RESUMO
Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1α accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1α target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. Intriguingly, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1α destabilization under normoxic conditions, suggesting that K8 may regulate HIF1α stability in a non-canonical pathway. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1α-mediated hypoxic gene activation.
Assuntos
Neuroblastoma , Humanos , Hipóxia Celular/genética , Linhagem Celular , Hipóxia/metabolismo , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel-Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.
Assuntos
Autofagia , Ciclopropanos , Macroautofagia , Pirrolidinas , Tiazóis , Humanos , Células HeLa , Homeostase , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
The primary cilium, an antenna-like structure on the cell surface, acts as a mechanical and chemical sensory organelle. Primary cilia play critical roles in sensing the extracellular environment to coordinate various developmental and homeostatic signaling pathways. Here, we showed that the depletion of heat shock protein family A member 9 (HSPA9)/mortalin stimulates primary ciliogenesis in SH-SY5Y cells. The downregulation of HSPA9 enhances mitochondrial stress by increasing mitochondrial fragmentation and mitochondrial reactive oxygen species (mtROS) generation. Notably, the inhibition of either mtROS production or mitochondrial fission significantly suppressed the increase in primary ciliogenesis in HSPA9-depleted cells. In addition, enhanced primary ciliogenesis contributed to cell survival by activating AKT in SH-SY5Y cells. The abrogation of ciliogenesis through the depletion of IFT88 potentiated neurotoxicity in HSPA9-knockdown cells. Furthermore, both caspase-3 activation and cell death were increased by MK-2206, an AKT inhibitor, in HSPA9-depleted cells. Taken together, our results suggest that enhanced primary ciliogenesis plays an important role in preventing neurotoxicity caused by the loss of HSPA9 in SH-SY5Y cells.
Assuntos
Neuroblastoma , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Apoptose , Estresse Oxidativo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Primary cilia help to maintain cellular homeostasis by sensing conditions in the extracellular environment, including growth factors, nutrients, and hormones that are involved in various signaling pathways. Recently, we have shown that enhanced primary ciliogenesis in dopamine neurons promotes neuronal survival in a Parkinson's disease model. Moreover, we performed fecal metabolite screening in order to identify several candidates for improving primary ciliogenesis, including L-carnitine and acetyl-L-carnitine. However, the role of carnitine in primary ciliogenesis has remained unclear. In addition, the relationship between primary cilia and neurodegenerative diseases has remained unclear. In this study, we have evaluated the effects of carnitine on primary ciliogenesis in 1-methyl-4-phenylpyridinium ion (MPP+)-treated cells. We found that both L-carnitine and acetyl-L-carnitine promoted primary ciliogenesis in SH-SY5Y cells. In addition, the enhancement of ciliogenesis by carnitine suppressed MPP+-induced mitochondrial reactive oxygen species overproduction and mitochondrial fragmentation in SH-SY5Y cells. Moreover, carnitine inhibited the production of pro-inflammatory cytokines in MPP+-treated SH-SY5Y cells. Taken together, our findings suggest that enhanced ciliogenesis regulates MPP+-induced neurotoxicity and inflammation.
Assuntos
Neuroblastoma , Síndromes Neurotóxicas , 1-Metil-4-fenilpiridínio/toxicidade , Acetilcarnitina/farmacologia , Apoptose , Carnitina/farmacologia , Linhagem Celular Tumoral , Neurônios Dopaminérgicos , Humanos , InflamaçãoRESUMO
Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A-D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation, O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Animais , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Ir is one of the most efficient oxygen evolution reaction (OER) catalysts; however, it is also one of the rarest and most expensive elements. Therefore, it is highly desirable to develop Ir catalysts with nanostructures that reduce Ir consumption by maximizing the surface-to-volume ratio without limiting the mass transport of reactants and products of reactions. Ir OER catalysts on a template that consisted of porous nanotubes (PNTs) based on Ni are fabricated. The Ir/Ni PNTs offer multiple benefits, including high catalytic performance (potential of 1.500 V vs. reversible hydrogen electrode (RHE) at an operating current density of 10 mA cm-2 and Tafel slope of 44.34 mV decade-1 ), minimal use of Ir (mass activity of 3273 A g-1 at 1.53 V vs RHE), and facile mass transport through the NT-sidewall pores (stable operation for more than 10 h). The Ir/Ni PNTs are also applied to a tandem device, consisting of a Cu(In,Ga)Se2 -based photocathode and halide perovskite photovoltaic cell, for unassisted water splitting. A solar-to-hydrogen conversion efficiency that exceeded 10% is also demonstrated, which is nearly 1% point greater than when a planar Ir film is used as the anode instead of Ir/Ni PNTs.
RESUMO
Selective autophagy controls cellular homeostasis by degrading unnecessary or damaged cellular components. Melanosomes are specialized organelles that regulate the biogenesis, storage, and transport of melanin in melanocytes. However, the mechanisms underlying melanosomal autophagy, known as the melanophagy pathway, are poorly understood. To better understand the mechanism of melanophagy, we screened an endocrine-hormone chemical library and identified nalfurafine hydrochlorides, a κ-opioid receptor agonist, as a potent inducer of melanophagy. Treatment with nalfurafine hydrochloride increased autophagy and reduced melanin content in alpha-melanocyte-stimulating hormone (α-MSH)-treated cells. Furthermore, inhibition of autophagy blocked melanosomal degradation and reversed the nalfurafine hydrochloride-induced decrease in melanin content in α-MSH-treated cells. Consistently, treatment with other κ-opioid receptor agonists, such as MCOPPB or mianserin, inhibited excessive melanin production but induced autophagy in B16F1 cells. Furthermore, nalfurafine hydrochloride inhibited protein kinase A (PKA) activation, which was notably restored by forskolin, a PKA activator. Additionally, forskolin treatment further suppressed melanosomal degradation as well as the anti-pigmentation activity of nalfurafine hydrochloride in α-MSH-treated cells. Collectively, our data suggest that stimulation of κ-opioid receptors induces melanophagy by inhibiting PKA activation in α-MSH-treated B16F1 cells.
Assuntos
Melaninas , alfa-MSH , alfa-MSH/farmacologia , Colforsina , Melaninas/metabolismo , Receptores Opioides kappa/agonistas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Animais , CamundongosRESUMO
Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.
Assuntos
Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Malatos/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Cílios/metabolismo , Cílios/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 1 da Matriz/genética , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.
Assuntos
Autofagia/efeitos dos fármacos , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Lisossomos/efeitos dos fármacos , Triantereno/farmacologia , Autofagia/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células HeLa , Células Hep G2 , Humanos , Lisossomos/metabolismoRESUMO
Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.
Assuntos
Trióxido de Arsênio/toxicidade , Células MCF-7/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Arsênio/metabolismo , Trióxido de Arsênio/metabolismo , Trióxido de Arsênio/farmacologia , Arsenicais/farmacologia , Neoplasias da Mama/genética , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Glândulas Mamárias Humanas/metabolismo , Cultura Primária de Células , Células Tumorais CultivadasRESUMO
The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. In contrast to melanosome biogenesis, the processes underlying melanosome degradation remain largely unknown. Autophagy is a process that promotes degradation of intracellular components' cooperative process between autophagosomes and lysosomes, and its role for process of melanosome degradation remains unclear. Here, we assessed the regulation of autophagy and its contributions to depigmentation associated with Melasolv (3,4,5-trimethoxycinnamate thymol ester). B16F1 cells-treated with Melasolv suppressed the α-MSH-stimulated increase of melanin content and resulted in the activation of autophagy. However, introduction of bafilomycin A1 strongly suppressed melanosome degradation in Melasolv-treated cells. Furthermore, inhibition of autophagy by ATG5 resulted in significant suppression of Melasolv-mediated depigmentation in α-MSH-treated cells. Taken together, our results suggest that treatment with Melasolv inhibits skin pigmentation by promoting melanosome degradation via autophagy activation.
Assuntos
Cinamatos/farmacologia , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cinamatos/metabolismo , Macrolídeos/farmacologia , Melaninas/metabolismo , Melanócitos/metabolismo , Camundongos , Pigmentação/efeitos dos fármacos , Transtornos da Pigmentação/metabolismo , Pigmentação da Pele/efeitos dos fármacos , alfa-MSH/efeitos dos fármacos , alfa-MSH/metabolismoRESUMO
Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.
Assuntos
Autofagia/efeitos dos fármacos , Melanoma Experimental/patologia , Melanossomas/patologia , Pigmentação da Pele/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Melaninas/biossíntese , Melanossomas/efeitos dos fármacos , Camundongos , Triterpenos/química , alfa-MSH/farmacologia , Ácido UrsólicoRESUMO
Mitochondria are essential for providing the energy necessary for neuronal function. Dysregulation of mitochondrial dynamics has been linked with the pathogenesis of many neurodegenerative diseases. Dynamin related protein 1 (Drp1) participates in fission activity in the mitochondria, and post-translational modifications to Drp1 modulate complex mitochondrial dynamics. However, the regulation of Drp1 at the post-transcriptional level remains poorly understood. In this study, we found that the RNA-binding protein Hu antigen R (HuR) post-transcriptionally regulates Drp1 expression. HuR interacts with Drp1 mRNA at its 3' untranslated region. Depletion of HuR reduces Drp1 expression, which leads to mitochondrial elongation in SH-SY5Y neuroblastoma cells. In contrast, ectopic expression of HuR enhances Drp1 expression, which promotes mitochondrial fragmentation in response to treatment with the mitochondrial complex 1 inhibitor MPP+. In addition, depletion of HuR suppressed the generation of mitochondrial ROS and cytotoxicity in MPP+ treated cells. Taken together, these findings suggest that HuR controls mitochondrial morphology via regulation of Drp1.
Assuntos
Dinaminas/genética , Proteína Semelhante a ELAV 1/genética , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Proteínas de Ligação a RNA/genética , 1-Metil-4-fenilpiridínio/farmacologia , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Dinaminas/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Herbicidas/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Exposure to fine particulate matter (PM) with diameter <2.5 µm (PM2.5) causes epithelium injury and endothelial dysfunction. Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses and have roles in physiology. To date, there have been no studies investigating whether PM2.5 affects primary cilia in skin. We addressed this in the present study using normal human epidermal keratinocytes (NHEKs) and retinal pigment epithelium (RPE) cells. We found that formation of primary cilium is increased in differentiated NHEKs. However, treatment with PM2.5 blocked increased ciliogenesis in NHEKs and RPE cells. Furthermore, PM2.5 transcriptionally upregulated small proline rich protein 3 (SPRR3) expression by activating c-Jun, and ectopic expression of SPRR3 inhibits suppressed the ciliogenesis. Accordingly, treatment with c-Jun activator (anisomycin) induced SPRR3 expression, whereas the inhibitor (SP600125) recovered the ciliated cells and cilium length in PM2.5-treated cells. Moreover, c-Jun inhibitor suppressed upregulation of SPRR3 in PM2.5-treated cells. Taken together, our finding suggested that PM2.5 inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and keratinocytes.
Assuntos
Cílios/efeitos dos fármacos , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Queratinócitos/efeitos dos fármacos , Material Particulado/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pele/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Cílios/metabolismo , Humanos , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pele/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-ß (Aß) production. Treatment with Aß increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Encéfalo/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas/metabolismo , Doença de Alzheimer/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/enzimologia , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular , Cisteína Endopeptidases/genética , Regulação para Baixo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The ultraviolent irradiation resistance-associated gene (UVRAG), a component of the Beclin 1/autophagy-related 6 complex, regulates the autophagy initiation step and functions in the DNA-damage response. UVRAG is frequently mutated in various cancer types, and mutations of UVRAG increase sensitivity to chemotherapy by impairing DNA-damage repair. In this study, we addressed the epigenetic regulation of UVRAG in colorectal cancer cells. UVRAG expression was increased in cells treated with histone deacetylase (HDAC) inhibitors, such as valproic acid and suberoylanilide hydroxamic acid. Down-regulation of HDAC1 enhanced UVRAG expression in colorectal cancer cells. In addition, both chemical and genetic inhibition of HDAC1 reduced the activation of caspase-3 and cytotoxicity in 5-fluorouracil (5FU)-treated cancer cells. In contrast, UVRAG overexpression inhibited caspase activation and cell death in 5FU-treated cells. Taken together, our findings suggest that up-regulation of UVRAG by HDAC1 inhibition potentiates DNA-damage-mediated cell death in colorectal cancer cells.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Fluoruracila/farmacologia , Histona Desacetilase 1/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Morte Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Dano ao DNA , Epigênese Genética , Células HCT116 , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Regulação para CimaRESUMO
Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Movimento Celular , Neoplasias Colorretais/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Antígenos CD/metabolismo , Proteínas Relacionadas à Autofagia/genética , Caderinas/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação para Baixo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Receptores de Hialuronatos/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transfecção , Proteínas de Transporte Vesicular/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
PURPOSE: We aimed to investigate the effectiveness of ferritin as a contrast agent and a potential reporter gene for tracking tumor cells or macrophages in mouse cancer models. MATERIALS AND METHODS: Adenoviral human ferritin heavy chain (Ad-hFTH) was administrated to orthotopic glioma models and subcutaneous colon cancer mouse models using U87MG and HCT116 cells, respectively. Brain MR images were acquired before and daily for up to 6 days after the intracranial injection of Ad-hFTH. In the HCT116 tumor model, MR examinations were performed before and at 6, 24, and 48 h after intratumoral injection of Ad-hFTH, as well as before and every two days after intravenous injection of ferritin-labeled macrophages. The contrast effect of ferritin in vitro was measured by MR imaging of cell pellets. MRI examinations using a 7T MR scanner comprised a T1-weighted (T1w) spin-echo sequence, T2-weighted (T2w) relaxation enhancement sequence, and T2*-weighted (T2*w) fast low angle shot sequence. RESULTS: Cell pellet imaging of Ad-hFTH in vitro showed a strong negatively enhanced contrast in T2w and T2*w images, presenting with darker signal intensity in high concentrations of Fe. T2w images of glioma and subcutaneous HCT116 tumor models showed a dark signal intensity around or within the Ad-hFTH tumor, which was distinct with time and apparent in T2*w images. After injection of ferritin-labeled macrophages, negative contrast enhancement was identified within the tumor. CONCLUSION: Ferritin could be a good candidate as an endogenous MR contrast agent and a potential reporter gene that is capable of maintaining cell labeling stability and cellular safety.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Rastreamento de Células/métodos , Neoplasias do Colo/diagnóstico por imagem , Meios de Contraste , Ferritinas , Glioma/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Neoplasias Cutâneas/diagnóstico por imagem , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Meios de Contraste/administração & dosagem , Modelos Animais de Doenças , Feminino , Ferritinas/administração & dosagem , Genes Reporter , Glioma/patologia , Humanos , Injeções Intravenosas , Macrófagos , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias Cutâneas/patologia , Fatores de TempoRESUMO
Mitochondrial dynamics control mitochondrial morphology and function, and aberrations in these are associated with various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. To identify novel regulators of mitochondrial dynamics, we screened a phytochemical library and identified liquiritigenin as a potent inducer of mitochondrial fusion. Treatment with liquiritigenin induced an elongated mitochondrial morphology in SK-N-MC cells. In addition, liquiritigenin rescued mitochondrial fragmentation induced by knockout of mitochondrial fusion mediators such as Mfn1, Mfn2, and Opa1. Furthermore, we found that treatment with liquiritigenin notably inhibited mitochondrial fragmentation and cytotoxicity induced by Aß in SK-N-MC cells.