RESUMEN
Blue light is part of the natural light spectrum that emits high energy. Currently, people are frequently exposed to blue light from 3C devices, resulting in a growing incidence of retinopathy. The retinal vasculature is complex, and retinal vessels not only serve the metabolic needs of the retinal sublayers, but also maintain electrolyte homeostasis by forming the inner blood-retinal barrier (iBRB). The iBRB, which is primarily composed of endothelial cells, has well-developed tight junctions. However, with exposure to blue light, the risks of targeting retinal endothelial cells are currently unknown. We found that endothelial claudin-5 (CLDN5) was rapidly degraded under blue light, coinciding with the activation of a disintegrin and metalloprotease 17 (ADAM17), even at non-cytotoxic lighting. An apparently broken tight junction and a permeable paracellular cleft were observed. Mice exposed to blue light displayed iBRB leakage, conferring attenuation of the electroretinogram b-wave and oscillatory potentials. Both pharmacological and genetic inhibition of ADAM17 remarkably alleviated CLDN5 degradation induced by blue light. Under untreated condition, ADAM17 is sequestered by GNAZ (a circadian-responsive, retina-enriched inhibitory G protein), whereas ADAM17 escapes from GNAZ by blue light illuminance. GNAZ knockdown led to ADAM17 hyperactivation, CLDN5 downregulation, and paracellular permeability in vitro, and retinal damage mimicked blue light exposure in vivo. These data demonstrate that blue light exposure might impair the iBRB by accelerating CLDN5 degradation through the disturbance of the GNAZ-ADAM17 axis.
Asunto(s)
Barrera Hematorretinal , Células Endoteliales , Ratones , Animales , Barrera Hematorretinal/metabolismo , Claudina-5/metabolismo , Células Endoteliales/metabolismo , Retina/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Lacticaseibacillus paracasei strain PS23 (PS23) exhibits some probiotic properties. In this study, a genomic analysis of PS23 revealed no genes related to virulence or antibiotic resistance. Moreover, ornithine decarboxylase activity was not detected in vitro. In addition, PS23 was sensitive to the tested antibiotics. Genotoxicity tests for PS23 including the Ames test and chromosomal aberrations in vitro using Chinese hamster ovary cells and micronuclei in immature erythrocytes of ICR mice were all negative. Moreover, following a 28-day study involving repeated oral dose toxicity tests (40, 400, and 4000 mg/kg equal 1.28 × 1010, 1.28 × 1011, and 1.28 × 1012 CFU/kg body weight, respectively) using an ICR mouse model, no adverse effects were observed from any doses. In addition, supplementation with live or heat-killed PS23 ameliorates DSS-induced colonic inflammation in mice. Our findings suggest that PS23 is safe and has anti-inflammatory effects and may therefore have therapeutic implications.
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Lacticaseibacillus paracasei , Cricetinae , Ratones , Animales , Lacticaseibacillus , Células CHO , Cricetulus , Ratones Endogámicos ICR , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéuticoRESUMEN
The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from â¼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 µM and an antiviral activity with EC50 of 49.89 µM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 µM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 µM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.
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Tratamiento Farmacológico de COVID-19 , Proteasas 3C de Coronavirus/química , Reposicionamiento de Medicamentos , SARS-CoV-2/enzimología , Animales , Antivirales/farmacología , Dominio Catalítico , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Nelfinavir/farmacología , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , Conformación Proteica , Glicoproteína de la Espiga del Coronavirus/química , Células VeroRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Recent studies have shown that AhR is a novel master regulator of the mucosal immune system, including lungs and intestine. To elucidate the role of AhR in chronic severe asthma, AhR wild-type and knockout mice (AhR-/- ) were sensitized and challenged with ovalbumin for 4 weeks. To uncover the underlying mechanisms, inflammatory cells profile and cytokines production were analyzed in bronchial lavage fluid (BALF) and lung tissue. Compared to wild-type mice, AhR-/- mice had exacerbated asthma symptoms, including airway inflammation, mucus production, airway hyperresponsiveness, and airway remodeling. BALF monocytes, neutrophils, eosinophils, and lymphocytes were all enhanced in OVA-immunized AhR-/- mice. In OVA-immunized AhR-/- mice, T helper (Th) 17 cell-specific cytokine IL-17A, as well as airway remodeling factors, including epithelial-mesenchymal transition (EMT) markers and vascular endothelial growth factor (VEGF), were all enhanced in lung tissue. Moreover, human cohort studies showed that AhR gene expression in bronchial epithelial cells decreases in severe asthma patients. Loss of AhR leads to worsening of allergic asthma symptoms, indicating its importance in maintaining normal lung function and mediating disease severity.
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Asma/complicaciones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Inflamación/etiología , Receptores de Hidrocarburo de Aril/fisiología , Hipersensibilidad Respiratoria/etiología , Células Th17/inmunología , Animales , Asma/inducido químicamente , Movimiento Celular , Citocinas/metabolismo , Femenino , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/toxicidad , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/patologíaRESUMEN
The aryl hydrocarbon receptor (AHR) is a ligand-activated receptor that regulates the metabolism of several xenobiotics and participates in ocular inflammation. Although severe inflammation is a major risk of retinal damage, the underlying mechanism is not well established. In this study, to elucidate how AHR mediates inflammation homeostasis, we hypothesized that AHR expression may diminish during long-term exposure to benzo [a] pyrene (B [a]P), a carcinogen in cigarette smoke. The blockage of AHR function considerably impaired suppressor of cytokine signaling 3 (SOCS3) negative feedback regulation and upregulated B [a]P-induced pro-inflammation. Signal transducer and activator of transcription 3 (STAT3) was activated by B [a] P due to AHR dysfunction in human adult retinal pigment epithelial cells (ARPE-19). The STAT3-inducible element revealed higher activity in AHR knockout cells with B [a] P treatment, but not in wild type ARPE-19 cells. Moreover, AHR dysfunction led to STAT3 hypo-ubiquitination and changed the STAT3-SOCS3 interaction. Increased STAT3-SOCS3 complex during AHR dysfunction by B [a] P was suppressed by nifuroxazide in ARPE-19 cells. Furthermore, the in vivo results showed that STAT3 inhibition during AHR impairment by long-term B [a] P exposure preserved the retina thickness and reversed the visual function in male C57Bl/6 mice. Overall, long-term B [a] P exposure may attenuate AHR function, dysregulating the homeostasis of the SOCS3-STAT3 axis with intensive STAT3 activation. This finding is significant given that the disintegration of the AHR-SOCS3 axis is a sensitive factor involved in AMD-like lesion development in the retina, revealing that the low AHR level may be associated with cigarette smoking or xenobiotics exposure, causing retina inflammation and damage.
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Receptores de Hidrocarburo de Aril/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Animales , Benzo(a)pireno/toxicidad , Carcinógenos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Hidrocarburo de Aril/genética , Factor de Transcripción STAT3/genética , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Activación TranscripcionalRESUMEN
Recently, histone deacetylase inhibitors (HDACi) have become widely used in anti-cancer treatment; however, due to acquired drug resistance and their relatively low specificity, they are largely ineffective against late-stage cancer. Thus, it is critical to elucidate the molecular mechanisms underlying these issues, so as to identify novel therapeutic targets to prevent late-stage cancer progression and resistance acquisition. The present study investigated the Aryl hydrocarbon receptor (AHR), that has been shown to mediate histone acetylation by regulating histone deacetylase (HDAC) activity during HDACi treatment in human gastric-cancer cell lines (i.e. AGS and NCI-N87 cells). The potent HDACi, Aza-PBHA, was thus shown to upregulate AHR expression in both AGS and NCI-N87 cell lines, and to increase histone acetylation levels by facilitating AHR/HDAC interactions. Conversely, AHR knockdown increased HDAC activity. Aza-PBHA also increased PKCα phosphorylation and membrane translocation; however, interestingly, PKCα inhibition reduced the Aza-PBHA-increased AHR and histone acetylation levels, and inhibited the formation of the AHR/HDAC complex, likely upregulating Aza-PBHA-inhibited cell migration. Thus, our results suggest that Aza-PBHA treatment increased AHR levels to suppress HDAC activity, and inhibited cell migration by activating PKCα activation. These findings support the use of drugs to control AHR-related epigenetic regulation as a promising potential method to prevent acquired resistance to cancer treatments.
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Movimiento Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Proteína Quinasa C-alfa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/química , Humanos , Fosforilación/efectos de los fármacos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
: Probiotics are extensively available to consumers; however, the use of probiotics may not always be safe, and there are few reports on their side effects, including those of Lactobacillus. Lactobacillus plantarum strain PS128TM isolated from spontaneously fermented mustard greens in Taiwan was recently reported to exhibit probiotic properties. In this study, we aimed to assess the safety of strain PS128TM for use in humans via examining genotoxic and oral toxic effects using in vitro and in vivo testing. Five strains of Salmonella typhimurium were evaluated by the Ames test; no signs of increased reverse mutation were observed following exposure to PS128TM. Additional testing of Chinese hamster ovary (CHO) cells exposed to PS128TM revealed that the incidence of chromosomal aberrations in CHO cells had not increased. PS128TM treatment also did not affect the proportion of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Moreover, following a 28 day study involving repeated oral dose toxicity tests (2400, 400, and 40 mg/kg body weight) utilizing an ICR mouse model, no observable adverse level (NOAEL) was found at any of the doses. PS128TM was sensitive to antibiotics; however, genes related to the production of biogenic amines were absent. While further research is required, these toxicological assessments suggest that PS128TM could be safe for human consumption.
RESUMEN
Hedychium coronarium has a long history of use worldwide as a food and in folk medicine. In this study, we aimed to investigate the effect of an aqueous extract of H. coronarium leaves (HC) on type 2 diabetes mellitus (T2DM). Two types of animal models were used in this study: Streptozotocin (STZ)-induced T2DM (Wistar rats; N = 8) and C57BKSdb/db mice (N = 5). After treatment with HC for 28 days, glucose tolerance improved in both of the diabetic animal models. As significant effects were shown after 14 days of treatment in the STZ-induced T2DM model, we carried out the experiments with it. After 28 days of treatment with HC, the levels of cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein were significantly improved in the STZ-induced T2DM model. The lesions degree of islet ß-cells was decreased after the HC treatment. Although the insulin level increased moderately, the aldosterone level was significantly decreased in the HC-treated groups, suggesting that aldosterone might play an important role in this effect. In summary, HC is a natural product and it is worth exploring its effect on T2DM.
Asunto(s)
Glucemia/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , Zingiberaceae/química , Aldosterona/sangre , Animales , Diabetes Mellitus Tipo 2/metabolismo , Insulina/sangre , Hojas de la Planta/química , Ratas , EstreptozocinaRESUMEN
Gastrointestinal mucositis is a serious side effect of chemotherapy. Currently, no effective treatment exists for chemotherapy-induced mucositis, prompting the need to develop an anti-mucositis agent for use in clinics. The present study investigated whether azatyrosine-PBHA (AzP), a histone deacetylase inhibitor, has a therapeutic effect on intestinal mucosa. The results indicated that AzP did not affect the proliferation and viability of cancer cells, outcomes that are achieved by suberoylanilide hydroxamic acid (SAHA). However, AzP could decrease production of the inflammatory mediators interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor-necrosis factor-α (TNF-α). In vivo histopathological assessment showed that AzP reduced cisplatin-induced injury to the jejunum villi and triggered weight loss in the C57BL/6 mice. Immunohistochemistry (IHC) results demonstrated that mice treated with AzP also recovered from cisplatin-induced injury to the intestinal mucosa. Mechanistic in vitro study using DAVID/KEGG enrichment analysis of microarray data and confirmation by a Western blot indicated the influence of AzP on the MEK/ERK and AKT-dependent pathway. In conclusion, the study demonstrated that AzP might regulate the MEK/ERK MAPK signaling pathway to attenuate MCP-1, TNF-α, and IL-6 production and provide opportunities for the development of new anti-inflammatory drugs targeting mucositis.
Asunto(s)
Alanina/análogos & derivados , Antineoplásicos/efectos adversos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos , Mucositis/etiología , Mucositis/patología , Alanina/química , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/química , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Masculino , Ratones , Estructura Molecular , Mucositis/tratamiento farmacológico , RatasRESUMEN
Glial activation and neuroinflammatory processes play important roles in the pathogenesis of brain abscess and neurodegenerative diseases. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which contribute to the exacerbation of neuronal cell death. The inhibition of glial activation has been shown to alleviate neurodegenerative conditions. The present study was to investigate the specific HDAC8 inhibitor WK2-16, especially its effects on the neuroinflammatory responses through glial inactivation. WK2-16 significantly reduced the gelatinolytic activity of MMP-9, and expression of COX-2/iNOS proteins in striatal lipopolysaccharide (LPS)-induced neuroinflammation in C57BL/6 mice. The treatment of WK2-16 markedly improved neurobehavioral deficits. Immunofluorescent staining revealed that WK2-16 reduced LPS-stimulated astrogliosis and microglial activation in situ. Consistently, cellular studies revealed that WK2-16 significantly suppressed LPS-induced mouse microglia BV-2 cell proliferation. WK2-16 was proven to concentration-dependently induce the levels of acetylated SMC3 in microglial BV-2 cells. It also reduced the expression of COX-2/iNOS proteins and TNF-α production in LPS-activated microglial BV-2 cells. The signaling studies demonstrated that WK2-16 markedly inhibited LPS-activated STAT-1/-3 and Akt activation, but not NF-κB or MAPK signaling. In summary, the HADC8 inhibitor WK2-16 exhibited neuroprotective effects through its anti-neuroinflammation and glial inactivation properties, especially in microglia in vitro and in vivo.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Inflamación/etiología , Lipopolisacáridos/efectos adversos , Microglía/efectos de los fármacos , Microglía/metabolismo , Enfermedades del Sistema Nervioso/etiología , Animales , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Microglía/patología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patología , Fármacos Neuroprotectores , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
Chloramphenicol is an inexpensive and excellent bactericidal antibiotic. It is used to combat anaerobic infections in the Third World countries, whereas its systemic application has been abandoned in developed countries. However, in recent years, clinicians have reintroduced chloramphenicol in clinical practice. In this study, chloramphenicol was found to repress the oxygen-labile transcription factor, hypoxia inducible factor-1 alpha (HIF-1α), in hypoxic A549 and H1299 cells. Furthermore, it suppressed the mRNA levels of vascular endothelial growth factor (VEGF) and glucose transporter 1, eventually decreasing VEGF release. Chloramphenicol initiated the autophagy pathway in treated cells, as observed by the increase in formation of Atg12-Atg5 conjugates, and in beclin-1 and LC3-II levels. The chloramphenicol-mediated HIF-1α degradation was completely reverted by autophagic flux blockage. In HIF-1α-overexpressing cells, the formation of HIF-1α/SENP-1 (Sentrin/SUMO-specific protease 1) protein complex seemed to facilitate the escape of HIF-1α from degradation. Chloramphenicol inhibited HIF-1α/SENP-1 protein interaction, thereby destabilizing HIF-1α protein. The enhancement in HIF-1α degradation due to chloramphenicol was evident during the incubation of the antibiotic before hypoxia and after HIF-1α accumulation. Since HIF-1α plays multiple roles in infections, inflammation, and cancer cell stemness, our findings suggest a potential clinical value of chloramphenicol in the treatment of these conditions.
Asunto(s)
Autofagia/efectos de los fármacos , Cloranfenicol/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células A549 , Beclina-1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Proteína Sequestosoma-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The pharmacological effects of Cistanches Herba, known as "Ginseng of the desert", have been extensively studied. In this study, we aimed to assess the genotoxic and oral toxic effects of the Cistanche tubulosa health food product Memoregain® using in vitro and in vivo tests. Ames tests using five strains of Salmonella typhimurium showed no signs of increased reverse mutation upon exposure to Memoregain® up to a concentration of 5 mg/plate. Exposure of Chinese hamster ovary (CHO-K1) cells to Memoregain® did not increase the frequency of chromosomal aberrations in vitro. Moreover, Memoregain® treatment did not affect the proportions of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Additionally, after 28-day repeated oral dose toxicity tests (0, 0.15, 0.3, and 0.5g/kg body weight) in rats, no observable adverse effects were found. These toxicological assessments supported the safety of Memoregain® for human consumption.
Asunto(s)
Cistanche/química , Animales , Células CHO , Cricetinae , Cricetulus , Ratones , Pruebas de Mutagenicidad , Nivel sin Efectos Adversos Observados , Ratas , Pruebas de ToxicidadRESUMEN
Diabetic retinopathy (DR), a major microvascular complication of diabetes, leads to retinal vascular leakage, neuronal dysfunction, and apoptosis within the retina. In this study, we combined STZ with whole-body hypoxia (10% O2) for quicker induction of early-stage retinopathy in C57BL/6 mice. We also compared the effects of a high glucose condition combined with hypoxia (1% O2) to a low glucose condition by using retinal pigment epithelial (RPE) cells, which are a crucial component of the outer blood-retinal barrier and the damage is related to retinopathy. In the retina of DM/hypoxic C57BL/6 mice, abnormal a-wave and b-wave activity, yellowish-white spots, hyperfluorescence, and reduced retinal thickness were found using electroretinography (ERG), fundus photography (FP), fundus fluorescein angiography (FFA), and optical coherence tomography (OCT). Shikonin dose-dependently (0.5-50 mg/kg, per os) prevented DM/hypoxia-induced lesions. In eye tissue, administration of shikonin also attenuated DM/hypoxia-induced pre-apoptotic protein BAX expression as well as the production of inflammatory proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). We also demonstrated that shikonin administration rescues high glucose/hypoxia (1% O2)-induced inflammation, decreased junction protein expression, and permeability in RPE cells. These results indicate that shikonin treatment may prevent the loss of vision associated with DR.
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Antiinflamatorios no Esteroideos/farmacología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Naftoquinonas/farmacología , Animales , Glucemia , Retinopatía Diabética/tratamiento farmacológico , Modelos Animales de Enfermedad , Electrorretinografía , Células Epiteliales , Angiografía con Fluoresceína , Inmunohistoquímica , Ratones , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia ÓpticaRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in multiple organs and tissues. Whereas AhR mediates the metabolism of xenobiotic and endogenous compounds, its novel function in cancer epithelial-mesenchymal transition (EMT) remains controversial. Autophagy also participates in tumour progression through its functions in cell homeostasis and facilitates adaptation to EMT progression. In the present study, we found that AhR-regulated autophagy positively modulates EMT in non-small cell lung cancer cells. The motility of A549, H1299, and CL1-5 cells were correlated with different AhR expression levels. Invasive potential and cell morphology also changed when AhR protein expression was altered. Moreover, AhR levels exerted a contrasting effect on autophagy potential. Autophagy was higher in CL1-5 and H1299 cells with lower AhR levels than in A549 cells. Both AhR overexpression and autophagy inhibition decreased CL1-5 metastasis in vivo. Furthermore, AhR promoted BNIP3 ubiquitination for proteasomal degradation. AhR silencing in A549 cells also reduced BNIP3 ubiquitination. Taken together, these results provide a novel insight into the cross-linking between AhR and autophagy, we addressed the mechanistic BNIP3 modulation by endogenous AhR, which affect cancer cell EMT progression.
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Autofagia , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular , Humanos , Neoplasias Pulmonares/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Proteínas Mitocondriales/metabolismo , Receptores de Hidrocarburo de Aril/genética , UbiquitinaciónRESUMEN
Blue light-induced phototoxicity plays an important role in retinal degeneration and might cause damage as a consequence of smartphone dependency. Here, we investigated the effects of periodic exposure to blue light-emitting diode in a cell model and a rat retinal damage model. Retinal pigment epithelium (RPE) cells were subjected to blue light in vitro and the effects of blue light on activation of key apoptotic pathways were examined by measuring the levels of Bcl-2, Bax, Fas ligand (FasL), Fas-associated protein with death domain (FADD), and caspase-3 protein. Blue light treatment of RPE cells increased Bax, cleaved caspase-3, FasL, and FADD expression, inhibited Bcl-2 and Bcl-xL accumulation, and inhibited Bcl-2/Bax association. A rat model of retinal damage was developed with or without continuous or periodic exposure to blue light for 28 days. In this rat model of retinal damage, periodic blue light exposure caused fundus damage, decreased total retinal thickness, caused atrophy of photoreceptors, and injured neuron transduction in the retina.
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Apoptosis/efectos de la radiación , Luz , Retina/efectos de la radiación , Teléfono Inteligente , Proteína X Asociada a bcl-2/metabolismo , Animales , Células Cultivadas , Unión Proteica , RatasRESUMEN
Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, has been studied extensively in carcinogenesis through the genomic pathway. In recent years, AHR has also been reported to exert positive or negative effects on epithelial-mesenchymal transition (EMT), the crucial step in tumor malignant progression. However, the detailed mechanism remains controversial. Analysis of AHR-expression levels in non-small cell lung cancer cell lines and lung cancer tissues revealed an inverse correlation between AHR protein levels and tumor cell invasion and metastasis. Overexpression of wild-type AHR in H1299 cells (AHR poorly expressed, potently invasive) not only accelerated mesenchymal vimentin degradation, but also prevented cell invasion in vitro and in vivo. In the absence of AHR agonists, the overexpressed AHR protein was predominantly localized in the cytoplasm, where it interacted with vimentin and functioned as an E3 ubiquitin ligase. A 6-h incubation with the proteasome inhibitor MG-132 fully rescued vimentin from AHR-mediated proteasomal degradation. In AHR-overexpressing H1299 cells, either vimentin degradation or invasive suppression could be reversed when glycogen synthase kinase 3 beta (GSK3ß) was inactivated by CHIR-99021 treatment. In contrast, silencing of AHR in A549 cells (AHR highly expressed, weakly invasive) resulted in the downregulation of epithelial biomarkers (E-cadherin and claudin-1), augmentation of mesenchymal vimentin level, and GSK3ß Ser-9 hyper-phosphorylation, which led to enhanced invasiveness. This work demonstrates that cytoplasmic, resting AHR protein may act as an EMT suppressor via a non-genomic pathway. Depletion of cytoplasmic AHR content represents a potential switch for EMT, thereby leading to the scattering of tumor cells.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Vimentina/metabolismo , Anciano , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Citoplasma/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Hidrocarburo de Aril/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-ß signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells.
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Adenocarcinoma/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Neoplasias Pulmonares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Proteína Smad4/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Complejo del Señalosoma COP9 , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Invasividad Neoplásica , Péptido Hidrolasas/metabolismo , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Interferencia de ARN , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal , Factores de Transcripción de la Familia Snail/metabolismo , Transfección , Ubiquitinación , Vimentina/metabolismoRESUMEN
The main concern for container closure systems of drugs is to ensure suitability for the intended use which is associated with issues regarding protection, compatibility, safety, and performance. Among various concerns, leachables may pose a safety hazard to patients, while risks might vary depending on the dosage form and the administration route. Stringent regulatory authorities such as the European Medicines Agency and the United States Food and Drug Administration have established risk-based regulatory requirements and published corresponding guidelines to facilitate implementation. Taiwan, a member of the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme, makes every effort to harmonize with international regulations and to strengthen protection of public health through regulatory controls. The aim of the present study was to investigate the regulatory framework and policies set by stringent regulatory authorities. The strategy proposed for the development of an eventual guideline was sent to the Taiwan Food and Drug Administration for decision. A risk-based, phased-in approach which was extensively discussed in the expert committee was proposed. The approach proposed herein could also serve as a starting point which is worth considered by other countries in which international harmonization is in process.
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Seguridad de Productos para el Consumidor/legislación & jurisprudencia , Contaminación de Medicamentos/legislación & jurisprudencia , Contaminación de Medicamentos/prevención & control , Embalaje de Medicamentos/legislación & jurisprudencia , Control de Medicamentos y Narcóticos/legislación & jurisprudencia , Regulación Gubernamental , Cooperación Internacional/legislación & jurisprudencia , Seguridad del Paciente/legislación & jurisprudencia , Seguridad de Productos para el Consumidor/normas , Embalaje de Medicamentos/normas , Diseño de Equipo , Guías como Asunto , Humanos , Seguridad del Paciente/normas , Formulación de Políticas , Control de Calidad , Medición de Riesgo , Taiwán , Estados Unidos , United States Food and Drug Administration/legislación & jurisprudenciaRESUMEN
High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with AhR nuclear translocator (ARNT) for complex formation with hypoxia-inducible factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In this study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia response element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2 between AhR and hypoxic conditions, thus providing a novel mechanism underlying the cross talk between AhR and hypoxia that affects disease development and progression.