RESUMEN
(-)-Epigallocatechin-3-gallate (EGCg), a major constituent of green tea extract, is well-known to exhibit many beneficial actions for human health by interacting with numerous proteins. In this study we identified synaptic vesicle membrane protein VAT-1 homolog (VAT1) as a novel EGCg-binding protein in human neuroglioma cell extracts using a magnetic pull-down assay and LC-tandem mass spectrometry. We prepared recombinant human VAT1 and analyzed its direct binding to EGCg and its alkylated derivatives using surface plasmon resonance. For EGCg and the derivative NUP-15, we measured an association constant of 0.02-0.85 ×103 M-1s-1 and a dissociation constant of nearly 8 × 10-4 s-1. The affinity Km(affinity) of their binding to VAT1 was in the 10-20 µM range and comparable with that of other EGCg-binding proteins reported previously. Based on the common structure of the compounds, VAT1 appeared to recognize a catechol or pyrogallol moiety around the B-, C- and G-rings of EGCg. Next, we examined whether VAT1 mediates the effects of EGCg and NUP-15 on expression of neprilysin (NEP). Treatments of mock cells with these compounds upregulated NEP, as observed previously, whereas no effect was observed in the VAT1-overexpressing cells, indicating that VAT1 prevented the effects of EGCg or NUP-15 by binding to and inactivating them in the cells overexpressing VAT1. Further investigation is required to determine the biological significance of the VAT1-EGCg interaction.
Asunto(s)
Catequina , Proteínas de Transporte Vesicular , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Vesículas Sinápticas/metabolismo , Té/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Brain gliomas are difficult in the field of tumor therapy because of their high recurrence rate, high mortality rate, and low selectivity of therapeutic agents. The efficacy of Traditional Chinese Medicine (TCM) in the treatment for tumours has been widely recognized. Here, three Chinese herb related molecules, namely Catechins, Caudatin and Cucurbitacin-I, were screened by bioinformatic means, and were found to inhibit the proliferation of glioblastoma T98G cells using Colony-forming and CCK-8 assays. Notably, the simultaneous use of all three molecules could more significantly inhibit the proliferation of glioma cells. Consistent with this, temozolomide, each in the combination with three molecules, could synergistically inhibit the proliferation of T98G cells. Results of qPCR assay was also showed that this inhibition was through the activation of the KDELR2-mediated endoplasmic reticulum stress (ER) pathway. Molecular docking experiments further revealed that Catechins, Caudatin and Cucurbitacin-I could activate ER stress might by targeting KDELR2. Taken together, these results suggest that these herbal molecules have the potential to inhibit the growth of glioma cells and could provide a reference for clinical therapeutic drug selection.
Asunto(s)
Antineoplásicos , Catequina , Glioblastoma , Glioma , Humanos , Glioblastoma/patología , Catequina/farmacología , Cucurbitacinas/farmacología , Cucurbitacinas/uso terapéutico , Simulación del Acoplamiento Molecular , Glioma/patología , Antineoplásicos/farmacología , Proliferación Celular , Estrés del Retículo Endoplásmico , Línea Celular Tumoral , Apoptosis , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Background: Vacuolar protein sorting 16 (VPS16) overexpression was recently considered related to cancer growth and drug resistance; however, little is known about whether VPS16 plays a vital role in liver hepatocellular carcinoma (LIHC). Methods: The TIMER2 online database was used to analyze the expression of VPS16 in pancancer, and the Xena Browser was used to explore the correlation between VPS16 expression level and survival time. R language was used to test the survival data of 374 LIHC cases in the TCGA database. DESeq2 was used for differentially expressed gene (DEG) analysis. The HPA database was used to verify the expression level of VPS16 in LIHC. The clusterProfiler package was used to analyze functions and related signaling pathways via GO/KEGG enrichment analysis. Drug sensitivity analysis and molecular docking technology were used to screen the most sensitive drugs targeting VPS16 molecules. Results: Pancancer analysis showed that VPS16 was highly expressed in various tumors, especially in LIHC. With the increase in the T stage and grade of LIHC, the expression level of VPS16 was also increased. The expression of VPS16 was negatively correlated with the overall survival of LIHC patients. The stage can be used as an independent prognostic factor. A total of 63 sensitive drugs were found, and 19 drugs were displaying strong molecular binding energy with VPS16. Conclusion: VPS16 may be a potential biomarker for the diagnosis and prognosis of LIHC. Drugs targeting VPS16 may be used in the treatment of LIHC in the future.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Pronóstico , Evaluación Preclínica de Medicamentos , Simulación del Acoplamiento Molecular , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Biología Computacional , Proteínas de Transporte Vesicular/genéticaRESUMEN
Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients.
Asunto(s)
Leishmania donovani , Leishmaniasis Visceral , Colesterol/metabolismo , Humanos , Macrófagos/metabolismo , Proteína Niemann-Pick C1 , Tretinoina/metabolismo , Tretinoina/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: YiQiFuMai lyophilized injection (YQFM) is derived from a traditional Chinese medicine prescription termed Shengmai San.YQFM is clinically applied to the treatment of cardiovascular and cerebrovascular diseases. It has been found that critical components of YQFM affect non-muscle myosin heavy chain IIA (NMMHC IIA), but its regulation in the excessive autophagy and the underlying mechanism has yet to be clarified. PURPOSE: To evaluate whether YQFM has neuroprotective effects on cerebral ischemia/reperfusion-induced injury by inhibiting NMMHC IIA-actin-ATG9A interaction for autophagosome formation. METHODS: The neuroprotective effects of YQFM were investigated in vivo in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) (n = 6) by detecting neurological deficits, infarct volume, and histopathological changes. The NMMHC IIA-actin-ATG9A interaction was determined using immunofluorescence co-localization, co-immunoprecipitation, and proximity ligation assay. Rat pheochromocytoma (PC12) cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic neurons in in vitro experiments. RESULTS: In MCAO/R model mice, YQFM (1.342 g/kg) attenuated brain ischemia/reperfusion-induced injury by regulating NMMHC IIA-actin-mediated ATG9A trafficking. YQFM (400 µg/ml) also exerted similar effects on OGD/R-induced PC12 cells. Furthermore, RNAi of NMMHC IIA weakened the NMMHC IIA-F-actin-dependent ATG9A trafficking and, therefore, attenuated the neuroprotective activities of YQFM in vitro. CONCLUSION: These findings demonstrated that YQFM exerted neuroprotective effects by regulating the NMMHC IIA-actin-ATG9A interaction for autophagosome formation. This evidence sheds new light on the potential mechanism of YQFM in the treatment of cerebral ischemia/reperfusion.
Asunto(s)
Isquemia Encefálica , Medicamentos Herbarios Chinos , Fármacos Neuroprotectores , Daño por Reperfusión , Actinas , Animales , Autofagia , Proteínas Relacionadas con la Autofagia , Isquemia Encefálica/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Proteínas de la Membrana , Ratones , Fármacos Neuroprotectores/farmacología , Ratas , Daño por Reperfusión/tratamiento farmacológico , Proteínas de Transporte VesicularRESUMEN
Pathological angiogenesis, as exhibited by aberrant vascular structure and function, has been well deemed to be a hallmark of cancer and various ischemic diseases. Therefore, strategies to normalize vasculature are of potential therapeutic interest in these diseases. Recently, identifying bioactive compounds from medicinal plant extracts to reverse abnormal vasculature has been gaining increasing attention. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza, has been shown to play significant roles in improving blood circulation and delaying tumor progression. However, the underlying mechanisms responsible for the therapeutic effects of Tan IIA are not fully understood. Herein, we established animal models of HT-29 human colon cancer xenograft and hind limb ischemia to investigate the role of Tan IIA in regulating abnormal vasculature. Interestingly, our results demonstrated that Tan IIA could significantly promote the blood flow, alleviate the hypoxia, improve the muscle quality, and ameliorate the pathological damage after ischemic insult. Meanwhile, we also revealed that Tan IIA promoted the integrity of vascular structure, reduced vascular leakage, and attenuated the hypoxia in HT-29 tumors. Moreover, the circulating angiopoietin 2 (Ang2), which is extremely high in these two pathological states, was substantially depleted in the presence of Tan IIA. Also, the activation of Tie2 was potentiated by Tan IIA, resulting in decreased vascular permeability and elevated vascular integrity. Mechanistically, we uncovered that Tan IIA maintained vascular stability by targeting the Ang2-Tie2-AKT-MLCK cascade. Collectively, our data suggest that Tan IIA normalizes vessels in tumors and ischemic injury via regulating the Ang2/Tie2 signaling pathway.
Asunto(s)
Abietanos/farmacología , Neoplasias del Colon/irrigación sanguínea , Regulación de la Expresión Génica/efectos de los fármacos , Isquemia/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Receptor TIE-2/antagonistas & inhibidores , Proteínas de Transporte Vesicular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Proliferación Celular , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Humanos , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Parkinson's disease (PD) is a disease that involves brain damage and is associated with neuroinflammation, mitochondrial damage, and cell aging. However, the pathogenic mechanism of PD is still unknown. Sequencing data and proteomic data can describe the fluctuation of molecular abundance in diseases at the mRNA level and protein level, respectively. In order to explore new targets in the pathogenesis of PD, the study analyzed molecular changes from the database by combining transcriptomic and proteomic analysis. Differentially expressed genes and differentially abundant proteins were summarized and analyzed. Enrichment and cluster analysis emphasized the importance of neurotransmitter release, mitochondrial damage, and vesicle transport. The molecular network revealed a subnetwork of 9 molecules related to SCNA and TH and revealed hub gene with differential expression at both mRNA and protein levels. It found that ACHE and CADPS could be used as new targets in PD, emphasizing that impaired nerve signal transmission and vesicle transport affect the pathogenesis of PD. Our research emphasized that the joint analysis and verification of transcriptomics and proteomics were devoted to understanding the comprehensive views and mechanism of pathogenesis in PD.
Asunto(s)
Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sustancia Negra/metabolismo , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Análisis por Conglomerados , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Enfermedad de Parkinson/patología , ARN Mensajero/genética , Sustancia Negra/patología , Transcriptoma , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.
Asunto(s)
Quitosano/análogos & derivados , Quitosano/uso terapéutico , Gentamicinas/uso terapéutico , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/uso terapéutico , Penaeidae/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Quitosano/síntesis química , Cisteína Endopeptidasas/metabolismo , Suplementos Dietéticos , Gentamicinas/síntesis química , Hemocitos/metabolismo , Hepatopáncreas/efectos de los fármacos , Hepatopáncreas/microbiología , Hepatopáncreas/patología , Factores Inmunológicos/síntesis química , Penaeidae/inmunología , Penaeidae/metabolismo , Penaeidae/microbiología , Fagocitos/metabolismo , Esterol Esterasa/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vibrio parahaemolyticus/patogenicidadRESUMEN
In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Pectinas/metabolismo , Tubo Polínico/crecimiento & desarrollo , Proteínas de Transporte Vesicular/genética , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cuerpos Multivesiculares/enzimología , Tubo Polínico/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
In eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A-mCherry and SEC31B-GFP exhibited a high co-localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a-1/+sec31b-3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Gametogénesis en la Planta , Polen/crecimiento & desarrollo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Fertilidad , Genes de Plantas/fisiología , Células Germinativas de las Plantas/metabolismo , Polen/metabolismo , Tubo Polínico/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiologíaRESUMEN
Pups born from females of the inbred mouse strain RR/Sgn tend to have low survival rates during rearing. We have previously identified Naq3, a quantitative trait locus underlying this low pup survival rate. In the present study, we confirmed the effect of Naq3 in congenic mice and investigated whether Vps8 is a candidate gene for Naq3. The survival rate of pups on the twelfth postpartum day was significantly decreased for mothers homozygous for the Naq3 allele. Hypothalamic expression of Vps8 was induced by nurturing in wild-type mice, and was significantly lower in Naq3 congenic mice than in wild-type mice. Thus, Vps8 is suggested to be involved in maternal nurturing, and therefore, as a plausible candidate gene for Naq3.
Asunto(s)
Expresión Génica , Genotipo , Hipotálamo/metabolismo , Nacimiento Vivo , Sitios de Carácter Cuantitativo , Proteínas de Transporte Vesicular/genética , Alelos , Animales , Femenino , Masculino , Ratones , Tasa de Supervivencia , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: The heart is one of the most commonly affected organs during sepsis. Mitsugumin-53 (MG53) has attracted attention in research due to its cardioprotective function. However, the role of MG53 in sepsis-induced myocardial dysfunction (SIMD) remains unknown. The purpose of this study was to explore the underlying mechanism of MG53 in SIMD and investigate its potential relationship with peroxisome proliferator-activated receptor-α (PPARα). METHODS: The cecal ligation and puncture (CLP) model was created to induce SIMD in rats. Protein levels of MG53 and PPARα, cardiac function, cardiomyocyte injury, myocardial oxidative stress and inflammatory indicators, and cardiomyocyte apoptosis were measured at 18 h after CLP. The effects of MG53 on PPARα in SIMD were investigated via preconditioning recombinant human MG53 (rhMG53) and PPARα antagonist GW6471. RESULTS: The expression of MG53 and PPARα sharply decreased in the myocardium at 18 h after CLP. Compared with the sham group, cardiac function was significantly depressed, which was associated with the destructed myocardium, upregulated oxidative stress indicators and proinflammatory cytokines, and excessive cardiomyocyte apoptosis in the CLP group. Supplementation with rhMG53 enhanced myocardial MG53, increased the survival rate with improved cardiac function, and reduced oxidative stress, inflammation, and myocardial apoptosis, which were associated with PPARα upregulation. Pretreatment with GW6471 abolished the abovementioned protective effects induced by MG53. CONCLUSIONS: Both MG53 and PPARα were downregulated after sepsis shock. MG53 supplement protects the heart against SIMD by upregulating PPARα expression. Our results provide a new treatment strategy for SIMD.
Asunto(s)
Proteínas Musculares/metabolismo , Miocardio/patología , PPAR alfa/genética , Sustancias Protectoras/metabolismo , Sepsis/complicaciones , Regulación hacia Arriba/genética , Proteínas de Transporte Vesicular/metabolismo , Animales , Apoptosis , Humanos , Inflamación/patología , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Oxazoles , Estrés Oxidativo , PPAR alfa/metabolismo , Ratas Sprague-Dawley , Análisis de Supervivencia , Tirosina/análogos & derivadosRESUMEN
Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Flavonoides/farmacología , Isoflurano/antagonistas & inhibidores , Isoflurano/toxicidad , Complejo Mayor de Histocompatibilidad/efectos de los fármacos , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/prevención & control , Polifenoles/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Hipocampo/efectos de los fármacos , Ratones , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy. One of the main proteins induced in response to hyperthermia is heat shock protein 70 (HSP70). The aim of our study was to investigate up- and down-regulated genes in response to (thermo)radiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line (Abrams), and functional role of HSP70 in the mechanism of thermoradiosensitization. Cells were transfected with negative control siRNA or siRNA targeting HSP70 and treated with hyperthermia (HT), radiotherapy (RT), and thermoradiotherapy (HTRT). RNA sequencing was used to analyze gene expression. Hyperthermia and thermoradiotherapy, but not radiotherapy alone, induced differential gene expression. We identified genes differentially expressed only in HSP70 knockdown (thus HSP70-dependent) cells in response to hyperthermia and thermoradiotherapy. Interestingly, cell proliferation but not clonogenicity and apoptosis/necrosis was affected by the HSP70 knockdown in response to thermoradiotherapy. The results suggest that HSP70 regulates expression of specific genes in response to hyperthermia and thermoradiotherapy. Further investigations into the role of specific genes regulated in a HSP70-dependent manner in response to thermoradiotherapy could pave a way into new, combinatorial treatment options for (canine) osteosarcoma and other cancer types.
Asunto(s)
Neoplasias Óseas/metabolismo , Neoplasias Óseas/radioterapia , Proteínas HSP70 de Choque Térmico/metabolismo , Osteosarcoma/metabolismo , Osteosarcoma/radioterapia , Animales , Proliferación Celular , Análisis por Conglomerados , Modelos Animales de Enfermedad , Perros , Regulación Neoplásica de la Expresión Génica , Hipertermia Inducida , Metaloproteinasa 1 de la Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Fotones , Análisis de Componente Principal , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , RNA-Seq , Radioterapia , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Coenzyme Q (CoQ) is an essential player in the respiratory electron transport chain and is the only lipid-soluble antioxidant synthesized endogenously in mammalian and yeast cells. In humans, genetic mutations, pathologies, certain medical treatments, and aging, result in CoQ deficiencies, which are linked to mitochondrial, cardiovascular, and neurodegenerative diseases. The only strategy available for these patients is CoQ supplementation. CoQ supplements benefit a small subset of patients, but the poor solubility of CoQ greatly limits treatment efficacy. Consequently, the efficient delivery of CoQ to the mitochondria and restoration of respiratory function remains a major challenge. A better understanding of CoQ uptake and mitochondrial delivery is crucial to make this molecule a more efficient and effective therapeutic tool. In this study, we investigated the mechanism of CoQ uptake and distribution using the yeast Saccharomyces cerevisiae as a model organism. The addition of exogenous CoQ was tested for the ability to restore growth on non-fermentable medium in several strains that lack CoQ synthesis (coq mutants). Surprisingly, we discovered that the presence of CoQ biosynthetic intermediates impairs assimilation of CoQ into a functional respiratory chain in yeast cells. Moreover, a screen of 40 gene deletions considered to be candidates to prevent exogenous CoQ from rescuing growth of the CoQ-less coq2Δ mutant, identified six novel genes (CDC10, RTS1, RVS161, RVS167, VPS1, and NAT3) as necessary for efficient trafficking of CoQ to mitochondria. The proteins encoded by these genes represent essential steps in the pathways responsible for transport of exogenously supplied CoQ to its functional sites in the cell, and definitively associate CoQ distribution with endocytosis and intracellular vesicular trafficking pathways conserved from yeast to human cells.
Asunto(s)
Enfermedades Mitocondriales , Proteínas de Saccharomyces cerevisiae , Animales , Proteínas de Unión al GTP , Humanos , Lípidos , Proteínas de Microfilamentos , Acetiltransferasa B N-Terminal , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquinona/metabolismo , Proteínas de Transporte VesicularRESUMEN
Some phytochemical-derived synthetic compounds have been shown to improve neurological disorders, especially in ischemic stroke. In this study, we identified a novel biscoumarin compound, known as COM 3, which had substantial antioxidant effects in neurons. Next, we found that COM 3 occupies a critical binding site between the Nrf2 and Keap1 dipolymer, impairing the inhibitory effects of Keap1 on Nrf2, both of which play central roles in increasing endogenous antioxidant activity. We verified that COM 3 could increase the survival of neurons experiencing oxygen and glucose deprivation (OGD) from 51.1% to 77.2% when exposure to 2.5 and 10 µg/mL of COM 3, respectively. In addition, the same concentrations of COM 3 could reduce brain infarct volumes by 33.8%to13.7%, respectively, while also reducing the neurobehavioral score from 3.3 to 1.4 on average in mice with a middle cerebral artery occlusion (MCAO). COM 3 reduced neuronal death from 36.5% to 13.9% and apoptosis from 35.1% to 18.2%. In addition, COM 3 could improve the neuronal mitochondrial energy metabolism after experiencing oxidative stress caused by OGD or MCAO. The present study suggests that COM 3 protects against OGD in neurons and MCAO in mice by interfering with the structure of Keap1 to activate the nuclear transcription of Nrf2, which balances endogenous redox activity and restores mitochondrial function. Hence, COM 3 might be a potential therapeutic agent for ischemic stroke in the clinic.
Asunto(s)
Isquemia Encefálica/metabolismo , Cumarinas/uso terapéutico , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Isquemia Encefálica/tratamiento farmacológico , Células Cultivadas , Cumarinas/química , Cumarinas/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Feto , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Complejo Mayor de Histocompatibilidad , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/fisiología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Cerebral ischemic stroke is a severe disease afflicting people worldwide. Phytochemicals play a pivotal role in the discovery of novel therapeutic approaches for the prevention of ischemic stroke. In our continual search for bioactive natural products for the treatment of ischemic stroke, we have evaluated the protective effects of theaflavic acid (TFA) from black tea using PC12 cells injured by oxygen and glucose deprivation/restoration (OGD/R), and investigated the possible mechanisms. The results showed that TFA can protect PC12 cells against OGD/R through increasing cell viability and decreasing intracellular lactate dehydrogenase (LDH) release. Further investigations found that TFA could inhibit the overproduction of intracellular reactive oxygen species (ROS), reduce malondialdehyde content, and elevate superoxide dismutase activity, which implied that TFA suppresses oxidative stress in PC12 cells induced by OGD/R. In addition, overload of intracellular calcium and collapse of the mitochondrial membrane potential were improved in the presence of TFA, and the activity of caspase-3 was significantly reduced by TFA. Western blot analysis showed that the expression of Bcl-2 was up-regulated while Bax was down-regulated. Therefore, it can be concluded that TFA can inhibit mitochondria-dependent apoptosis of PC12 cells induced by OGD/R. In addition, activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathway was explored to elucidate the mechanism by which TFA inhibits ROS-mediated apoptosis in PC12 cells. The results revealed that TFA promoted the translocation of Nrf2 into nuclei, enhanced the transcriptional activity of ARE, and up-regulated expression of downstream HO-1, which indicates that the Nrf2/ARE signaling pathway is involved in the protection by TFA of PC12 cells injured by OGD/R.
Asunto(s)
Anaerobiosis/fisiología , Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Glucosa/deficiencia , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Elementos de Respuesta Antioxidante/efectos de los fármacos , Caspasa 3 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Glucosa/metabolismo , Hemo-Oxigenasa 1/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Malondialdehído/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxígeno/metabolismo , Células PC12 , Fitoquímicos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Té/química , Proteínas de Transporte Vesicular/metabolismoRESUMEN
RESEARCH QUESTION: Do low doses of dietary nitrate help to attenuate the progression of diabetic reproductive disorders in streptozotocin-induced diabetic male rats? DESIGN: Fifty male Wistar rats were divided into five groups: controls receiving distilled water; controls receiving 100 mg/l nitrate in distilled water; diabetic rats receiving distilled water; diabetic rats receiving insulin 2-4 U/day of neutral protamine hagedorn insulin; and diabetic rats receiving 100 mg/l nitrate in distilled water. Diabetes was induced by 45 mg/kg streptozotocin. Nitrate and insulin treatment were started 4 weeks after diabetes induction for 8 weeks. Serum insulin, nitrogen oxide, stereology of testis, apoptosis, sperm parameters, and mRNA expression of Pdcd4, Pacs2, p53 and miR-449a were assessed at the end of the study. RESULTS: Blood glucose, apoptotic index of seminiferous tubules and expression of p53, Pdcd4, and Pacs2 mRNA were significantly higher in the diabetic rats (P < 0.001). Decreased body weight, serum insulin and nitrogen oxide level, and miR-449a were observed in the diabetic group (P < 0.01 for insulin; P < 0.001 for others). Most sperm parameters and stereological results differed between diabetic and control rats; nitrate recovered almost all these alterations, including dead spermatozoa, sperm motility grade, sperm deformity index, spermatozoa with damaged DNA, malformations in abnormal spermatozoa, total volume of seminiferous tubule, germinal epithelium, capsule, lumen, interstitial tissue, seminiferous tubule diameter, germinal epithelium height, the number of spermatogenic, Sertoli and Leydig cells. CONCLUSIONS: Treatment with sodium nitrate could modulate apoptosis, which is a major cause of diabetic testicular disorder. These experiments suggest that nitric oxide plays an important role in the function of the reproductive system.
Asunto(s)
Apoptosis/efectos de los fármacos , Diabetes Mellitus Experimental/dietoterapia , Nitratos/uso terapéutico , Motilidad Espermática/efectos de los fármacos , Enfermedades Testiculares/dietoterapia , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Suplementos Dietéticos , Masculino , MicroARNs/metabolismo , Nitratos/farmacología , Distribución Aleatoria , Ratas Wistar , Enfermedades Testiculares/etiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: KChIP2 (K+ channel interacting protein) is the auxiliary subunit of the fast transient outward K+ current ( Ito,f) in the heart, and insufficient KChIP2 expression induces Ito,f downregulation and arrhythmogenesis in cardiac hypertrophy. Studies have shown muscle-specific mitsugumin 53 (MG53) has promiscuity of function in the context of normal and diseased heart. This study investigates the possible roles of cardiac MG53 in regulation of KChIP2 expression and Ito,f, and the arrhythmogenic potential in hypertrophy. METHODS: MG53 expression is manipulated by genetic ablation of MG53 in mice and adenoviral overexpression or knockdown of MG53 by RNA interference in cultured neonatal rat ventricular myocytes. Cardiomyocyte hypertrophy is produced by phenylephrine stimulation in neonatal rat ventricular myocytes, and pressure overload-induced mouse cardiac hypertrophy is produced by transverse aortic constriction. RESULTS: KChIP2 expression and Ito,f density are downregulated in hearts from MG53-knockout mice and MG53-knockdown neonatal rat ventricular myocytes, but upregulated in MG53-overexpressing cells. In phenylephrine-induced cardiomyocyte hypertrophy, MG53 expression is reduced with concomitant downregulation of KChIP2 and Ito,f, which can be reversed by MG53 overexpression, but exaggerated by MG53 knockdown. MG53 knockout enhances Ito,f remodeling and action potential duration prolongation and increases susceptibility to ventricular arrhythmia in mouse cardiac hypertrophy. Mechanistically, MG53 regulates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity and subsequently controls KChIP2 transcription. Chromatin immunoprecipitation demonstrates NF-κB protein has interaction with KChIP2 gene. MG53 overexpression decreases, whereas MG53 knockdown increases NF-κB enrichment at the 5' regulatory region of KChIP2 gene. Normalizing NF-κB activity reverses the alterations in KChIP2 in MG53-overexpressing or knockdown cells. Coimmunoprecipitation and Western blotting assays demonstrate MG53 has physical interaction with TAK1 (transforming growth factor-b [TGFb]-activated kinase 1) and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), critical components of the NF-κB pathway. CONCLUSIONS: These findings establish MG53 as a novel regulator of KChIP2 and Ito,f by modulating NF-κB activity and reveal its critical role in electrophysiological remodeling in cardiac hypertrophy.