RESUMEN
Spermatogenesis is a finely regulated process that involves the interaction of several cellular mechanisms to ensure the proper development and maturation of germ cells. This study assessed autophagy contribution and its relation to apoptosis in fish spermatogenesis during starvation. To that end, Nile tilapia males were subjected to 0 (control), 7, 14, 21, and 28 days of starvation to induce autophagy. Testes samples were obtained for analyses of spermatogenesis by histology, electron microscopy, immunohistochemistry, and western blotting. Sperm quality was assessed using a computer-assisted sperm analysis (CASA) system. Data indicated a significant reduction in gonadosomatic index, seminiferous tubule area, and spermatozoa proportion in fish subject to starvation compared to the control group. Immunoblotting revealed a reduction of Bcl2 and Beclin 1 associated with increased Bax and Caspase-3, mainly after 21 and 28 days of starvation. LC3 and P62 indicated reduced autophagic flux in these starvation times. Immunolabeling for autophagic and apoptotic proteins occurred in all development stages of the germ cells, but protein expression varied throughout starvation. Beclin 1 and Cathepsin D decreased while Bax and Caspase-3 increased in spermatocytes, spermatids, and spermatozoa after 21 and 28 days. Autophagic and lysosomal proteins colocalization indicated the fusion of autophagosomes with lysosomes and lysosomal degradation in spermatogenic cells. The CASA system indicated reduced sperm motility and velocity in animals subjected to 21 and 28 days of starvation. Altogether, the data support autophagy acting at different spermatogenesis stages in Nile tilapia, with decreased autophagy and increased apoptosis after 21 and 28 days of starvation, which results in a decrease in the spermatozoa number and sperm quality.
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Cíclidos , Masculino , Animales , Caspasa 3/metabolismo , Cíclidos/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Motilidad Espermática , Semen/metabolismo , Espermatozoides/metabolismo , Espermatogénesis , Espermátides , AutofagiaRESUMEN
Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles maintaining cellular integrity. It seems to be essential for cell survival during stress, starvation, hypoxia, and consequently to the placenta implantation and development. Preeclampsia (PE) is a multisystemic disorder characterized by the onset of hypertension associated or not with proteinuria and other maternal complications. Considering that the placenta seems to play an important role in the pathogenesis of PE, the objective of the present study was to evaluate protein levels of light chain protein (LC3), beclin-1, and the mammalian target of rapamycin (mTOR) in the placenta of pregnant women with PE. Placental tissues collected from 20 women with PE and 20 normotensive (NT) pregnant women were evaluated for LC3, beclin-1, and mTOR expression by qPCR and immunohistochemistry. The mRNA for LC3 and beclin-1 were significantly lower, while mTOR gene expression was significantly higher in the placenta of pregnant women with PE than in the NT group. Placentas of PE women showed significantly decreased protein expression of LC3-II and beclin-1, whereas mTOR was significantly increased compared with the NT pregnant women. There was a negative correlation between protein expression of mTOR and LC3-II in the placental tissue of PE women. In conclusion, the results showed autophagy deficiency suggesting that failure in this degradation process may contribute to the pathogenesis of PE; however, new studies involving cross-talk between autophagy and inflammatory molecular mechanisms might help to better understand the autophagy process in this obstetric pathology.
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Placenta , Preeclampsia , Femenino , Embarazo , Humanos , Mujeres Embarazadas , Preeclampsia/genética , Beclina-1/genética , Beclina-1/metabolismo , Regulación hacia Abajo , Serina-Treonina Quinasas TOR/metabolismo , Autofagia/fisiologíaRESUMEN
OBJECTIVES: This study aims to explore the effect of silencing Beclin-1 gene on autophagy and apoptosis of Benign Prostatic Hyperplasia (BPH) (BPH-1) cells under the condition of Androgen Deprivation (AD) and Autophagy Inhibition (AI). METHODS: Control group (BPH-1 group), empty carrier group (sh-RNA-BPH-1 group) and Beclin-1 silenced group (sh-Beclin1-BPH-1 group) were set. The Beclin-1 gene silencing efficiency was detected by RT-PCR and Western blot. Autophagic flux was monitored by GFP-LC3 cleavage assay and cell apoptosis was analyzed by flow cytometry. The protein expression levels of LC3, Caspase-3, PARP-1, Bcl-2, and Bax were detected by Western blot. RESULTS: The transfection of sh-Beclin-1 obviously down-regulated the expression of Beclin-1 at both mRNA and protein levels. Under the conditions of AD and AI, silencing of Beclin-1 restrained the autophagy of BPH-1 cells, as evidenced by a decreased number of autophagosomes and down-regulation of LC3-II protein (p < 0.001). The results of flow cytometry showed that the apoptotic rate of sh-Beclin-1 group was elevated significantly compared to the other two groups (p < 0.01). Western blot results showed that silencing of Beclin-1 promoted 89 kd fragmentation of PARP-1 (p < 0.001) and Caspase-3 activation (p < 0.01). Moreover, silencing of Beclin-1 resulted in declined Bcl-2 and augmented Bax protein expression in BPH-1 cells (p < 0.01), which ultimately led to a decreased Bcl-2/Bax ratio. CONCLUSIONS: The results indicated that the silencing of Beclin-1 gene hampered autophagy while activating apoptosis in BPH-1 cells. Thus, Beclin-1 may participate in an antagonistic relationship between autophagy and apoptosis in BPH.
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Hiperplasia Prostática , Neoplasias de la Próstata , Antagonistas de Andrógenos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/farmacología , Autofagia , Beclina-1/genética , Beclina-1/metabolismo , Beclina-1/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 3/farmacología , Células Epiteliales/metabolismo , Silenciador del Gen , Humanos , Masculino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacologíaRESUMEN
In insects the reserve proteins are stored in the oocytes into endocytic-originated vesicles named yolk organelles. VPS38/UVRAG and ATG14 are the variant regulatory subunits of two class-III ATG6/Beclin1 PI3K complexes that regulate the recruitment of the endocytic (complex II) and autophagic (complex I) machineries. In a previous work from our group, we found that the silencing of ATG6/Beclin1 resulted in the formation of yolk-deficient oocytes due to defects in the endocytosis of the yolk proteins. Because ATG6/Beclin1 is present in the two above-described PI3K complexes, we could not identify the contributions of each complex to the yolk defective phenotypes. To address this, here we investigated the role of the variant subunits VPS38/UVRAG (complex II, endocytosis) and ATG14 (complex I, autophagy) in the biogenesis of the yolk organelles in the insect vector of Chagas Disease Rhodnius prolixus. Interestingly, the silencing of both genes phenocopied the silencing of ATG6/Beclin1, generating 1) accumulation of yolk proteins in the hemolymph; 2) white, smaller, and yolk-deficient oocytes; 3) abnormal yolk organelles in the oocyte cortex; and 4) unviable F1 embryos. However, we found that the similar phenotypes were the result of a specific cross-silencing effect among the PI3K subunits where the silencing of VPS38/UVRAG and ATG6/Beclin1 resulted in the specific silencing of each other, whereas the silencing of ATG14 triggered the silencing of all three PI3K components. Because the silencing of VPS38/UVRAG and ATG6/Beclin1 reproduced the yolk-deficiency phenotypes without the cross silencing of ATG14, we concluded that the VPS38/UVRAG PI3K complex II was the major contributor to the previously observed phenotypes in silenced insects. Altogether, we found that class-III ATG6/Beclin1 PI3K complex II (VPS38/UVRAG) is essential for the yolk endocytosis and that the subunits of both complexes are under an unknown transcriptional regulatory system.
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Beclina-1/metabolismo , Proteínas de Insectos/metabolismo , Oocitos/fisiología , Orgánulos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Rhodnius/fisiología , Animales , Beclina-1/genética , Enfermedad de Chagas/transmisión , Yema de Huevo/fisiología , Regulación de la Expresión Génica , Silenciador del Gen , Proteínas de Insectos/genética , Insectos Vectores/fisiología , Oocitos/citología , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
There is currently no clear understanding on the pathways involved in the process of cell inhibition by photobiomodulation (PBM). The present study evaluated the influence of PBM on the expression of autophagy markers in vitro in an in situ model of oral carcinoma. Oral squamous cell carcinoma (Cal27) and stromal fibroblasts (FG) cultures were used. The independent variables were 'cell type' (FG and CAL27) 'culture condition' (monocultures or co-cultures) and PBM (placebo and 36 J/cm2). The cultures were irradiated from a red LED source for mRNA expression and protein expression analyses. The autophagy markers evaluated were Beclin-1, LC3B and p62 as well as adjuvant markers (BAX Bcl-2, VEGF, CD105, CD34, PRDX1, PRDX4 and GRP78). The Cal27 cells upregulated the autophagy markers upon exposure to PBM both at the mRNA and protein expression levels, providing evidence to explain malignant cell inhibition by PBM.
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Autofagia/genética , Luz , Regulación hacia Arriba/efectos de la radiación , Beclina-1/genética , Beclina-1/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular , Técnicas de Cocultivo , Chaperón BiP del Retículo Endoplásmico , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismoRESUMEN
Colorectal cancer (CRC) is among the top three most deadly cancers worldwide. The survival rate for this disease has not been reduced despite the treatments, the reason why the search for therapeutic alternatives continues to be a priority issue in oncology. In this research work, we tested our successful pharmacological combination of three drugs, metformin, doxorubicin, and sodium oxamate (triple therapy, or TT), as an autophagy inducer. Firstly, we employed western blot (WB) assays, where we observed that after 8 h of stimulation with TT, the proteins Unc-51 like autophagy activating kinase 1(ULK1), becline-1, autophagy related 1 protein (Atg4), and LC3 increased in the CRC cell lines HCT116 and SW480 in contrast to monotherapy with doxorubicin. The overexpression of these proteins indicated the beginning of autophagy flow through the activation of ULK1 and the hyperlipidation of LC3 at the beginning of this process. Moreover, we confirm that ULK1 is a bona fide target of hsa-miR-106a-5p (referred to from here on as miR-106a) in HCT116. We also observed through the GFP-LC3 fusion protein that in the presence of miR-106a, the accumulation of autophagy vesicles in cells stimulated with TT is inhibited. These results show that the TT triggered autophagy to modulate miR-106a/ULK1 expression, probably affecting different cellular pathways involved in cellular proliferation, survivance, metabolic maintenance, and cell death. Therefore, considering the importance of autophagy in cancer biology, the study of miRNAs that regulate autophagy in cancer will allow a better understanding of malignant tumors and lead to the development of new disease markers and therapeutic strategies.
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Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Neoplasias Colorrectales/tratamiento farmacológico , Doxorrubicina/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , MicroARNs/genética , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/efectos de los fármacos , Beclina-1/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Combinación de Medicamentos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Metformina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Despite recent progressions in the treatment of melanoma, the response to conventional therapies and the long-term survival in melanoma patients still remain poor. Recently, the use of nanoparticles (NPs) has been highlighted for promoting the chemotherapeutic effects of cytotoxic drugs in melanoma. The aim of this study is to mechanistically evaluate the potential of titanium dioxide (TiO2) nanoparticles (NPs) for enhancing chemotherapy effects in in vitro and in vivo models of murine melanoma. METHODS: The F10 melanoma cells were exposed to different concentrations of TiO2 NPs and/or cisplatin, then cell growth, cell viability, and cell death were evaluated. In parallel, C57BL/6 syngeneic melanoma mice were treated by TiO2 NPs and/or cisplatin, and then drug responses, tumor size and mice's organs were studied pathologically. Autophagy was examined by evaluating the formation of autophagosomes and gene expression levels of autophagy markers (ATG5 and ATG6) by fluorescent microscopy and qPCR, respectively. RESULTS: Nontoxic concentrations of TiO2 NPs (50 µg/ml) promote anti-proliferative and cytotoxic effects of cisplatin in F10 melanoma cells, which is mediated through the induction of autophagy and necrotic cell death. Whereas TiO2 NPs have no cytotoxic or metastatic effects in melanoma mice, its combination with cisplatin enhances drug responses (up to 50%), leading to higher inhibition of tumor growth compared with each monotherapy. CONCLUSION: The combination of TiO2 NP with cisplatin enhances chemotherapy response in both in vitro and in vivo melanoma models. In addition, autophagy plays an essential role during sensitizing melanoma cells to chemotherapy.
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Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Cisplatino/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Nanopartículas/uso terapéutico , Titanio/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Autofagosomas , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Beclina-1/genética , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Combinación de Medicamentos , Sinergismo Farmacológico , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , Necroptosis/efectos de los fármacos , Tamaño de la Partícula , Distribución Aleatoria , Bazo/efectos de los fármacos , Titanio/administración & dosificación , Carga Tumoral/efectos de los fármacosRESUMEN
P2Et extract obtained from the Caesalpinia spinosa plant is abundant in phenolic compounds such as gallic acid and ethyl gallate and can generate signals to activate the immune response by inducing a mechanism known as immunogenic cell death in murine models of breast cancer and melanoma. Immunogenic cell death involves mechanisms such as autophagy, which can be modulated by various natural compounds, including phenolic compounds with a structure similar to those found in P2Et extract. Here, we determine the role of autophagy in apoptosis and the generation of immunogenic signals using murine wild-type B16-F10 melanoma cells and cells with beclin-1 gene knockout. We show that P2Et extract and ethyl gallate induced autophagy, partially protecting tumor cells from death and promoting calreticulin exposure and the release of ATP. Although ethyl gallate showed a mechanism similar to that of P2Et, the induction of apoptosis and immunogenic signals was significantly weaker. In contrast, gallic acid-induced autophagy acted by blocking autophagic flux, which was associated with increased cell death. However, this compound did not induce any of the immunogenic death signals evaluated. Therefore, the complex extract has greater antitumor potential than isolated compounds. Here, we show that inducing autophagic flux with P2Et protects cancer cells from cell death and that this delay in cell death is required for the generation of immunogenic signals.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Beclina-1/genética , Caesalpinia/química , Línea Celular Tumoral , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Ácido Gálico/uso terapéutico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéuticoRESUMEN
Rhodnius prolixus is an obligatorily hematophagous insect known as an important vector of Chagas disease. Autophagy is a conserved cellular mechanism that acts in response to nutrient starvation, where components of the cytoplasm are sequestered by a double membrane organelle, named autophagosome, which is targeted to fuse with the lysosome for degradation. Lipophagy is the process of lipid degradation by selective autophagy, where autophagosomes sequester lipid droplets and degrade triacylglycerol (TAG) generating free fatty acids for ß-oxidation. Here, two essential genes of the autophagic pathway, Atg6/Beclin1 (RpAtg6) and Atg8/LC3 (RpAtg8), were silenced and the storage of lipids during starvation in Rhodnius prolixus was monitored. We found that RNAi knockdown of both RpAtg6 and RpAtg8 resulted in higher levels of TAG in the fat body and the flight muscle, 24 days after the blood meal, as well as a larger average diameter of the lipid droplets in the fat body, as seen by Nile Red staining under the confocal fluorescence microscope. Silenced starved insects had lower survival rates when compared to control insects. Accordingly, when examined during the starvation period for monitored activity, silenced insects had lower spontaneous locomotor activity and lower forced flight rates. Furthermore, we found that some genes involved in lipid metabolism had their expression levels altered in silenced insects, such as the Brummer lipase (down regulated) and the adipokinetic hormone receptor (up regulated), suggesting that, as previously observed in mammalian models, the autophagy and neutral lipolysis machineries are interconnected at the transcriptional level. Altogether, our data indicate that autophagy in the fat body is important to allow insects to mobilize energy from lipid stores.
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Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Beclina-1/genética , Silenciador del Gen , Proteínas de Insectos/genética , Insectos Vectores/genética , Rhodnius/genética , Triglicéridos/metabolismo , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Beclina-1/metabolismo , Enfermedad de Chagas , Cuerpo Adiposo/metabolismo , Femenino , Privación de Alimentos , Proteínas de Insectos/metabolismo , Insectos Vectores/crecimiento & desarrollo , Insectos Vectores/metabolismo , Ninfa/crecimiento & desarrollo , Ninfa/metabolismo , Rhodnius/crecimiento & desarrollo , Rhodnius/metabolismoRESUMEN
Autophagy is upregulated in adipose tissue (AT) from people with obesity. We showed that activation of the calcium-sensing receptor (CaSR) elevates proinflammatory cytokines through autophagy in preadipocytes. Our aim is to understand the role of CaSR on autophagy in AT from humans with obesity. We determined mRNA and protein levels of CaSR and markers of autophagy by qPCR and western blot in human visceral AT explants or isolated primary preadipocytes (60 donors: 72% female, 23-56% body fat). We also investigated their association with donors' anthropometric variables. Donors' % body fat and CaSR mRNA expression in AT were correlated (r = 0.44, p < 0.01). CaSR expression was associated with mRNA levels of the autophagy markers atg5 (r = 0.37, p < 0.01), atg7 (r = 0.29, p < 0.05) and lc3b (r = 0.40, p < 0.01). CaSR activation increased becn and atg7 mRNA expression in AT. CaSR activation also upregulated LC3II by ~50%, an effect abolished by the CaSR inhibitor. Spermine (CaSR agonist) regulates LC3II through the ERK1/2 pathway. Structural equation model analysis suggests a link between donors' AT CaSR expression, AT autophagy and expression of Tumor Necrosis Factor alpha TNF-α. CaSR expression in visceral AT is directly associated with % body fat, and CaSR activation may contribute to obesity-related disruption in AT autophagy.
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Autofagia , Grasa Intraabdominal/metabolismo , Obesidad/metabolismo , Receptores Sensibles al Calcio/metabolismo , Adipocitos/metabolismo , Adulto , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Células Cultivadas , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
INTRODUCTION AND OBJECTIVES: The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing. Previous studies indicated that Liraglutide, glucagon-like peptide-1 analogue, could regulate glucose homeostasis as a valuable treatment for Type 2 Diabetes. However, the precise effect of Liraglutide on NAFLD model in rats and the mechanism remains unknown. In this study, we investigated the molecular mechanism by which Liraglutide ameliorates hepatic steatosis in a high-fat diet (HFD)-induced rat model of NAFLD in vivo and in vitro. MATERIALS AND METHODS: NALFD rat models and hepatocyte steatosis in HepG2 cells were induced by HFD and palmitate fatty acid treatment, respectively. AMPK inhibitor, Compound C was added in HepG2 cells. Autophagy-related proteins LC3, Beclin1 and Atg7, and AMPK pathway-associated proteins were evaluated by Western blot and RT-PCR. RESULTS: Liraglutide enhanced autophagy as showed by the increased expression of the autophagy markers LC3, Beclin1 and Atg7 in HFD rats and HepG2 cells treated with palmitate fatty acid. In vitro, The AMPK inhibitor exhibited an inhibitory effect on Liraglutide-induced autophagy enhancement with the deceased expression of LC3, Beclin1 and Atg7. Additionally, Liraglutide treatment elevated AMPK levels and TSC1, decreased p-mTOR expression. CONCLUSIONS: Liraglutide could upregulate autophagy to decrease lipid over-accumulation via the AMPK/mTOR pathway.
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Autofagia/efectos de los fármacos , Liraglutida/farmacología , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Adenilato Quinasa/efectos de los fármacos , Adenilato Quinasa/metabolismo , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/efectos de los fármacos , Beclina-1/genética , Beclina-1/metabolismo , Dieta Alta en Grasa , Células Hep G2 , Humanos , Técnicas In Vitro , Hígado/metabolismo , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Palmitatos/farmacología , Ratas , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa/efectos de los fármacos , Proteína 1 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
The first-line chemotherapy treatment for Glioblastoma (GBM) - the most aggressive and frequent brain tumor - is temozolomide (TMZ). The Sonic hedgehog (SHH) pathway is involved with GBM tumorigenesis and TMZ chemoresistance. The role of SHH pathway inhibition in the potentiation of TMZ's effects using T98G, U251, and GBM11 cell lines is investigated herein. The combination of GANT-61 and TMZ over 72 hr suggested a synergistic effect. All TMZ-resistant cell lines displayed a significant decrease in cell viability, increased DNA fragmentation and loss of membrane integrity. For T98G cells, G2 /M arrest was observed, while U251 cells presented a significant increase in reactive oxygen species production and catalase activity. All the cell lines presented acidic vesicles formation correlated to Beclin-1 overexpression. The combined treatment also enhanced GLI1 expression, indicating the presence of select resistant cells. The selective inhibition of the SHH pathway potentiated the cytotoxic effect of TMZ, thus becoming a promising in vitro strategy for GBM treatment.
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Beclina-1/genética , Glioblastoma/tratamiento farmacológico , Proteínas Hedgehog/genética , Temozolomida/farmacología , Proteína con Dedos de Zinc GLI1/genética , Animales , Antineoplásicos Alquilantes/farmacología , Autofagia/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Humanos , Ratones , Estrés Oxidativo/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Autophagy is a catabolic pathway required for cellular and organism homeostasis. Autophagy participates in the innate and adaptive immune responses at different levels. Xenophagy is a class of selective autophagy that involves the elimination of intracellular pathogens. Trypanosoma cruzi is the causative agent of Chagas, a disease that affects 8 million individuals worldwide. Previously, our group has demonstrated that autophagy participates in the invasion of T. cruzi in non-phagocytic cells. In this work we have studied the involvement of autophagy in the development of T. cruzi infection in mice. Beclin-1 is a protein essential for autophagy, required for autophagosome biogenesis and maturation. We have performed an acute model of infection on the autophagic deficient Beclin-1 heterozygous knock-out mice (Bcln±) and compared to control Bcln+/+ animals. In addition, we have analyzed the infection process in both peritoneal cells and RAW macrophages. Our results have shown that the infection was more aggressive in the autophagy-deficient mice, which displayed higher numbers of parasitemia, heart´s parasitic nests and mortality rates. We have also found that peritoneal cells derived from Bcln± animals and RAW macrophages treated with autophagy inhibitors displayed higher levels of infection compared to controls. Interestingly, free cytosolic parasites recruited LC3 protein and other markers of xenophagy in control compared to autophagy-deficient cells. Taken together, these data suggest that autophagy plays a protective role against T. cruzi infection in mice, xenophagy being one of the processes activated as part of the repertoire of immune responses generated by the host.
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Autofagia , Enfermedad de Chagas/inmunología , Interacciones Huésped-Patógeno , Animales , Beclina-1/genética , Beclina-1/inmunología , Enfermedad de Chagas/mortalidad , Modelos Animales de Enfermedad , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células RAW 264.7 , Trypanosoma cruziRESUMEN
In oviparous animals, the egg yolk is synthesized by the mother in a major metabolic challenge, where the different yolk components are secreted to the hemolymph and delivered to the oocytes mostly by endocytosis. The yolk macromolecules are then stored in a wide range of endocytic-originated vesicles which are collectively referred to as yolk organelles and occupy most of the mature oocytes cytoplasm. After fertilization, the contents of these organelles are degraded in a regulated manner to supply the embryo cells with fundamental molecules for de novo synthesis. Yolk accumulation and its regulated degradation are therefore crucial for successful development, however, most of the molecular mechanisms involved in the biogenesis, sorting and degradation of targeted yolk organelles are still poorly understood. ATG6 is part of two PI3P-kinase complexes that can regulate the recruitment of the endocytic or the autophagy machineries. Here, we investigate the role of RpATG6 in the endocytosis of the yolk macromolecules and in the biogenesis of the yolk organelles in the insect vector Rhodnius prolixus. We found that vitellogenic females express high levels of RpATG6 in the ovaries, when compared to the levels detected in the midgut and fat body. RNAi silencing of RpATG6 resulted in yolk proteins accumulated in the vitellogenic hemolymph, as a consequence of poor uptake by the oocytes. Accordingly, the silenced oocytes are unviable, white (contrasting to the control pink oocytes), smaller (62% of the control oocyte volume) and accumulate only 40% of the yolk proteins, 80% of the TAG and 50% of the polymer polyphosphate quantified in control oocytes. The cortex of silenced oocytes present atypical smaller vesicles indicating that the yolk organelles were not properly formed and/or sorted, which was supported by the lack of endocytic vesicles near the plasma membrane of silenced oocytes as seen by TEM. Altogether, we found that RpATG6 is central for the mechanisms of yolk accumulation, emerging as an important target for further investigations on oogenesis and, therefore, reproduction of this vector.
Asunto(s)
Beclina-1/genética , Yema de Huevo/metabolismo , Proteínas de Insectos/genética , Insectos Vectores/embriología , Rhodnius/embriología , Animales , Beclina-1/metabolismo , Femenino , Silenciador del Gen , Proteínas de Insectos/metabolismo , Insectos Vectores/metabolismo , Orgánulos/genética , Orgánulos/metabolismo , Rhodnius/genética , Rhodnius/metabolismoRESUMEN
Coxiella burnetii, the etiologic agent of Q fever, is a Gram-negative obligate intracellular bacterium. It has been previously described that both the endocytic and autophagic pathways contribute to the Coxiella replicative vacuole (CRV) generation. Galectins are ß-galactoside-binding lectins that accumulate in the cytosol before being secreted via a non-conventional secretory pathway. It has been shown that Galectin-3, -8, -9 monitor bacteria vacuolar rupture and endosomal and lysosomal loss of membrane integrity through binding of host glycans exposed in the cytoplasm after membrane damage. Using microinjection of fluorescence-coupled dextrans, a FRET assay, and galectins distribution, we demonstrate that Coxiella infection actually result in transient phagosomal/CRV membrane damage in a Dot/Icm-dependent manner. We also show the association of different adaptor molecules involved in autophagy and of LC3 to the limiting membrane of the CRV. Moreover, we show that upon autophagy inhibition, the proportion of CRVs labeled with galectins and less acidified increases which is associated with bacteria replication impairment. Based on these observations, we propose that autophagy can facilitate resealing of intracellular damaged membranes.
Asunto(s)
Autofagia/fisiología , Coxiella burnetii/fisiología , Citosol/metabolismo , Galectinas/metabolismo , Vacuolas/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Beclina-1/genética , Células CHO , Membrana Celular , Chlorocebus aethiops , Coxiella burnetii/genética , Coxiella burnetii/crecimiento & desarrollo , Coxiella burnetii/patogenicidad , Cricetulus , Técnicas de Silenciamiento del Gen , Células HeLa , Interacciones Huésped-Patógeno/fisiología , Humanos , Concentración de Iones de Hidrógeno , Viabilidad Microbiana , Fagosomas/metabolismo , Polisacáridos/metabolismo , Fiebre Q/microbiología , Fiebre Q/patología , Células VeroRESUMEN
Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. We first used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila melanogaster eye. Subsequently, we inhibited their expression in D. melanogaster neurons under the direction of a Dopa decarboxylase (Ddc) transgene, and examined the effects on longevity and motor function. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, we investigated the roles of these genes in the well-studied α-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies.
Asunto(s)
Autofagia/genética , Beclina-1/genética , Dopa-Decarboxilasa/genética , Proteínas de Drosophila/genética , Enfermedad de Parkinson/genética , Animales , Animales Modificados Genéticamente , Beclina-1/biosíntesis , Modelos Animales de Enfermedad , Dopa-Decarboxilasa/biosíntesis , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/genética , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Longevidad/genética , Actividad Motora/genética , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/patología , Interferencia de ARN , alfa-Sinucleína/genéticaRESUMEN
Doxorubicin (Doxo) is one of the most effective anti-neoplastic agents but its cardiotoxicity has been an important clinical limitation. The major mechanism of Doxo-induced cardiotoxicity is associated to its oxidative capacity. However, other processes are also involved with significant consequences for the cardiomyocyte. In recent years, a number of studies have investigated the role of autophagy on Doxo-induced cardiotoxicity but to date it is not clear how Doxo alters that process and its consequence on cardiomyocytes viability. Here we investigated the effect of Doxo 1uM for 24h of stimulation on cultured neonatal rat cardiomyocytes. We showed that Doxo inhibits basal autophagy. This inhibition is due to both Akt/mTOR signaling pathway activation and Beclin 1 level decrease. To assess the role of autophagy on Doxo-induced cardiomyocyte death, we evaluated the effects 3-methyladenine (3-MA), bafilomycin A1 (BafA), siRNA Beclin 1 (siBeclin 1) and rapamycin (Rapa) on cell viability. Inhibition of autophagy with 3-MA, BafA and siBeclin 1 increased lactate dehydrogenase (LDH) release but, when autophagy was induced by Rapa, Doxo-induced cardiomyocyte death was decreased. These results suggest that Doxo inhibits basal autophagy and contributes to cardiomyocyte death. Activation of autophagy could be used as a strategy to protect the heart against Doxo toxicity.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Autofagia , Cardiotoxinas/toxicidad , Doxorrubicina/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Animales , Apoptosis/efectos de los fármacos , Beclina-1/genética , Beclina-1/metabolismo , Supervivencia Celular/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Macrólidos/farmacología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Miocitos Cardíacos/citología , Ratas , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The aim of this study was to evaluate the effects of miR-26a on Beclin 1 expression in retinoblastoma (RB) cell lines (Y79 and WERi-RB-1). RB cells were transfected with miR-26a mimic, antagomir-26a, or control mimic. The Beclin 1 mRNA and protein levels were detected by quantitative polymerase chain reaction and western blot, respectively. The activity of Beclin 1 3ê-UTR reporter gene was detected with the luciferase assay. After transfection with miR-26a mimic, Beclin 1 mRNA and protein levels as well as the activity of the 3'-UTR reporter gene decreased. However, all were increased upon inhibition of miR-26a with antagomir-26a. Beclin 1 is the target of miR-26a in human RB cell lines Y79 and WERi-RB-1, and miR-26a inhibits the expression of Beclin 1 by reducing its mRNA and protein levels.
Asunto(s)
Beclina-1/biosíntesis , MicroARNs/genética , Neoplasias de la Retina/genética , Retinoblastoma/genética , Regiones no Traducidas 3' , Beclina-1/antagonistas & inhibidores , Beclina-1/genética , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/metabolismo , Retinoblastoma/patología , TransfecciónRESUMEN
We investigated the associations between Beclin-1 and microRNA-30a (miR-30a) expression and the severity and treatment response in colorectal cancer (CRC). Our sample size consisted of 139 CRC patients who were treated with surgery alone. Immunohistochemistry was used to investigate the expression and prognostic significance of Beclin-1 in CRC, while the weak expression of Beclin-1 in normal tissue was used as the basis for assessing tumors (control group). Real-time reverse transcription-polymerase chain reaction quantified miR-30a levels. The expression levels of Beclin-1 and miR-30a were associated with clinical variables and prognoses. Beclin-1 was expressed more highly in CRC tissues than in controls. This expression was related to gender (P = 0.023), histological grade (P = 0.006), M stage (P = 0.004), tumor node metastasis stage (P = 0.020), vascular invasion, and nodal involvement. Patients with higher Beclin-1 expression levels had higher survival rates (P = 0.08) than patients with lower Beclin-1 expression levels. Beclin-1 was a prognostic indicator (P < 0.05) in a multivariate analysis. Beclin-1 was overexpressed in CRC tissues and was correlated with lower levels of miR-30a (P < 0.05, r = -0. 4189). In conclusion, Beclin-1 was a good prognostic indicator in CRC and was correlated with survival rate. Beclin-1 is important in the growth and metastasis of CRC. Apoptosis in CRC might be due to the increased autophagy induced by decreased levels of miR-30a.