RESUMEN
Systemic treatment with hedgehog inhibitors (HHis) is available to treat basal cell carcinomas but their utility is limited by adverse effects. Topical delivery methods may reduce adverse effects, but successful topical treatment depends on sufficient skin uptake, biological response, and time in tumor tissue. The aim of this review was to evaluate the current status of topical HHi delivery for BCCs and discuss barriers for translating systemic HHis into topical treatments. A literature search identified 16 preclinical studies and 7 clinical trials on the topical delivery of 12 HHis that have been clinically tested on BCCs. Preclinical studies on drug uptake demonstrated that novel formulations, and delivery- and pre-treatment techniques enhanced topical HHi delivery. Murine studies showed that the topical delivery of sonidegib, itraconazole, vitamin D3 and CUR-61414 led to biological responses and tumor remission. In clinical trials, only topical patidegib and sonidegib led to at least a partial response in 26/86 BCCs and 30/34 patients, respectively. However, histological clearance was not observed in the samples analyzed. In conclusion, the incomplete clinical response could be due to poor HHi uptake, biodistribution or biological response over time. Novel topical delivery techniques may improve HHi delivery, but additional research on cutaneous pharmacokinetics and biological response is needed.
Asunto(s)
Administración Cutánea , Carcinoma Basocelular , Proteínas Hedgehog , Animales , Humanos , Ratones , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/patología , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Distribución Tisular , ItraconazolRESUMEN
Autophagy is an evolutionarily conserved mechanism of cellular self-digestion in which proteins and organelles are degraded through delivery to lysosomes. Defects in this process are implicated in numerous human diseases including cancer. To further elucidate regulatory mechanisms of autophagy, we performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we identified three novel miR-101 targets, STMN1, RAB5A and ATG4D. siRNA-mediated depletion of these genes phenocopied the effect of miR-101 overexpression, demonstrating their importance in autophagy regulation. Importantly, overexpression of STMN1 could partially rescue cells from miR-101-mediated inhibition of autophagy, indicating a functional importance for this target. Finally, we show that miR-101-mediated inhibition of autophagy can sensitize breast cancer cells to 4-hydroxytamoxifen (4-OHT)-mediated cell death. Collectively, these data establish a novel link between two highly important and rapidly growing research fields and present a new role for miR-101 as a key regulator of autophagy.
Asunto(s)
Autofagia , MicroARNs/genética , MicroARNs/metabolismo , Estatmina/metabolismo , Proteínas Relacionadas con la Autofagia , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Etopósido/farmacología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Interferente Pequeño , Sirolimus/farmacología , Estatmina/biosíntesis , Estatmina/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.
Asunto(s)
Autofagia , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Pruebas Genéticas , Fagosomas/metabolismo , Proteínas/metabolismo , Proteómica , Aminoácidos/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antivirales/farmacología , Neoplasias de la Mama/patología , Electroforesis en Gel de Poliacrilamida , Femenino , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Inmunosupresores/farmacología , Marcaje Isotópico , Lisosomas/metabolismo , Macrólidos/farmacología , Fagosomas/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sirolimus/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Inanición , Células Tumorales CultivadasRESUMEN
The capacity of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) to trigger apoptosis preferentially in cancer cells, although sparing normal cells, has motivated clinical development of TRAIL receptor agonists as anti-cancer therapeutics. The molecular mechanisms responsible for the differential TRAIL sensitivity of normal and cancer cells are, however, poorly understood. Here, we show a novel signalling pathway that activates cytoprotective autophagy in untransformed human epithelial cells treated with TRAIL. TRAIL-induced autophagy is mediated by the AMP-activated protein kinase (AMPK) that inhibits mammalian target of rapamycin complex 1, a potent inhibitor of autophagy. Interestingly, the TRAIL-induced AMPK activation is refractory to the depletion of the two known AMPK-activating kinases, LKB1 and Ca(2+)/calmodulin-dependent kinase kinase-beta, but depends on transforming growth factor-beta-activating kinase 1 (TAK1) and TAK1-binding subunit 2. As TAK1 and AMPK are ubiquitously expressed kinases activated by numerous cytokines and developmental cues, these data are most likely to have broad implications for our understanding of cellular control of energy homoeostasis as well as the resistance of untransformed cells against TRAIL-induced apoptosis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Citoprotección/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Quinasas Quinasa Quinasa PAM/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Mama/citología , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Modelos Biológicos , Complejos Multiproteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas , Epitelio Pigmentado de la Retina/citología , Serina-Treonina Quinasas TOR , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Immunodeficient mice engrafted with psoriatic human skin are widely used for the preclinical evaluation of new drug candidates. However, the T-cell activity, including the IL23/IL17 pathway, declines in the graft over time after engraftment, which likely affects the study data. Here, we investigated whether the T-cell activity could be sustained in xenografted psoriatic skin by local stimulation of T cells or systemic injection of autologous CD4 + T cells. We surgically transplanted human psoriatic skin from 5 untreated patients onto female NOG mice. Six days after surgery, mice received an intraperitoneal injection of autologous human CD4+ T cells, a subcutaneous injection under the grafts of a T-cell stimulation cocktail consisting of recombinant human IL2, human IL23, antihuman CD3, and antihuman CD28, or saline. Mice were euthanized 21 d after surgery and spleens and graft biopsies were collected for analysis. Human T cells were present in the grafts, and 60% of the grafts maintained the psoriatic phenotype. However, neither local T-cell stimulation nor systemic injection of autologous CD4+ T cells affected the protein levels of human IL17A, IL22, IFN γ, and TNF α in the grafts. In conclusion, NOG mice seem to accept psoriatic skin grafts, but the 2 approaches studied here did not affect human T-cell activity in the grafts. Therefore, NOG mice do not appear in this regard to be superior to other immunodeficient mice used for psoriasis xenografts.
Asunto(s)
Psoriasis , Linfocitos T , Humanos , Ratones , Femenino , Animales , Xenoinjertos , Piel/patología , Psoriasis/tratamiento farmacológico , Psoriasis/patología , Linfocitos T CD4-PositivosRESUMEN
Xenografting of psoriasis skin onto immune deficient mice has been widely used to obtain proof-of-principle of new drug candidates. However, the lack of human T-cell activity in the grafts limits the use of the model. Here, we show that xenografting of lesional skin from psoriasis patients onto human IL-2 NOG mice results in increased numbers of human CD3+ cells in the grafts, axillary lymph nodes and blood from human IL-2 NOG mice compared to C.B-17 scid and NOG mice. In addition, disease relevant human cytokine levels were higher in graft lysates and serum from human IL-2 NOG mice. However, the epidermis was lacking and no efficacy of ustekinumab, a human anti-P40 antibody targeting both IL-12 and IL-23, was shown. Thus, despite the sustained T-cell activity, the model needs further investigations and validation to capture more aspects of psoriasis.
Asunto(s)
Interleucina-2 , Psoriasis , Humanos , Ratones , Animales , Trasplante Heterólogo , Linfocitos T/patología , Piel/patología , Psoriasis/patologíaRESUMEN
Human immune system (HIS) mouse models can be valuable when cross-reactivity of drug candidates to mouse systems is missing. However, no HIS mouse models of psoriasis have been established. In this study, it was investigated if imiquimod (IMQ) induced psoriasis-like skin inflammation was driven by human immune cells in human FMS-related tyrosine kinase 3 ligand (hFlt3L) boosted (BRGSF-HIS mice). BRGSF-HIS mice were boosted with hFlt3L prior to two or three topical applications of IMQ. Despite clinical skin inflammation, increased epidermal thickness and influx of human immune cells, a human derived response was not pronounced in IMQ treated mice. However, the number of murine neutrophils and murine cytokines and chemokines were increased in the skin and systemically after IMQ application. In conclusion, IMQ did induce skin inflammation in hFlt3L boosted BRGSF-HIS mice, although, a limited human immune response suggest that the main driving cellular mechanisms were of murine origin.
Asunto(s)
Dermatitis , Psoriasis , Humanos , Ratones , Animales , Imiquimod/efectos adversos , Piel , Psoriasis/tratamiento farmacológico , Inflamación/inducido químicamente , Modelos Animales de EnfermedadRESUMEN
We investigated the proposed necrotic mechanism of ingenol mebutate, a natural compound with anti-cancer properties in human keratinocytes, the human squamous cell carcinoma cell line HSC-5, and HeLa cervix carcinoma cells. Topical application of a clinical dose of ingenol mebutate 0.05% (1.15 mM) gel to human reconstituted full-thickness skin equivalents strongly reduced epidermal, but not dermal viability. Ingenol mebutate showed cytotoxic potency between 200-300 M on normal and cancer cells. When keratinocytes were induced to differentiate, they became significantly less sensitive to ingenol mebutate and half-maximal induction of cell death required more than 300 M ingenol mebutate. Cytotoxic concentrations of ingenol mebutate caused rupture of the mitochondrial network within minutes paralleled by cytosolic calcium release in all cells. Subsequently, plasma membrane integrity was lost as seen by propidium uptake into the cells. This was in sharp contrast to lysis of cells with low concentrations of the detergent Triton X-100 that permeabilized the plasma membrane within minutes without affecting organelle morphology. Buffering of intracellular calcium and inhibition of the mitochondrial permeability transition pore reduced the cytotoxic effect of ingenol mebutate in cancer cells, but not in normal keratinocytes. However, these inhibitors could not prevent cell death subsequent to prolonged incubation. Our findings reveal that ingenol mebutate does not mediate cytotoxicity by a simple lytic, necrotic mechanism, but activates distinct processes involving multiple cell organelles in a cell-type and differentiation-dependent manner. These data improve our understanding of ingenol mebutate-target cell interactions and offer new insights relevant to the removal of aberrant cells in human skin.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Escamosas/patología , Diferenciación Celular , Diterpenos/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Piel/patología , Calcio/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Detergentes/farmacología , Células HeLa , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Mitocondrias/efectos de los fármacos , Necrosis , Octoxinol/farmacología , Piel/efectos de los fármacosRESUMEN
Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c-induced, apoptosome-dependent caspase activation in vitro and Fas ligand-induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.
Asunto(s)
Apoptosis/fisiología , Permeabilidad de la Membrana Celular/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Lisosomas/fisiología , Animales , Caspasas/metabolismo , Catepsinas/metabolismo , Supervivencia Celular/fisiología , Células HeLa , Humanos , Immunoblotting , Lisosomas/metabolismo , Ratones , Microscopía Inmunoelectrónica , Células Tumorales CultivadasRESUMEN
Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route. Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.
Asunto(s)
Aminoácidos/deficiencia , Autofagia , Orgánulos/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Análisis por Conglomerados , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mitocondrias/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/química , Fagosomas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.
Asunto(s)
Apoptosis/fisiología , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caspasa 9/metabolismo , Citocromos c/metabolismo , Lisosomas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Factor Apoptótico 1 Activador de Proteasas/genética , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/genética , Células Cultivadas , Cicloheximida/metabolismo , Citocromos c/genética , Activación Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Vincristine is a microtubule-destabilizing antimitotic drug that has been used in cancer therapy for over 40 years. However, the knowledge on vincristine-induced cell death pathways is still sparse. Here, we show that vincristine induces dramatic changes in the lysosomal compartment and sensitizes cells to lysosomal membrane permeabilization. In HeLa cervix carcinoma cells, vincristine induced mitotic arrest and massive cell death associated with an early increase in the lysosomal volume and lysosomal leakage followed by the activation of the intrinsic apoptosis program. In contrast, the majority of vincristine-treated MCF-7 breast carcinoma cells resisted apoptosis. Instead, they adapted to the spindle assembly checkpoint and escaped the mitotic arrest as micronucleated and senescent cells with an increase in the volume and the activity of their lysosomal compartment. Consistent with its substantial effects on the lysosomes, vincristine greatly sensitized cultured cancer cells as well as orthotopic breast cancer xenografts in mice to the cytotoxicity induced by siramesine, a sigma-2 receptor ligand that kills cancer cells by destabilizing their lysosomes. Importantly, the combination of nontoxic concentrations of vincristine and siramesine resulted in massive cell death even in MCF-7 cells that were capable of escaping vincristine-induced spindle assembly checkpoint and cell death. Similar synergism was observed when siramesine was combined with a semisynthetic vincristine analogue, vinorelbine, or with microtubule-stabilizing paclitaxel. These data strongly suggest that combination therapies consisting of microtubule-disturbing and lysosome-destabilizing drugs may prove useful in the treatment of otherwise therapy-resistant human cancers.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Indoles/farmacología , Lisosomas/efectos de los fármacos , Compuestos de Espiro/farmacología , Vincristina/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Células HeLa , Humanos , Indoles/administración & dosificación , Ratones , Ratones SCID , Compuestos de Espiro/administración & dosificación , Células Tumorales Cultivadas , Vincristina/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Heat shock protein 70-2 (Hsp70-2) is a chaperone protein essential for the growth of spermatocytes and cancer cells. Here, we show that Hsp70-2 depletion triggers lysosomal membrane permeabilization and cathepsin-dependent cell death and identify lens epithelium-derived growth factor (LEDGF) as an Hsp70-2-regulated guardian of lysosomal stability in human cancer. Knockdown of LEDGF in cancer cells induces destabilization of lysosomal membranes followed by caspase-independent and Bcl-2-resistant cell death. Accordingly, ectopic LEDGF stabilizes lysosomes and protects cancer cells against cytotoxicity induced by anticancer agents that trigger the lysosomal cell death pathway. Remarkably, ectopic LEDGF also increases the tumorigenic potential of human cancer cells in immunodeficient mice, and LEDGF expression is increased in human breast and bladder carcinomas correlating with that of Hsp70-2 in invasive bladder cancer. Taken together, these data reveal LEDGF as an oncogenic protein that controls a caspase-independent lysosomal cell death pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Neoplasias de la Mama/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Lisosomas/metabolismo , Factores de Transcripción/fisiología , Neoplasias de la Vejiga Urinaria/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Muerte Celular/fisiología , Linaje de la Célula , Permeabilidad de la Membrana Celular , Regulación hacia Abajo , Femenino , Proteínas HSP70 de Choque Térmico/deficiencia , Células HeLa , Humanos , Lisosomas/patología , Ratones , Ratones SCID , Factores de Transcripción/biosíntesis , Factores de Transcripción/deficiencia , Transfección , Trasplante Heterólogo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Acquired resistance to classic caspase-mediated apoptosis is a common problem for the treatment of human cancer. Here, we show that siramesine, a novel sigma-2 receptor ligand, effectively induces caspase-independent programmed cell death in immortalized and transformed cells of various origins. Siramesine-treated tumor cells displayed increased levels of reactive oxygen species, lysosomal membrane permeabilization, chromatin condensation, and shrinkage and detachment of cells. Lipid antioxidants (alpha-tocopherol and gamma-tocopherol), but not other tested antioxidants (butylated hydroxyanisol or N-acetyl cysteine), effectively inhibited siramesine-induced morphologic changes and cell death. Cathepsin B inhibitors (CA-074-Me and R-2525) conferred similar, but less pronounced protection, whereas ectopic expression of antiapoptotic protein Bcl-2, lack of wild-type p53 as well as pharmacologic inhibitors of caspases (zVAD-fmk, DEVD-CHO, and LEHD-CHO), calpains (PD150606), and serine proteases (N-tosyl-L-phenylalanine chloromethyl ketone and pefabloc) failed to protect cells against siramesine-induced death. Importantly, transformation of murine embryonic fibroblasts with activated c-src or v-Ha-ras oncogenes greatly sensitized them to siramesine-induced cytotoxicity. Furthermore, p.o. administration of well-tolerated doses of siramesine had a significant antitumorigenic effect in orthotopic breast cancer and s.c. fibrosarcoma models in mice. These results present siramesine as a promising new drug for the treatment of tumors resistant to traditional therapies.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Indoles/farmacología , Lisosomas/efectos de los fármacos , Receptores sigma/metabolismo , Compuestos de Espiro/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasas/metabolismo , Catepsinas/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Citocromos c/metabolismo , Femenino , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Humanos , Ligandos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Estrés Oxidativo/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.
Asunto(s)
Aminoácidos/deficiencia , Autofagia , Biosíntesis de Proteínas , Proteolisis , Autofagosomas/metabolismo , Humanos , Marcaje Isotópico , Células MCF-7RESUMEN
Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation.
Asunto(s)
Autofagia , Pruebas Genéticas/métodos , Fosfotransferasas/metabolismo , ARN Interferente Pequeño/metabolismo , Mama/patología , Línea Celular Tumoral , Proliferación Celular , Endocitosis , Células Epiteliales/metabolismo , Femenino , Humanos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos , Fosfatidilinositol 3-Quinasas , Transporte de Proteínas , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Autophagy is an evolutionarily conserved lysosomal self-digestion process essential for cellular homeostasis, differentiation and survival. As an adaptive response, it protects organisms against a wide range of pathologies, including cancer, infection, neurodegeneration, heart disease and ageing. Thus, compounds activating autophagy could have great potential in the prevention of common diseases. Interestingly, recent data link autophagy to two functions of the active form of vitamin D (VD): the induction of cancer cell death and the clearance of Mycobacterium tuberculosis in macrophages. Because VD deficiency is associated with many pathologies resembling those induced by defective autophagy, it is tempting to speculate that autophagy plays a more general role in the multiple health-promoting effects of VD.
Asunto(s)
Autofagia/efectos de los fármacos , Salud , Vitamina D/farmacología , Animales , Humanos , Macrófagos/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Neoplasias/metabolismo , Neoplasias/patología , Tuberculosis/patologíaRESUMEN
A complex of human alpha-lactalbumin and oleic acid (HAMLET) was originally isolated from human milk as a potent anticancer agent. It kills a wide range of transformed cells of various origins while leaving nontransformed healthy cells largely unaffected both in vitro and in vivo. Importantly, purified alpha-lactalbumins from other mammals form complexes with oleic acid that show biological activities similar to that of HAMLET. The mechanism by which these protein-lipid complexes kill tumor cells is, however, largely unknown. Here, we show that complex of bovine alpha-lactalbumin and oleic acid (BAMLET), the bovine counterpart of HAMLET, kills tumor cells via a mechanism involving lysosomal membrane permeabilization. BAMLET shows potent cytotoxic activity against eight cancer cell lines tested, whereas nontransformed NIH-3T3 murine embryonic fibroblasts are relatively resistant. BAMLET accumulates rapidly and specifically in the endolysosomal compartment of tumor cells and induces an early leakage of lysosomal cathepsins into the cytosol followed by the activation of the proapoptotic protein Bax. Ectopic expression of three proteins known to stabilize the lysosomal compartment, i.e. heat shock protein 70 (Hsp70), Hsp70-2, and lens epithelium-derived growth factor, confer significant protection against BAMLET-induced cell death, whereas the antiapoptotic protein Bcl-2, caspase inhibition, and autophagy inhibition fail to do so. These data indicate that BAMLET triggers lysosomal cell death pathway in cancer cells, thereby clarifying the ability of alpha-lactalbumin:oleate complexes to kill highly apoptosis-resistant tumor cells.
Asunto(s)
Lactalbúmina/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Neoplasias/patología , Ácido Oléico/farmacología , Animales , Autofagia/efectos de los fármacos , Inhibidores de Caspasas , Bovinos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lactalbúmina/química , Ratones , Neoplasias/metabolismo , Ácido Oléico/química , Ácidos Oléicos/farmacología , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Macroautophagy (hereafter referred to as autophagy) has recently emerged as an attractive target for the treatment of various degenerative diseases and cancer. The discovery of effective pharmaceutical regulators of autophagy has, however, been hindered by a lack of feasible assay systems for autophagic flux. Here, we present a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrate that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics. Furthermore, by screening a small molecule kinase inhibitor library containing 80 compounds we identified 12 compounds as inducers of autophagic flux. Importantly, six inhibitors of the class I phosphoinositide 3-kinase -- protein kinase B -- mammalian target of rapamycin complex 1 axis, the central signaling pathway repressing autophagy, scored as autophagy inducers adequately validating the screen. We conclude that the assay system presented here allows easy and rapid monitoring of autophagy kinetics and is suitable for screening of small molecule libraries.