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1.
Chem Biol Interact ; 312: 108751, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31369747

RESUMO

OBJECTIVES: To explore the effects of carbon disulfide (CS2) and N-carbamoyl glutamate (NCG) on autophagy during the window of embryo implantation in mice and whether dietary NCG supplementation can promote embryo implantation in case of CS2 exposure. METHODS: Pregnant mice that received single intraperitoneal injection of CS2 on Gestational day (GD)4 were fed basal diet with or without NCG supplementation from GD1 to endpoints. The control mice were injected solvents. There were four endpoints (GD5, GD6, GD7 and GD9 endpoints) in each group. The uterus was collected on endpoints to detect autophagy-related markers by using the methods of transmission electron microscopy (TEM), immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR) and ELISA. RESULTS: The P62 brown punctate staining increased in CS2 exposure group and reduced after dietary NCG supplementation, which was opposite with LC3B, Beclin1 and ATG5 on GD5 endpoint. Simultaneously, P62 protein expression raised 43.33% on GD5 endpoint (p < 0.01) when exposed to CS2 and descended to the control level after NCG supplementation. The rate of decline of LC3B and Beclin1 proteins were 27.04% (p < 0.01) and 23.27% (p < 0.05) on GD5 endpoint, 20.20% (p < 0.05) and 11.30% on GD7 endpoint in CS2 exposure group, respectively, then NCG supplementation caused the LC3B and Beclin1 protein expression to rise in different degrees. Comparatively, the mRNA expression of all autophagy-related gene changed more apparently on three endpoints than the protein expression. The images of TEM showed that nearly no autophagosome could be seen in CS2 exposure group, while dietary NCG supplementation increased the number of autophagosome obviously on GD5 endpoint. The number of implanted embryos which declined due to CS2 exposure returned to normal in NCG supplementation group. CONCLUSIONS: Dietary NCG supplementation could rescue the suppressed autophagy induced by CS2 in the window of implantation and increase the number of implanted embryos.


Assuntos
Autofagia/efeitos dos fármacos , Dissulfeto de Carbono/toxicidade , Glutamatos/farmacologia , Animais , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Implantação do Embrião , Endométrio/ultraestrutura , Feminino , Masculino , Camundongos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismo
2.
Eur J Histochem ; 63(2)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31189296

RESUMO

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Cóclea/embriologia , Cóclea/metabolismo , Animais , Anticorpos/imunologia , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proteína Beclina-1/metabolismo , Caspase 3/genética , Caspase 3/imunologia , Caspase 3/metabolismo , Cóclea/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/imunologia , Proteína Sequestossoma-1/metabolismo , Fatores de Tempo
3.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088931

RESUMO

The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) screening in a unique model of matched primary and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells and identified genes that were growth promoting and growth suppressive for both types of cells, among which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by small interfering RNA (siRNA) knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells and in cell lines of other cancers, including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation, but the mechanisms of p53 activation differed among the different types of cancer cells. p53 activation depended on the formation of promyelocytic leukemia (PML) nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken the data together, we have identified novel growth-promoting and growth-suppressive genes of primary and cancer cells and have demonstrated that XPO1 is a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML- and p62-dependent mechanism of p53 activation in some types of cancer cells.IMPORTANCE Using a model of oncogenic virus KSHV-driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both growth promoting and growth suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated that the exportin XPO1 is a critical factor for the survival of transformed cells. Using a XPO1 inhibitor (KPT-8602) and siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and in growth transformation of KSHV-transformed cells, as well as of gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53, but the mechanisms of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells.


Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Herpesvirus Humano 8/patogenicidade , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/genética , Sistemas CRISPR-Cas , Pontos de Checagem do Ciclo Celular , Detecção Precoce de Câncer , Genes p53 , Humanos , Carioferinas/antagonistas & inibidores , Leucemia Promielocítica Aguda , Neoplasias Hepáticas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Proteína Sequestossoma-1/genética , Neoplasias Gástricas , Células Tumorais Cultivadas
4.
Anticancer Res ; 39(3): 1233-1242, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30842153

RESUMO

AIM: To investigate clinicopathological significance of autophagy and its association with genetic alterations in gliomas. MATERIALS AND METHODS: The expression of three autophagy-related proteins, light chain-3 (LC3), beclin 1, and p62 was immunohistochemically analyzed in 32 low-grade gliomas and 65 high-grade gliomas. RESULTS: LC3, beclin 1, and p62 expression was positive in 70/94 (74%), 51/94 (54%) and 55/96 (57%) gliomas, respectively. High expression of LC3, beclin 1 and p62 was significantly more frequent in high-grade gliomas than in low-grade. Positive expression of LC3, beclin 1 and p62 were significantly positively correlated with overall survival, methylation of O6-methylyguanine-DNA methyltransferase (MGMT) promoter, mutations of isocitrate dehydrogenase 1 (IDH1) and telomerase reverse transcriptase (TERT) promoter, and 1p/19q co-deletion. Kaplan-Meier analyses revealed that LC3, p62 and autophagy status (positivity for at least two of the three proteins) were significantly associated with poorer survival. CONCLUSION: Autophagy might be associated with the progression of glioma, particularly high-grade, and thus might be a useful prognostic factor in patients with glioma.


Assuntos
Proteína Beclina-1/genética , Neoplasias Encefálicas/genética , Glioma/genética , Proteínas Associadas aos Microtúbulos/genética , Proteína Sequestossoma-1/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Autofagia , Proteína Beclina-1/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Feminino , Glioma/metabolismo , Glioma/patologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Proteína Sequestossoma-1/metabolismo , Adulto Jovem
5.
Mol Cell ; 74(2): 330-346.e11, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853400

RESUMO

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.


Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genética
6.
Cell Prolif ; 52(3): e12585, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30793399

RESUMO

OBJECTIVES: The role of p62 in cancer is controversial. Evidence has shown that p62 is upregulated in different cancers and promotes tumour growth, such as in liver cancer and lung cancer. However, a recent study showed that the downregulation of p62 in hepatic stellate cells (HSCs) promotes hepatocellular carcinoma (HCC) development. How p62 is regulated in colorectal cancer (CRC) remains largely unknown. In this study, we aimed to investigate the roles and molecular mechanisms of p62 in CRC. MATERIALS AND METHODS: The expression levels of p62 in CRC tissues and adjacent non-tumour tissues were determined by immunohistochemistry (IHC). Stable p62-overexpression HCT116 cells and p62-knockdown SW480 cells were established with lentiviral vectors. The role of p62 in CRC was investigated in in vitro and in vivo functional studies. The relationship between p62 and the vitamin D receptor (VDR) was investigated by coimmunoprecipitation (Co-IP) assays. RESULTS: p62 was significantly upregulated in CRC, and a high p62 level was an independent risk factor for a poor prognosis in CRC patients. p62 promoted CRC migration and invasion by inhibiting apoptosis and promoting cell proliferation in vitro, and p62 aggravated tumour growth and metastasis in vivo. Co-IP assays indicated that p62 interacts with the VDR and may target the NRF2-NQO1 axis. CONCLUSIONS: Our study suggested that p62 functions as an oncogene in CRC through inhibiting apoptosis and promoting cell proliferation by interacting with the VDR.


Assuntos
Neoplasias Colorretais/genética , Oncogenes , Receptores de Calcitriol/metabolismo , Proteína Sequestossoma-1/genética , Animais , Apoptose/genética , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Técnicas de Silenciamento de Genes , Células HCT116 , Células HT29 , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Invasividade Neoplásica/genética , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Sequestossoma-1/antagonistas & inibidores , Proteína Sequestossoma-1/metabolismo , Regulação para Cima
7.
Methods Mol Biol ; 1880: 3-15, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610687

RESUMO

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.


Assuntos
Escherichia coli/genética , Microscopia Eletrônica/métodos , Coloração Negativa/métodos , Proteína Sequestossoma-1/ultraestrutura , Autofagia , Cromatografia de Afinidade/métodos , Expressão Gênica , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/isolamento & purificação , Solubilidade , Regulação para Cima
8.
Mol Med Rep ; 19(2): 984-993, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569150

RESUMO

Tumor necrosis factor­related apoptosis-inducing ligand (TRAIL) is well known as a transmembrane cytokine and has been proposed as one of the most effective anti­cancer therapeutic agents, owing to its efficiency to selectively induce cell death in a variety of tumor cells. Suppression of autophagy flux has been increasingly acknowledged as an effective and novel therapeutic intervention for cancer. The present study demonstrated that the anti­cancer and anti­inflammatory drug celastrol, through its anti­metastatic properties, may initiate TRAIL­mediated apoptotic cell death in lung cancer cells. This sensitization was negatively affected by N­acetyl­l­cysteine, which restored the mitochondrial membrane potential (ΔΨm) and inhibited reactive oxygen species (ROS) generation. Notably, treatment with celastrol caused an increase in microtubule­associated proteins 1A/1B light chain 3B­II and p62 levels, whereas co­treatment of celastrol and TRAIL increased active caspase 3 and 8 levels compared with the control, confirming inhibited autophagy flux. The combined use of TRAIL with celastrol may serve as a safe and adequate therapeutic technique for the treatment of TRAIL­resistant lung cancer, suggesting that celastrol­mediated autophagy flux inhibition sensitized TRAIL­initiated apoptosis via regulation of ROS and ΔΨm.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/agonistas , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Triterpenos/farmacologia , Células A549 , Acetilcisteína/farmacologia , Antineoplásicos Fitogênicos/antagonistas & inibidores , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Triterpenos/antagonistas & inibidores
9.
Immun Inflamm Dis ; 7(1): 7-21, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30569531

RESUMO

INTRODUCTION: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency. METHODS: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei. RESULTS: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient. CONCLUSION: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.


Assuntos
Autofagia/genética , Burkholderia mallei/fisiologia , Burkholderia pseudomallei/fisiologia , Dessensibilização Imunológica/métodos , Mormo/metabolismo , Melioidose/metabolismo , Animais , Quimases/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo
10.
Viruses ; 11(1)2018 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-30586869

RESUMO

Previous studies have indicated that cytotoxic treatments may induce or not activate viral lytic cycle activation in cancer cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV). To investigate the molecular mechanisms responsible for such an effect, we compared two cytotoxic treatments able to induce the viral lytic cycle, named 12-O-tetradecanoylphorbol 13-acetate (TPA) (T) in combination with sodium butyrate (B) and bortezomib (BZ), with two cytotoxic treatments that did not activate this process, named metformin (MET) and quercetin (Q). Our results indicated that TB and bortezomib increased levels of oxygen reactive species (ROS) while metformin and quercetin reduced them. The finding that N-acetylcysteine (NAC), a reactive oxigen species (ROS) scavenger, counteracted K-bZIP expression induced by TB or bortezomib, confirmed that an ROS increase played a role in KSHV lytic cycle activation. Moreover, we found that TB and bortezomib up-regulated p62/Sequestosome1(p62/SQSTM1) protein, while metformin and quercetin down-regulated it. p62/SQSTM1 silencing or the inhibition of NF-E2-related factor 2 (NRF2) or Heat Shock Factor 1 (HSF1), that mediate p62/SQSTM1 transcription, also reduced KSHV lytic antigen expression induced by TB or bortezomib. Interestingly, such combination treatments further increased intracellular ROS and cytotoxicity induced by the single TB or bortezomib treatment, suggesting that NRF2, HSF1 and p62/SQSTM1 keep the ROS level under control, allowing primary effusion lymphoma (PEL) cells to continue to survive and KSHV to replicate.


Assuntos
Fatores de Transcrição de Choque Térmico/genética , Herpesvirus Humano 8/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/genética , Latência Viral/efeitos dos fármacos , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Ácido Butírico/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo , Herpesvirus Humano 8/fisiologia , Humanos , Metformina/farmacologia , Quercetina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/genética , Ativação Transcricional , Ativação Viral/efeitos dos fármacos
11.
Life Sci ; 211: 270-278, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30240580

RESUMO

AIMS: The aims of this study were to evaluate the effects of p62/SQSTM1 expression levels on lipopolysaccharide (LPS)-induced mucus secretion in BEAS-2B bronchial epithelial cells by measuring expression levels of the MUC5AC gene and the Mucin-5AC (MUC5AC) protein. MATERIALS AND METHODS: Bronchial epithelial cells, BEAS-2B, were treated with LPS at different time points. Rapamycin, an autophagy agonist, was added to the BEAS-2B cells 30 min before LPS treatment. Lentivirus transfection was then used to knock down the expression of p62/SQSTM1 (Sequestosome 1) to investigate changes in the downstream signaling pathway. Western blotting and immunofluorescence were used to study the expression levels of MUC5AC, and reverse transcription-polymerase chain reaction (RT-PCR) was used to study the expression of MUC5AC mRNA. KEY FINDINGS: LPS treatment of BEAS-2B cells inhibited autophagy, activated the nuclear factor kappa B (NF-κB) signaling pathway and increased the expression of MUC5AC. The autophagy agonist, rapamycin, activated autophagy, inhibited the NF-κB signaling pathway and decreased LPS-induced expression of MUC5AC. Knockdown of p62/SQSTM1 expression reduced activation of the NF-κB signaling pathway and reduced LPS-induced mucus secretion by BEAS-2B cells in vitro. SIGNIFICANCE: In this in vitro study, which utilized BEAS-2B bronchial epithelial cells, p62/SQSTM1 was shown to have a role in LPS-induced mucus hypersecretion by activating the NF-κB signaling pathway.


Assuntos
Brônquios/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Mucina-5AC/metabolismo , Muco/metabolismo , Proteína Sequestossoma-1/metabolismo , Brônquios/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Mucina-5AC/genética , Proteína Sequestossoma-1/genética , Transdução de Sinais
12.
Molecules ; 23(8)2018 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-30060618

RESUMO

Obesity and nonalcoholic fatty liver disease (NAFLD) are highly prevalent and cause numerous metabolic diseases. However, drugs for the prevention and treatment of obesity and NAFLD remain unavailable. In this study, we investigated the effects of mogrosides (luo han guo, LH) in Siraitia grosvenorii saponins on high-fat-diet-induced obesity and NAFLD in mice. We found that compared with the negative control, LH reduced body and liver weight. LH also decreased fat accumulation and increased AMP-activated protein kinase (AMPK) phosphorylation (pAMPK) levels in mouse livers. We also found that high-purity mogroside V upregulated pAMPK expression in HepG2 cells. In addition, high-purity mogroside V inhibited reactive oxygen species production and upregulated sequestosome-1 (SQSTM1, p62) expression in THP-1 cells. These results suggest that LH may affect obesity and NAFLD by enhancing fat metabolism and antioxidative defenses. Mogroside V may be a main component of LH. However, the exact molecular mechanisms and active components responsible for the inhibitory effects of LH on obesity and NAFLD require further investigation.


Assuntos
Fármacos Antiobesidade/farmacologia , Anticolesterolemiantes/farmacologia , Momordica/química , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Triterpenos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/isolamento & purificação , Anticolesterolemiantes/química , Anticolesterolemiantes/isolamento & purificação , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Tamanho do Órgão/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Saponinas/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Proteína Sequestossoma-1/agonistas , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Células THP-1 , Triterpenos/química , Triterpenos/isolamento & purificação
13.
Mol Med Rep ; 18(2): 1485-1494, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29901107

RESUMO

In recent years, the association between saturated fatty acids (FA) and bone cells has received a high level of attention. Previous studies have shown that palmitate (PA), a common saturated FA, can cause apoptosis in bone marrow mesenchymal stem cells (BMSCs). However, whether PA can induce autophagy, an important intracellular protection mechanism that is closely associated with apoptosis, in BMSCs is still unknown; the association between autophagy and apoptosis is also unclear. The aim of the present study was to determine whether PA can induce autophagy in BMSCs. When BMSCs were treated with PA for >18 h, p62 began to accumulate, indicating that autophagic flux was impaired by prolonged exposure to PA. In addition, the proportion of apoptotic cells was increased when autophagy was inhibited by the autophagy inhibitor 3­methyladenine. Furthermore, inducing autophagy by pretreating cells with rapamycin, a known inducer of autophagy, markedly reduced PA­induced apoptosis, suggesting that autophagy may serve a protective role in PA­induced apoptosis in BMSCs. PA also increased intracellular reactive oxygen species (ROS) production, which was decreased by the antioxidant N­Acetyl­cysteine, and promoted the activation of c­Jun N­terminal kinases (JNKs) and p38 mitogen­activated protein kinase (MAPK). The addition of JNK and p38 MAPK inhibitors substantially reduced autophagy. Therefore, the results indicated that PA can induce autophagy in BMSCs and protect cells from PA­induced apoptosis through the ROS­JNK/p38 MAPK signaling pathways. These results may improve the general understanding of the mechanisms through which BMSCs adapt to PA­induced apoptosis. The present study also provides a novel approach for the prevention and treatment of PA­induced lipotoxicity.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Palmitatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Diagn Pathol ; 13(1): 28, 2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29747676

RESUMO

BACKGROUND: Epithelioid cell histiocytoma (ECH), which is also known as epithelioid benign fibrous histiocytoma, has been classified as a rare variant of fibrous histiocytoma (FH). However, the recent detection of ALK protein expression and/or ALK gene rearrangement in ECH suggests that it might be biologically different from conventional FH. CASE PRESENTATION: A 27-year-old male presented with nodule on his left foot, which had been present for 5 years. A macroscopic examination revealed an exophytic, hyperkeratotic nodule on the dorsum of the left foot. Tumorectomy was performed, and a microscopic examination showed a subepidermal lesion composed of sheets of tumor cells with oval to round nuclei and ill-defined eosinophilic cytoplasm. The tumor cells were diffusely positive for factor XIIIa and ALK, but were negative for AE1/AE3 keratin, alpha-smooth muscle actin, CD30, CD34, CD68, PU.1, melan A, MITF, and S-100 protein. ALK immunostaining showed a diffuse cytoplasmic staining pattern. ALK fluorescence in situ hybridization demonstrated break-apart signals, which was suggestive of ALK rearrangement. A 5'-rapid amplification of cDNA ends assay detected SQSTM1-ALK fusion, in which exon 5 of the SQSTM1 gene was fused to exon 20 of the ALK gene. The patient was free from recurrence and distant metastasis at the 1-year of follow-up. CONCLUSION: We were able to demonstrate the SQSTM1-ALK fusion gene in ECH. Practically, detecting immunopositivity for ALK and appropriate cell-lineage markers are the key to diagnosing ECH.


Assuntos
Histiocitoma Fibroso Benigno/genética , Receptores Proteína Tirosina Quinases/genética , Proteína Sequestossoma-1/genética , Neoplasias Cutâneas/genética , Adulto , Quinase do Linfoma Anaplásico , Histiocitoma Fibroso Benigno/diagnóstico , Histiocitoma Fibroso Benigno/patologia , Humanos , Masculino , Fusão Oncogênica , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia
15.
Int J Mol Sci ; 19(5)2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29738493

RESUMO

Sequestosome1 (p62/SQSTM 1) is a multidomain protein that interacts with the autophagy machinery as a key adaptor of target cargo. It interacts with phagophores through the LC3-interacting (LIR) domain and with the ubiquitinated protein aggregates through the ubiquitin-associated domain (UBA) domain. It sequesters the target cargo into inclusion bodies by its PB1 domain. This protein is further the central hub that interacts with several key signaling proteins. Emerging evidence implicates p62 in the induction of multiple cellular oncogenic transformations. Indeed, p62 upregulation and/or reduced degradation have been implicated in tumor formation, cancer promotion as well as in resistance to therapy. It has been established that the process of autophagy regulates the levels of p62. Autophagy-dependent apoptotic activity of p62 is recently being reported. It is evident that p62 plays a critical role in both autophagy and apoptosis. Therefore in this review we discuss the role of p62 in autophagy, apoptosis and cancer through its different domains and outline the importance of modulating cellular levels of p62 in cancer therapeutics.


Assuntos
Autofagia/genética , Neoplasias/tratamento farmacológico , Proteína Sequestossoma-1/genética , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/genética , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/genética , Neoplasias/genética , Proteínas Nucleares/genética , Domínios Proteicos/genética , Fatores de Transcrição/genética , Ubiquitina/genética , Ubiquitinação/genética
16.
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
17.
Mol Med Rep ; 17(6): 7618-7626, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29620262

RESUMO

Acute kidney injury (AKI) is one of the most common complications of sepsis. The roles of autophagy in AKI have been demonstrated in previous studies. Sequestosome­1 (p62) has been demonstrated to serve essential roles in autophagy. The dysregulation of autophagy causes p62 accumulation, which is associated with increased inflammation and tumorigenesis. However, the expression patterns and role of p62 in septic AKI remain unknown. The present study detected the renal autophagy level, and the expression and localization of p62, in a lipopolysaccharide (LPS)­induced AKI mouse model. The results demonstrated that autophagy was induced in the kidneys of LPS­treated mice. The mRNA and protein levels of p62 were decreased in whole renal tissue samples and increased in mice treated with LPS. Immunohistochemistry indicated that p62 protein was predominantly expressed in the cytoplasm of proximal tubules under normal conditions and was significantly decreased following LPS injection into the cortex. In addition, p62 protein was gradually redistributed to the outer and inner medullas following treatment with LPS. In vitro experiments demonstrated that overexpression of p62 significantly decreased the viability and increased the lactate dehydrogenase (LDH) release and apoptosis rate, of renal tubular epithelial cells. By contrast, interference with p62 expression using small interfering RNA increased the cell viability and decreased the LDH release and apoptosis rate. The results of the present study demonstrated that p62 may aggravate LPS­induced acute kidney injury in mice by promoting apoptosis in renal tubular epithelial cells.


Assuntos
Lesão Renal Aguda/etiologia , Lesão Renal Aguda/metabolismo , Regulação da Expressão Gênica , Lipopolissacarídeos/efeitos adversos , Proteína Sequestossoma-1/genética , Lesão Renal Aguda/patologia , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Endotoxemia/complicações , Endotoxemia/genética , Endotoxemia/metabolismo , Células Epiteliais/metabolismo , Imuno-Histoquímica , Córtex Renal/metabolismo , Testes de Função Renal , Túbulos Renais/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico , Proteína Sequestossoma-1/metabolismo
18.
Handb Clin Neurol ; 148: 409-430, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29478591

RESUMO

Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by progressive changes in behavior, personality, and language with involvement of the frontal and temporal regions of the brain. About 40% of FTD cases have a positive family history, and about 10% of these cases are inherited in an autosomal-dominant pattern. These gene defects present with distinct clinical phenotypes. As the diagnosis of FTD becomes more recognizable, it will become increasingly important to keep these gene mutations in mind. In this chapter, we review the genes with known associations to FTD. We discuss protein functions, mutation frequencies, clinical phenotypes, imaging characteristics, and pathology associated with these genes.


Assuntos
Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , Proteína C9orf72/genética , Proteínas de Ligação a DNA/genética , Estudos de Associação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Glicoproteínas de Membrana/genética , Proteínas Mitocondriais/genética , Progranulinas , Receptores Imunológicos/genética , Proteína Sequestossoma-1/genética , Proteína com Valosina/genética , Proteínas tau/genética
19.
J Therm Biol ; 71: 142-152, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29301683

RESUMO

Hyperthermia causes oxidative stress in testes, which triggers antioxidant signals including autophagy and nuclear factor erythroid 2-related factor 2 (Nrf2). However, their relationship in testes under oxidative stress is unclear. In this study, we conducted testes injection for autophagy alteration and heat exposure to reveal the interaction between autophagy and the Nrf2-antioxidant system. Male mice were injected once with normal saline as control (Cont group), autophagy inhibitor 3-methyladenine (3-MA group) or autophagy inducer rapamycin (Rapa group). Then, each group was divided into two parts: one received a 2-h 42°C heat treatment for eight days (HT groups), and the other was kept thermal neutral (NT groups). Heat-exposed mice showed significantly increased rectal, scrotal surface and body surface temperatures. Histology of the testes revealed many vacuoles inserted in the seminiferous tubules in the HT Cont group and two 3-MA groups. Ultrastructural changes in germ cells revealed autophagosomes in two 3-MA groups. Immunohistochemical detection of Nrf2 and p62/SQSTM1 proteins showed prominent expression in Leydig cells. Heat exposure increased Nrf2 protein and mRNA levels. 3-MA and Rapa testes injection also resulted in Nrf2 cytoplasm accumulation. Massive conversion of LC3 (microtubule-associated protein light chain 3)Ⅰ to LC3Ⅱ was detected in two 3-MA groups, accompanied by decreased ATG5 (autophagy related gene 5) mRNA levels in the HT 3-MA group. These results indicated autophagy alteration triggered the Nrf2 signaling pathway with consequences such that the autophagy inducer protected the testes and the autophagy inhibitor enhanced the detrimental effects caused by heat exposure.


Assuntos
Autofagia , Resposta ao Choque Térmico , Fator 2 Relacionado a NF-E2/metabolismo , Túbulos Seminíferos/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Temperatura Alta , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/genética , Túbulos Seminíferos/efeitos dos fármacos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Sirolimo/farmacologia
20.
Nat Commun ; 9(1): 256, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343728

RESUMO

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.


Assuntos
Autofagia , Proteostase , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos Knockout , Oxidantes/farmacologia , Oxirredução , Homologia de Sequência de Aminoácidos , Proteína Sequestossoma-1/genética
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