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1.
Nat Commun ; 12(1): 2587, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972537

RESUMO

Host cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/- and RhoB-/- mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.


Assuntos
Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Proteína rhoB de Ligação ao GTP/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/ultraestrutura , Proteína Beclina-1/genética , Linhagem Celular , Epitélio/metabolismo , Epitélio/microbiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Estabilidade Proteica , RNA Interferente Pequeno , Proteínas Recombinantes , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Proteína rhoB de Ligação ao GTP/genética
2.
Nat Commun ; 12(1): 1564, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33692360

RESUMO

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.


Assuntos
Membrana Celular/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/química , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rab1 de Ligação ao GTP/química , Proteínas rab1 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteína Beclina-1/química , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estrutura Secundária de Proteína , Tomografia , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína VPS15 de Distribuição Vacuolar/química , Proteína VPS15 de Distribuição Vacuolar/genética , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética
3.
J Steroid Biochem Mol Biol ; 208: 105823, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33484844

RESUMO

Polycystic ovarian syndrome (PCOS) is a multi-factorial gynecological endocrine disorder. It affects fertility in women and also predisposes to insulin resistance, type 2 diabetes mellitus, obesity etc. Earlier, significance of autophagy has been explored in PCOS-related metabolic disorders and during normal folliculogenesis. Increasing evidences reveal connection of autophagy with chronic inflammatory behaviour, an associated phenomena in polycystic ovaries. However, understanding of the association of autophagy with PCOS is still obscure. This study reveals that increased autophagy in mifepristone (RU486) treated KK-1 cells and in vivo PCO rat model is characterized by upregulated Androgen Receptor (AR) expression and downregulated PCO biomarker aromatase. The prevalence of autophagy has been observed to be concomitant with increased expression of two autophagic markers Beclin1 and MAP-LC3-II while the autophagy substrate p62/SQSTM1 was downregulated. Immunohistochemical staining revealed increased localization of MAP-LC3 in the compacted granulosa layers of the follicular cysts in the PCO model. The PCO rat models also demonstrated augmented levels of p65, the active subunit of NF-κB, which acts as a transcriptional regulator of several pro-inflammatory factors. NF-κB repressor and anti-inflammatory herbal drug thymoquinone, known to alleviate PCO condition, downregulated autophagy modules substantially. Pre-treatment with thymoquinone upregulated aromatase, reduced AR levels and decreased autophagic markers as well as p65 levels, simulating super-ovulated condition. In conclusion, the anti-inflammatory phytochemical thymoquinone alleviated PCO condition.


Assuntos
Autofagia/efeitos dos fármacos , Benzoquinonas/farmacologia , Mifepristona/farmacologia , Síndrome do Ovário Policístico/tratamento farmacológico , Receptores Androgênicos/genética , Androgênios/metabolismo , Animais , Autofagia/genética , Proteína Beclina-1/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Humanos , Resistência à Insulina/genética , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovulação/efeitos dos fármacos , Ovulação/genética , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/patologia , Ratos , eIF-2 Quinase/genética
4.
Med Sci Monit ; 27: e927850, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33510126

RESUMO

BACKGROUND Several studies have suggested the importance of autophagy during esophageal adenocarcinoma (EAC) development. This study aimed to explore the autophagy-related genes correlated with overall survival in patients with EAC. MATERIAL AND METHODS The RNA-seq expression profiles and clinical data of patients with EAC were screened using The Cancer Genome Atlas (TCGA) database. Screening of autophagy-related genes was conducted using the human autophagy database (HADb). Bioinformatic analysis was conducted and included the following: univariate cox, lasso regression, and multivariate cox regression analysis; building overall survival assessment of the prognosis model; drawing the model of receiver operating characteristic (ROC) curve and determining the area under the curve; and a C-index reliability index assessment model through Kaplan-Meier screening of statistically significant genes in the model. The screening results were verified via Oncomine differential expression analysis. Gene set enrichment analysis (GSEA) was further used to analyze the molecular biological functions and related pathways of the gene model. RESULTS Through cox regression and ROC analysis, the model showed that the risk score could accurately and independently predict the prognosis of EAC. The screening identified 4 genes: DAPK1, BECN1, ATG5, and VAMP7. GSEA showed that the high and low expression levels of the 4 genes were mainly enriched in biological functions, such as cell production and regulation, and metabolic pathways that maintain cell activity. CONCLUSIONS Our research found that autophagy was involved in the process of EAC development and that several autophagy-related genes may provide prognostic information and clinical application value for patients with EAC.


Assuntos
Adenocarcinoma/genética , Autofagia/genética , Biologia Computacional/métodos , Neoplasias Esofágicas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Proteína 5 Relacionada à Autofagia/genética , Proteína Beclina-1/genética , Biomarcadores Tumorais/genética , Bases de Dados Genéticas , Proteínas Quinases Associadas com Morte Celular/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidade , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Prognóstico , Proteínas R-SNARE/genética , Curva ROC , Reprodutibilidade dos Testes , Fatores de Risco , Transcriptoma/genética
5.
Mol Biol Rep ; 48(2): 1423-1431, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33507476

RESUMO

Periodontal ligament fibroblasts (PDLFs) are integral to the homeostasis of periodontal tissue. The transcription factor Dec1 functions to modulate Porphyromonas gingivalis-induced periodontal inflammation. Here, we aimed to characterize the Dec1-mediated autophagy in PDLFs under inflammatory conditions. Human PDLFs were subjected to an inflammatory environment using P. gingivalis Lipopolysaccaride (LPS) along with Dec1 siRNA in vitro. Quantitative real-time polymerase chain reaction and Western blot analyses were used to evaluate the expression levels of autophagy-related genes and their upstream AKT/mTOR signaling pathways. An experimental P. gingivalis-treated Dec1 knockout (Dec1KO) mouse model was used to confirm the expression of autophagy in PDLFs in vivo. Treatment with P. gingivalis LPS induced the expression of ATG5, Beclin1 and microtubule-associated protein 1 light chain 3 (LC3) and elevated the expression of pro-inflammatory cytokine IL-1ß and Dec1 in human PDLFs. Knockdown of Dec1 partly reversed the detrimental influences of LPS on these autophagy markers in human PDLFs. The inhibition of autophagy with Dec1 siRNA suppressed the inflammatory effect of AKT/mTOR signaling pathways following treatment with P. gingivalis LPS. P. gingivalis-treated Dec1KO mice partly reduced autophagy expression. These findings suggest that a Dec1 deficiency can modulate the interaction between autophagy and inflammation in PDLFs.


Assuntos
Autofagia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Inflamação/genética , Ligamento Periodontal/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Proteína 5 Relacionada à Autofagia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Proteína Beclina-1/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Ligamento Periodontal/microbiologia , Ligamento Periodontal/patologia , Porphyromonas gingivalis/patogenicidade , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
6.
PLoS One ; 16(1): e0245694, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465164

RESUMO

Autophagy is a conserved cellular process playing a role in maintenance of cellular homeostasis and response to changing nutrient conditions via degradation and recirculation of cellular redundant components. Autophagy-related proteins (Atg) play important function in autophagy pathway. Aedes albopictus mosquito is an effective vector transmitting multiple viruses which cause serious human diseases. Moreover, Aedes albopictus mosquito is becoming a serious threat to human health due to its widening distribution in recent years and thus worth of more research attention. It was reported that autophagy might play a role in viral infection in Aedes mosquito. To better understand the interaction between autophagy and arbovirus infection in mosquito system, it is necessary to identify autophagy pathway in the system. However, autophagy in Aedes albopictus mosquito is still poorly understood so far. We recently identified AaAtg8, the first Atg protein reported in Aedes albopictus mosquito. This work further identified twelve atg genes in Aedes albopictus mosquito. Sequence and phylogenetic analysis of the twelve atg genes were performed. Expression profiles of all the twelve Aaatg genes in different developmental stages and genders of Aedes albopictus mosquito were conducted. Effects of chemicals inhibiting or inducing autophagy on the levels of eight identified AaAtg proteins were examined. The function of two identified AaAtg proteins AaAtg6 and AaAtg16 and their response to arbovirus SINV infection were studied preliminarily. Taken together, this work systematically identified Aedes albopictus atg genes and provided basic information which might help to elucidate the autophagy pathway and the role of autophagy in arbovirus infection in Aedes mosquito system.


Assuntos
Aedes/metabolismo , Infecções por Arbovirus/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Adenina/análogos & derivados , Adenina/farmacologia , Aedes/genética , Aedes/crescimento & desenvolvimento , Aedes/virologia , Animais , Infecções por Arbovirus/genética , Arbovírus/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Linhagem Celular , Cloroquina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Filogenia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real
7.
Curr Pharm Des ; 27(7): 996-1005, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33349212

RESUMO

BACKGROUND: Hypopharyngeal carcinoma is characterized by a high degree of malignancy. The most common pathological type is squamous cell carcinoma (HSCC). Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most widely used chemotherapeutic drugs nowadays and cisplatin resistance is a major problem in current treatment strategies. Clinical researchers have reported that high autophagy levels often caused insensitivity to chemotherapy, a common phenomenon that greatly reduces the therapeutic effect in cisplatin- resistant tumor cell lines. 3-methyladenine (3-MA), an inhibitor of PI3K, plays a vital role in forming and developing autophagosomes. Therefore, we speculate that the use of 3-MA may reduce cisplatin resistance in hypopharyngeal squamous cell carcinoma (HSCC). METHODS: Part I: Cisplatin-resistant FaDu cell line (Human hypopharyngeal squamous cell carcinoma cells) was established and cultured. Cell counting kit-8 was used to detect drug resistance. An inverted microscope was used to observe the morphological changes at different concentrations, then the survival rate was calculated. After MDC staining, the autophagic vacuoles were observed by fluorescence microscopy. The expression of Beclin1 from each group was confirmed by RT-PCR and Western blot method. Part II: 3-MA was applied for cisplatin-resistant cells intervention, Beclin1 was knocked down by plasmid transfection. Cell cycle was detected using flow cytometry assay, apoptosis with necrosis was detected by staining with propidium iodide (PI). CCK-8 was used to observe the cell survival rate in each group. The expression of autophagy-related protein Beclin1, LC3I, LC3II, Atg-5 and P62 in each group was verified by Western blot analysis. RESULTS: Cisplatin-resistant FaDu cell line can be stably constructed by cisplatin intervention. Compared with normal group, autophagy and its related protein Beclin1 expression were enhanced in cisplatin resistant FaDu cells. Autophagy inhibition group showed significant cell cycle changes, mainly manifested by G1 arrest, increased apoptosis rate and significantly decreased survival rate at 24h level. The number of autophagy vacuoles were significantly reduced in the 3-MA group. Furthermore, Western blot showed that expression of Beclin1, lc3-I, lc3-II, atg-5 protein decreased significantly after 3-MA intervention, while the expression of p62 upregulated, which also confirmed autophagy flow was blocked. CONCLUSION: Our work confirmed that enhanced autophagy is an important cause of cisplatin resistance in FaDu cells. The use of 3-MA can significantly reduce autophagy level and arresting its cell cycle, promote apoptosis and reverse the cisplatin resistance condition, this effect is partly mediated by inhibition of Beclin-1 expression. Our data provide a theoretical basis for the application of 3-MA in overcoming cisplatin resistance in hypopharyngeal cancer.


Assuntos
Antineoplásicos , Carcinoma de Células Escamosas , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Autofagia , Proteína Beclina-1/genética , Proteína Beclina-1/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética
8.
Biomed Pharmacother ; 133: 111023, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378941

RESUMO

Long noncoding RNAs (lncRNAs) have been identified to play increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. LncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is demonstrated to decrease in several cancers. However, it is still unknown whether lncRNA NBR2 is involved in hepatocellular carcinoma and autophagy. We found that HCC cases with lower NBR2 expression had significantly worse overall survival than those with higher NBR2 expression in advanced patients. And the expression of NBR2 was negatively correlated with the degree of malignancy of HCC cell lines and differentiation of hepatocellular carcinoma. Besides, NBR2 inhibited the proliferation, invasion, and migration of liver cancer cells. We further found that NBR2 repressed cytoprotective autophagy to restrain HCC cell proliferation. Moreover, NBR2 inhibited Beclin 1-dependent autophagy through ERK and JNK pathways. Taken together, NBR2 suppressed autophagy-induced cell proliferation at least partly through ERK and JNK pathways. These data indicated that NBR2 served as a tumor suppressor gene in hepatocellular carcinoma. The current study provides a novel insight and treatment strategy for hepatocellular carcinoma.


Assuntos
Autofagia , Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Movimento Celular , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , RNA Longo não Codificante/genética , Transdução de Sinais
9.
Toxicol Lett ; 332: 155-163, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32645460

RESUMO

Chronic exposure to arsenic increases the risk of developing a variety of human cancers including lung carcinomas. However, the exact molecular mechanism underlying arsenic carcinogenicity remains largely unknown. Autophagy is a conserved catabolic process for maintaining cellular protein homeostasis whose defects might result in accumulation of dysfunctional organelles and damaged proteins thus promoting tumorigenesis. In the present study, we found that chronic exposure of human bronchial epithelial BEAS-2B cells to sub-lethal dose of sodium arsenite led to autophagy activation and induced an epithelial-to-mesenchymal transition (EMT) to enhance cell migratory and invasive capability. The malignant transformation was mediated via activation of MEK/ERK1/2 signaling. Importantly, inhibition of autophagy in these arsenic-exposed cells by pharmacological intervention or genetic deletion further promoted the EMT and increased the generation of inflammasomes. Both autophagy inhibitor and genetic deletion of autophagy core gene Beclin-1 produced similar effects. These results may suggest the important role of autophagy in sodium arsenite-induced lung tumorigenesis which may serve as a potential target in prevention and treatment of arsenic-imposed lung cancer.


Assuntos
Arsênio/toxicidade , Autofagia/fisiologia , Brônquios/patologia , Neoplasias Brônquicas/induzido quimicamente , Neoplasias Brônquicas/patologia , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Beclina-1/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Inflamassomos/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
10.
Gene ; 754: 144775, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32428696

RESUMO

Myocardial ischemia/reperfusion (I/R) injury is a common consequence of restored blood supply after acute myocardial infarction (AMI), but its underlying mechanisms remain largely elusive. In this study, we aimed to investigate the functional role of long non-coding RNA PVT1 in hypoxia/reoxygenation (H/R)-treated AC16 cardiomyocytes. Our experimental results demonstrated that H/R treatment impaired the viability and increased the apoptosis of AC16 cells, and knockdown of PVT1 blocked the H/R injury. Besides, PVT1 knockdown also reduced excessive autophagy in H/R-treated AC16 cells. Furthermore, we confirmed that PVT1 might serve as a ceRNA for miR-186 in AC16 cells, and rescue experiments showed that miR-186 inhibition blocked the effects of PVT1 knockdown in H/R-treated AC16 cells. In summary, this study implied that PVT1 might be a promising therapeutic target for treating myocardial I/R injury.


Assuntos
Apoptose , Autofagia , Proteína Beclina-1/metabolismo , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Proteína Beclina-1/genética , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Hipóxia/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Substâncias Protetoras/metabolismo , RNA Longo não Codificante/genética
11.
J Nanobiotechnology ; 18(1): 65, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345308

RESUMO

Nanoparticle based gene delivery systems holds great promise. Superparamagnetic iron oxide nanoparticles (SPIONs) are being heavily investigated due to good biocompatibility and added diagnostic potential, rendering such nanoparticles theranostic. Yet, commonly used cationic coatings for efficient delivery of such anionic cargos, results in significant toxicity limiting translation of the technology to the clinic. Here, we describe a highly biocompatible, small and non-cationic SPION-based theranostic nanoparticles as novel gene therapy agents. We propose for the first-time, the usage of the microRNA machinery RISC complex component Argonaute 2 (AGO2) protein as a microRNA stabilizing agent and a delivery vehicle. In this study, AGO2 protein-conjugated, anti-HER2 antibody-linked and fluorophore-tagged SPION nanoparticles were developed (SP-AH nanoparticles) and used as a carrier for an autophagy inhibitory microRNA, MIR376B. These functionalized nanoparticles selectively delivered an effective amount of the microRNA into HER2-positive breast cancer cell lines in vitro and in a xenograft nude mice model of breast cancer in vivo, and successfully blocked autophagy. Furthermore, combination of the chemotherapy agent cisplatin with MIR376B-loaded SP-AH nanoparticles increased the efficacy of the anti-cancer treatment both in vitro in cells and in vivo in the nude mice. Therefore, we propose that AGO2 protein conjugated SPIONs are a new class of theranostic nanoparticles and can be efficiently used as innovative, non-cationic, non-toxic gene therapy tools for targeted therapy of cancer.


Assuntos
Proteínas Argonauta/química , Autofagia , Materiais Biocompatíveis/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Nanopartículas de Magnetita/química , MicroRNAs/metabolismo , Animais , Anticorpos/química , Anticorpos/imunologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cisplatino/química , Cisplatino/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Nus , MicroRNAs/química , Receptor ErbB-2/imunologia , Transplante Heterólogo
12.
Circ J ; 84(4): 616-625, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32115441

RESUMO

BACKGROUND: Ischemic postconditioning (IPostC) is an endogenous protective mechanism to reduce ischemia-reperfusion (I/R) injury. However, whether IPostC protects aged cardiomyocytes against I/R injury is not fully understood. Considering the protective function of microRNA 30a (miR-30a) against ischemia-induced injury in H9C2 cells, its role in the protective effects of IPostC on I/R injury of aged cardiomyocytes was investigated further.Methods and Results:To mimic I/R and IPostC in vitro, the aged cardiomyocyte model for hypoxia postconditioning (HPostC) treatment was established by 9 days of incubation with 8 mg/mL D-galactose and then followed by exposure to hypoxic environment. HPostC significantly alleviated hypoxia/reoxygenation (H/R) injury and reduced autophagy of aged cardiomyocytes, as evidenced by decreased LC3B-II expression and increased p62 by Western blot. Quantified by quantitative real-time polymerase chain reaction (qRT-PCR), miR-30a was increased in aged cardiomyocytes treated with HPostC compared with I/R injury group. Overexpression of miR-30a by LV3-rno-miR-30a mimic promoted cardioprotective effect of HPostC in aged cardiomyocytes by suppressing BECN1-mediated autophagy, all of which was abrogated by knockdown of miR-30a expression. Epigenetic analyses demonstrated that HPostC reduced DNA methyltransferase 3b-mediated DNA hypomethylation levels at miR-30a promoter, leading to upregulation of miR-30a. CONCLUSIONS: HPostC protected aged cardiomyocytes survival against H/R injury via DNMT3b-dependent activation of miR-30a. miR-30a could be a potential therapeutic target for ischemic myocardial infarction.


Assuntos
Autofagia , Senescência Celular , Metilação de DNA , Epigênese Genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Hipóxia Celular , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Regiões Promotoras Genéticas , Ratos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
13.
Int J Mol Sci ; 21(3)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046105

RESUMO

Autophagy is a catabolic process that enables cells to degrade obsolete content and refuel energy depots. In colorectal cancer (CRC) autophagy has been shown to promote tumorigenesis through energy delivery in the condition of uncontrolled proliferation. With this study, we aimed at evaluating whether autophagy sustains CRC cell viability and if it impacts therapy resistance. Initially, a colorectal cancer tissue micro array, containing mucosa (n = 10), adenoma (n = 18) and adenocarcinoma (n = 49) spots, was stained for expression of essential autophagy proteins LC3b, Atg7, p62 and Beclin-1. Subsequently, central autophagy proteins were downregulated in CRC cells using siRNA technology. Viability assays, flow cytometry and immunoblotting were performed and three-dimensional cell culture was utilized to study autophagy in a tissue mimicking environment. In our study we found an upregulation of Atg7 in CRC. Furthermore, we identified Atg7 as crucial factor within the autophagy network for CRC cell viability. Its disruption induced cell death via triggering apoptosis and in combination with conventional chemotherapy it exerted synergistic effects in inducing CRC cell death. Cell death was strictly dependent on nuclear LC3b, since simultaneous knockdown of Atg7 and LC3b completely restored viability. This study unravels a novel cell death preventing function of Atg7 in interaction with LC3b, thereby unmasking a promising therapeutic target in CRC.


Assuntos
Adenocarcinoma/metabolismo , Apoptose , Proteína 7 Relacionada à Autofagia/metabolismo , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Adenocarcinoma/genética , Antineoplásicos/farmacologia , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Neoplasias Colorretais/genética , Fluoruracila/farmacologia , Células HT29 , Humanos , Irinotecano/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo
14.
Life Sci ; 248: 117466, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101760

RESUMO

AIMS: Nanoparticles (NPs)-based drugs have been recently introduced to improve the efficacy of current therapeutic strategies for the treatment of cancer; however, the molecular mechanisms by which a NP interacts with cellular systems still need to be delineated. Here, we utilize the autophagic potential of TiO2 NPs for improving chemotherapeutic effects of 5-fluorouracil (5-FU) in human AGS gastric cells. MATERIALS AND METHODS: Cell growth and viability were determined by trypan blue exclusion test and MTT assay, respectively. Vesicular organelles formation was evaluated by acridine orange staining of cells. Cell cycle and apoptosis were monitored by flow cytometry. Reactive oxygen species (ROS) level were measured by DCHF-DA staining. Autophagy was examined by q-PCR and western blotting. Molecular docking was used for studying NP interaction with autophagic proteins. KEY FINDINGS: TiO2 NPs increase ROS production, impair lysosomal function and subsequently block autophagy flux in AGS cells. In addition, the autophagy blockade induced by non-toxic concentrations of TiO2 NPs (1 µg/ml) can promote cytotoxic and apoptotic effects of 5-FU in AGS cells. SIGNIFICANCE: These results confirm the beneficial effects of TiO2 NPs in combination with chemotherapy in in vitro model of gastric cancer, which may pave the way to develop a possible solution to circumvent chemoresistance in cancer.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Nanopartículas/química , Titânio/farmacologia , Antimetabólitos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fluoruracila/química , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Nanopartículas/ultraestrutura , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/antagonistas & inibidores , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Titânio/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
15.
Int J Mol Sci ; 21(4)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32102330

RESUMO

Hepatic disorders have been increasing in recent years because of high carbohydrate diets. Hepatocytes depend mainly on the basal autophagy to maintain hepatic glucose/lipid homeostasis in mammals. However, the regulatory mechanisms of autophagy in hepatic energy metabolism are still unknown in fish species. Accordingly, mutant zebrafish lines of autophagy-related genes beclin1 and atg7 were generated by CRISPR/Cas9 gene-editing technology. Interestingly, unlike atg7+/-, male beclin1+/- zebrafish displayed liver defects in the morphology and histology, including abnormal hepatocyte proliferation, hemorrhagic and inflammatory phenotypes. A significant decrease in hepatocyte glycogen and an increase in hepatocyte lipids were detected in the histological assay that coincidence with the hepatic gene expression. Meanwhile, loss of heterozygosity for beclin1 creates a suitable microenvironment for hepatic tumorigenesis via phosphorylation of Akt kinase, which in turn affects liver autophagy. The reduction in autophagy activity in male beclin1+/- liver leads to a disturbance in the glucose/lipid metabolism and negatively regulates apoptosis accompanied by the induction of cellular proliferation and acute inflammatory response. Our findings highlight an important role of beclin1 in zebrafish liver development and energy metabolism, suggesting the crucial role of autophagy in maintaining homeostasis of the nutrient metabolism in fish species.


Assuntos
Proteína Beclina-1/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Hepatopatias/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Metabolismo dos Carboidratos , Proliferação de Células/genética , Heterozigoto , Hepatopatias/genética , Masculino , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
16.
FASEB J ; 34(2): 3129-3150, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908069

RESUMO

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.


Assuntos
Envelhecimento/metabolismo , Autofagia , Glucuronidase/metabolismo , Fosfatos/metabolismo , Animais , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Feminino , Glucuronidase/genética , Células HEK293 , Humanos , Rim/metabolismo , Masculino , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
17.
Nat Microbiol ; 5(2): 272-281, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959973

RESUMO

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5-12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200-but not of other essential autophagy components ATG5, ATG16L1 or ATG7-in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.


Assuntos
Autofagia/genética , Listeria monocytogenes/patogenicidade , Macrófagos Peritoneais/imunologia , Animais , Autofagia/imunologia , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proliferação de Células , Suscetibilidade a Doenças/imunologia , Feminino , Predisposição Genética para Doença , Interferon gama/imunologia , Listeria monocytogenes/imunologia , Listeriose/etiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
18.
Am J Physiol Renal Physiol ; 318(3): F772-F792, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984794

RESUMO

Klotho- and beclin 1-driven autophagy extends life. We examined the role of beclin 1 in modifying acute kidney injury (AKI) and whether beclin 1 mediates Klotho's known renoprotective action in AKI. AKI was induced by ischemia-reperfusion injury in mice with different levels of autophagy activity by genetic manipulation: wild-type (WT) mice with normal beclin 1 expression and function, mice with normal beclin 1 levels but high activity through knockin of gain-of-function mutant beclin 1 (Becn1F121A), mice with low beclin 1 levels and activity caused by heterozygous global deletion of beclin 1 (Becn1+/-), or mice with extremely low beclin 1 activity from knockin of the mutant constitutively active beclin 1 inhibitor Bcl-2 (Bcl2AAA). Klotho was increased by transgenic overexpression (Tg-Kl) or recombinant Klotho protein administration. After ischemia-reperfusion injury, Becn1F121A mice (high autophagy) had milder AKI and Becn1+/- and Bcl2AAA mice (low autophagy) had more severe AKI than WT mice. Tg-Kl mice had milder AKI, but its renoprotection was partially attenuated in Becn1+/-;Tg-Kl mice and was significantly reduced, although not completely abolished, in Bcl2AAA;Tg-Kl mice. Recombinant Klotho protein conferred more renoprotection from AKI in WT mice than in Becn1+/- or Bcl2AAA mice. Klotho reduced beclin 1/Bcl-2 protein complexes and increased autophagy activity, but this effect was less prominent in mice or cells with Bcl2AAA. Transfected Bcl2AAA or Becn1F123A decreased or increased autophagy activity and rendered cells more susceptible or more resistant to oxidative cytotoxicity, respectively. In conclusion, beclin 1 confers renoprotection by activating autophagy. Klotho protects the kidney partially via disruption of beclin 1/Bcl-2 interactions and enhancement of autophagy activity.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Glucuronidase/metabolismo , Nefropatias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismo por Reperfusão , Animais , Proteína Beclina-1/genética , Linhagem Celular , Regulação da Expressão Gênica , Genótipo , Glucuronidase/genética , Peróxido de Hidrogênio , Nefropatias/etiologia , Camundongos , Gambás , Proteínas Proto-Oncogênicas c-bcl-2/genética
19.
PLoS One ; 15(1): e0227603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940411

RESUMO

In-vitro studies with different Fanconi anemia (FA) cell lines and FANC gene silenced cell lines indicating involvement of mitochondria function in pathogenesis of FA have been reported. However, in-vivo studies have not been studied so far to understand the role of mitochondrial markers in pathogenesis of FA. We have carried out a systematic set of biomarker studies for elucidating involvement of mitochondrial dysfunction in disease pathogenesis for Indian FA patients. We report changes in the mtDNA number in 59% of FA patients studied, a high frequency of mtDNA variations (37.5% of non-synonymous variations and 62.5% synonymous variations) and downregulation of mtDNA complex-I and complex-III encoding genes of OXPHOS (p<0.05) as strong biomarkers for impairment of mitochondrial functions in FA. Deregulation of expression of mitophagy genes (ATG; p>0.05, Beclin-1; p>0.05, and MAP1-LC3, p<0.05) has also been observed, suggesting inability of FA cells to clear off impaired mitochondria. We hypothesize that accumulation of such impaired mitochondria in FA cells therefore may be the principal cause for bone marrow failure (BMF) and a plausible effect of inefficient clearance of impaired mitochondria in FA.


Assuntos
DNA Mitocondrial/genética , Anemia de Fanconi/genética , Adolescente , Adulto , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Criança , DNA Mitocondrial/metabolismo , Anemia de Fanconi/metabolismo , Feminino , Variação Genética/genética , Humanos , Índia , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia , Espécies Reativas de Oxigênio/metabolismo
20.
FASEB J ; 34(2): 2055-2074, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908016

RESUMO

In spinal cord ischemia-reperfusion (I/R) injury, large amounts of reactive oxygen species can cause mitochondrial damage. Therefore, mitophagy acts as the main mechanism for removing damaged mitochondria and protects nerve cells. This study aimed to illustrate the important role of GPCR kinase 2-interacting protein-1 (GIT1) in mitophagy in vivo and in vitro. The level of mitophagy in the neurons of Git1 knockout mice was significantly reduced after ischemia-reperfusion. However, the overexpression of adeno-associated virus with Git1 promoted mitophagy and inhibited the apoptosis of neurons. GIT1 regulated the phosphorylation of Beclin-1 in Thr119, which could promote the translocation of Parkin to the mitochondrial outer membrane. This process was independent of PTEN-induced kinase 1 (PINK1), but it could not rescue the role in the absence of PINK1. Overall, GIT1 enhanced mitophagy and protected neurons against ischemia-reperfusion injury and, hence, might serve as a new research site for the protection of ischemia-reperfusion injury.


Assuntos
Proteína Beclina-1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mitofagia , Traumatismo por Reperfusão , Doenças da Medula Espinal , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína Beclina-1/genética , Proteínas de Ciclo Celular/genética , Proteínas Ativadoras de GTPase/genética , Camundongos , Camundongos Knockout , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Doenças da Medula Espinal/genética , Doenças da Medula Espinal/metabolismo , Doenças da Medula Espinal/patologia , Doenças da Medula Espinal/prevenção & controle , Ubiquitina-Proteína Ligases/genética
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