácidos Cetoglutáricos/química , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Mitocondrias/metabolismo , ARN Interferente Pequeño/metabolismo
7.
Autophagy ; 9(5): 714-29, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23519090

RESUMEN

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K (+) /H (+) ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH (+) population in HMLER cells than in the ALDH (-) population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH (+) population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH (+) population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.


Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/patología , Células Madre Neoplásicas/patología , Piranos/farmacología , Naranja de Acridina/metabolismo , Aldehído Deshidrogenasa/metabolismo , Apoptosis/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Células MCF-7 , Fusión de Membrana/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Coloración y Etiquetado , Enzimas Activadoras de Ubiquitina/metabolismo
8.
Neurobiol Aging ; 34(3): 770-90, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22892312

RESUMEN

Using cultured cortical neurons, we show that the blockade of protein phosphatase 2A (PP2A), either pharmacologically by okadaic acid or by short hairpin RNA (shRNA)-mediated silencing of PP2A catalytic subunit, inhibited basal autophagy and autophagy induced in several experimental settings (including serum deprivation, endoplasmic reticulum stress, rapamycin, and proteasome inhibition) at early stages before autophagosome maturation. Conversely, PP2A upregulation by PP2A catalytic subunit overexpression stimulates neuronal autophagy. In addition, PP2A blockade resulted in the activation of the negative regulator of autophagy mammalian target of rapamycin complex 1 and 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) and led to intraneuronal accumulation of p62- and ubiquitin-positive protein inclusions, likely due to autophagy downregulation. These data are consistent with previous findings showing that specific invalidation of the autophagy process in the nervous system of mouse resulted in the accumulation of p62- and ubiquitin-positive protein inclusion bodies. Furthermore, we showed that PP2A inhibition alters the distribution of the microtubule-associated protein 1 light chain(LC) 3-I (MAP LC3-I), a key component of the autophagy molecular machinery. Whether MAP LC3-I distribution in the cell accounts for autophagy regulation remains to be determined. These data are important to human neurodegenerative diseases, especially Alzheimer's disease, because they provide links for the first time between the pathological features of Alzheimer's disease:PP2A downregulation, autophagy disruption, and protein aggregation.


Asunto(s)
Autofagia/fisiología , Neuronas/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteínas Ubiquitinadas/metabolismo , Animales , Células Cultivadas , Silenciador del Gen , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , ARN Interferente Pequeño , Ratas , Ratas Wistar
9.
J Biol Chem ; 285(33): 25570-81, 2010 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-20529838

RESUMEN

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Gosipol/farmacología , Proteínas de la Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/química , Androstadienos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Western Blotting , Línea Celular Tumoral , Anticonceptivos Masculinos/farmacología , Gosipol/química , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de la Membrana/genética , Microscopía Electrónica , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Wortmanina
10.
J Biol Chem ; 285(14): 10850-61, 2010 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-20123989

RESUMEN

A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.


Asunto(s)
Adenina/análogos & derivados , Autofagia , Embrión de Mamíferos/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenina/farmacología , Androstadienos/farmacología , Animales , Proteína 7 Relacionada con la Autofagia , Western Blotting , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Fibroblastos/citología , Fibroblastos/enzimología , Inmunoprecipitación , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/clasificación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Wortmanina
11.
Cancer Res ; 69(24): 9346-53, 2009 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-19934311

RESUMEN

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl1(-/-)) was compared with that of cells from heterozygous (Sgpl1(+/-)) or wild-type (Sgpl1(+/+)) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl1 gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl1(-/-) cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl1(-/-) cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA-mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S1P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant.


Asunto(s)
Aldehído-Liasas/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteína bcl-X/biosíntesis , Aldehído-Liasas/biosíntesis , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Línea Celular Tumoral , Regulación hacia Abajo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Dosificación de Gen , Humanos , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Regulación hacia Arriba , Proteína bcl-X/genética
12.
J Biol Chem ; 284(5): 2719-2728, 2009 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-19029119

RESUMEN

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C(2)-ceramide and C(6)-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr(69), Ser(70), and Ser(87), located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.


Asunto(s)
Autofagia/fisiología , Ceramidas/fisiología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Western Blotting , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Humanos , Inmunoprecipitación , Fosforilación
13.
Autophagy ; 3(4): 390-2, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17471012

RESUMEN

The NF-kappaB transcription factor is an important anti-apoptotic factor, which is frequently deregulated in cancer cells. We have recently demonstrated that NF-kappaB activation mediates the repression of autophagy in response to TNFa in three models of cancer cell lines. In contrast, in the absence of NF-kappaB activation, TNFa induces macroautophagy (autophagy), which requires reactive oxygen species (ROS) production and participates in the TNFalpha-induced apoptotic signaling pathway. Autophagy-dependent apoptosis was also observed following direct addition of ROS to cells. Moreover, addition of rapamycin to TNFalpha renders these cells susceptible to the cytotoxic effect of this cytokine. These findings highlight the regulation of autophagy by oxidative stress and support the idea that repression of autophagy by NF-kappaB may constitute a novel anti-apoptotic function of this transcription factor. We also bring evidence that direct stimulation of autophagy may represent a new therapeutic strategy for overcoming the NF-kappaB-dependent chemoresistance of cancer cells.


Asunto(s)
Autofagia/fisiología , Regulación de la Expresión Génica/fisiología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Factor de Necrosis Tumoral alfa/farmacología
14.
J Biol Chem ; 281(41): 30373-82, 2006 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-16857678

RESUMEN

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.


Asunto(s)
Autofagia , Subunidad p50 de NF-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antineoplásicos/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Cadaverina/análogos & derivados , Cadaverina/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno
15.
J Biol Chem ; 279(18): 18384-91, 2004 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-14970205

RESUMEN

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.


Asunto(s)
Autofagia/efectos de los fármacos , Ceramidas/farmacología , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas , Proteínas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis , Beclina-1 , Línea Celular Tumoral , Ceramidas/fisiología , Inhibidores Enzimáticos/farmacología , Glucosiltransferasas/antagonistas & inhibidores , Humanos , Proteínas de la Membrana , Microscopía Electrónica , Proteínas Proto-Oncogénicas c-akt , Tamoxifeno/farmacología , Regulación hacia Arriba , Vacuolas
16.
J Biol Chem ; 278(23): 20995-1002, 2003 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-12642577

RESUMEN

AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the alpha-subunit of the trimeric Gi3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation. Together with the full-length form, minute amounts of the mRNA encoding a NH2-terminal truncated form of AGS3, previously characterized in cardiac tissues, were also detected. Both the endogenous form of AGS3 and a tagged expressed form have a localization compatible with a role in the Galphai3-dependent control of autophagy. Accordingly, expressing its non-Galphai3-interacting NH2-terminal domain or its Galphai3-interacting COOH-terminal domain reversed the stimulatory role of AGS3 on autophagy. On the basis of biochemical and morphometric analysis, we conclude that AGS3 is involved in an early event during the autophagic pathway probably prior to the formation of the autophagosome. These data demonstrate that AGS3 is a novel partner of the Galphai3 protein in the control of a major catabolic pathway.


Asunto(s)
Proteínas Portadoras/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Fagocitosis/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Expresión Génica , Células HT29 , Humanos , Intestinos/citología , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/análisis
17.
J Biol Chem ; 278(19): 16667-74, 2003 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-12609989

RESUMEN

Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Galpha-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090-39095). Here we show that ERK1/2 activation by aurintricarboxylic acid (ATA) treatment induces the phosphorylation of GAIP in an amino acid-dependent manner. Accordingly, ATA challenge increased the rate of macroautophagy, whereas epidermal growth factor did not significantly affect macroautophagy and GAIP phosphorylation status. In fact, ATA activated the ERK1/2 signaling pathway, whereas epidermal growth factor stimulated both the ERK1/2 pathway and the class I phosphoinositide 3-kinase pathway, known to decrease the rate of macroautophagy. Amino acids interfered with the ATA-induced macroautophagy by inhibiting the activation of the kinase Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIP- and amino acid-dependent control of macroautophagy was confirmed in HT-29 cells expressing the Ras(G12V,T35S) mutant. Similar to the protein phosphatase 2A inhibitor okadaic acid, amino acids sustained the phosphorylation of Ser(259), which is involved in the negative regulation of Raf-1. In conclusion, these results add a novel target to the amino acid signaling-dependent control of macroautophagy in intestinal cells.


Asunto(s)
Autofagia/fisiología , Neoplasias del Colon/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ácido Aurintricarboxílico/farmacología , Autofagia/efectos de los fármacos , Neoplasias del Colon/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas RGS , Células Tumorales Cultivadas
18.
J Biol Chem ; 277(31): 27613-21, 2002 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-12000750

RESUMEN

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr(308) and Ser(473). However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1(A280V)) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1(K114G)) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC(50) = 3.5 microm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells.


Asunto(s)
Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sulfonamidas/farmacología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Celecoxib , Neoplasias del Colon , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Quinasas/metabolismo , Pirazoles , Células Tumorales Cultivadas
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