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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 323-327, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631597

RESUMO

Objective: To determine the effects of autophagy inhibitor hydroxychloroquine (HCQ) on chemosensitivity of castration-resistant prostate cancer 22RV1 cell line in vitro and in vivo, and changes in its mRNA expressions of autophagy gene Bcelin-1, autophagy specific substrate P62 gene, pro-apoptotic gene Bax. Methods: 22RV1 cells were cultured in vitro and divided into blank control (no drug), DOC, and HCQ (20 µmol/L)+DOC groups. The concentration of DOC was set at 10 -6 mol/L, 10 -7 mol/L, and 10 -8 mol/L in the tests. Cell proliferation activities were detected by CCK-8 method 72 h after drug treatments. The 22RV1 cell suspension was injected subcutaneously into nude mice to establish transplanted tumor. The successfully modeled mice were randomly divided into three groups (five each) treated by physiological saline, DOC and HCQ+DOC (injected intraperitoneally for 4 weeks), respectively. Changes in growth of the transplanted tumor were observed. The mRNA expressions of Beclin-1, P62, and Bax were detected by qPCR. The protein expressions of Beclin-1, LC3B, and Bax were detected by Western blot. Results: In vitro: compared with the blank control, the DOC and HCQ+DOC groups showed decrease proliferation of cells( P<0.05); HCQ further lowered cell proliferation in the presence of DOC ( P<0.05), resulting in reduced half maximal inhibitory concentration (IC 50) of DOC. In vivo: compared with the model mice, the DOC and HCQ+DOC groups had decreased volume of transplanted tumor. HCQ slowed the weekly growth of tumor in the presence of DOC ( P<0.05), most obvious at the 4th week. In vitro and in vivo, HCQ+DOC upregulated the mRNA and protein expressions of Beclin-1, P62 and Bax ( P<0.05). Conclusion: HCQ can interfere with the autophagy of castration-resistant prostate cancer cells, inhibiting its proliferation and enhancing its sensitivity to chemotherapeutic drugs.


Assuntos
Autofagia/efeitos dos fármacos , Hidroxicloroquina/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Animais , Apoptose , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Distribuição Aleatória , Proteína Sequestossoma-1/metabolismo , Proteína X Associada a bcl-2/metabolismo
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 619-624, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537247

RESUMO

Objective To investigate the effects of Astragalus polysaccharide (APS) on autophagy and expression of microtubule-associated protein 1 light chain 3B (LC3B), mammalian target of rapamycin (mTOR) and beclin1 in xanthine oxidase (XOD)-induced autophagic model of non-small cell lung cancer A549 cells. Methods A549 cells were divided into five groups: control group, model group, 100, 200 and 400 µg/mL APS-treated group. Except for control group, all groups were administered XOD for 24 hours to establish autophagic models. Morphology of autophagosome was detected by transmission electron microscopy (TEM) and the number was counted by monodansylcadaverine (MDC) staining. The expression levels of LC3B, beclin1 and mTOR were detected by Western blot analysis. Results Compared with the control group, the number of autophagosome in the model group increased; the expression of LC3B and beclin1 significantly increased; while the expression of mTOR significantly decreased. Compared with the model group, the number of autophagosome decreased remarkably; the expression of LC3B and beclin1 severely decreased, and the expression of mTOR obviously increased in 200 or 400 µg/mL APS-treated group. Conclusion APS reduces the level of autophagy, down-regulates the expression of LC3B and beclin1, and increases mTOR expression in the autophagic model of A549 cells induced by XOD.


Assuntos
Astrágalo (Planta)/química , Autofagia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Polissacarídeos/farmacologia , Células A549 , Proteínas Relacionadas à Autofagia , Proteína Beclina-1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Xantina Oxidase
3.
J Agric Food Chem ; 67(41): 11364-11372, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31542934

RESUMO

The extensive use of pesticide caused an amount of pressure on the environment and increased the potential human health risk. Glyphosate-based herbicide (GBH) is one of the most widely used pesticides based on a 5-enolpyruvylshikimate-3-phosphate synthase target, which does not exist in vertebrates. Here, we study autophagic effects of the most famous commercial GBH Roundup (RDP) on human A549 cells in vitro. Intracellular biochemical assay indicated opening of mitochondrial permeability transition pore, LC3-II conversion, up-regulation of beclin-1, down-regulation of p62, and the changes in the phosphorylation of AMPK and mTOR induced by RDP in A549 cells. Further experimental results indicated that all the effects induced by RDP were related to its adjuvant polyethoxylated tallow amine, not its herbicidal active ingredient glyphosate isopropylamine salt. All these results showed that RDP has the ability to induce AMPK/mTOR-mediated cell autophagy in human A549 cells. This study would provide a theoretical basis for understanding RDP's autophagic effects on human A549 cells and attract attention on the potential human health risks induced by the adjuvant.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Glicina/análogos & derivados , Herbicidas/toxicidade , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Proteínas Quinases Ativadas por AMP/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Linhagem Celular , Glicina/toxicidade , Humanos , Serina-Treonina Quinases TOR/genética
4.
APMIS ; 127(12): 746-752, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31520423

RESUMO

Meningiomas are common intracranial tumors, and most exhibit optimal prognosis; however, some meningiomas still recur and even develop malignant transformation in the following years, regardless of initial pathological grade. During these years, autophagy raises its significance in tumorigenesis and tumor suppression, both important for tumor development. The aim of this study was to elucidate the relationship between two autophagy markers, LC3B and beclin 1, with clinical and pathological parameters in patients with meningiomas. A total of 77 thin-sectioned slides, retrospectively collected from meningioma patients, were analyzed and correlated with clinicopathological parameters. We found that expression of beclin 1 rather than LC3B correlated to better prognosis, lower pathological grade, and longer survival. Furthermore, intensity of beclin 1 was also found to be significantly related to the pathological grade. These findings indicated that beclin 1 as a protective factor predicts better prognosis and plays the role of tumor suppression in meningiomas.


Assuntos
Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Neoplasias Meníngeas/fisiopatologia , Meningioma/fisiopatologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/mortalidade , Meningioma/metabolismo , Meningioma/mortalidade , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
5.
Life Sci ; 233: 116709, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31369760

RESUMO

BACKGROUND: Cisplatin resistance has been found to contribute to the failure of ovarian carcinoma treatment. Oridonin is a natural en-kaurane tetracyclic diterpenoid compound discovered in Rabdosia rubescene (Henmsl.) Hara. Herein, we tested whether oridonin could exert chemo-sensitization activity on cisplatin-resistant ovarian carcinoma cells. METHODS: Firstly, A2780CP cells and SKOV3/DDP cells were exposed to cisplatin and/or oridonin treatment. Cell counting kit-8 (CCK-8) kit and Dead Cell Apoptosis Kit with Annexin V-FITC and PI were carried out to test cell viability and apoptosis, respectively. Then, pBeclin-1 was transfected to overexpress Beclin-1. 3-Methyladenine (3-MA) acted as autophagy inhibitor, while rapamycin acted as autophagy activator. Finally, the influence of cisplatin and/or oridonin treatment on human normal ovarian epithelial IOSE 364 cell viability and apoptosis were also detected. RESULTS: Oridonin incubation notably elevated the cisplatin-caused reduction of A2780CP and SKOV3/DDP cell viabilities and enhancement of cell apoptosis. Cisplatin-caused A2780CP and SKOV3/DDP cell autophagy was dramatically inhibited by oridonin. Overexpression of Beclin-1 mitigated the influence of oridonin on cisplatin-caused A2780CP and SKOV3/DDP cell autophagy. Inhibition of cell autophagy by 3-MA promoted the oridonin + cisplatin-caused A2780CP and SKOV3/DDP cell apoptosis, while activation of cell autophagy by rapamycin had opposite effects. Oridonin and cisplatin co-treatment had no significant effects on IOSE 364 cell viability and apoptosis. CONCLUSION: The chemo-sensitization activity of oridonin on cisplatin-resistant ovarian carcinoma cells was verified in this study. Oridonin elevated sensitivity of ovarian carcinoma cells to cisplatin via suppressing cisplatin-mediated autophagy.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia , Cisplatino/farmacologia , Diterpenos de Caurano/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias Ovarianas/patologia , Antineoplásicos/farmacologia , Proteína Beclina-1/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
6.
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
7.
Ecotoxicol Environ Saf ; 183: 109480, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31382115

RESUMO

Spinetoram is one of the most extensively used bio-pesticide in the world. The effects of pesticide in human health are mainly caused by its residue in food. The liver is the direct target of pesticides exposure, however the study of cytotoxicity on human liver cells caused by spinetoram remains unclear. The aim of the present study was to evaluate the cytotoxic effects of the spinetoram in human liver cells in vitro. We demonstrated that spinetoram could inhibit the proliferation of human liver HepG2 cells and induce the oxidative DNA damage. Intracellular biochemical assay indicated that decrease of mitochondrial membrane potential, LC3-II conversion, accumulation of Beclin-1, degradation of p62 and the changes in the phosphorylation of AMPK, mTOR are contributed to the toxic effects of Spinetoram on HepG2 cells. These results showed that the cytotoxicity of spinetoram may be associated with the activity of AMPK/mTOR-mediated autophagy pathway. Meanwhile, the generation of 8-oxodG caused by the spinetoram suggested it has a potential genotoxic effect on human liver cells. We conclude that spinetoram has a significant cytotoxic effect by inducing AMPK/mTOR-mediated autophagy and oxidative DNA damage. This study would provide a theoretical basis for understanding its mechanisms of toxicity and supply an indication for recognizing the safety of spinetoram to human beings.


Assuntos
Autofagia/efeitos dos fármacos , Dano ao DNA , Inseticidas/toxicidade , Macrolídeos/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Proliferação de Células/efeitos dos fármacos , Citotoxinas/toxicidade , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo , Fosforilação/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
8.
Anticancer Res ; 39(7): 3687-3695, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262895

RESUMO

BACKGROUND: Neuroblastoma is the main solid extracranial tumor of childhood. The amplification of N-myc oncogene (MYCN) and 1p deletion are the main molecular alterations. These features are what make treatment impossible, especially in high-risk patients with metastases. MATERIALS AND METHODS: Our study investigated the processes undergone by CHP-212 neuroblastoma cells, after being treated with Casiopeínas® (Cas) IIgly, IIIEa, and IIIia for 2, 10, and 24 h. RESULTS: At 2 h, all the treatments Ied to apoptosis [defined by the presence of B-cell lymphoma 2 (BCL2), BCL2-associated X protein, cytochrome c, and caspase-3]. In addition, autophagy with specific molecules beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I (ratio >1). Later at 10 h, autophagy-associated proteins were observed, and at 24 h, only survival proteins nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), and extracellular signal-regulated kinases (ERK)2/ERK1>1 were found. Another relevant finding was the presence of caspase-10 throughout the study, especially in cells treated with CasIIgly and CasIIIEa. CONCLUSION: These relationships indicate a possible mechanism of action of Casiopeínas on neuroblastoma.


Assuntos
Complexos de Coordenação/farmacologia , Neuroblastoma/metabolismo , Fenantrolinas/farmacologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
9.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(2): 165-168, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-31250610

RESUMO

OBJECTIVE: To investigate the effects of Notch signal on hypoxic induction factor (HIF-1α) and autophagy-associated genes Beclin1, LC3I, LC3II in oxygen-glucose deprivation (OGD) induced myocardial cell injury. METHODS: The OGD model was established using hypoxic culture box and hypoglycemic DMEM medium. The cells were divided into normal control group, OGD group, OGD + NC siRNA group, OGD + Notch1 siRNA group and OGD + HIF-1α siRNA group. Western blot was used to detect the interference effects of HIF-1α siRNA and Notch1 siRNA. The effects of Notch1 siRNA and HIF-1α siRNA on the activity of myocardial cells in OGD model were detected by the CCK-8 assay. The effects of Notch1 siRNA and HIF-1α siRNA on autophage-associated genes Beclin1, LC3I and LC3II expression were detected by Western blot. RESULTS: The results of Western blot showed that HIF-1α siRNA could effectively knock down the expression of HIF-1α in myocardial cells in OGD model, and Notch1 siRNA could effectively knock down the expression of Notch1 and HIF-1α in myocardial cells in OGD model. The result of CCK-8 assay showed that Notch1 siRNA and HIF-1α siRNA reduced the activity of myocardial cells in OGD model, and there was no statistical difference between the two groups. Western blot results showed that Notch1 siRNA and HIF-1α siRNA could reduce the expressions of the autophagy-associated genes Beclin1, LC3I and LC3II, and reduce the ratio of LC3II to LC3I at mRNA level. CONCLUSION: Notch1 plays a role in myocardial protection by regulating the expression of HIF-1α to regulate the autophagy in OGD model cells.


Assuntos
Autofagia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miócitos Cardíacos/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Proteína Beclina-1/metabolismo , Hipóxia Celular , Células Cultivadas , Glucose , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/patologia , Oxigênio
10.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 37(2): 168-173, 2019 Apr 01.
Artigo em Chinês | MEDLINE | ID: mdl-31168983

RESUMO

OBJECTIVE: To investigate the expression of autophagy-related protein Beclin-1 and microtubule-associated protein 2 light chain 3 (LC3Ⅱ) in periodontal ligament cells in orthodontic tooth pressure areas. METHODS: Sixty male SD rats were randomly divided into a blank control group and nine experimental groups. In the experimental groups, 0.392 N orthodontic force was used to move the first right upper molars for 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 1 d, 3 d, or 7 d. The blank control group did not receive any treatment. The rats were euthanized. Changes in the morphology of the periodontal membrane in the pressure areas were observed through hematoxylin and eosin (HE) staining. The expression levels of Beclin-1 and LC3Ⅱ were detected by immunohistochemical staining, and tartrate-resistant acid phosphatase (TRAP) staining was performed for the counting of osteoclasts. RESULTS: The HE stains showed that the hyalinization of the periodontal ligament appeared in the pressure areas after 1 day of exertion and was gradually aggravated. The immunohistochemical stains showed that the expression levels of Beclin-1 and LC3Ⅱ in the experimental groups gradually increased, peaked after 1 h, and then gradually decreased. The expression levels peaked again after 1 d, then decreased to baseline levels at 7 d of exertion. Beclin-1 and LC3Ⅱ were expressed in the osteoclasts. The TRAP stains indicated that the number of osteoclasts started to increase after 1 day. CONCLUSIONS: Autophagy may participate in the process of periodontal ligament reconstruction in orthodontic tooth pressure areas by mediating the hyalinization of periodontal ligament and affecting the biological effects of osteoclasts.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Ligamento Periodontal , Animais , Proteína Beclina-1/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Osteoclastos , Ligamento Periodontal/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Técnicas de Movimentação Dentária
11.
Life Sci ; 231: 116578, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31211996

RESUMO

AIMS: The aim of the present study is to shed light on the modulating action of selenium on two of the most crucial cellular pathways; apoptosis and autophagy and the possible interplay between them in determining the pituitary fate in the context of mercury intoxication through demonstration of the molecular, histopathological, immunohistochemical, and ultrastructural features of selenium mercury-treated adenohypophysis. METHODS: Thirty adult Sprague Dawley male albino rats were assigned into control group, mercury-treated group and mercury­selenium concomitantly-treated group. The adenohypophysis was subjected to structural, molecular and protein expression assessment of autophagy and apoptotic markers and western blotted analysis of Beclin 1 as a key cross-regulator of autophagy and apoptosis. KEY FINDINGS: Selenium treatment ameliorated the mercury-induced apoptosis detected by improvement in PCR and immunohistochemical expression of the apoptotic markers Bax, Bcl-2 and Caspase-3. Selenium also improved mercury-induced autophagic dysfunction with statistically significant improvement in western blotted levels of the autophagy markers LC3I, LC3II and Beclin1. The histopathological and ultrastructural studies strongly confirmed those findings. SIGNIFICANCE: The crosstalk between the apoptotic Bcl-2 family of proteins and the autophagic Beclin-1LC3 pathway in the context of mercury intoxication paves the way for developing novel effective treatment strategies for several mercury-induced pituitary diseases.


Assuntos
Intoxicação por Mercúrio/prevenção & controle , Mercúrio/toxicidade , Hipófise/efeitos dos fármacos , Selênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Masculino , Intoxicação por Mercúrio/metabolismo , Intoxicação por Mercúrio/patologia , Hipófise/metabolismo , Hipófise/patologia , Adeno-Hipófise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismo
12.
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
13.
Chemosphere ; 233: 261-272, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31176127

RESUMO

Aflatoxin B1 (AFB1), a potential endocrine disrupter, has been shown to induce hepatotoxicity in animal models, but the effects of AFB1 on Leydig cell function are unclear. In this study, in vivo exposure to AFB1 at 15 and 150 µg/kg/day lowered serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels, reduced Leydig cell number, and down-regulated the expression of testosterone biosynthesis-related genes. In vitro study showed that AFB1 (10 µM) significantly increased ROS levels, and decreased T production in Leydig cells by suppressing certain T-biosynthesis gene expressions. Moreover, AFB1 induced Leydig cell apoptosis through lowering pAMPK/AMPK ratio and increasing pmTOR/mTOR ratio, and then further up-regulating autophagy and apoptosis proteins, LC3, BECLIN 1, and BAX, as well as down-regulating autophagy flux protein P62 and anti-apoptosis protein BCL-2. AFB1-induced toxicity in Leydig cells was characterized by inhibiting T-biosynthesis gene expression, reducing Leydig cell number, promoting ROS production, and inducing cell apoptosis via suppressing AMPK/mTOR-mediated autophagy flux pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aflatoxina B1/toxicidade , Autofagia/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Hormônio Luteinizante/sangue , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Testosterona/sangue , Testosterona/genética , Testosterona/metabolismo
14.
World J Gastroenterol ; 25(15): 1840-1853, 2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31057298

RESUMO

BACKGROUND: Colorectal cancer (CRC) is one of the main causes of cancer-related deaths in China and around the world. Advanced CRC (ACRC) patients suffer from a low cure rate though treated with targeted therapies. The response rate is about 50% to chemotherapy and cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR) and used for ACRC with wild-type KRAS. It is important to identify more predictors of cetuximab efficacy to further improve precise treatment. Autophagy, showing a key role in the cancer progression, is influenced by the EGFR pathway. Whether autophagy can predict cetuximab efficacy in ACRC is an interesting topic. AIM: To investigate the effect of autophagy on the efficacy of cetuximab in colon cancer cells and ACRC patients with wild-type KRAS. METHODS: ACRC patients treated with cetuximab plus chemotherapy, with detailed data and tumor tissue, at Sun Yat-sen University Cancer Center from January 1, 2005, to October 1, 2015, were studied. Expression of autophagy-related proteins [Beclin1, microtubule-associated protein 1A/B-light chain 3 (LC3), and 4E-binding protein 1 (4E-BP1)] was examined by Western blot in CRC cells and by immunohistochemistry in cancerous and normal tissues. The effect of autophagy on cetuximab-treated cancer cells was confirmed by MTT assay. The associations between Beclin1, LC3, and 4E-BP1 expression in tumor tissue and the efficacy of cetuximab-based therapy were analyzed. RESULTS: In CACO-2 cells exposed to cetuximab, LC3 and 4E-BP1 were upregulated, and P62 was downregulated. Autophagosome formation was observed, and autophagy increased the efficacy of cetuximab. In 68 ACRC patients, immunohistochemistry showed that Beclin1 levels were significantly correlated with those of LC3 (0.657, P < 0.001) and 4E-BP1 (0.211, P = 0.042) in ACRC tissues. LC3 was significantly overexpressed in tumor tissues compared to normal tissues (P < 0.001). In 45 patients with wild-type KRAS, the expression levels of these three proteins were not related to progression-free survival; however, the expression levels of Beclin1 (P = 0.010) and 4E-BP1 (P = 0.005), pathological grade (P = 0.002), and T stage (P = 0.004) were independent prognostic factors for overall survival (OS). CONCLUSION: The effect of cetuximab on colon cancer cells might be improved by autophagy. LC3 is overexpressed in tumor tissues, and Beclin1 and 4E-BP1 could be significant predictors of OS in ACRC patients treated with cetuximab.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia , Biomarcadores Tumorais/metabolismo , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína Beclina-1/metabolismo , Cetuximab/farmacologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fosfoproteínas/metabolismo , Prognóstico , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas p21(ras)/genética , Regulação para Cima , Adulto Jovem
15.
Cell Mol Life Sci ; 76(17): 3311-3322, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31062072

RESUMO

Oxygen deprivation affects human health by modulating system as well as cellular physiology. Hypoxia generates reactive oxygen species (ROS), causes oxidative stress and affects female reproductive health by altering ovarian as well as oocyte physiology in mammals. Hypoxic conditions lead to several degenerative changes by inducing various cell death pathways like autophagy, apoptosis and necrosis in the follicle of mammalian ovary. The encircling somatic cell death interrupts supply of nutrients to the oocyte and nutrient deprivation may result in the generation of ROS. Increased level of ROS could induce granulosa cells as well as oocyte autophagy. Although autophagy removes damaged proteins and subcellular organelles to maintain the cell survival, irreparable damages could induce cell death within intra-follicular microenvironment. Hypoxia-induced autophagy is operated through 5' AMP activated protein kinase-mammalian target of rapamycin, endoplasmic reticulum stress/unfolded protein response and protein kinase C delta-c-junN terminal kinase 1 pathways in a wide variety of somatic cell types. Similar to somatic cells, we propose that hypoxia may induce granulosa cell as well as oocyte autophagy and it could be responsible at least in part for germ cell elimination from mammalian ovary. Hypoxia-mediated germ cell depletion may cause several reproductive impairments including early menopause in mammals.


Assuntos
Autofagia , Células da Granulosa/citologia , Animais , Proteína Beclina-1/metabolismo , Hipóxia Celular , Feminino , Células da Granulosa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
Virology ; 533: 34-44, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31082732

RESUMO

Coronavirus infection induces the generation of autophagosomes, and certain host proteins regulating cellular autophagy are hijacked by some coronaviruses to facilitate the formation of double membrane vesicles. However, mechanisms underlying coronavirus-induced autophagy remain largely unknown. In this study, we demonstrate that autophagosome formation and apparent autophagic flux are induced in cells infected with infectious bronchitis virus (IBV) - a gammacoronavirus. Notably, IBV-induced autophagy was dependent on autophagy related 5 (ATG5) but not beclin1 (BECN1), although both are essential proteins in the canonical autophagy pathway. Moreover, the ER stress sensor inositol requiring enzyme 1 (IRE1), but not its substrate X-box protein 1 (XBP1), was also essential for the induction of autophagy during IBV infection. Finally, the anti-apoptotic extracellular signal-regulated kinase 1/2 (ERK1/2) also contributed to IBV-induced autophagy. Our findings add new knowledge to the regulatory mechanisms governing coronavirus-induced autophagy, highlighting an extensive cross-talk among cellular signaling pathways during coronavirus infection.


Assuntos
Autofagia , Infecções por Coronavirus/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Vírus da Bronquite Infecciosa/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Infecções por Coronavirus/genética , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Humanos , Vírus da Bronquite Infecciosa/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Serina-Treonina Quinases/genética
17.
Medicine (Baltimore) ; 98(20): e15402, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31096436

RESUMO

Adenoid cystic carcinoma (ACC) is an uncommon salivary gland malignancy with a poor long-term prognosis. Clinical reports show the high rates of local recurrences and distant metastases. This study aimed to investigate the expression of MIF, Beclin1, and light-chain 3 (LC3) in salivary adenoid cystic carcinoma (SACC).Tissue specimens were obtained from 48 salivary glands adenoid cystic carcinoma (SACC) patients and 15 oral squamous cell carcinoma (OSCC) patients. Immunohistochemical staining was performed to estimate the level of LC3, Beclin1, and MIF. All SACC patients were followed up. The Kaplan-Meier method was used to compare the prognosis of patients after treatment.The 3-year, 5 year-, and 10 year-survival rates of the SACC patients were 83.9%, 69.9%, and 46.6%, respectively. MIF, LC3, and Beclin1 in SACC were all obviously over-expressed. MIF showed an increased tendency in cases with advanced TNM stages, and at the same time, there was an inversely proportional relationship between MIF and LC3, Beclin1.The long-term survival of SACC patients is poor. MIF might be a risk factor for SACC patients, whereas, LC3 and Beclin1 might be an effective strategy for treatment of SACC.


Assuntos
Proteína Beclina-1/metabolismo , Carcinoma Adenoide Cístico/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Patologia Molecular/métodos , Neoplasias das Glândulas Salivares/metabolismo , Adulto , Idoso , Carcinoma Adenoide Cístico/mortalidade , Carcinoma Adenoide Cístico/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Estadiamento de Neoplasias/métodos , Prognóstico , Neoplasias das Glândulas Salivares/mortalidade , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares/patologia , Taxa de Sobrevida
18.
Mol Med Rep ; 19(6): 5453-5463, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059044

RESUMO

To assess the effect of cluster of differentiation (CD47) downregulation on autophagy in hypoxia/reoxygenation (H/R)­treated H9c2 cardiomyocytes. H9c2 cells were maintained in normoxic conditions (95% air, 5% CO2, 37˚C) without CD47 antibodies, Si­CD47 or chloroquine (CQ) treatment; H9c2 cells in the H/R group were subjected to 24 h of hypoxia (1% O2, 94% N2, 5% CO2, 37˚C) followed by 12 h of reoxygenation (95% air, 5% CO2, 37˚C). All assays were controlled, triplicated and repeated on three separately initiated cultures. The biochemical parameters in the medium supernatant were measured to evaluate the oxidative stress in cardiomyocytes. The Annexin V­fluorescein isothiocyanate assay was used to detect the apoptotic rate in the H9c2 cells. Transmission electron microscope, immunofluorescent staining and western blot analysis were performed to detect the effect of the CD47 antibody on autophagic flux in H/R­treated H9c2 cardiomyocytes. The cardiomyocytic oxidative stress and apoptotic rate decreased and autophagic clearance increased after CD47 downregulation. H/R triggered cell autophagy, autophagosome accumulation and apoptosis in H9c2 cell lines. However, these effects can be attenuated by CD47 downregulation. This study demonstrates its clinical implications in ischemia/reperfusion injury treatment.


Assuntos
Autofagia , Antígeno CD47/genética , Hipóxia Celular , Oxigênio/metabolismo , Animais , Apoptose , Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Agregados Proteicos/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo
19.
Cell Physiol Biochem ; 52(6): 1325-1338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050281

RESUMO

BACKGROUND/AIMS: Atherosclerosis is a chronic inflammatory cardiovascular disease. Macrophages are major components of atherosclerotic plaques and play a key role in the development of atherosclerosis by secreting a variety of pro-inflammatory factors. Our previous studies have confirmed that upconversion nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) mediated photodynamic therapy (PDT) can promote cholesterol efflux and induce apoptosis in THP-1 macrophages. In this study, we investigated whether reactive oxygen species (ROS) generated by UCNPs-Ce6-mediated PDT can induce autophagy to inhibit the expression of pro-inflammatory factor in M1 peritoneal macrophages. METHODS: Peritoneal macrophages were collected from C57/BL6 mice injected with 3% thioglycollate broth medium and induced by lipopolysaccharides and interferon-γ. Intracellular ROS production was assessed by 2'-7'-dichloroflorescein diacetate and flow cytometry. Autophagy was assayed by western blot, transmission electron microscopy and immunofluorescence. Pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay and western blot. RESULTS: Model M1 peritoneal macrophages were established after 24 h induction. Protein expression levels of LC3 II and Beclin1, and degradation of p62 increased and peaked at 2 h in the PDT group. Meanwhile, levels of inflammatory cytokines iNOS, IL-12, and TNF-α markedly decreased after PDT. The increase in autophagy levels and decrease in pro-inflammatory cytokines were significantly inhibited by 3-methyladenine. Furthermore, ROS generated by UCNPs- Ce6 mediated PDT activated autophagy. The expression of autophagy related-protein and inflammatory cytokines iNOS, IL-12, and TNF-α were inhibited by the ROS inhibitor N-acetyl cysteine. CONCLUSION: ROS generated by UCNPs-Ce6-mediated PDT activated autophagy and inhibited the expression of pro-inflammatory factors of M1 peritoneal macrophage via the PI3K/AKT/mTOR signaling pathway.


Assuntos
Autofagia/efeitos dos fármacos , Nanopartículas Metálicas/química , Porfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteína Beclina-1/metabolismo , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fotoquimioterapia , Porfirinas/química , Porfirinas/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
Mar Drugs ; 17(4)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991714

RESUMO

In the study, the protective effect of plasma protein from Tachypleus tridentatus (PPTT) on acute kidney injury (AKI) and the related molecular mechanisms were first investigated by Western blotting analyses, TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and immunohistochemistry. It was found that PPTT had an obviously inhibitory effect on Reactive oxygen species (ROS) in cyclophosphamide (CTX)-exposed mice. Furthermore, results demonstrated that the renal cell death mode is due to inducing apoptosis and autophagy inhibited by dose-dependent PPTT in mice treated with CTX by decreasing the protein expression of bax, beclin-1, and LC3 and increasing the expression of bcl-2. Moreover, the p38 MAPK and PI3K/Akt signaling pathways were observed to take part in the PPTT-induced renal cell growth effect by enhancing the upregulation of the expression of Akt and p-Akt as well as the downregulation of the expression of p38 and p-p38, which indicated a PPTT ameliorating effect on AKI CTX-induced in mice through p38 MAPK and PI3K/Akt signaling pathways. Briefly, this article preliminarily studies the mechanism of the PPTT ameliorating effect on AKI CTX-induced in mice, which helps to provide a reference for PPTT clinical application in AKI therapy.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Proteínas Sanguíneas/farmacologia , Caranguejos Ferradura/química , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteínas Sanguíneas/isolamento & purificação , Ciclofosfamida/farmacologia , Modelos Animais de Doenças , Feminino , Caranguejos Ferradura/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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