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1.
Int J Cancer ; 146(6): 1652-1666, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31180579

RESUMO

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T-antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T-antigens induce miR-375, miR-30a-3p and miR-30a-5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV-positive tumors. Ectopic expression of MCPyV small T-antigen and truncated large T-antigen (LT), but not the wild-type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV-mediated tumorigenesis. Torin-1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan-caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV-mediated tumorigenesis and potential MCC treatment.


Assuntos
Carcinoma de Célula de Merkel/virologia , Poliomavírus das Células de Merkel/metabolismo , MicroRNAs/biossíntese , Neoplasias Cutâneas/virologia , Antígenos Virais de Tumores/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/biossíntese , Proteína 7 Relacionada à Autofagia/genética , Proteína Beclina-1/biossíntese , Proteína Beclina-1/genética , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Linhagem Celular Tumoral , Humanos , Macrolídeos/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Naftiridinas/farmacologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia , Processamento Pós-Transcricional do RNA , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia
2.
J Neuroimmune Pharmacol ; 13(3): 383-395, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29790105

RESUMO

Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.


Assuntos
Autofagia/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Lipopolissacarídeos/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Fisostigmina/farmacologia , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/biossíntese , Proteína Beclina-1/metabolismo , Inflamação/genética , Inflamação/patologia , Inflamação/psicologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Microglia/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeo Sintases/biossíntese , Período Pós-Operatório , Ratos , Ratos Wistar , Proteína Sequestossoma-1/biossíntese
3.
Gynecol Oncol ; 150(1): 143-150, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29699801

RESUMO

OBJECTIVE: Multidrug resistance is the major cause of treatment failure in ovarian cancer. p62 (SQSTM1) is a multifunctional protein involved in multiple cellular processes including proliferation, drug sensitivity and autophagy-associated cancer cell growth. However, the role of p62 in drug resistance remains controversial. METHODS: In this study, we examined p62 expression by immunohistochemistry in a unique ovarian cancer tissue microarray (TMA), which was constructed with paired primary, metastatic, and recurrent tumor tissues. The expression levels of p62 and autophagy related proteins were evaluated in two panels of human cancer cell lines by western blot. Cell viabilities were determined by MTT assay after exposure ovarian cancer cells to different concentrations of paclitaxel alone or in combination with autophagy inhibitors. RESULTS: Both the metastatic and recurrent tumor tissues expressed less p62 than the patient-matched primary tumor. A significant inverse correlation has been found between p62 expression and both the disease-free survival and overall survival. Additionally, multidrug resistant cancer cell lines expressed lower levels of p62 as compared with their parental drug sensitive cell lines. Importantly, inhibition of autophagy enhanced paclitaxel sensitivity in drug resistant ovarian cancer cells. Furthermore, the wound healing assay exhibited that the inhibition of autophagy significantly decreased resistant ovarian cancer cell migration in vitro. CONCLUSION: Our findings highlight the potential of p62 as a new prognostic marker for ovarian cancer patients and p62's associated autophagy pathway may be a promising therapeutic target to prevent metastasis, recurrence and to reverse drug resistance in ovarian cancer.


Assuntos
Neoplasias Ovarianas/metabolismo , Proteína Sequestossoma-1/biossíntese , Adenina/administração & dosagem , Adenina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Hidroxicloroquina/administração & dosagem , Imuno-Histoquímica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo
4.
Mod Pathol ; 31(5): 753-762, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29327718

RESUMO

Epithelioid fibrous histiocytoma is a rare and distinctive cutaneous neoplasm. Most cases harbor ALK rearrangement and show ALK overexpression, which distinguish this neoplasm from conventional cutaneous fibrous histiocytoma and variants. SQSTM1 and VCL have previously been shown to partner with ALK in one case each of epithelioid fibrous histiocytoma. The purpose of this study was to examine a large cohort of epithelioid fibrous histiocytomas by next-generation sequencing to characterize the nature and prevalence of ALK fusion partners. A retrospective archival review was performed to identify cases of epithelioid fibrous histiocytoma (2012-2016). Immunohistochemistry was performed to confirm ALK expression. Targeted next-generation sequencing was applied on RNA extracted from formalin-fixed paraffin-embedded tissue to identify the fusion partners. Twenty-three cases fulfilled inclusion criteria. The mean patient age was 39 years (range, 8-74), there was no sex predilection, and >75% of cases involved the lower extremities. The most common gene fusions were SQSTM1-ALK (N=12; 52%) and VCL-ALK (N=7; 30%); the other four cases harbored novel fusion partners (DCTN1, ETV6, PPFIBP1, and SPECC1L). The pattern of ALK immunoreactivity was usually granular cytoplasmic (N=12; 52%) or granular cytoplasmic and nuclear (N=10; 43%); the case containing an ETV6 fusion partner showed nuclear staining alone. There was no apparent relationship between tumor morphology and the ALK fusion partner. In summary, SQSTM1 and VCL are the most common ALK fusion partners in epithelioid fibrous histiocytoma; DCTN1, ETV6, PPFIBP1, and SPECC1L represent rare fusion partners. The proteins encoded by these genes play diverse roles in scaffolding, cell adhesion, signaling, and transcription (among others) without clear commonalities. These findings expand the oncogenic promiscuity of many of these ALK fusion genes, which drive neoplasia in tumors of diverse lineages with widely varied clinical behavior. This is the first documented account of ETV6-ALK and SPECC1L-ALK translocations in neoplasms.


Assuntos
Quinase do Linfoma Anaplásico/biossíntese , Histiocitoma Fibroso Benigno/metabolismo , Histiocitoma Fibroso Benigno/patologia , Adolescente , Adulto , Idoso , Quinase do Linfoma Anaplásico/genética , Criança , Estudos de Coortes , Células Epitelioides/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Estudos Retrospectivos , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/metabolismo , Adulto Jovem , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genética
5.
Hum Pathol ; 73: 156-163, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29241738

RESUMO

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide, and is associated with poor prognosis. Autophagy is a programmed cell survival mechanism involved in physiologic processes and various diseases including cancer. However, the relationship between autophagy and cancer is controversial. Several studies have claimed that the expression of autophagy-related proteins, namely microtubule-associated protein light chain3 (LC3) and p62/SQSTM1 (p62), is associated with poor prognosis in OSCC. In this study, we evaluated the expression of the autophagy-related markers LC3A/B and p62 by immunohistochemistry in 71 OSCC patient samples, especially focusing on surgical margins. Results were correlated with clinical characteristics. The expression of LC3A and LC3B was correlated with tumor recurrence and poor overall survival based on multivariate analysis, whereas the expression of p62 was correlated with only tumor recurrence and not prognosis. Thus, we suggest that the expression of autophagy-related markers at the surgical margins might be an indicator of local recurrence and poor prognosis in human OSCC. These results will aid in the development of new therapeutics and diagnostics for OSCC.


Assuntos
Autofagia/fisiologia , Biomarcadores Tumorais/análise , Margens de Excisão , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/biossíntese , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Recidiva Local de Neoplasia/patologia , Prognóstico , Proteína Sequestossoma-1/análise , Proteína Sequestossoma-1/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade
6.
ACS Chem Neurosci ; 8(11): 2381-2392, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28605578

RESUMO

Inhibition of phosphodiesterase 4 (PDE4) suppressed the inflammatory responses in the brain. However, the underlying mechanisms are poorly understood. Roflupram (ROF) is a novel PDE4 inhibitor. In the present study, we found that ROF enhanced the level of microtubule-associated protein 1 light chain 3 II (LC3-II) and decreased p62 in microglial BV-2 cells. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by Lysotracker red and acridine orange staining. In addition, immunofluorescence indicated a significant increase in punctate LC3. Moreover, ß amyloid 25-35 (Aß25-35) or lipopolysaccharide (LPS) with ATP was used to activate inflammasome. We found that both LPS plus ATP and Aß25-35 enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1ß in BV-2 cells. Interestingly, these effects were blocked by the treatment of ROF. Consistently, knocking down the expression of PDE4B in primary microglial cells led to enhanced level of LC-3 II and decreased activation of inflammasome. What's more, Hoechst staining showed that ROF decreased the apoptosis of neuronal N2a cells in conditioned media from microglia. Our data also showed that ROF dose-dependently enhanced autophagy, reduced the activation of inflammasome and suppressed the production of IL-1ß in mice injected with LPS. These effects were reversed by inhibition of microglial autophagy. These results put together demonstrate that ROF inhibits inflammasome activities and reduces the release of IL-1ß by inducing autophagy. Therefore, ROF could be used as a potential therapeutic compound for the intervention of inflammation-associated diseases in the brain.


Assuntos
Anti-Inflamatórios/farmacologia , Autofagia/efeitos dos fármacos , Derivados de Benzeno/farmacologia , Furanos/farmacologia , Inflamassomos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Peptídeos beta-Amiloides/farmacologia , Animais , Anti-Inflamatórios/química , Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Proteína 7 Relacionada à Autofagia/genética , Derivados de Benzeno/química , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , Furanos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Estrutura Molecular , Fragmentos de Peptídeos/farmacologia , Inibidores da Fosfodiesterase 4/química , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética
7.
Theranostics ; 7(7): 1890-1900, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28638475

RESUMO

Long-term application of Tamoxifen (TAM) is usually recommended for hormone receptor positive breast cancer patients. Unfortunately, TAM will inevitably increase the incidence of endometrial hyperplasia even endometrial cancer. Despite of substantial investigations, no effective approaches to prevent TAM-induced endometrial carcinogenesis have been acknowledged. In this study, we found that inhibition of Nrf2 could be valuable to prevent TAM-induced endometrial hyperplasia. Upon TAM treatment, the mRNA and protein expression of autophagy adaptor SQSTM1 was specifically increased in endometrial cells but not breast cancer cells. Knocking-down of SQSTM1 expression retarded TAM-promoted growth of endometrial cancer cells. TAM stimulated SQSTM1 transcription specifically in endometrial cells by enhancing phosphorylation and nuclear translocation of Nrf2. Indeed, the expression of Nrf2 and SQSTM1 were positively correlated in primary endometrial tissues. In rats with TAM-induced endometrial hyperplasia, both Nrf2 and SQSTM1 expression were increased. Nrf2 inhibitor brusatol effectively attenuated TAM-induced SQSTM1 upregulation and endometrial hyperplasia. The kinase of Nrf2, PRKCD, was activated by TAM. Once PRKCD was depleted, TAM failed to promote Nrf2 phosphorylation and SQSTM1 expression. In summary, TAM stimulated Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia by activating PRKCD. Therefore, blocking PRKCD-Nrf2-SQSTM1 signaling could be useful to prevent TAM-induced endometrial hyperplasia.


Assuntos
Antineoplásicos Hormonais/efeitos adversos , Hiperplasia Endometrial/induzido quimicamente , Fator 2 Relacionado a NF-E2/metabolismo , Proteína Sequestossoma-1/biossíntese , Tamoxifeno/efeitos adversos , Transcrição Genética , Animais , Antineoplásicos Hormonais/administração & dosagem , Feminino , Humanos , Ratos , Tamoxifeno/administração & dosagem
8.
Autophagy ; 13(7): 1205-1220, 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28548877

RESUMO

Enhanced macroautophagy/autophagy is recognized as a component of the pathogenesis of smoking-induced airway disease. Based on the knowledge that enhanced autophagy is linked to oxidative stress and the DNA damage response, both of which are linked to smoking, we used microarray analysis of the airway epithelium to identify smoking upregulated genes known to respond to oxidative stress and the DNA damage response. This analysis identified OSGIN1 (oxidative stress induced growth inhibitor 1) as significantly upregulated by smoking, in both the large and small airway epithelium, an observation confirmed by an independent small airway microarray cohort, TaqMan PCR of large and small airway samples and RNA-Seq of small airway samples. High and low OSGIN1 expressors have different autophagy gene expression patterns in vivo. Genome-wide correlation of RNAseq analysis of airway basal/progenitor cells showed a direct correlation of OSGIN1 mRNA levels to multiple classic autophagy genes. In vitro cigarette smoke extract exposure of primary airway basal/progenitor cells was accompanied by a dose-dependent upregulation of OSGIN1 and autophagy induction. Lentivirus-mediated expression of OSGIN1 in human primary basal/progenitor cells induced puncta-like staining of MAP1LC3B and upregulation of MAP1LC3B mRNA and protein and SQSTM1 mRNA expression level in a dose and time-dependent manner. OSGIN1-induction of autophagosome, amphisome and autolysosome formation was confirmed by colocalization of MAP1LC3B with SQSTM1 or CD63 (endosome marker) and LAMP1 (lysosome marker). Both OSGIN1 overexpression and knockdown enhanced the smoking-evoked autophagic response. Together, these observations support the concept that smoking-induced upregulation of OSGIN1 is one link between smoking-induced stress and enhanced-autophagy in the human airway epithelium.


Assuntos
Autofagia , Fumar Cigarros , Proteínas/fisiologia , Mucosa Respiratória/metabolismo , Proteínas Reguladoras de Apoptose , Autofagossomos/ultraestrutura , Autofagia/genética , Células Cultivadas , Humanos , Lisossomos/ultraestrutura , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Estresse Oxidativo , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Regulação para Cima
9.
Invest Ophthalmol Vis Sci ; 57(10): 4356-66, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27564518

RESUMO

PURPOSE: Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG). METHODS: Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours. RESULTS: High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity. CONCLUSIONS: High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.


Assuntos
Autofagia/fisiologia , Diabetes Mellitus Experimental , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , RNA Interferente Pequeno/genética , Retina/metabolismo , Proteína Sequestossoma-1/genética , Animais , Apoptose , Autofagia/efeitos dos fármacos , Western Blotting , Células Cultivadas , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Glucose/farmacologia , Microscopia Eletrônica de Transmissão , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Retina/patologia , Proteína Sequestossoma-1/biossíntese , Edulcorantes/farmacologia
10.
FEBS Lett ; 590(15): 2375-97, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27404485

RESUMO

p62/SQSTM1 is a multifunctional signaling hub and autophagy adaptor with many binding partners, which allow it to activate mTORC1-dependent nutrient sensing, NF-κB-mediated inflammatory responses, and the NRF2-activated antioxidant defense. p62 recognizes polyubiquitin chains via its C-terminal domain and binds to LC3 via its LIR motif, thereby promoting the autophagic degradation of ubiquitinated cargos. p62 accumulates in many human liver diseases, including nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), where it is a component of Mallory-Denk bodies and intracellular hyaline bodies. Chronic p62 elevation contributes to HCC development by preventing oncogene-induced senescence and death of cancer-initiating cells and enhancing their proliferation. In this review, we discuss p62-mediated signaling pathways and their roles in liver pathophysiology, especially NASH and HCC.


Assuntos
Carcinoma Hepatocelular/genética , Inflamação/genética , Neoplasias Hepáticas/genética , Proteína Sequestossoma-1/biossíntese , Carcinoma Hepatocelular/metabolismo , Alimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/metabolismo , Neoplasias Hepáticas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , NF-kappa B/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo/genética , Proteína Sequestossoma-1/genética , Serina-Treonina Quinases TOR/genética
11.
J Neurochem ; 139(2): 285-293, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27456931

RESUMO

Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis. Read the Editorial Highlight for this article on page 159.


Assuntos
Proteínas de Ciclo Celular/biossíntese , Neurônios , Ubiquitinas/biossíntese , Animais , Autofagia/genética , Encéfalo/patologia , Proteínas de Ciclo Celular/genética , Morte Celular , Humanos , /psicologia , Mutação/genética , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Aprendizagem Espacial , Ubiquitinas/genética
12.
Neoplasia ; 18(6): 347-55, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27292024

RESUMO

Enhanced autophagy has been observed in hypoxic regions of solid tumors. Here we address the hypothesis that autophagy is required for survival of hypoxic cells. We evaluated sensitivity to hypoxia of three human tumor cell lines (MCF7, PC3, and LNCaP) and their autophagy-deficient variants with shRNA knockdown of the genes ATG7 and BECLIN1. Hypoxia-induced cell death was more rapid for autophagy-deficient cells and was increased in the presence of the proton pump inhibitor pantoprazole that inhibits autophagy. Autophagy-deficient cells had a lower rate of oxygen consumption than wild-type cells. In xenografts derived from the three cell lines, autophagy (as determined by increased LC3 and reduced p62/SQSTM1) colocalized with hypoxic regions (identified by EF5). Xenografts derived from autophagy-deficient cells grew more slowly than wild-type tumors. Both LC3 expression and hypoxia were decreased in xenografts generated from single-knockdown cells and absent in double-knockdown tumors. Our results are consistent with the hypothesis that autophagy facilitates the survival of hypoxic cells, although reduced oxygen consumption of autophagy-deficient cells may contribute to lack of hypoxia in tumors derived from them. Because hypoxia is associated with resistance to anticancer therapy, inhibition of autophagy has potential to enhance the effectiveness of cancer treatment.


Assuntos
Autofagia/fisiologia , Hipóxia Celular/fisiologia , Neoplasias/patologia , Consumo de Oxigênio/fisiologia , 2-Piridinilmetilsulfinilbenzimidazóis , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Transplante de Neoplasias , Pantoprazol , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Sequestossoma-1/biossíntese , Transplante Heterólogo
13.
Neurochem Int ; 96: 62-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26930584

RESUMO

Autophagy is a homeostatic process for recycling proteins and organelles that is increasingly being proposed as a therapeutic target for acute and chronic neurodegenerative diseases, including stroke. Confirmation that autophagy is present in the human brain after stroke is imperative before prospective therapies can begin the translational process into clinical trials. Our current study using human post-mortem tissue observed an increase in staining in microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1; also known as p62) and the increased appearance of autophagic vesicles after stroke. These data confirm that alterations in autophagy take place in the human brain after stroke and suggest that targeting autophagic processes after stroke may have clinical significance.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/biossíntese , Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/biossíntese , Proteína Sequestossoma-1/biossíntese , Acidente Vascular Cerebral/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteína Beclina-1/análise , Encéfalo/patologia , Química Encefálica/fisiologia , Feminino , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/análise , Proteína Sequestossoma-1/análise , Acidente Vascular Cerebral/patologia
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