RESUMEN
Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Neoplasias Pulmonares , Ubiquitina-Proteína Ligasas , Humanos , Regiones no Traducidas 5' , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína 1 Similar a ELAV/metabolismoRESUMEN
Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.
Asunto(s)
Elementos Transponibles de ADN , Proteínas Fluorescentes Verdes , Ratas Transgénicas , Animales , Elementos Transponibles de ADN/genética , Proteínas Fluorescentes Verdes/genética , Ratas , Técnicas de Transferencia de Gen/veterinaria , Transgenes , Masculino , Silenciador del Gen , Femenino , Regiones Promotoras GenéticasRESUMEN
The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a pandemic, prompting rapid vaccine development. Although vaccines are effective, the occurrence of rare adverse events following vaccination highlights the necessity of determining whether the benefits outweigh the risks posed by the infection itself. The recombinant Vesicular Stomatitis Virus (rVSV) platform is a promising vector for vaccines against emerging viruses. However, limited studies have evaluated the genotoxicity and safety pharmacology of this viral vector vaccine, which is crucial to ensure the safety of vaccines developed using this platform. Hence, the present study aimed to assess the genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc COVID-19 vaccine using micronucleus and comet assays, as well as neurobehavioral, body temperature, respiratory, and cardiovascular assessments in Sprague-Dawley rats and beagle dogs. The intramuscular administration of rVSVInd(GML)-mspSGtc at doses up to 1.5 × 109 PFU/animal did not increase the number of bone marrow micronucleated polychromatic erythrocytes or cause liver DNA damage. Additionally, it had no significant impact on neurobehavioral functions in rats and showed marginal temporary changes in body temperature, respiratory rate, heart rate, and electrocardiogram parameters in rats and dogs, all of which resolved within 24 h. Overall, following genotoxicity and pharmacological safety assessments, rVSVInd(GML)-mspSGtc displayed no notable systemic adverse effects in rats and dogs, suggesting its potential as a vaccine candidate for human clinical trials.
Asunto(s)
Vacunas contra la COVID-19 , Pruebas de Micronúcleos , Ratas Sprague-Dawley , SARS-CoV-2 , Animales , Perros , Vacunas contra la COVID-19/toxicidad , Ratas , Masculino , SARS-CoV-2/inmunología , SARS-CoV-2/efectos de los fármacos , COVID-19/prevención & control , Femenino , Daño del ADN/efectos de los fármacos , Ensayo Cometa , Vesiculovirus/efectos de los fármacos , Vacunas Sintéticas/inmunología , Temperatura Corporal/efectos de los fármacosRESUMEN
We identified a gene, subunit C3 (ATP5G3) of mitochondrial ATP synthase, that displayed changes in gene expression under oxidative stress. We examined the role of ATP5G3 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using ATP5G3 small interfering RNA (siATP5G3)-transfected HeLa cells. A significant increase in cytotoxicity was observed in the transfected cells treated with SNP, which suggests a protective role of ATP5G3 in SNP-induced cytotoxicity in the cells. The transfected cells treated with photodegraded SNP showed equal cytotoxicity to SNP, and pretreatment with deferoxamine (DFO) completely inhibited this cytotoxicity. Further, cytotoxicity was significantly inhibited by pretreatment with a p38 inhibitor and was accentuated by the p38 activator in cells. Pretreatment with the Bcl-xL inhibitor also significantly accentuated cytotoxicity. The increase in p38 phosphorylation was significantly higher in siATP5G3-transfected cells treated with SNP in immunoblotting, which was inhibited by pretreatment with DFO. The increase in cytotoxicity with siATP5G3 transfection was completely blocked by cotransfection with sip38, and the blocking effect disappeared by cotransfection with additional siBcl-xL, which suggests that the protective role of ATP5G3 is mediated by Bcl-xL via the inhibition of p38 activity. Cytotoxicity was completely blocked by the cotransfection of siATP5G3 with siBax. No change in apoptotic parameters was observed during cytotoxicity. However, pretreatment with lysosomal inhibitors significantly inhibited cytotoxicity and increased p62 protein levels. These findings suggest that ATP5G3 plays a protective role in autophagic cell death/lysosome-associated cell death induced by SNP via the sequential signaling of ROS/p38/Bcl-xL/Bax in HeLa cells.
Asunto(s)
Carcinoma , Humanos , Apoptosis , Muerte Celular , Línea Celular Tumoral , Células HeLa , Nitroprusiato/farmacologíaRESUMEN
Bcl-2-interacting cell death suppressor (BIS), also called BAG3, plays a role in physiological functions such as anti-apoptosis, cell proliferation, autophagy, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality accompanied by abnormalities in cardiac and skeletal muscles, suggesting the critical role of BIS in these muscles. In this study, we generated skeletal muscle-specific Bis-knockout (Bis-SMKO) mice for the first time. Bis-SMKO mice exhibit growth retardation, kyphosis, a lack of peripheral fat, and respiratory failure, ultimately leading to early death. Regenerating fibers and increased intensity in cleaved PARP1 immunostaining were observed in the diaphragm of Bis-SMKO mice, indicating considerable muscle degeneration. Through electron microscopy analysis, we observed myofibrillar disruption, degenerated mitochondria, and autophagic vacuoles in the Bis-SMKO diaphragm. Specifically, autophagy was impaired, and heat shock proteins (HSPs), such as HSPB5 and HSP70, and z-disk proteins, including filamin C and desmin, accumulated in Bis-SMKO skeletal muscles. We also found metabolic impairments, including decreased ATP levels and lactate dehydrogenase (LDH) and creatine kinase (CK) activities in the diaphragm of Bis-SMKO mice. Our findings highlight that BIS is critical for protein homeostasis and energy metabolism in skeletal muscles, suggesting that Bis-SMKO mice could be used as a therapeutic strategy for myopathies and to elucidate the molecular function of BIS in skeletal muscle physiology.
Asunto(s)
Músculo Esquelético , Enfermedades Musculares , Animales , Ratones , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Atrofia Muscular/metabolismo , Metabolismo Energético , Fosforilación , Ratones NoqueadosRESUMEN
Bcl-2-interacting cell death suppressor (BIS), also called as BAG3, regulates numerous physiological processes, such as apoptosis, protein quality control, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality following cardiac and skeletal muscle dysfunction. The first attempt to generate organ-specific knockout mice resulted in constitutive or inducible heart-specific Bis-knockout mice, which exhibited cardiac dilation and underwent premature death. Here, we generated hepatocyte-specific Bis-knockout (Bis-HKO) mice and found no abnormalities in metabolic function and survival. However, depletion of HSPB8 and accumulation of p62 indicated impaired autophagy in Bis-HKO livers. Interestingly, the number of peroxisomes wrapped by phagophore membranes increased as evidenced by transmission electron microscopy analysis, indicating defects in the progression of pexophagy. In addition, increased dihydroethidine intensities and histone H3 K9me3-positive nuclei indicated increased oxidative stress and senescence induction in Bis-HKO livers. Mechanistically, p27 was upregulated in Bis-HKO livers. In SNU368 hepatocellular carcinoma cells, BIS depletion led to p27 upregulation, and increase in histone H3 K9me3 levels and senescence-associated ß-galactosidase staining; therefore, reproducing the in vivo senescence phenotype. Despite the observation of no metabolic abnormalities, BIS depletion led to defective autophagy, increased oxidative stress, and senescence in Bis-HKO livers. Collectively, our results suggest a role for BIS in maintaining liver regeneration potential under pathological conditions.
Asunto(s)
Histonas , Neoplasias Hepáticas , Animales , Senescencia Celular/genética , Hepatocitos/metabolismo , Histonas/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas/patología , Regeneración Hepática/fisiología , Ratones , Ratones NoqueadosRESUMEN
Alterations in sialylation of terminal residues of glycoproteins have been implicated in forming tumor-associated glycans. ST6GALNAC transfers sialyl moiety to N-acetylgalactosamine residue via α2,6 linkage. Although the oncogenic characteristics of ST6GALNACI or II have been demonstrated in various cancer cells, the impact of ST6GALNACIII on tumor progression remains undefined. In this study, we evaluated the effect of ST6GALNACIII knockdown on the growth of A549 non-small cell lung cancer cells. ST6GALNACIII depletion resulted in significant retardation in growth of A549 cells under various culture conditions, including collagen-supported 3D culture and anchorage-independent soft agar culture conditions. Liquid chromatography with tandem mass spectrometry revealed that two glycopeptides of transferrin receptor protein 1 (TFR1) containing N-acetylhexosamine-sialic acid were not detected in ST6GALNACIII-depleted A549 cells compared with control cells. Subsequent lectin binding assay, western blotting, and real-time RT-PCR indicated that TFR1 sialylation was not significantly changed, but TFR1 protein and mRNA expressions were decreased after ST6GALNACIII knockdown. However, cell growth retardation by ST6GALNACIII knockdown was partially rescued by TFR1 overexpression. Additionally, TFR1 mRNA degradation was accelerated following ST6GALNACIII knockdown with concomitant reduction in mRNA levels of iron regulatory protein 1 and 2, the upstream regulators of TFR1 mRNA stability. Therefore, our results indicated an important role of ST6GALNACIII in promoting A549 cell growth through quantitative regulation of TFR1 expression and provided therapeutic implications for ST6GALNACIII targeting in tumor growth suppression in vivo.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/prevención & control , Hierro/metabolismo , Neoplasias Pulmonares/prevención & control , Estabilidad del ARN , Receptores de Transferrina/antagonistas & inhibidores , Sialiltransferasas/deficiencia , Antígenos CD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptores de Transferrina/metabolismoRESUMEN
The reaction of ß-ketonitrile and N-substituted pyrrole-2-carboxaldehyde in the presence of piperidinium acetate in CH3CN permitted regioselective access to 5-acylindolizine-7-carbonitrile through a domino Knoevenagel condensation-intramolecular aldol cyclization sequence. A wide range of new indolizine derivatives with a poly-functionalized pyridine moiety were readily synthesized in an atom-economical fashion, thereby expanding the indolizine chemical space for further applications.
RESUMEN
A new pyrrolo[1,2-a]pyrazine chemical space with a poly-substituted pyrazine unit was readily accessed by Sc(OTf)3-catalyzed one-pot three-component coupling of a pyrrole derivative, amine, and trialkylphosphite under environment-friendly conditions. The formation of multiple bonds (two C-N and one C-P) via a domino process consisting of the chemoselective Kabachnik-Fields reaction and intramolecular cyclodehydration allowed for the construction of highly functionalized pyrazines.
RESUMEN
BCL-2 interacting cell death suppressor (BIS) is a multifunctional protein that has been implicated in cancer and myopathy. Various mutations of the BIS gene have been identified as causative of cardiac dysfunction in some dilated cardiomyopathy (DCM) patients. This was recently verified in cardiac-specific knock-out (KO) mice. In this study, we developed tamoxifen-inducible cardiomyocyte-specific BIS-KO (Bis-iCKO) mice to assess the role of BIS in the adult heart using the Cre-loxP strategy. The disruption of the Bis gene led to impaired ventricular function and subsequent heart failure due to DCM, characterized by reduced left ventricular contractility and dilatation that were observed using serial echocardiography and histology. The development of DCM was confirmed by alterations in Z-disk integrity and increased expression of several mRNAs associated with heart failure and remodeling. Furthermore, aggregation of desmin was correlated with loss of small heat shock protein in the Bis-iCKO mice, indicating that BIS plays an essential role in the quality control of cardiac proteins, as has been suggested in constitutive cardiac-specific KO mice. Our cardiac-specific BIS-KO mice may be a useful model for developing therapeutic interventions for DCM, especially late-onset DCM, based on the distinct phenotypes and rapid progressions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Cardiomiopatía Dilatada/genética , Animales , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Noqueados , Miocardio/patologíaRESUMEN
KRIBB11, an HSF1 inhibitor, was shown to sensitize various types of cancer cells to treatment with several anticancer drugs. However, the exclusive effects of KRIBB11 in preventing the growth of glioblastoma cells and the related mechanisms have not been elucidated yet. Herein, we aimed to examine the potential of KRIBB11 as an anticancer agent for glioblastoma. Using MTT and colony formation assays and Western blotting for c-PARP, we demonstrated that KRIBB11 substantially inhibits the growth of A172 glioma cells by inducing apoptosis. At the molecular level, KRIBB11 decreased anti-apoptotic protein MCL-1 levels, which was attributable to the increase in MULE ubiquitin ligase levels. However, the constitutive activity of HSF1 in A172 cells was not influenced by the exclusive treatment with KRIBB11. Additionally, based on cycloheximide chase assay, we found that KRIBB11 markedly retarded the degradation of MULE. In conclusion, stabilization of MULE upon KRIBB11 treatment is apparently an essential step for degradation of MCL-1 and the subsequent induction of apoptosis in A172 cells. Our results have expanded the knowledge on molecular pathways controlled by KRIBB11 and could be potentially effective for developing an inhibitory therapeutic strategy for glioblastoma.
Asunto(s)
Aminopiridinas/farmacología , Glioblastoma/tratamiento farmacológico , Indazoles/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Aminopiridinas/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Glioblastoma/metabolismo , Glioma/tratamiento farmacológico , Glioma/metabolismo , Humanos , Indazoles/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Supresoras de Tumor/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Digoxin, a compound of the cardiac glycoside family, was originally prescribed for heart failure but has recently been rediscovered for its potent antitumor activity. However, it has a narrow therapeutic margin due to its cardiotoxicity, limiting its safe use as an antitumor agent in clinical practice. To widen its therapeutic margin, we investigated whether the antitumor effect of digoxin is potentiated by the depletion of BCL-2-interacting cell death suppressor (BIS) in A549 lung cancer cells. BIS is a multifunctional protein that is frequently overexpressed in most human cancers including lung cancer. Our results demonstrated that the inhibitory potential of digoxin on the migratory behavior of A549 cells is significantly enhanced by BIS depletion as assessed by transwell assay and collagen-incorporated 3D spheroid culture. Western blotting revealed that combination treatment significantly reduces p-STAT3 expression. In addition, a STAT3 inhibitor substantially suppressed the aggressive phenotypes of A549 cells. Thus, our results suggest that loss of STAT3 activity is a possible molecular mechanism for the synergistic effect of digoxin and BIS depletion. Our findings suggest the sensitizing role of BIS silencing to reduce the dose of digoxin for treatment of lung cancer with a high metastatic potential.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Movimiento Celular/efectos de los fármacos , Digoxina/farmacología , Regulación hacia Abajo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Células A549 , Supervivencia Celular/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Invasividad NeoplásicaRESUMEN
As a means to make new benzofuran-embedded polycyclic structures, we established two efficient one-pot sequential coupling routes to 2-amino-3-arylbenzofurans and 2-amino-3-arylnaphtho[2,1-b]furans. Further ring formation (six- and seven-membered rings) with the resulting amine moiety at the C2 position of benzofurans was realized, leading to further expansion of benzofuran-based chemical space.
RESUMEN
Chemical territory bearing a 2,2-dimethyl-2H-chromene motif was expanded by utilizing an o-hydroxy aldehyde group of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde as a synthetic handle to install distinctive morphology and functionality of each scaffold. Cell based assays and in silico docking analysis led us to discover that these new compounds exhibit inhibitory effect on anoctamin1 (ANO1). ANO1 is amplified and highly expressed in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer. Biological assays revealed that (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (3n, Ani-FCC) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 µM. 3n showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, 3n strongly decreased cell viability of PC-3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, 3n significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC-3 and FaDu cells. This study shows that a novel ANO1 inhibitor, 3n, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.
Asunto(s)
Anoctamina-1/antagonistas & inhibidores , Benzopiranos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Anoctamina-1/metabolismo , Apoptosis/efectos de los fármacos , Benzopiranos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Proteínas de Neoplasias/metabolismoRESUMEN
Autophagy, a lysosomal self-degradative process of cellular components, is essential for cellular homeostasis to response cellular stress and is tightly controlled by autophagy-related genes (ATGs). Autophagy-related gene 6 (ATG6, also known as Beclin-1 in human) is an essential factor regulating autophagy and apoptosis. RNA binding proteins (RBPs) regulate gene expression at the post-transcriptional level and their differential expression is linked to the pathogenesis of several human diseases. Here, we demonstrate the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a novel factor regulating ATG6 expression. hnRNPA1 associates with the 3' untranslated region (3'UTR) of ATG6 mRNA and promotes its expression without significant changes at the mRNA level. Knockdown of hnRNPA1 decreases ATG6 expression, which is enhanced by the overexpression of hnRNPA1. Also, we show augmented expression of both hnRNPA1 and ATG6 in the colorectal cancer (CRC) tissues obtained from patients and demonstrate a positive correlation of their expression in CRC tissues. Our results suggest the potential role of hnRNPA1-mediated ATG6 regulation in the pathogenesis of CRC.
Asunto(s)
Beclina-1/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea A1/genética , Regiones no Traducidas 3' , Autofagia , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Células HCT116 , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Osteopontin (OPN, SPP1) is upregulated in response to acute brain injury, and based on its immunoreactivity, two distinct forms have been identified: intracellular OPN within brain macrophages and small granular OPN, identified as OPN-coated degenerated neurites. This study investigates the spatiotemporal relationship between punctate OPN deposition and astroglial and microglial reactions elicited by 3-nitropropionic acid (3-NP). METHODS: Male Sprague-Dawley rats were intraperitoneally injected with mitochondrial toxin 3-NP and euthanized at 3, 7, 14, and 28 days. Quantitative and qualitative light and electron microscopic techniques were used to assess the relationship between OPN and glial cells. Statistical significance was determined by Student's t test or a one-way analysis of variance followed by Tukey's multiple comparisons test. RESULTS: Punctate OPN-immunoreactive profiles were synthesized and secreted by amoeboid-like brain macrophages in the lesion core, but not by reactive astrocytes and activated microglia with a stellate shape in the peri-lesional area. Punctate OPN accumulation was detected only in the lesion core away from reactive astrocytes in the peri-lesional area at day 3, but had direct contact with, and even overlapped with astroglial processes at day 7. The distance between the OPN-positive area and the astrocytic scar significantly decreased from days 3 to 7. By days 14 and 28 post-lesion, when the glial scar was fully formed, punctate OPN distribution mostly overlapped with the astrocytic scar. Three-dimensional reconstructions and quantitative image analysis revealed numerous granular OPN puncta inside the cytoplasm of reactive astrocytes and brain macrophages. Reactive astrocytes showed prominent expression of the lysosomal marker lysosomal-associated membrane protein 1, and ultrastructural analysis confirmed OPN-coated degenerating neurites inside astrocytes, suggesting the phagocytosis of OPN puncta by reactive astrocytes after injury. CONCLUSIONS: Punctate OPN-immunoreactive profiles corresponded to OPN-coated degenerated neurites, which were closely associated with, or completely engulfed by, the reactive astrocytes forming the astroglial scar in 3-NP lesioned striatum, suggesting that OPN may cause astrocytes to migrate towards these degenerated neurites in the lesion core to establish physical contact with, and possibly, to phagocytose them. Our results provide novel insights essential to understanding the recovery and repair of the central nervous system tissue.
Asunto(s)
Cuerpo Estriado/metabolismo , Mitocondrias/metabolismo , Neuroglía/metabolismo , Nitrocompuestos/toxicidad , Osteopontina/metabolismo , Fagocitosis/fisiología , Propionatos/toxicidad , Animales , Cuerpo Estriado/química , Cuerpo Estriado/efectos de los fármacos , Masculino , Mitocondrias/química , Mitocondrias/efectos de los fármacos , Neuroglía/química , Neuroglía/efectos de los fármacos , Osteopontina/análisis , Fagocitosis/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Efficient construction of new chemical space by way of strategic use of tandem reactions is highly important in drug discovery. Described herein is an atom-economical [4+1+1] annulation approach to 3-(hetero)aryl-4-acyl-3,4-dihydropyrrolo[1,2- a]pyrazines, a new chemical space, via a one-pot three-component reaction under mild reaction conditions. Formation of multiple bonds (one C-C and two C-N) was achieved by a cascade reaction sequence consisting of generation of a Schiff base, a diastereoselective Mannich reaction, and intramolecular imine formation. This modular and environment-friendly process allowed rapid access to a wide range of 4-acylated 3,4-dihydropyrrolo[1,2- a]pyrazines and their analogues, opening opportunity to explore biological activity associated with this scaffold.
RESUMEN
Golgi S-nitro-N-acetylpenicillamine receptor complex 1 (GS28) has been implicated in Golgi vesicle transport. We examined the role of GS28 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using GS28 siRNA (siGS28)-transfected HeLa cells. Significant inhibition of cytotoxicity was observed in the cells treated with SNP, and photodegraded SNP showed equal cytotoxicity to SNP. Pretreatment with an ERK inhibitor or siErk1 cotransfection blocked the inhibition in cytotoxicity. Additionally, increased phosphorylation of ERK was maintained in the cells treated with SNP, and Nrf2 level was dependent on ERK phosphorylation. However, pretreatment with a pan-caspase inhibitor had no effect on cytotoxicity or procaspase-3 level. Pretreatment with an autophagy inhibitor or siATG5 cotransfection blocked the inhibition of cytotoxicity. The changes of LC3 corresponded to that in siErk1-cotransfected cells. These data suggest that GS28 has an inductive role in SNP-induced cell death via inhibition of ERK, leading to inhibition of autophagic processes in HeLa cells.
Asunto(s)
Muerte Celular/efectos de los fármacos , Nitroprusiato/farmacología , Proteínas Qb-SNARE/metabolismo , Neoplasias del Cuello Uterino/patología , Autofagia/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Células HeLa , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/metabolismoRESUMEN
RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA.
Asunto(s)
Proteínas Argonautas/fisiología , Proteínas Portadoras/fisiología , MicroARNs/fisiología , Proteínas Nucleares/fisiología , Interferencia de ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Sitios de Unión , Factores Eucarióticos de Iniciación/metabolismo , Células HCT116 , Células HEK293 , Humanos , Secuencias Invertidas Repetidas , Células MCF-7 , Unión Proteica , Biosíntesis de Proteínas , Dominios Proteicos , Estabilidad del ARN , ARN Mensajero/genética , Proteínas de Unión al ARNRESUMEN
PURPOSE: We investigated the changes of tissue oxygen saturation (sO2) in sorafenib-treated HCC (hepatocelluar carcinoma) mouse models using photoacoustic imaging (PI). MATERIALS AND METHODS: Nude mice, implanted with human HCC (HepG2-RFP) cells in the liver, were randomised to the sorafenib-treated group (n = 21) or the control group (n = 20). Tumour volume and sO2 were measured by PI at baseline and then one week later, and radiant efficiency (RE) and therapeutic response were analysed by fluorescence imaging and histologic analysis. RESULTS: Sorafenib was effective in treating HCC by evaluating necrotic fraction, apoptosis index, and microvessel density (MVD). One week after treatment, the sO2 of HCC and residual healthy liver tissue decreased, and the hypoxia inducible factor-1α (HIF-1α) protein expression of HCC increased, correlating with the apoptosis index. The ΔsO2 in HCC showed a significantly positive correlation with the necrotic fraction and the apoptosis index of tumour and a negative correlation with the MVD of tumour. CONCLUSION: Sorafenib treatment results in changes of sO2 in HCC and liver parenchyma and induces the accumulation of HIF-1α by hypoxic environment. sO2 as measured by PI, can be a useful marker for non-invasive monitoring of the therapeutic response in orthotopic HCC mouse models. KEY POINTS: ⢠Hypoxia is a characteristic feature of the tumour microenvironment ⢠It is important to monitor sO 2 in HCC during sorafenib treatment ⢠PI is useful for non-invasive monitoring of sO 2 in HCC. ⢠ΔsO 2 in HCC showed a significantly correlation with tumour response.