RESUMEN
Studies in targeting metabolism in cancer cells have shown the flexibility of cells in reprogramming their pathways away from a given metabolic block. Such behavior prompts a combination drug approach in targeting cancer metabolism, as a single compound may not address the tumor intractability. Overall, mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated as enabling metabolic escape in the case of a glycolysis block. From a library of compounds, the tyrosine kinase inhibitor ponatinib was screened to provide optimal reduction in metabolic activity in the production of adenosine triphosphate (ATP), pyruvate, and lactate for multiple myeloma cells; however, these cells displayed increasing levels of oxidative phosphorylation (OXPHOS), enabling them to continue generating ATP, although at a slower pace. The combination of ponatinib with the mTORC1 inhibitor, sirolimus, blocked OXPHOS; an effect also manifested in activity reductions for hexokinase 2 (HK2) and glucose-6-phosphate isomerase (GPI) glycolysis enzymes. There were also remarkably higher levels of reactive oxygen species (ROS) produced in mouse xenografts, on par with increased glycolytic block. The combination of ponatinib and sirolimus resulted in synergistic inhibition of tumor xenografts with no overt toxicity in treated mice for kidney and liver function or maintaining weight.
RESUMEN
In MYCN-amplified neuroblastoma (NB), we noticed that the single compound treatment with the HDAC inhibitor vorinostat led to a reprogramming of the glycolytic pathway in these cells. This reprogramming was upregulation of fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS), allowing the cells to generate ATP, albeit at a reduced rate. This behavior was dependent on reduced levels of MYCN and a corresponding increase in the levels of PPARD transcription factors. By integrating metabolic and functional studies in NB cells and mouse xenografts, we demonstrate a compensatory upregulation of FAO/OXPHOS metabolism that promotes resistance to HDAC inhibitors. From the additional compounds that could reverse this metabolic reprogramming, the mTORC1 inhibitor sirolimus was selected. Besides both a block of glycolysis and OXPHOS, the HDAC/mTORC1 inhibitor combination produced significantly higher levels of reactive oxygen species (ROS) in the treated cells and in xenograft tumor samples, also a consequence of increased glycolytic block. The lead compounds were also tested for changes in the message levels of the glycolytic enzymes and their pathway activity, and HK2 and GPI glycolytic enzymes were most affected at their RNA message level. This combination was seen with no overall toxicity in treated mice in terms of weight loss or liver/kidney function.
Asunto(s)
Inhibidores de Histona Desacetilasas , Neuroblastoma , Animales , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismoRESUMEN
BACKGROUND: Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied. METHODS: RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells. RESULTS: In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts. CONCLUSIONS: HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína 4 Similar a ELAV/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Animales , Autofagia , Humanos , Masculino , Ratones , Ratones Desnudos , TransfecciónRESUMEN
Prostate cancer (PCa) is a common type of cancer among males, with a relatively high mortality rate. Tumor necrosis factorrelated apoptosisinducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, initiates the apoptosis of certain cancer cells. Neferine, a primary ingredient of bisbenzylisoquinoline alkaloids, has various antitumor activities. The present study examined the effects of neferine treatment on human PCa cells. Human prostate cancer (DU145) cells were treated with neferine for 18 h, and subsequently treated with TRAIL for 2 h. Combined treatment with neferine and TRAIL significantly decreased cell viability compared to treatment with TRAIL alone. Furthermore, neferine treatment decreased the expression of p62 and increased LC3BII expression, as assessed by western blot analysis and immunocytochemistry. It was alsp demonstrated that neferine and TRAIL act synergistically to trigger autophagy in PCa cells, as revealed by autophagosome formation, LC3BII accumulation demonstrated by transmission electron microscopy (TEM) analysis and phosphorylated cJun Nterminal kinase (pJNK) upregulation. When the autophagic flux was attenuated by the inhibitor, chloroquine, or by genetically modified ATG5 siRNA, the enhancement of TRAILinduced autophagy by neferineinduced was also attenuated. Furthermore, treatment with the JNK inhibitor, SP600125, distinctly increased the viability of the cells treated with neferine and TRAIL. On the whole, the findings of the present study demonstrate that neferine treatment effectively promotes TRAILmediated cell death and this effect likely occurs via the autophagic flux and the JNK pathway.
Asunto(s)
Bencilisoquinolinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Antracenos/farmacología , Autofagia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cloroquina/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
Tumor necrosis factor (TNF)related apoptosisinducing ligand (TRAIL), a type II transmembrane protein, is a part of the TNF superfamily of cytokines. Cantharidin, a type of terpenoid, is extracted from the blister beetles (Mylabris genus) used in Traditional Chinese Medicine. Cantharidin elicits antibiotic, antiviral and antitumor effects, and can affect the immune response. The present study demonstrated that a cantharidin and TRAIL combination treatment regimen elicited a synergistic outcome in TRAILresistant DU145 cells. Notably, it was also identified that cantharidin treatment initiated the downregulation of cellular FLICElike inhibitory protein (cFLIP) and upregulation of death receptor 5 (DR5), and sensitized cells to TRAILmediated apoptosis by initiating autophagy flux. In addition, cantharidin treatment increased lipidmodified microtubuleassociated proteins 1A/1B light chain 3B expression and significantly attenuated sequestosome 1 expression. Attenuation of autophagy flux by a specific inhibitor such as chloroquine and genetic modification using ATG5 small interfering RNA abrogated the cantharidinmediated TRAILinduced apoptosis. Overall, the results of the present study revealed that cantharidin effectively sensitized cells to TRAILmediated apoptosis and its effects are likely to be mediated by autophagy, the downregulation of cFLIP and the upregulation of DR5. They also suggested that the combination of cantharidin and TRAIL may be a successful therapeutic strategy for TRAILresistant prostate cancer.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Cantaridina/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/genética , Autofagia/fisiología , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Sinergismo Farmacológico , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Microscopía Electrónica de Transmisión , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Proteína Sequestosoma-1/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) is well known as a transmembrane cytokine and has been proposed as one of the most effective anticancer therapeutic agents, owing to its efficiency to selectively induce cell death in a variety of tumor cells. Suppression of autophagy flux has been increasingly acknowledged as an effective and novel therapeutic intervention for cancer. The present study demonstrated that the anticancer and antiinflammatory drug celastrol, through its antimetastatic properties, may initiate TRAILmediated apoptotic cell death in lung cancer cells. This sensitization was negatively affected by Nacetyllcysteine, which restored the mitochondrial membrane potential (ΔΨm) and inhibited reactive oxygen species (ROS) generation. Notably, treatment with celastrol caused an increase in microtubuleassociated proteins 1A/1B light chain 3BII and p62 levels, whereas cotreatment of celastrol and TRAIL increased active caspase 3 and 8 levels compared with the control, confirming inhibited autophagy flux. The combined use of TRAIL with celastrol may serve as a safe and adequate therapeutic technique for the treatment of TRAILresistant lung cancer, suggesting that celastrolmediated autophagy flux inhibition sensitized TRAILinitiated apoptosis via regulation of ROS and ΔΨm.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/agonistas , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Triterpenos/farmacología , Células A549 , Acetilcisteína/farmacología , Antineoplásicos Fitogénicos/antagonistas & inhibidores , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Caspasa 8/genética , Caspasa 8/metabolismo , Línea Celular Tumoral , Cloroquina/farmacología , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Triterpenos Pentacíclicos , Especies Reactivas de Oxígeno/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Triterpenos/antagonistas & inhibidoresRESUMEN
The tumor necrosis factorrelated apoptosisinducing ligand (TRAIL) is a dynamic cytokine that initiates the apoptosis of cancer cells, but exhibits little or no toxicity in normal cells. Luteolin is a flavonoid compound frequently used in the treatment of cancer. In the current study, we demonstrate that treatment with luteolin and TRAIL exerts a synergistic effect and the mechanisms on TRAILresistant Huh7 cells. The results demonstrated that luteolin induced an autophagic flux in human liver cancer cells. The attenuation of the autophagic flux by applying the specific inhibitor of autophagy, chloroquine, significantly suppressed DR5 expression. Treatment with genetically modified autophagyrelated 5 siRNA abrogated the luteolinmediated sensitizing effect of TRAIL. Furthermore, pretreatment with the cJun Nterminal kinase (JNK) inhibitor, SP600125, significantly attenuated the luteolininduced upregulation of DR5 expression, thereby suggesting that JNK activation promotes DR5 expression. Our findings also revealed that Akt phosphorylation was required for TRAIL sensitization. On the whole, the findings of this study indicated that luteolin effectively enhanced TRAILinitiated apoptosis, and that these effects were likely to be mediated by autophagy and JNKmediated DR5 expression.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/genética , Neoplasias Hepáticas/tratamiento farmacológico , Luteolina/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Antracenos/farmacología , Apoptosis/genética , Autofagia/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteína Oncogénica v-akt/genética , Fosforilación/efectos de los fármacos , ARN Interferente PequeñoRESUMEN
Tumor necrosis factor (TNF)related apoptosisinducing ligand (TRAIL) is a member of the TNF superfamily and is an antitumor drug that induces apoptosis in tumor cells with minimal or no effects on normal cells. Here, it is demonstrated that 6shogaol (6sho), a bioactive component of ginger, exerted antiinflammatory and anticancer properties, attenuated tumor cell propagation and induced TRAILmediated cell death in liver cancer cells. The current study identified a potential pathway by revealing that TRAIL and 6sho or chloroquine acted together to trigger reactive oxygen species (ROS) production, to upregulate tumorsuppressor protein 53 (p53) expression and to change the mitochondrial transmembrane potential (MTP). Treatment with NacetylLcysteine reversed these effects, restoring the MTP and attenuated ROS production and p53 expression. Interestingly, treatment with 6sho increased p62 and microtubuleassociated proteins 1A/1B light chain 3BII levels, indicating an inhibited autophagy flux. In conclusion, attenuation of 6shoinduced autophagy flux sensitized cells to TRAILinduced apoptosis via p53 and ROS, suggesting that the administration of TRAIL in combination with 6sho may be a suitable therapeutic method for the treatment of TRAILresistant Huh7 liver cells.
Asunto(s)
Autofagia/efectos de los fármacos , Catecoles/farmacología , Neoplasias Hepáticas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Caspasas Iniciadoras/metabolismo , Catecoles/uso terapéutico , Línea Celular Tumoral , Cloroquina/metabolismo , Cloroquina/farmacología , Cloroquina/uso terapéutico , Sinergismo Farmacológico , Quimioterapia Combinada , Zingiber officinale/química , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mutágenos/farmacología , Mutágenos/uso terapéutico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéuticoRESUMEN
Telmisartan broadly used for the treatment of hypertension that is also known for its anticancer properties. TRAIL has the potential to kill tumor cells with minimal toxicity in normal cells by binding to death receptors, DR4 and DR5. Unfortunately, these TRAIL-death receptors have failed as most human cancers are resistant to TRAIL-mediated apoptosis. In this study, we evaluated telmisartan as a novel TRAIL-DR5-targeting agent with the aim of rendering TRAIL-based cancer therapies more active. Herein, we demonstrated that telmisartan could sensitize TRAIL and enhance NSCLC tumor cell death. The molecular mechanism includes the blocking of AMPK phosphorylation causes inhibition of autophagy flux by telmisartan resulting in ROS generation leading to death receptor (DR5) upregulation and subsequent activation of the caspase cascade by TRAIL treatment. Furthermore, using chloroquine and siATG5 significantly enhances ROS production and application of the ROS scavenger N-acetyl-cysteine (NAC) rescues the cells undergoing apoptosis by abrogating the expression of DR5 and finally the caspase cascade. Additionally, NAC treatment also maintains autophagy flux and makes the cells unresponsive to TRAIL. In summary, telmisartan in combination with TRAIL exhibits enhanced cytotoxic capacity toward lung cancer cells, thereby providing the potential for effective and novel therapeutic approaches to treat lung cancer.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Telmisartán/farmacología , Regulación hacia Arriba/efectos de los fármacos , Células A549 , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Terapia Molecular Dirigida , Fosforilación/efectos de los fármacos , Telmisartán/uso terapéuticoRESUMEN
Tumor necrosis factor related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein, belongs to the TNF superfamily. Compared to other family members, TRAIL is a promising anti-cancer agent that can selectively induce apoptosis of various types of transformed cells and xenografts, with negligible cytotoxicity against normal tissues. Ophiopogonin B is a bioactive ingredient of Radix Ophiopogon japonicus, which is frequently used in traditional Chinese medicine to treat cancer. In this study, we report that Cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein (c-FLIP) is the key determinant mediating TRAIL resistance in A549 cells and Ophiopogonin B downregulates c-FLIP and enhances TRAIL-induced apoptosis by activating autophagy flux. In addition, treatment with Ophiopogonin B resulted in a slight increase in the conversion of LC3-I to LC3-II and significantly decreased p62 expression levels in a dose-dependent manner. This indicates that Ophiopogonin B induces autophagy flux activation in human lung cancer cells. Inhibiting autophagy flux by applying a specific inhibitor ATG5 siRNA with Ophiopogonin B mediated enhancement of TRAIL effects. These data demonstrate that downregulation of c-FLIP by Ophiopogonin B enhances TRAIL-induced tumor cell death by activating autophagy flux in TRAIL-resistant A549 cells, and also suggests that Ophiopogonin B combined with TRAIL may be a successful therapeutic strategy for TRAIL-resistant lung cancer cells.
RESUMEN
The combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with subsidiary agents is a promising anticancer strategy to conquer TRAIL resistance in malignant cells. Glipizide is a second-generation oral hypoglycemic medicine for the cure of type II diabetes because of its capability to selectively stimulate insulin secretion from ß-cells. In this study, we revealed that glipizide could trigger TRAIL-mediated apoptotic cell death in human lung adenocarcinoma cells. Pretreatment with glipizide downregulation of p-Akt and p-mTOR in different concentrations. In addition, LC3-II and p-Akt was suppressed in the presence of LY294002, a well-known inhibitor of P13K. Treatment with glipizide commenced in a slight increase in conversion rate of LC3-I to LC3-II and significantly decreased p62 expression levels in a dose-dependent manner. This indicates that glipizide encouraged autophagy flux activation in human lung cancer cells. Inhibition of autophagy flux applying a specific inhibitor and genetically modified ATG5 siRNA enclosed glipizide-mediated enhancing effect of TRAIL. These data demonstrate that inhibition of Akt/mTOR by glipizide sensitizes TRAIL-induced tumor cell death through activating autophagy flux and also suggest that glipizide may be a combination therapeutic target with TRAIL protein in TRAIL-resistant cancer cells.
RESUMEN
Members of the tumor necrosis factor (TNF) transmembrane cytokine superfamily, such as TNFα and Fas ligand (FasL), play crucial roles in inflammation and immunity. TRAIL is a member of this superfamily with the ability to selectively trigger cancer cell death but does not motive cytotoxicity to most normal cells. Troglitazone are used in the cure of type II diabetes to reduce blood glucose levels and improve the sensitivity of an amount of tissues to insulin. In this study, we revealed that troglitazone could trigger TRAIL-mediated apoptotic cell death in human lung adenocarcinoma cells. Pretreatment of troglitazone induced activation of PPARγ in a dose-dependent manner. In addition conversion of LC3-I to LC3-II and PPARγ was suppressed in the presence of GW9662, a well-characterized PPARγ antagonist. Treatment with troglitazone resulted in a slight increase in conversion rate of LC3-I to LC3-II and significantly decreased p62 expression levels in a dose-dependent manner. This indicates that troglitazone induced autophagy flux activation in human lung cancer cells. Inhibition of autophagy flux applying a specific inhibitor and genetically modified ATG5 siRNA enclosed troglitazone-mediated enhancing effect of TRAIL. These data demonstrated that activation of PPARγ mediated by troglitazone enhances human lung cancer cells to TRAIL-induced apoptosis via autophagy flux and also suggest that troglitazone may be a combination therapeutic target with TRAIL protein in TRAIL-resistant cancer cells.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Cromanos/farmacología , Neoplasias Pulmonares/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Tiazolidinedionas/farmacología , Línea Celular Tumoral , Humanos , TroglitazonaRESUMEN
Lung cancer, especially lung adenocarcinoma, is one of the main causes of death worldwide. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a primary anticancer agent and a member of the tumor necrosis factor family that selectively induces apoptosis in various tumor cells, but not in normal cells. Combination chemotherapy can be used for treating specific cancer types even at progressive stages. In the present study, we observed that 5-fluorouracil, which exerts anticancer effects by inhibiting tumor cell proliferation, enhanced TRAIL-induced apoptosis of TRAIL-resistant human adenocarcinoma A549 cells. Interestingly, 5-fluorouracil treatment markedly increased Bax and p53 levels and 5-fluorouracil and TRAIL cotreatment increased Ac-cas3 and Ac-cas8 levels compared with those in control cells. Taken together, the present study demonstrated that 5-fluorouracil enhances TRAIL-induced apoptosis in TRAIL-resistant lung adenocarcinoma cells by activating Bax and p53, and also suggest that TRAIL and 5-fluorouracil cotreatment can be used as an adequate therapeutic strategy for TRAIL-resistant human cancers.
Asunto(s)
Adenocarcinoma/patología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Fluorouracilo/farmacología , Neoplasias Pulmonares/patología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Apoptosis/efectos de los fármacos , Western Blotting , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2/metabolismoRESUMEN
An unusual molecular structure of the prion protein, PrPsc is found only in mammals with transmissible prion diseases. Prion protein stands for either the infectious pathogen itself or a main component of it. Recent studies suggest that autophagy is one of the major functions that keep cells alive and has a protective effect against the neurodegeneration. In this study, we investigated that the effect of human prion protein on autophagy-lysosomal system of primary neuronal cells. The treatment of human prion protein induced primary neuron cell death and decreased both LC3-II and p62 protein amount indicating autophagy flux activation. Electron microscope pictures confirmed the autophagic flux activation in neuron cells treated with prion protein. Inhibition of autophagy flux using pharmacological and genetic tools prevented neuron cell death induced by human prion protein. Autophagy flux induced by prion protein is more activated in prpc expressing cells than in prpc silencing cells. These data demonstrated that prion protein-induced autophagy flux is involved in neuron cell death in prion disease and suggest that autophagy flux might play a critical role in neurodegenerative diseases including prion disease.
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Apoptosis/fisiología , Autofagia , Neuronas/fisiología , Fragmentos de Péptidos/fisiología , Enfermedades por Prión/metabolismo , Priones/fisiología , Animales , Células Cultivadas , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/ultraestructura , Cultivo Primario de Células , Proteína Sequestosoma-1/metabolismoRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL is regarded as one of the most promising anticancer agents, because it can destruct cancer cells without showing any toxicity to normal cells. Metformin is an anti-diabetic drug with anticancer activity by inhibiting tumor cell proliferation. In this study, we demonstrated that metformin could induce TRAIL-mediated apoptotic cell death in TRAIL-resistant human lung adenocarcinoma A549 cells. Pretreatment of metformindownregulation of c-FLIP and markedly enhanced TRAIL-induced tumor cell death by dose-dependent manner. Treatment with metformin resulted in slight increase in the accumulation of microtubule-associated protein light chain LC3-II and significantly decreased the p62 protein levels by dose-dependent manner indicated that metformin induced autophagy flux activation in the lung cancer cells. Inhibition of autophagy flux using a specific inhibitor and genetically modified ATG5 siRNA blocked the metformin-mediated enhancing effect of TRAIL. These data demonstrated that downregulation of c-FLIP by metformin enhanced TRAIL-induced tumor cell death via activating autophagy flux in TRAIL-resistant lung cancer cells and also suggest that metformin may be a successful combination therapeutic strategy with TRAIL in TRAIL-resistant cancer cells including lung adenocarcinoma cells.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Neoplasias Pulmonares/patología , Metformina/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Células Tumorales CultivadasRESUMEN
Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease.
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Peróxido de Hidrógeno/farmacología , Queratinocitos/efectos de los fármacos , Melatonina/farmacología , Sirtuina 1/metabolismo , Autofagia/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/efectos de los fármacos , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismo , TransfecciónRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a transmembrane cytokine that is a promising anticancer agent as it selectively induces apoptosis in various types of tumor cells. Autophagic flux, which includes the complete process of autophagy, and suppression of autophagic flux has been increasingly recognized as a favorable and novel therapeutic approach for cancer treatment. Here, we showed that genistein, a major isoflavone compound that exerts its anticancer properties by inhibiting tumor cell proliferation, can induce TRAIL-mediated apoptotic cell death in TRAILresistant human adenocarcinoma A549 cells. Notably, genistein treatment led to a marked increase in the accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II and p62 protein levels. The combination of genistein and TRAIL increased LC3-II, p62, activated caspase-3 and activated caspase-8 accumulation, confirming the inhibition of autophagic flux. Taken together, our results revealed that genistein enhanced TRAIL-induced tumor cell death in TRAIL-resistant A549 adenocarcinoma cells by inhibiting autophagic flux.
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Anticarcinógenos/farmacología , Autofagia/efectos de los fármacos , Genisteína/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Prion diseases caused by aggregated misfolded prion protein (PrP) are transmissible neurodegenerative disorders that occur in both humans and animals. Epigallocatechin-3-gallate (EGCG) has preventive effects on prion disease; however, the mechanisms related to preventing prion diseases are unclear. We investigated whether EGCG, the main polyphenol in green tea, prevents neuron cell damage induced by the human prion protein. We also studied the neuroprotective mechanisms and proper signals mediated by EGCG. The results showed that EGCG protects the neuronal cells against human prion protein-induced damage through inhibiting Bax and cytochrome c translocation and autophagic pathways by increasing LC3-II and reducing and blocking p62 by using ATG5 small interfering (si) RNA and autophagy inhibitors. We further demonstrated that the neuroprotective effects of EGCG were exhibited by a class III histone deacetylase; sirt1 activation and the neuroprotective effects attenuated by sirt1 inactivation using sirt1 siRNA and sirtinol. We demonstrated that EGCG activated the autophagic pathways by inducing sirt1, and had protective effects against human prion protein-induced neuronal cell toxicity. These results suggest that EGCG may be a therapeutic agent for treatment of neurodegenerative disorders including prion diseases.
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Autofagia , Catequina/análogos & derivados , Histona Desacetilasas/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Apoptosis , Catequina/química , Línea Celular , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Neuroprotección , Priones/metabolismo , ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Sirtuina 1/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a primary anticancer agent and a member of the tumor necrosis factor family that selectively induces apoptosis in various tumor cells, but not in normal cells. Gingerol is a major ginger component with anti-inflammatory and antitumorigenic activities. Autophagy flux is the complete process of autophagy, in which the autophagosomes are lysed by lysosomes. The role of autophagy in cell death or cell survival is controversial. A549 adenocarcinoma cells are TRAIL-resistant. In the present study, we showed that treatment with TRAIL slightly induced cell death, but gingerol treatment enhanced the TRAIL-induced cell death in human lung cancer cells. The combination of gingerol and TRAIL increased accumulation of microtubule-associated protein light chain 3-II and p62, confirming the inhibited autophagy flux. Collectively, our results suggest that gingerol sensitizes human lung cancer cells to TRAIL-induced apoptosis by inhibiting the autophagy flux.