RESUMEN
BACKGROUND: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that has no specific treatment except for supportive medical care. JEV is a neurotropic virus that affects the nervous system and triggers inflammation in the brain. METHODS: Melatonin is used as a sleep-inducing agent in neurophysiology and may serve as a protective agent against neurological and neurodegenerative diseases. Herein, we investigated the effects of melatonin and the critical roles of the serine/threonine protein phosphatase calcineurin during JEV infection in SK-N-SH neuroblastoma cells. RESULTS: Melatonin treatment decreased JEV replication and JEV-mediated neurotoxicity. Calcineurin activity was increased by JEV infection and inhibited by melatonin treatment. Through calcineurin regulation, melatonin decreased the JEV-mediated neuroinflammatory response and attenuated JEV-induced autophagy. CONCLUSIONS: Calcineurin inactivation has a protective effect in JEV-infected neuronal cells, and melatonin is a novel resource for the development of anti-JEV agents.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Melatonina , Animales , Humanos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Calcineurina/farmacología , Melatonina/farmacología , AutofagiaRESUMEN
Globally, the demand for masks has increased due to the COVID-19 pandemic, resulting in 490,201 tons of waste masks disposed of per month. Since masks are used in places with a high risk of virus infection, waste masks retain the risk of virus contamination. In this study, a 1 kg/h lab-scale (diameter: 0.114 m, height: 1 m) bubbling fluidized bed gasifier was used for steam gasification (temperature: 800 °C, steam/carbon (S/C) ratio: 1.5) of waste masks. The use of a downstream reactor with activated carbon (AC) for tar cracking and the enhancement of hydrogen production was examined. Steam gasification with AC produces syngas with H2, CO, CH4, and CO2 content of 38.89, 6.40, 21.69, and 7.34 vol%, respectively. The lower heating value of the product gas was 29.66 MJ/Nm3 and the cold gas efficiency was 74.55 %. This study showed that steam gasification can be used for the utilization of waste masks and the production of hydrogen-rich gas for further applications.
RESUMEN
BACKGROUND: The distinctive molecular structure of the prion protein, PrPsc, is established only in mammals with infectious prion diseases. Prion protein characterizes either the transmissible pathogen itself or a primary constituent of the disease. Our report suggested that prion protein-mediated neuronal cell death is triggered by the autophagy flux. However, the alteration of intracellular calcium levels, AMPK activity in prion models has not been described. This study is focused on the effect of the changes in intracellular calcium levels on AMPK/autophagy flux pathway and PrP (106-126)-induced neurotoxicity. METHODS: Western blot and Immunocytochemistry was used to detect AMPK and autophagy-related protein expression. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells. Calcium measurement was employed using fluo-4 by confocal microscope. RESULTS: We examined the effect of calcium homeostasis alterations induced by human prion peptide on the autophagy flux in neuronal cells. Treatment with human prion peptide increased the intracellular calcium concentration and induced cell death in primary neurons as well as in a neuronal cell line. Using pharmacological inhibitors, we showed that the L-type calcium channel is involved in the cellular entry of calcium ions. Inhibition of calcium uptake prevented autophagic cell death and reduction in AMP-activated protein kinase (AMPK) activity induced by human prion peptide. CONCLUSION: Our data demonstrated that prion peptide-mediated calcium inflow plays a pivotal role in prion peptide-induced autophagic cell death, and reduction in AMPK activity in neurons. Altogether, our results suggest that calcium influx might play a critical role in neurodegenerative diseases, including prion diseases. Video Abstract.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Calcio/metabolismo , Neuronas/metabolismo , Neuronas/patología , Péptidos/farmacología , Priones/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Espacio Intracelular/metabolismo , Ratones Endogámicos ICR , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacosRESUMEN
Activation of ß-catenin in neurons regulates mitochondrial function and protects against protein misfolding disorders, including Alzheimer's disease and Huntington's disease. Melatonin, a natural secretory product of the pineal gland, exerts neuroprotective effects through the activation of ß-catenin. In this study, melatonin increased ß-catenin protein expression and activation in human neuroblastoma cell lines SH-SY5Y cells. Melatonin also inhibited PrP (106-126)-induced neurotoxicity and the inhibition attenuated by treatment of ß-catenin inhibitor ICG-001. Activation of ß-catenin blocked PrP (106-126)-mediated downregulation of anti-apoptotic protein survivin and Bcl-2. Reduction of mitochondrial membrane potential, translocation of Bax, and cytochrome c release which induced by PrP (106-126) treatment were inhibited by ß-catenin activation, which contributed to prevented PrP (106-126)-induced neuronal cell death. In conclusion, ß-catenin activation by melatonin prevented PrP (106-126)-induced neuronal cell death through regulating anti-apoptotic proteins and mitochondrial pathways. These results also suggest the therapeutic value of Wnt/ß-catenin signaling in prion-related disorders as influenced by melatonin.
Asunto(s)
Melatonina/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Priones/toxicidad , beta Catenina/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neuronas/patología , ARN Interferente Pequeño , TransfecciónRESUMEN
A solid tumor is often exposed to hypoxic or anoxic conditions; thus, tumor cell responses to hypoxia are important for tumor progression as well as tumor therapy. Our previous studies indicated that tumor cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis under hypoxic conditions. Melatonin inhibits cell proliferation in many cancer types and induces apoptosis in some particular cancer types. Here, we examined the effects of melatonin on hypoxic resistant cells against TRAIL-induced apoptosis and the possible mechanisms of melatonin in the hypoxic response. Melatonin treatment increased TRAIL-induced A549 cell death under hypoxic conditions, although hypoxia inhibited TRAIL-mediated cell apoptosis. In a mechanistic study, hypoxia inducible factor-1α and prolyl-hydroxylase 2 proteins, which increase following exposure to hypoxia, were dose-dependently down-regulated by melatonin treatment. Melatonin also blocked the hypoxic responses that reduced pro-apoptotic proteins and increased anti-apoptotic proteins including Bcl-2 and Bcl-xL. Furthermore, melatonin treatment reduced TRAIL resistance by regulating the mitochondrial transmembrane potential and Bax translocation. Our results first demonstrated that melatonin treatment induces apoptosis in TRAIL-resistant hypoxic tumor cells by diminishing the anti-apoptotic signals mediated by hypoxia and also suggest that melatonin could be a tumor therapeutic tool by combining with other apoptotic ligands including TRAIL, particularly in solid tumor cells exposed to hypoxia.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Melatonina/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Alzheimer's disease (AD) is the most common age-related progressive neurodegenerative disorder. The accumulation of amyloid beta-peptide is a neuropathological marker of AD. While melatonin is recognized to have protective effects on aging and neurodegenerative disorders, the therapeutic effect of melatonin on calcineurin in AD is poorly understood. In this study, we examined the effect and underlying molecular mechanisms of melatonin treatment on amyloid beta-mediated neurotoxicity in neuroblastoma cells. Melatonin treatment decreased calcineurin and autophagy in neuroblastoma cells. Electron microscopy images showed that melatonin inhibited amyloid beta-induced autophagic vacuoles. The increase in the amyloid beta-induced apoptosis rate was observed more in PrPC-expressing ZW cells than in PrPC-silencing Zpl cells. Taken together, the results suggest that by mitigating the effect of calcineurin and autophagy flux activation, melatonin could also rescue amyloid beta-induced neurotoxic effects. These findings may be relevant to therapy for neurodegenerative diseases, including AD.
RESUMEN
Flaviviruses are a major cause of viral diseases worldwide, for which effective treatments have yet to be discovered. The prion protein (PrPc) is abundantly expressed in brain cells and has been shown to play a variety of roles, including neuroprotection, cell homeostasis, and regulation of cellular signaling. However, it is still unclear whether PrPc can protect against flaviviruses. In this study, we investigated the role of PrPc in regulating autophagy flux and its potential antiviral activity during Japanese encephalitis virus (JEV) infection. Our in vivo experiment showed that JEV was more lethal to the PrPc knocked out mice which was further supported by histological analysis, western blot and rtPCR results from infected mice brain samples. Role of PrPc against viral propagation in vitro was verified through cell survival study, protein expression and RNA replication analysis, and adenoviral vector assay by overexpressing PrPc. Further analysis indicated that after virus entry, PrPc inhibited autophagic flux that prevented JEV replication inside the host cell. Our results from in vivo and in vitro investigations demonstrate that prion protein effectively inhibited JEV propagation by regulating autophagy flux which is used by JEV to release its genetic material and replication after entering the host cell, suggesting that prion protein may be a promising therapeutic target for flavivirus infection.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Animales , Ratones , Proteínas Priónicas/genética , Proteínas Priónicas/farmacología , Línea Celular , Antivirales/farmacología , Antivirales/uso terapéutico , Replicación ViralRESUMEN
In this study, we have examined the anticancer effects of SH005S7 on MET-amplified and (HCC827GR) NSCLC cells and their primary HCC827 cells. In vitro, first of all, cell viability and colony formation assay confirmed the growth inhibitory effects of SH005S7 on both cells. Second, SH005S7 inactivated EGFR-related multiple cell signaling, which was associated with a marked decrease in the constitutive phosphorylation of EGFR, HER3, MET, AKT, and ERK. Third, SH005S7 attenuated the anchorage-independent cell growth. Fourth, SH005S7 blocked invasive and metastatic capability by downregulation of mesenchymal markers-vimentin, snail, and MMP-9. Fifth, BrdU assay confirmed the cell cycle arrest of SH005S7 on these cells. When administered orally to nude mice xenografically transplanted human NSCLC, SH005S7 inhibited the growth of tumor and did not cause hepatotoxicity and nephrotoxicity in animals. Immunohistochemical and Western blot analyses of tissue showed that the suppression of growth correlated with inhibition of proliferation (Ki-67, PCNA), invasiveness (vimentin, snail), and angiogenesis (CD31) marker and decrement in the constitutive and phosphorylation of EGFR, HER3, MET, AKT, and ERK. Additionally, SH005S7 had immune stimulatory effects by TNF-α cytokine release on macrophage, without cell cytotoxicity. Overall, our results suggest that SH005S7 can inhibit the growth of MET-amplified and gefitinib-resistant NSCLC cells through the suppression of EGFR-related multiple targets linked to overcome gefitinib resistance.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Apoptosis , Bromodesoxiuridina/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Gefitinib/farmacología , Humanos , Antígeno Ki-67 , Neoplasias Pulmonares/patología , Metaloproteinasa 9 de la Matriz , Ratones , Ratones Desnudos , Antígeno Nuclear de Célula en Proliferación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinazolinas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , VimentinaRESUMEN
Prion diseases, which involve the alteration of cellular prion protein into a misfolded isoform, disrupt the central nervous systems of humans and animals alike. Prior research has suggested that peroxisome proliferatoractivator receptor (PPAR)γ and autophagy provide some protection against neurodegeneration. PPARs are critical to lipid metabolism regulation and autophagy is one of the main cellular mechanisms by which cell function and homeostasis is maintained. The present study examined the effect of troglitazone, a PPARγ agonist, on autophagy flux in a prion peptide (PrP) (106126)mediated neurodegeneration model. Western blot analysis confirmed that treatment with troglitazone increased LC3II and p62 protein expression, whereas an excessive increase in autophagosomes was verified by transmission electron microscopy. Troglitazone weakened PrP (106126)mediated neurotoxicity via PPARγ activation and autophagy flux inhibition. A PPARγ antagonist blocked PPARγ activation as well as the neuroprotective effects induced by troglitazone treatment, indicating that PPARγ deactivation impaired troglitazonemediated protective effects. In conclusion, the present study demonstrated that troglitazone protected primary neuronal cells against PrP (106126)induced neuronal cell death by inhibiting autophagic flux and activating PPARγ signals. These results suggested that troglitazone may be a useful therapeutic agent for the treatment of neurodegenerative disorders and prion diseases.
Asunto(s)
Autofagia/efectos de los fármacos , Hipoglucemiantes/farmacología , Neuronas/metabolismo , PPAR gamma/metabolismo , Fragmentos de Péptidos/efectos adversos , Priones/efectos adversos , Troglitazona/farmacología , Animales , Proteína 5 Relacionada con la Autofagia/genética , Línea Celular , Humanos , Ratones , Ratones Endogámicos ICR , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , PPAR gamma/agonistas , Proteínas PriónicasRESUMEN
Prion diseases are caused by PrPsc accumulation in the brain, which triggers dysfunctional mitochondrial injury and reactive oxygen species (ROS) generation in neurons. Recent studies on prion diseases suggest that endoplasmic reticulum (ER) stress induced by misfolding proteins such as misfolded prion protein results in activation of calcineurin. Calcineurin is a calcium-related protein phosphatase of type 2B that exists in copious quantities in the brain and acts as a critical nodal component in the control of cellular functions. To investigate the relationship between calcineurin and intracellular ROS, we assessed the alteration of CaN and ROS induced by prion peptide (PrP) 106-126. Human prion peptide increased mitochondrial ROS by activating calcineurin, and the inhibition of calcineurin activity protected mitochondrial function and neuronal apoptosis in neuronal cells. These results suggest that calcineurin plays a pivotal role in neuronal apoptosis by mediating mitochondrial injury and ROS in prion diseases.
Asunto(s)
Calcineurina/metabolismo , Mitocondrias/efectos de los fármacos , Péptidos/farmacología , Proteínas Priónicas/química , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Mitocondrias/metabolismo , Péptidos/síntesis química , Tacrolimus/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Prion diseases are mortal neurodegenerative pathologies that are caused by the accumulation of abnormal prion protein (PrPSc) in the brain. Recent advances reveal that calcineurin may play a critical role in regulating nuclear factor kappa B (NF-κB) in the calcium-calmodulin pathway. However, the exact mechanism by calcineurin remains unclear. In the present study, we observed that the prion peptide induces calcineurin and autophagy activation. Also, NF-κB and proinflammatory cytokines like interleukin (IL)-6 and tumor necrosis factor (TNF)-α are upregulated upon exposure to prion peptide in human neuroblastoma. The results show that the prion peptide induces calcineurin activation, leading to the activation of NF-κB transcription factor via autophagy signaling. Expression of TNF-α and IL-6 was increased by calcineurin activation and blocked by calcineurin inhibitor and autophagy inhibitor treatments. Collectively, these findings indicate that calcineurin activation mediated by prion protein induces NF-κB-driven neuroinflammation via autophagy pathway, suggesting that calcineurin and autophagy may be possible therapeutic targets for neuroinflammation in neurodegeneration diseases including prion disease.
Asunto(s)
FN-kappa B , Priones , Autofagia , Calcineurina , Calcio , Humanos , PéptidosRESUMEN
It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPSc-neurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106-126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Calcineurina/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Priones/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Inhibidores de la Calcineurina/farmacología , Línea Celular Tumoral , Humanos , Neuroblastoma/enzimología , Neuroblastoma/metabolismo , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/patología , Fosforilación/efectos de los fármacos , Tacrolimus/farmacologíaRESUMEN
Accumulation of prion protein (PrPc) into a protease-resistant form (PrPsc) in the brains of humans and animals affects the central nervous system. PrPsc occurs only in mammals with transmissible prion diseases. Prion protein refers to either the infectious pathogen itself or the main component of the pathogen. Recent studies suggest that autophagy is one of the major functions that keep cells alive and which has a protective effect against neurodegeneration. In this study, we investigated whether the anti-hypertensive drug, captopril, could attenuate prion peptide PrP (106-126)-induced calcium alteration-mediated neurotoxicity. Treatment with captopril increased both LC3-II (microtubule-associated protein 1A/1B-light chain 3-II) and p62 protein levels, indicating autophagy flux inhibition. Electron microscopy confirmed the occurrence of autophagic flux inhibition in neuronal cells treated with captopril. Captopril attenuated PrP (106-126)-induced neuronal cell death via AMPK activation and autophagy inhibition. Compound C suppressed AMPK activation as well as the neuroprotective effects of captopril. Thus, these data showed that an anti-hypertensive drug has a protective effect against prion-mediated neuronal cell death via autophagy inhibition and AMPK activation, and also suggest that anti-hypertensive drugs may be effective therapeutic agents against neurodegenerative disorders, including prion diseases.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Captopril/farmacología , Neuronas/enzimología , Neuronas/patología , Péptidos/metabolismo , Priones/metabolismo , Animales , Calcio/metabolismo , Activación Enzimática/efectos de los fármacos , Ratones , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Neurotoxinas/toxicidadRESUMEN
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
Skin inflammation is a response of the immune system to infection and injury. In this study, we report that hinokitiol, a tropolone-related natural compound that exhibits antioxidant, anti-inflammatory, and anticancer properties in various cell types, can modulate the inflammatory responses of primary human keratinocytes challenged with lipopolysaccharide (LPS). Hinokitiol treatment inhibited LPS-mediated up-regulation of proinflammatory factors including tumor necrosis factor alpha, IL-6, and prostaglandin E2 (PGE2). NF-κB activation and cell migration induced by LPS were blocked in keratinocytes treated with hinokitiol. Sirt1, a class â ¢ histone deacetylase, was up-regulated by hinokitiol treatment, and the inhibition of Sirt1 activity using a pharmacological inhibitor or genetic silencing blocked hinokitiol-mediated anti-inflammatory effects. Further, hyperactivation of Sirt1 deacetylase using an adenoviral vector also attenuated LPS-induced inflammatory responses. We thus show that hinokitiol can attenuate LPS-mediated proinflammatory signals via Sirt1 histone deacetylase activation in primary human keratinocytes and suggest that hinokitiol may be a potential therapeutic agent in skin inflammatory diseases like psoriasis.
Asunto(s)
Antiinflamatorios/farmacología , Inflamación/prevención & control , Lipopolisacáridos/farmacología , Monoterpenos/farmacología , Sirtuina 1/metabolismo , Tropolona/análogos & derivados , Análisis de Varianza , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Inmunohistoquímica , Inflamación/patología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sirtuina 1/efectos de los fármacos , Tropolona/farmacologíaRESUMEN
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.
Asunto(s)
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
Epigallocatechin gallate (EGCG) is a major polyphenol in green tea. Recent studies have reported that EGCG can inhibit TRAIL-induced apoptosis and activate autophagic flux in cancer cells. However, the mechanism behind these processes is unclear. The present study found that EGCG prevents tumor cell death by antagonizing the TRAIL pathway and activating autophagy flux. Our results indicate that EGCG dose-dependently inhibits TRAIL-induced apoptosis and decreases the binding of death receptor 4 and 5 (DR4 and 5) to TRAIL. In addition, EGCG activates autophagy flux, which is involved in the inhibition of TRAIL cell death. We confirmed that the protective effect of EGCG can be reversed using genetic and pharmacological tools through re-sensitization to TRAIL. The inhibition of autophagy flux affects not only the re-sensitization of tumor cells to TRAIL, but also the restoration of death receptor proteins. This study demonstrates that EGCG inhibits TRAIL-induced apoptosis through the manipulation of autophagic flux and subsequent decrease in number of death receptors. On the basis of these results, we suggest further consideration of the use of autophagy activators such as EGCG in combination anti-tumor therapy with TRAIL.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Catequina/análogos & derivados , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Receptores de Muerte Celular/antagonistas & inhibidores , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Anticarcinógenos/farmacología , Catequina/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células Tumorales CultivadasRESUMEN
Mitochondrial quality control is a process by which mitochondria undergo successive rounds of fusion and fission with dynamic exchange of components to segregate functional and damaged elements. Removal of mitochondrion that contains damaged components is accomplished via autophagy. In this study, we investigated whether ginsenoside Rg3, an active ingredient of the herbal medicine ginseng that is used as a tonic and restorative agent, could attenuate prion peptide, PrP (106-126)-induced neurotoxicity and mitochondrial damage. To this end, western blot and GFP-LC3B puncta assay were performed to monitor autophagy flux in neuronal cells; LC3B-II protein level was found to increase after Rg3 treatment. In addition, electron microscopy analysis showed that Rg3 enhanced autophagic vacuoles in neuronal cells. By using autophagy inhibitors wortmannin and 3-methyladenine (3MA) or autophagy protein 5 (Atg5) small interfering RNA (siRNA), we demonstrated that Rg3 could protect neurons against PrP (106-126)-induced cytotoxicity via autophagy flux. We found that Rg3 could also attenuate PrP (106-126)-induced mitochondrial damage via autophagy flux. Taken together, our results suggest that Rg3 is a possible therapeutic agent in neurodegenerative disorders, including prion diseases.
Asunto(s)
Autofagia/efectos de los fármacos , Ginsenósidos/farmacología , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Priones/toxicidad , Adenina/análogos & derivados , Adenina/farmacología , Apoptosis/efectos de los fármacos , Autofagia/fisiología , Línea Celular Tumoral , Humanos , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
Prion diseases are fatal neurodegenerative disorders that are derived from structural changes of the native PrPc. Recent studies indicated that hinokitiol induced autophagy known to major function that keeps cells alive under stressful conditions. We investigated whether hinokitiol induces autophagy and attenuates PrP (106-126)-induced neurotoxicity. We observed increase of LC3-II protein level, GFP-LC3 puncta by hinokitiol in neuronal cells. Addition to, electron microscopy showed that hinokitiol enhanced autophagic vacuoles in neuronal cells. We demonstrated that hinokitiol protects against PrP (106-126)-induced neurotoxicity via autophagy by using autophagy inhibitor, wortmannin and 3MA, and ATG5 small interfering RNA (siRNA). We checked hinokitiol activated the hypoxia-inducible factor-1α (HIF-1α) and identified that hinokitiol-induced HIF-1α regulated autophagy. Taken together, this study is the first report demonstrating that hinokitiol protected against prion protein-induced neurotoxicity via autophagy regulated by HIF-1α. We suggest that hinokitiol is a possible therapeutic strategy in neuronal disorders including prion disease.