sirolimus and Neuroblastoma

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; 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A phase II study of temsirolimus was conducted in children and adolescents with high-grade glioma, neuroblastoma or rhabdomyosarcoma.. Temsirolimus 75 mg/m(2) was administered once weekly until disease progression or intolerance. Using the Simon 2-stage design, further enrolment in each disease cohort required ≥ 2 objective responses within the first 12 weeks for the first 12 evaluable patients (those who received ≥ 3 temsirolimus doses).. Fifty-two heavily pretreated patients with relapsed (12%) or refractory (88%) disease, median age 8 years (range 1-21 years), were enroled and treated. One patient with neuroblastoma achieved confirmed partial response within the first 12 weeks; thus, none of the 3 cohorts met the criterion for continued enrolment. Disease stabilisation at week 12 was observed in 7 of 17 patients (41%) with high-grade glioma (5 diffuse pontine gliomas, 1 glioblastoma multiforme and 1 anaplastic astrocytoma), 6 of 19 (32%) with neuroblastoma and 1 of 16 (6%) with rhabdomyosarcoma (partial response confirmed at week 18). In the three cohorts, median duration of stable disease or better was 128, 663 and 75 d, respectively. The most common treatment-related adverse events were thrombocytopaenia, hyperlipidaemia and aesthenia. Pharmacokinetic findings were similar to those observed in adults.. Temsirolimus administered weekly at the dose of 75 mg/m(2) did not meet the primary objective efficacy threshold in children with high-grade glioma, neuroblastoma or rhabdomyosarcoma; however, meaningful prolonged stable disease merits further evaluation in combination therapy.

Topics: Adolescent; Adult; Antineoplastic Agents; Brain Neoplasms; Child; Child, Preschool; Cohort Studies; Disease Progression; Female; Glioma; Humans; Infusions, Intravenous; Male; Neuroblastoma; Rhabdomyosarcoma; Sirolimus

To explore the intrinsic mechanism of the mammalian target of rapamycin (mTOR) pathway activation and promotion of neuronal axon growth.. Human neuroblastoma cells, SH-SY5Y, were induced with all-trans retinoic acid (ATRA; 10 μM for three days) which differentiated the cell line into a neuronal-like state. Immunohistochemical staining was used to detect the differentiation status of the neuronal-like cells. Phosphatase and tensin homolog (PTEN) RNA interference (RNAi) experiments were performed on the differentiated cells; reverse transcription-polymerase chain reaction (RT-PCR) detected transcriptional levels of PTEN following 24 h of interference. After 36 h, western blot assay was used to detect expression levels of ribosomal protein S6 kinase (pS6k) and mTOR. To downregulate the expression of PTEN and cluster of differentiation 44 (CD44), a cell-surface glycoprotein, simultaneously, PTEN siRNA and CD44 siRNA sequences were mixed in equal proportions in co-interference experiments. RT-PCR detected the transcription level of CD44, and the relationship between the CD44 and axonal growth was observed after 48 h of interference.. Microtubule-associated protein 2 (MAP2) expression was enhanced after three days of induction in SH-SY5Y cells. RT-PCR showed the transcription level of PTEN was significantly downregulated after 24 h of PTEN knockdown. mTOR and pS6k protein expression levels were significantly upregulated after 36 h of interference. CD44 transcription levels were upregulated after PTEN gene interference. The neurite length of the cells in the experimental interference group was significantly longer than that in the control group, and the expression level of CD44 was positively correlated with neurite growth. The neurite length of the PTEN-only interference group was significantly greater than that of the co-interference and ATRA groups.. Activation of the mTOR pathway promoted neurite growth through upregulation of CD44 expression, thus promoting neuronal regeneration.

Topics: Humans; Hyaluronan Receptors; Neurites; Neuroblastoma; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

T14 modulates calcium influx via the α-7 nicotinic acetylcholine receptor to regulate cell growth. Inappropriate triggering of this process has been implicated in Alzheimer's disease (AD) and cancer, whereas T14 blockade has proven therapeutic potential in in vitro, ex vivo and in vivo models of these pathologies. Mammalian target of rapamycin complex 1 (mTORC1) is critical for growth, however its hyperactivation is implicated in AD and cancer. T14 is a product of the longer 30mer-T30. Recent work shows that T30 drives neurite growth in the human SH-SY5Y cell line via the mTOR pathway. Here, we demonstrate that T30 induces an increase in mTORC1 in PC12 cells, and ex vivo rat brain slices containing substantia nigra, but not mTORC2. The increase in mTORC1 by T30 in PC12 cells is attenuated by its blocker, NBP14. Moreover, in post-mortem human midbrain, T14 levels correlate significantly with mTORC1. Silencing mTORC1 reverses the effects of T30 on PC12 cells measured via AChE release in undifferentiated PC12 cells, whilst silencing mTORC2 does not. This suggests that T14 acts selectively via mTORC1. T14 blockade offers a preferable alternative to currently available blockers of mTOR as it would enable selective blockade of mTORC1, thereby reducing side effects associated with generalised mTOR blockade.

Topics: Alzheimer Disease; Animals; Humans; Mammals; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Neuroblastoma; Peptides; Rats; Sirolimus; TOR Serine-Threonine Kinases

Prolonged sevoflurane exposure leads to neurotoxicity. Autophagy plays an important role in promoting cell survival in different conditions. However, the role and mechanism of autophagy in sevoflurane-induced neurotoxicity were not fully elucidated. We attempted to indicate whether sevoflurane could activate the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR)-mediated autophagy to attenuate anesthetics-induced neuronal injury in this study. Sevoflurane treatment significantly decreased the cell viability and induced apoptosis of SH-SY5Y cells. The expression level of Bcl-2 decreased, while that of Bax remarkably increased. Meanwhile, autophagy was activated by sevoflurane exposure as evidenced by increased expression levels of autophagy-related proteins (LC3-II and Atg5), decreased expression level of autophagic substrate P62, and increased autophagosomes and autolysosomes. Further autophagosomes and fewer autolysosomes were observed in the presence of Bafilomycin A1, an autolysosomes degradation inhibitor, suggesting that sevoflurane induced autophagic flux rather than inhibiting degradation of autophagy. Activation of autophagy by rapamycin partly reversed the sevoflurane-decreased cell viability. In contrast, inhibition of autophagy by 3-Methyladenine (3-MA) or Atg5-targeted small interfering RNA (siRNA) aggravated the sevoflurane-induced neurotoxicity. Further examination revealed that sevoflurane-induced autophagy was mediated by the AMPK/mTOR signaling pathway, with increased p-AMPK expression and decreased p-mTOR expression. Collectively, these results indicated that sevoflurane activates autophagy by regulating the AMPK/mTOR signaling pathway, which is protective against sevoflurane-induced damage in SH-SY5Y cells. Our results may assist clinicians to develop further promising therapeutic strategies for the neurotoxicity induced by inhaled anesthetics.

Topics: AMP-Activated Protein Kinases; Apoptosis; Autophagy; Cell Line, Tumor; Humans; Neuroblastoma; Sevoflurane; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB.. Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses.. Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy.. Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.

Topics: Acetanilides; Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Azepines; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Down-Regulation; Drug Synergism; Eukaryotic Initiation Factor-4E; G1 Phase Cell Cycle Checkpoints; Heterocyclic Compounds, 3-Ring; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Spheroids, Cellular; TOR Serine-Threonine Kinases; Transcription Factors; Triazoles

Deregulation of v-myc avian myelocytomatosis viral oncogene homolog (MYC) occurs in a broad range of human cancers and often predicts poor prognosis and resistance to therapy. However, directly targeting oncogenic MYC remains unsuccessful, and indirectly inhibiting MYC emerges as a promising approach. Checkpoint kinase 1 (CHK1) is a protein kinase that coordinates the G2/M cell cycle checkpoint and protects cancer cells from excessive replicative stress. Using c-MYC-mediated T-cell acute lymphoblastic leukemia (T-acute lymphoblastic leukemia) and N-MYC-driven neuroblastoma as model systems, we reveal that both c-MYC and N-MYC directly bind to the CHK1 locus and activate its transcription. CHIR-124, a selective CHK1 inhibitor, impairs cell viability and induces remarkable synergistic lethality with mTOR inhibitor rapamycin in MYC-overexpressing cells. Mechanistically, rapamycin inactivates carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD), the essential enzyme for the first three steps of de novo pyrimidine synthesis, and deteriorates CHIR-124-induced replicative stress. We further demonstrate that dual treatments impede T-acute lymphoblastic leukemia and neuroblastoma progression in vivo. These results suggest simultaneous targeting of CHK1 and mTOR as a novel and powerful co-treatment modality for MYC-mediated tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Checkpoint Kinase 1; Datasets as Topic; Disease Progression; Drug Synergism; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; N-Myc Proto-Oncogene Protein; Neuroblastoma; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Proto-Oncogene Proteins c-myc; Quinolines; Quinuclidines; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The prognosis for patients with relapsed or refractory high-risk neuroblastoma remains dismal and novel therapeutic options are urgently needed. The RIST treatment protocol has a multimodal metronomic therapy design combining molecular-targeted drugs (Rapamycin and Dasatinib) with chemotherapy backbone (Irinotecan and Temozolomide), which is currently verified in a phase II clinical trial (NCT01467986). With the availability of novel and more potent ATP competitive mTOR inhibitors, we expect to improve the RIST combination therapy. By comparing the IC

Topics: Administration, Metronomic; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Survival; Dasatinib; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Indoles; Inhibitory Concentration 50; Irinotecan; Naphthyridines; Neuroblastoma; Purines; Pyrimidines; Signal Transduction; Sirolimus; Temozolomide; Thiourea; TOR Serine-Threonine Kinases

The ubiquitous proteoglycan, biglycan (BGN) acts as an important modulator, regulating key molecular pathways of metabolism and brain function. Autophagy is documented as a defining feature of neurodegeneration in Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). In the present study, we found that BGN protected neuronal cells from nitric oxide (NO)-induced cell apoptosis. However, it is still unclear that whether the neuroprotective effect of BGN relates to autophagy. Here, we discovered that an NO donor, sodium nitroprusside (SNP) induced autophagy in human SH-SY5Y neuroblastoma cells, including activating LC3B and inhibiting p62. Inhibiting autophagy by 3MA aggravated NO-induced cell death, otherwise promoting autophagy by Rapamycin rescued NO-triggered cell death. Notably, BGN downregulated by NO, significantly protected SH-SY5Y cells against NO-induced neurotoxicity by inhibiting the activation of autophagy-dependent AMPK signaling pathway. Moreover, BGN overexpression also diminished NO-induced the elevation of intracellular reactive oxygen species (ROS) level, but not NO content. These findings suggest that BGN protects neuroblastoma cells from NO-induced death by suppressing autophagy-dependent AMPK-mTOR signaling and intracellular ROS level.

Topics: AMP-Activated Protein Kinases; Autophagy; Biglycan; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Neuroblastoma; Nitric Oxide; Nitroprusside; Reactive Oxygen Species; RNA-Binding Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM).. Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated.. The IC. MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Biomarkers; Cell Cycle; Cell Line, Tumor; Disease Models, Animal; Drug Interactions; Drug Resistance, Neoplasm; Gene Amplification; Humans; Mice; Molecular Structure; N-Myc Proto-Oncogene Protein; Neuroblastoma; Pyrroles; Sirolimus; Spectrum Analysis; Xenograft Model Antitumor Assays

Previous studies in various rodent epilepsy models have suggested that mammalian target of rapamycin (mTOR) inhibition with rapamycin has anti-epileptogenic potential. Since treatment with rapamycin produces unwanted side effects, there is growing interest to study alternatives to rapamycin as anti-epileptogenic drugs. Therefore, we investigated curcumin, the main component of the natural spice turmeric. Curcumin is known to have anti-inflammatory and anti-oxidant effects and has been reported to inhibit the mTOR pathway. These properties make it a potential anti-epileptogenic compound and an alternative for rapamycin.. To study the anti-epileptogenic potential of curcumin compared to rapamycin, we first studied the effects of both compounds on mTOR activation, inflammation, and oxidative stress in vitro, using cell cultures of human fetal astrocytes and the neuronal cell line SH-SY5Y. Next, we investigated the effects of rapamycin and intracerebrally applied curcumin on status epilepticus (SE)-induced inflammation and oxidative stress in hippocampal tissue, during early stages of epileptogenesis in the post-electrical SE rat model for temporal lobe epilepsy (TLE).. Rapamycin, but not curcumin, suppressed mTOR activation in cultured astrocytes. Instead, curcumin suppressed the mitogen-activated protein kinase (MAPK) pathway. Quantitative real-time PCR analysis revealed that curcumin, but not rapamycin, reduced the levels of inflammatory markers IL-6 and COX-2 in cultured astrocytes that were challenged with IL-1β. In SH-SY5Y cells, curcumin reduced reactive oxygen species (ROS) levels, suggesting anti-oxidant effects. In the post-SE rat model, however, treatment with rapamycin or curcumin did not suppress the expression of inflammatory and oxidative stress markers 1 week after SE.. These results indicate anti-inflammatory and anti-oxidant properties of curcumin, but not rapamycin, in vitro. Intracerebrally applied curcumin modified the MAPK pathway in vivo at 1 week after SE but failed to produce anti-inflammatory or anti-oxidant effects. Future studies should be directed to increasing the bioavailability of curcumin (or related compounds) in the brain to assess its anti-epileptogenic potential in vivo.

Topics: Animals; Anti-Inflammatory Agents; Astrocytes; Brain; Cells, Cultured; Curcumin; Cytokines; Disease Models, Animal; Fetus; Gene Expression Regulation; Humans; Inflammation; Male; Neuroblastoma; Neurons; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Status Epilepticus

Rapamycin (RAPA), an inhibitor of mammalian target of rapamycin (mTOR), exhibits a high neuroprotective action against neurodegenerative diseases in mouse models. Since neuroinflammation has been shown to be involved in Alzheimer's disease (AD) development and progression, the aim of this study was to examine the anti-inflammatory role of RAPA in AD in vivo and in vitro, and investigate the underlying mechanisms. We found that amyloid-β (Aβ) induced neuronal inflammation and a remarkable increase in mTOR activity in in-vivo and in-vitro models of inflammation, suggesting the critical role of mTOR signaling in neuronal inflammation. In addition, administration of RAPA was found to down-regulate mTOR, p-mTOR, Nuclear factor kappa B (NF-κB) p65, p-p65, TNF-α, IL-1β and Bax protein expression in Aβ

Topics: Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Brain; Cell Line, Tumor; Cell Proliferation; Cytokines; Disease Models, Animal; Inflammation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neuroblastoma; Neuroglia; NF-kappa B; Peptide Fragments; Signal Transduction; Sirolimus; Transfection

Alzheimer's disease (AD) is a complex and progressive neurological disorder, and amyloid-β (Aβ) has been recognized as the major cause of AD. Inhibiting Aβ production and/or enhancing the clearance of Aβ to reduce its levels are still the effective therapeutic strategies pursued in anti-AD research. In previous studies, we have reported that selenomethionine (Se-Met), a major form of selenium in animals and humans with significant antioxidant capacity, can reduce both amyloid-β (Aβ) deposition and tau hyperphosphorylation in a triple transgenic mouse model of AD. In this study, a Se-Met treatment significantly decreased the Aβ levels in Neuron-2a/AβPPswe (N2asw) cells, and the anti-amyloid effect of Se-Met was attributed to its ability to inhibit Aβ generation by suppressing the activity of BACE1. Furthermore, both the LC3-II/LC3-I ratio and the number of LC3-positive puncta were significantly decreased in Se-Met-treated cells, suggesting that Se-Met also promoted Aβ clearance by modulating the autophagy pathway. Subsequently, Se-Met inhibited the initiation of autophagy through the AKT-mTOR-p70S6K signaling pathway and enhanced autophagic turnover by promoting autophagosome-lysosome fusion and autophagic clearance. Our results further highlight the potential therapeutic effects of Se-Met on AD.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Autophagy; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation; Humans; Immunosuppressive Agents; Lactosylceramides; Macrolides; Microscopy, Electron, Transmission; Neuroblastoma; Oncogene Protein v-akt; Selenomethionine; Signal Transduction; Sirolimus; Transfection

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Neuroblastoma; para-Aminobenzoates; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

It is generally believed that gene-environment interaction may contribute to neurodegeneration. Of particular note is that iron overload may be one of the risk factors for neurodegeneration. However, the mechanisms underlying iron-associated neurotoxicity are not fully understood. Here we explored the effects of mechanistic target of rapamycin (mTOR) inhibition in iron-stressed human neuroblastoma cells. Two mTOR inhibitors, rapamycin and Torin 1, had similar effects in cells exposed to a relatively low concentration of iron. At a higher concentration of iron, Torin 1, instead of rapamycin, could further aggravate iron-induced cytotoxicity, and mitochondrial ROS levels were significantly higher in Torin 1-treated cells. These results suggest that mTOR inhibition may not be able to alleviate iron-induced neurotoxicity.

Topics: Cell Line, Tumor; Humans; Iron; Mitochondria; Naphthyridines; Neuroblastoma; Neuroprotective Agents; Reactive Oxygen Species; Sirolimus; TOR Serine-Threonine Kinases

Topics: Adult; Carcinoma, Hepatocellular; Esophagitis; Fatal Outcome; Humans; Liver Neoplasms; Male; Neuroblastoma; Radiation Injuries; Radiation-Sensitizing Agents; Sirolimus

Mammalian target of rapamycin (mTOR) inhibitors exert significant antitumor effects on several cancer cell types. In this study, we investigated the effects of mTOR inhibitors, in particular the regulation of the microRNA, in neuroblastoma cells.. AZD8055 (a new mTOR inhibitor)- or rapamycin-induced cytotoxic effects on neuroblastoma cells were studied. Western blotting was used to investigate the expression of various proteins in the mTOR pathway. MicroRNA precursors and antagomirs were transfected into cells to manipulate the expression of target microRNA.. AZD8055 exerted stronger cytotoxic effects than rapamycin in neuroblastoma cells (p<0.03). In addition, AZD8055 suppressed the mTOR pathway and increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the neuroblastoma cells. AZD8055 significantly decreased miR-19b expression (p<0.005); in contrast, rapamycin increased miR-19b expression (p<0.05). Transfection of miR-19b antagomir into the neuroblastoma cells mimicked the effects of AZD8055 treatment, whereas miR-19b overexpression reversed the effects of AZD8055. Combination of miR-19b knockdown and rapamycin treatment significantly improved the sensitivity of neuroblastoma cells to rapamycin (p<0.02).. Suppression of miR-19b may enhance the cytotoxic effects of mTOR inhibitors in neuroblastoma cells.

Topics: Blotting, Western; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Immunosuppressive Agents; MicroRNAs; Morpholines; Neuroblastoma; Real-Time Polymerase Chain Reaction; RNA, Neoplasm; Sirolimus; TOR Serine-Threonine Kinases

The insulin-like growth factors (IGFs), IGF-1 and IGF-2, have been implicated in the growth, survival and metastasis of a broad range of malignancies including pediatric tumors. They bind to the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR) which are overexpressed in many types of solid malignancies. Activation of the IR by IGF-2 results in increased survival of tumor cells. We have previously identified a novel human monoclonal antibody, m708.5, which binds with high (pM) affinity to both human IGF-1 and IGF-2, and potently inhibits phosphorylation of the IGF-1R and the IR in tumor cells. m708.5 exhibited strong antitumor activity as a single agent against most cell lines derived from neuroblastoma, Ewing family of tumor, rhabdomyosarcoma and osteosarcoma. When tested in neuroblastoma cell lines, it showed strong synergy with temsirolimus and synergy with chemotherapeutic agents in vitro. In xenograft models, the combination of m708.5 and temsirolimus significantly inhibited neuroblastoma growth and prolonged mouse survival. Taken together, these results support the clinical development of m708.5 for pediatric solid tumors with potential for synergy with chemotherapy and mTOR inhibitors.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Drug Synergism; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Mice, SCID; Neuroblastoma; Protein Binding; Sirolimus; Xenograft Model Antitumor Assays

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

Topics: Animals; Brain; Glucose Transporter Type 1; Glucose Transporter Type 3; MEF2 Transcription Factors; Mice; Naphthyridines; Neurites; Neuroblastoma; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Valerates

Current hypothesis suggest that tumors can originate from adult cells after a process of 'reprogramming' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic 'Warburg' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that 'in vitro' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate 'stem' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate.

Topics: Antibiotics, Antineoplastic; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; K562 Cells; Neoplastic Stem Cells; Neuroblastoma; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

To explore the effects of modulating autophagy on neuroamyloidogenesis in an ischemic stroke model of cultured neuroblastoma 2a (N2a)/Amyloid precursor protein (APP)695 cells.. The ischemic stroke model of N2a/APP695 cells was made by 6h oxygen-glucose deprivation/12h reperfusion (OGDR). Drug administration of 3-methyladenine (3-MA), rapamycin or dl-3-n-butylphthalide (NBP) was started at the beginning of the OGDR and lasted until the end of reperfusion, in order to explore their effects on N2a/APP695 cells under OGDR conditions. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 12h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-κB) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-β (Aβ)1-42 with Beclin 1 was performed to investigate their cellular co-localization relationship; β-secretase and γ-secretase activity assay and Aβ1-42 enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis.. The results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and Aβ generation in N2a/APP695 cells; down-regulating autophagy by 3-MA and NBP increased cell viability, decreased lactate dehydrogenase (LDH) production, inhibited the activation of NF-κB pathway, suppressed β- and γ-secretase activities and Aβ generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated Aβ1-42(+) signal was co-localized with Beclin 1(+) signal.. Our data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-κB pathway. This study might help us to find a new therapeutic strategy to prevent brain ischemic damage and depress the risk of post-stroke dementia.

Topics: Adenine; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Autophagy; Benzofurans; Brain Ischemia; Cell Survival; Mice; Neuroblastoma; Sirolimus; Stroke; Tumor Cells, Cultured

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Down-Regulation; ErbB Receptors; Genistein; Humans; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Neuroblastoma; Plasmids; Protein Kinase Inhibitors; RNA Interference; RNA, Small Interfering; Sirolimus; Transfection; Up-Regulation

Osteoprotegerin (OPG) is a decoy receptor for the Receptor of NF-κB (RANK) ligand that can inhibit osteoclastogenesis. Previous studies have suggested that Mammalian Target of Rapamycin (mTOR) inhibition upregulates OPG production. We tested the hypothesis that the mTOR inhibitor rapamycin could inhibit neuroblastoma bone metastases through its action on OPG.. An orthotopic model of bone metastasis was established. Mice with established disease were subsequently treated with rapamycin (5mg/kg IP daily) or vehicle control (DMSO 1:1000). X-rays were obtained twice a week to detect pathologic fractures. Serum OPG levels were measured by ELISA after two weeks of treatment.. Mice with bone disease receiving rapamycin had increased serum levels of OPG in the CHLA-20 mice compared to controls (36.89 pg/mL ± 3.90 vs 18.4 pg/mL ± 1.67, p=0.004) and NB1691 tumor-bearing groups (46.03 ± 2.67 pg/mL vs 17.96 ± 1.84pg/mL, p=0.001), and a significantly longer median time to pathologic fractures with CHLA-20 (103 days vs 74.5 days, p=0.014) and NB1691 xenografts.. In a xenograft model, increased OPG expression correlated with a delay to pathologic fracture suggesting a potential role for mTOR inhibitors in the treatment of neuroblastoma bone metastases.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Bone Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme-Linked Immunosorbent Assay; Fractures, Spontaneous; Humans; Injections, Intraperitoneal; Mice; Mice, SCID; Neuroblastoma; Osteoprotegerin; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Treatment Outcome

Children with high-risk and recurrent neuroblastoma have poor survival rates, and novel therapies are needed. Many cancer cells have been found to preferentially employ the glycolytic pathway for energy generation, even in the presence of oxygen. 3-BrOP is a novel inhibitor of glycolysis, and has demonstrated efficacy against a wide range of tumor types. To determine whether human neuroblastoma cells are susceptible to glycolysis inhibition, we evaluated the role of 3-BrOP in neuroblastoma model systems. Neuroblastoma tumor cell lines demonstrated high rates of lactate accumulation and low rates of oxygen consumption, suggesting a potential susceptibility to inhibitors of glycolysis. In all ten human tested neuroblastoma tumor cell lines, 3-BrOP induced cell death via apoptosis in a dose and time dependent manner. Furthermore, 3-BrOP-induced depletion of ATP levels correlated with decreased neuroblastoma cell viability. In a mouse neuroblastoma xenograft model, glycolysis inhibition with 3-BrOP demonstrated significantly reduced final tumor weight. In neuroblastoma tumor cells, treatment with 3-BrOP induced mTOR activation, and the combination of 3-BrOP and mTOR inhibition with rapamycin demonstrated synergistic efficacy. Based on these results, neuroblastoma tumor cells are sensitive to treatment with inhibitors of glycolysis, and the demonstrated synergy with rapamycin suggests that the combination of glycolysis and mTOR inhibitors represents a novel therapeutic approach for neuroblastoma that warrants further investigation.

Topics: Adenosine Triphosphate; Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Glycolysis; HEK293 Cells; Humans; Hydrocarbons, Brominated; Lactic Acid; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Oxygen Consumption; Propionates; Protein Kinase Inhibitors; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Cediranib (AZD2171) is a potent small molecule inhibitor of vascular endothelial growth factor (VEGF) receptors. Cediranib has demonstrated single agent activity in several adult cancers and is being studied in combination with standard cytotoxic agents in multiple disease settings.. Cediranib was tested in vivo against six childhood tumor xenograft models (four sarcomas, one glioblastoma, one neuroblastoma) alone or combined with cyclophosphamide (two models), vincristine (three models) or cisplatin (one model), each administered at its maximum tolerated dose, or rapamycin (six models).. The combination of cediranib with standard cytotoxic agents was superior to the cytotoxic agent used alone for a single xenograft (one of the three xenografts evaluated for the vincristine-cediranib combination). The cediranib-cyclophosphamide combination was inferior to single agent cyclophosphamide in time to event for both models studied and was significantly inferior for one of the models. Cediranib combined with rapamycin was superior to each of the agents used alone in two of the six models and was determined to be additive or supra-additive with rapamycin in four models, although the effects were not large.. Cediranib combined with cytotoxic chemotherapy agents demonstrated little or no benefit (and in one case was significantly inferior) compared to chemotherapy alone for the six pediatric cancer xenografts studied. By contrast, the combination of cediranib with rapamycin was additive or supra-additive in four of the six models in terms of prolongation of time to event, though tumor regressions were not observed for this combination.

Topics: Adolescent; Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Female; Glioblastoma; Humans; Infant; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Neuroblastoma; Quinazolines; Receptors, Vascular Endothelial Growth Factor; Sarcoma; Sirolimus; Transplantation, Heterologous; Xenograft Model Antitumor Assays

Activation of Akt is a marker of decreased event-free or overall survival in neuroblastoma patients. MK-2206, a novel allosteric Akt inhibitor, is now tested in clinical trials in adult cancers. In this study, effect of MK-2206 on tumor growth and murine survival, alone or in combination, with etoposide or rapamycin was evaluated.. The anticell proliferation effect of MK-2206 was tested in eight neuroblastoma cell lines by MTS assay. Caspase-3/7 activity, cell-cycle analysis, and reactive oxygen species (ROS) production were determined. Effect of MK-2206 combined with etoposide or rapamycin was evaluated in vitro and in vivo. Akt phosphorylation was measured by Western blotting in neuroblastoma cells and tumors.. In vitro, MK-2206 treatment inhibited neuroblastoma cell proliferation, which was accompanied by a cell line selective G(1) arrest of cell cycle or production of ROS. A synergistic effect between MK-2206 and etoposide was detected in four tested neuroblastoma cell lines via caspase-dependent apoptosis, whereas increased inhibition of cell growth induced by combination of MK-2206 and rapamycin was mediated by ROS production. In vivo, MK-2206 alone decreased tumor growth and increased murine survival at dose that inhibited Akt phosphorylation in tumors. MK-2206, in combination with etoposide or rapamycin, caused a significant decrease in tumor growth and increase of murine survival compared with MK-2206 alone.. Akt inhibition by MK-2206 increased the efficacy of etoposide or rapamycin. Our study supports future clinical evaluation of MK-2206 in combination with conventional cytotoxic therapy or with rapamycin in high-risk neuroblastoma patients.

Topics: Allosteric Regulation; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Etoposide; Female; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Mice; Mice, Nude; Neuroblastoma; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Sirolimus; Tumor Burden; Xenograft Model Antitumor Assays

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.

Topics: Acetylcysteine; Adaptor Proteins, Signal Transducing; AMP-Activated Protein Kinases; Apoptosis; Autophagy; Cell Line; Gene Expression Regulation; Humans; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Neuroblastoma; Oxidopamine; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Sequestosome-1 Protein; Sirolimus; TOR Serine-Threonine Kinases

Using a neuronal model of serum starved SK-N-SH neuroblastoma cells, we showed previously that the phosphorylation of Akt and the mTOR substrates S6K and S6 through the vascular endothelial growth factor receptor VEGFR2 was enhanced by treatments with the phosphatase PP2A inhibitor okadaic acid (OA). These findings suggested that PP2A inhibition uncouples the regulation of Akt signaling by mTOR and affects cell survival. We therefore examined the effects of mTOR inhibition on Akt phosphorylation at sites threonine 308 (T308) and serine 473 (S473) and survival in OA treated cells. OA induced a loss in cell viability, the accumulation of hyperactivated Akt as monomeric and ubiquitinated forms and an increase in the total levels of ubiquitinated proteins. These events were exacerbated by treatments with an allosteric (rapamycin) but not an active-site inhibitor (PP242) of mTOR. Notably, rapamycin augmented the OA-induced hyperphosphorylation of Akt by suppressing a negative feedback loop of Akt activation through VEGFR2 and its downstream target phosphatidylinositol 3-kinase (PI3K). Treatments with the antioxidant N-acetlycysteine but not the pan caspase inhibitor Z-VAD-FMK promoted survival. Unlike reports that rapamycin promotes survival through increased Akt activation, these findings show that rapamycin-induced hyperphosphorylation of Akt fails to rescue our neuronal model from an oxidative stress-induced and caspase-independent cell death mediated by PP2A inhibition. Moreover, the exacerbation of OA-induced events by rapamycin suggests that mTOR and PP2A work in concert to regulate cell survival, activated Akt and the levels of ubiquitinated proteins.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; Immunoblotting; Immunoprecipitation; Neuroblastoma; Okadaic Acid; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor Receptor-2

Tumor angiogenesis in childhood neuroblastoma is an important prognostic factor suggesting a potential role for antiangiogenic agents in the treatment of high-risk disease. Within the KidsCancerKinome project, we evaluated the new oral selective pan-VEGFR tyrosine kinase inhibitor axitinib (AG-013736) against neuroblastoma cell lines and the subcutaneous and orthotopic xenograft model IGR-N91 derived from a primary bone marrow metastasis. Axitinib reduced cell proliferation in a dose-dependent manner with IC(50) doses between 274 and >10,000 nmol/l. Oral treatment with 30 mg/kg BID for 2 weeks in advanced tumors yielded significant tumor growth delay, with a median time to reach five times initial tumor volume of 11.4 days compared to controls (p = 0.0006) and resulted in significant reduction in bioluminescence. Simultaneous inhibition of VEGFR downstream effector mTOR using rapamycin 20 mg/kg q2d×5 did not statistically enhance tumor growth delay compared to single agent activities. Axitinib downregulated VEGFR-2 phosphorylation resulting in significantly decreased microvessel density (MVD) and overall surface fraction of tumor vessels (OSFV) in all xenografts as measured by CD34 immunohistochemical staining (mean MVD ± SD and OSFV at 14 days 21.27 ± 10.03 in treated tumors vs. 48.79 ± 17.27 in controls and 0.56% vs. 1.29%; p = 0.0006, respectively). We further explored the effects of axitinib on circulating mature endothelial cells (CECs) and endothelial progenitor cells (CEPs) measured by flow cytometry. While only transient modification was observed for CECs, CEP counts were significantly reduced during and up to 14 days after end of treatment. Axitinib has potent antiangiogenic properties that may warrant further evaluation in neuroblastoma.

Topics: Animals; Apoptosis; Axitinib; Blotting, Western; Bone Marrow Neoplasms; Child; Drug Synergism; Endothelium, Vascular; Humans; Imidazoles; Indazoles; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Neuroblastoma; Phosphorylation; Sirolimus; Stem Cells; Tumor Cells, Cultured; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factor Receptor-3; Xenograft Model Antitumor Assays

Neuroblastoma is a malignant pediatric tumor with poor survival. The phosphatidylinositol 3'-kinase/AKT pathway is a crucial regulator of cellular processes including apoptosis. Thioredoxin 1, an inhibitor of tumor-suppressor phosphatase and tensin homolog, is overexpressed in many tumors. The objective of this study was to explore phosphatidylinositol 3'-kinase/AKT pathway activation and regulation by thioredoxin 1 to identify potential therapeutic targets. Immunohistochemical analysis was done on tissue microarrays from tumor samples of 101 patients, using antibodies against phosphatidylinositol 3'-kinase, AKT, activated AKT, phosphatase and tensin homolog, phosphorylated phosphatase and tensin homolog, thioredoxin 1, epidermal growth factor receptor, vascular endothelial growth factor and receptors (vascular endothelial growth factor 1 and vascular endothelial growth receptor 2), platelet-derived growth factor receptors, insulin-like growth factor 1 receptor, neurotrophic tyrosine kinase receptor type 2, phosphorylated 70-kd S6 protein kinase, 4E-binding protein 1, and phosphorylated mammalian target of rapamycin. Using 3 neuroblastoma cell lines, we investigated cell viability with AKT-specific inhibitors (LY294002, RAD001) and thioredoxin 1 alone or in combination. We found activated AKT and AKT expressed in 97% and 98%, respectively, of neuroblastomas, despite a high expression of phosphatase and tensin homolog correlated with thioredoxin 1. AKT expression was greater in metastatic than primary tumors. Insulin-like growth factor 1 receptor, tyrosine kinase receptor type 2, vascular endothelial growth receptor 1, and downstream phosphorylated 70-kd S6 protein kinase were correlated with activated AKT. LY294002 and RAD001 significantly reduced AKT activity and cell viability and induced a G(1) cell cycle arrest. Thioredoxin 1 decreased cytotoxicity of AKT inhibitors and doxorubicin, up-regulated AKT activation, and induced cell growth. Thus, vascular endothelial growth receptor 1, tyrosine kinase receptor type 2, insulin-like growth factor 1 receptor, and thioredoxin 1 emerged as preferentially committed to phosphatidylinositol 3'-kinase/AKT pathway activation as observed in neuroblastoma. Thioredoxin 1 is a potential target for therapeutic intervention.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Chromones; Doxorubicin; Enzyme Activation; Everolimus; Humans; Infant; Morpholines; Neuroblastoma; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; Thioredoxins; Vascular Endothelial Growth Factor A

Our previous microarray analysis identified a neuroprotective protein Oxi-alpha, that was down-regulated during oxidative stress (OS)-induced cell death in dopamine neurons [Neurochem. Res. (2004) vol. 29, pp. 1223]. Here we find that the phylogenetically conserved Oxi-alpha protects against OS by a novel mechanism: activation of the mammalian target of rapamycin (mTOR) kinase and subsequent repression of autophagic vacuole accumulation and cell death. To the best of our knowledge, Oxi-alpha is the first molecule discovered in dopamine neurons, which activates mTOR kinase. Indeed, the down-regulation of Oxi-alpha by OS suppresses the activation of mTOR kinase. The pathogenic effect of down-regulated Oxi-alpha was confirmed by gene-specific knockdown experiment, which resulted in not only the repression of mTOR kinase and the subsequent phosphorylation of p70 S6 kinase and 4E-BP1, but also enhanced susceptibility to OS. In accordance with these observations, treatment with rapamycin, an mTOR inhibitor and autophagy inducer, potentiated OS-induced cell death, while similar treatment with an autophagy inhibitor, 3-methyladenine protected the dopamine cells. Our findings present evidence for the presence of a novel class of molecule involved in autophagic cell death triggered by OS in dopamine neurons.

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy; Carrier Proteins; Cell Cycle Proteins; Cell Line, Transformed; Cell Line, Tumor; Dopamine; Down-Regulation; Eukaryotic Initiation Factors; Green Fluorescent Proteins; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Mice; Microscopy, Confocal; Neuroblastoma; Neurons; Oxidative Stress; Phosphoproteins; Phylogeny; Protein Kinases; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

Resistance to angiogenesis inhibition can occur through the upregulation of alternative mediators of neovascularization. We used a combination of angiogenesis inhibitors with different mechanisms of action, interferon-beta (IFN-beta) and rapamycin, to target multiple angiogenic pathways to treat neuroblastoma xenografts.. Subcutaneous and retroperitoneal neuroblastoma xenografts (NB-1691 and SK-N-AS) were used. Continuous delivery of IFN-beta was achieved with adeno-associated virus vector-mediated, liver-targeted gene transfer. Rapamycin was delivered intraperitoneally (5 mg/kg per day). After 2 weeks of treatment, tumor size was measured, and tumor vasculature was evaluated with intravital microscopy and immunohistochemistry.. Rapamycin and IFN-beta, alone and in combination, had little effect on tumor cell viability in vitro. In vivo, combination therapy led to fewer intratumoral vessels (69% of control), and the remaining vessels had an altered phenotype, being covered with significantly more pericytes (13x control). Final tumor size was significantly less than controls in all tumor models, with combination therapy having a greater antitumor effect than either monotherapy.. The combination of IFN-beta and rapamycin altered the vasculature of neuroblastoma xenografts and resulted in significant tumor inhibition. The use of combinations of antiangiogenic agents should be further evaluated for the treatment of neuroblastoma and other solid tumors.

Topics: Actins; Animals; Antibiotics, Antineoplastic; Antigens, CD34; Cluster Analysis; Humans; Immunohistochemistry; Immunologic Factors; Injections, Intraperitoneal; Interferon-beta; Male; Mice; Mice, SCID; Neoplasms, Experimental; Neovascularization, Pathologic; Neuroblastoma; Retroperitoneal Neoplasms; Sirolimus; Transplantation, Heterologous; Tumor Cells, Cultured

Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Dequalinium; Electron Transport; Gene Expression Profiling; Humans; Mice; Mice, Inbred NOD; Mitochondria; Neoplastic Stem Cells; Neuroblastoma; Sirolimus; Small Molecule Libraries; Xenograft Model Antitumor Assays

It has been previously shown that blockade of the type 1 insulin-like growth factor receptor (IGF1R) signaling combined with mTOR inhibition decreased neuroblastoma proliferation in vitro. MYC-N inactivation occurs through phosphorylation by downstream elements of the IGF1R signaling pathway. It was hypothesized that inhibition of IGF1R signaling would increase the inactivation of MYC-N.. BE-2(c) and IMR-32 neuroblastoma cell lines were treated with varying concentrations of alphaIR3, rapamycin and temsirolimus either alone or in combination and the expression of MYC-N and phosphorylated MYC-N proteins were evaluated by Western blotting. The number of apoptotic cells was evaluated through cleaved caspase-3 expression.. IGF1R signaling blockade in combination with mTOR inhibition decreased MYC-N protein expression, increased MYC-N phosphorylation and significantly increased cleaved caspase-3 expression in treated cells.. The combination of rapamycin or temsirolimus with alphaIR3 decreases MYC-N expression, increases MYC-N phosphorylation and induces apoptosis in vitro which may have clinical relevance to children with neuroblastoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Phosphorylation; Receptor, IGF Type 1; Signal Transduction; Sirolimus; Tumor Cells, Cultured

SIMCA classification can be applied to 2D-PAGE maps to identify changes occurring in cellular protein contents as a consequence of illnesses or therapies. These data sets are complex to treat due to the large number of proteins detected. A method for identifying relevant proteins from SIMCA discriminating powers is proposed, based on the Box-Cox transformation coupled to probability papers. The method successfully allowed the identification of the relevant spots from 2D maps.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Endothelium; Humans; Hydroxamic Acids; Mice; Neoplasms; Neuroblastoma; Pancreas; Pancreatic Neoplasms; Probability; Proteins; Proteomics; Sirolimus; Statistical Distributions; Vinblastine

Autophagy was induced in human neuroblastoma SH-SY5Y cells by two different procedures: deprivation of fetal serum in culture medium, or treatment with dopamine. 3-methyladenine prevented autophagy in the two procedures. Although it is usually considered that the conversion of soluble LC3-I to lipid bound LC3-II is associated with the formation of autophagosomes, the inhibition of autophagy with 3-methyladenine prevented this transformation in serum-deprived but not in dopamine-treated cells. While the PI3K-mTOR pathway was inhibited by serum deprivation, dopamine increased the phosphorylation of Akt but inhibited mTOR activity in a similar way to rapamycin. Dopamine and rapamycin increased LC3-II levels by a mechanism not prevented by 3-methyladenine. The activation of LC3-I to LC3-II may then be necessary but not sufficient to trigger cell autophagy. Thus, the increase in LC3-II, as the main biochemical parameter for autophagy at present, should be considered with caution.

Topics: Adenine; Autophagy; Blotting, Western; Dopamine; Humans; Microscopy, Electron; Microscopy, Fluorescence; Microtubule-Associated Proteins; Neuroblastoma; Phosphatidylinositol 3-Kinases; Protein Kinases; Serum; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Mammalian target of rapamycin (mTOR) has been shown to play an important function in cell proliferation, metabolism and tumorigenesis, and proteins that regulate signaling through mTOR are frequently altered in human cancers. In this study we investigated the phosphorylation status of key proteins in the PI3K/AKT/mTOR pathway and the effects of the mTOR inhibitors rapamycin and CCI-779 on neuroblastoma tumorigenesis. Significant expression of activated AKT and mTOR were detected in all primary neuroblastoma tissue samples investigated, but not in non-malignant adrenal medullas. mTOR inhibitors showed antiproliferative effects on neuroblastoma cells in vitro. Neuroblastoma cell lines expressing high levels of MYCN were significantly more sensitive to mTOR inhibitors compared to cell lines expressing low MYCN levels. Established neuroblastoma tumors treated with mTOR inhibitors in vivo showed increased apoptosis, decreased proliferation and inhibition of angiogenesis. Importantly, mTOR inhibitors induced downregulation of vascular endothelial growth factor A (VEGF-A) secretion, cyclin D1 and MYCN protein expression in vitro and in vivo. Our data suggest that mTOR inhibitors have therapeutic efficacy on aggressive MYCN amplified neuroblastomas.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Down-Regulation; Female; Growth Inhibitors; Humans; Infant; Infant, Newborn; Male; Mice; Mice, Nude; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.

Topics: Animals; Calcium; Calcium Channels; Electrophysiology; Humans; Immunophilins; Immunosuppressive Agents; Ligands; Models, Chemical; Neurites; Neuroblastoma; Neurons; Patch-Clamp Techniques; Protein Binding; Rats; Sirolimus; Stroke; Tacrolimus Binding Proteins

Cell differentiation is often associated with decreased cell growth, indicating an altered rate of macromolecule synthesis and degradation. In this study, we present evidence that autophagy, a process for bulk degradation of cytoplasm, is activated during retinoic acid-induced neuronal differentiation of neuroblastoma N2a cells. Chemical inhibitors of autophagy, including 3-MA and LY294002, abrogate cell differentiation. RNA interference of autophagy gene beclin 1 markedly delays the process of differentiation. We also find that cell differentiation is accompanied by decreased activity of mTOR, a major controller of cell growth and a negative regulator of autophagy. However, completely inhibiting mTOR by rapamycin decreases neurite outgrowth, cell size and the immunoreactivity for neuronal markers. Our study suggests that an appropriate level of mTOR activity is important in cell differentiation for a balance between macromolecule synthesis and degradation.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Size; Chromones; Dose-Response Relationship, Drug; Down-Regulation; Mice; Morpholines; Neurites; Neuroblastoma; Neurons; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tretinoin

Vinblastine and rapamycin displayed synergistic inhibition of human neuroblastoma-related angiogenesis. Here, we studied the antitumor activity of vinblastine and rapamycin against human neuroblastoma.. Cell proliferation, cell cycle progression, and apoptosis were evaluated by measuring (3)H-thymidine incorporation, bromodeoxyuridine uptake, and phosphatidylserine exposure, respectively. The in vivo sensitivity of neuroblastoma cells to vinblastine and rapamycin was determined in orthotopic neuroblastoma-engrafted mice. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay.. Each compound alone was able to induce a dose-dependent significant inhibition of cell proliferation, with a dramatically enhanced antiproliferative effect for the drugs used in combination. A marked G(2)-M cell cycle arrest with a nearly complete depletion of S phase was associated. The combined treatment triggered an increased apoptosis compared with either drug tested alone. A significant inhibition of tumor growth and microvessel area was obtained in neuroblastoma-bearing mice when treated with vinblastine or rapamycin alone, and a more dramatic effect with the combined treatment, compared with control mice. The therapeutic effectiveness, expressed as increased life span, was statistically improved by the combined therapy, compared with mice treated with either drug tested separately. Histologic evaluation of primary tumors showed that the combined treatment inhibited proliferation and angiogenesis and induced apoptosis. Combined treatment of neuroblastoma cells and neuroblastoma-bearing mice with vinblastine and rapamycin induced the down-modulation of both vascular endothelial growth factor production and vascular endothelial growth factor receptor 2 expression. In the chorioallantoic membrane assay, angiogenesis induced by human neuroblastoma biopsy specimens was significantly inhibited by vinblastine and rapamycin.. These results may be relevant to design new therapeutic strategies against neuroblastoma.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Mice; Neovascularization, Pathologic; Neuroblastoma; Phosphatidylserines; Sirolimus; Vascular Endothelial Growth Factor Receptor-2; Vinblastine

RB1-inducible Coiled-Coil 1 (RB1CC1) has been shown to be a novel tumor suppressor regulating RB1 expression. Neuronal abundance of RB1CC1 is reported to contribute to the non-proliferating enlarged cell phenotype through the maintenance of RB1 and mTOR. To clarify whether RB1CC1 insufficiency is involved in neuronal atrophy and Alzheimer's pathology, we investigated modifications of RB1CC1 as a possible cause of atrophy or death through the disturbance of mTOR signaling in Neuro-2a neuroblastoma cells. We also evaluated the correlation between RB1CC1 and mTOR signaling in a series of Alzheimer's brain tissues. Though RB1CC1 introduction enhanced neurite growth, RNAi-mediated knockdown of RB1CC1 or rapamycin treatment caused neurite atrophy and apoptosis due to mTOR signaling reduction in the differentiated Neuro-2a cells. Both TSC1 and RB1CC1 were equally functional and maintained mTOR signaling, indicated by phospho-S6 (Ser240/244) expression in 69% of Alzheimer's (9/13 cases) and 100% of normal brains (6/6 cases). However, scanty RB1CC1 expression, less than TSC1, caused phospho-S6 disappearance in 31% of Alzheimer's tissues (4/13 cases). These findings suggest that RB1CC1 insufficiency may result in mTOR signaling repression through unbalanced TSC1 abundance and may induce neuronal atrophy. These observations may have implications for the pathogenesis of Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Atrophy; Autophagy-Related Proteins; Brain; Cell Cycle; Cell Death; Cell Line, Tumor; Embryo, Mammalian; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Mice; Neuroblastoma; Neurons; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cholesterol; Cholesterol Esters; Dose-Response Relationship, Drug; Esterification; Everolimus; Fibroblasts; Genotype; Lymphocytes; Mice; Neuroblastoma; Pioglitazone; Prions; Scrapie; Sheep; Sheep Diseases; Sirolimus; Thiazolidinediones

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system characterized by two major lesions: extracellular senile plaques and intraneuronal neurofibrillary tangles. beta-Amyloid (Abeta) is known to play a major role in the pathogenesis of AD. Protein synthesis and especially translation initiation are modulated by different factors, including the PKR/eIF2 and the mTOR/p70S6K pathways. mRNA translation is altered in the brain of AD patients. Very little is known about the translation control mediated by mTOR in AD, although mTOR is a central regulator of translation initiation and also ribosome biogenesis and cell growth and proliferation. In this study, by using Western blotting, we show that mTOR pathway is down-regulated by Abeta treatment in human neuroblastoma cells, and the underlying mechanism explaining a transient activation of p70S6K is linked to cross-talk between mTOR and ERK1/2 at this kinase level. This phenomenon is associated with caspase-3 activation, and inhibition of mTOR by the inhibitor rapamycin enhances Abeta-induced cell death. Moreover, in our cell model, insulin-like growth factor-1 is able to increase markedly the p70S6K phosphorylation controlled by mTOR and reduces the caspase-3 activity, but its protective effect on Abeta cell death is mediated via an mTOR-independent pathway. These results demonstrate that mTOR plays an important role as a cellular survival pathway in Abeta toxicity and could represent a possible target for modulating Abeta toxicity.

Topics: Amyloid beta-Peptides; Blotting, Western; Brain Neoplasms; Caspase 3; Cell Line, Tumor; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Fluorescent Dyes; Humans; Immunosuppressive Agents; Indoles; Insulin-Like Growth Factor I; Neuroblastoma; Neurotoxins; Peptide Fragments; Protein Kinases; Receptor Cross-Talk; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.

Topics: Analysis of Variance; Animals; Antibiotics, Antineoplastic; Blotting, Western; Brain; Cell Line, Tumor; Cell Survival; Culture Media, Serum-Free; Cytoplasm; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunohistochemistry; In Vitro Techniques; Microscopy, Immunoelectron; Models, Biological; Neuroblastoma; Phosphorylation; rab5 GTP-Binding Proteins; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; tau Proteins; Tetrazolium Salts; Thiazoles; Time Factors; Zinc; Zinc Sulfate

Hyperphosphorylated neurofilaments are a part of neurofibrillary tangles in Alzheimer's disease brains. Zinc has been shown to be increased in the brain areas heavily affected by Alzheimer pathologies. Zinc could induce tau hyperphosphorylation in SH-SY5Y and N2a cells, and tau phosphorylation may be mediated by p70 S6 kinase activation. Many of the tau kinases can also phosphorylate neurofilaments, and in this study we wanted to see whether neurofilament phosphorylation is regulated by p70 S6 kinase in N2a cells. We found that zinc induces rapamycin-dependent p70 S6 kinase phosphorylation at Thr421/Ser424 and Thr389, and rapamycin-independent phosphorylation of neurofilaments at the SMI34 epitope. Although zinc could induce cell proliferation and cell growth, and increased phosphorylation of neurofilaments, only cell growth appeared to be related to p7056kinase activation.

Topics: Antibiotics, Antineoplastic; Cell Division; Cell Line, Tumor; Culture Media, Serum-Free; Enzyme Activation; Humans; Neuroblastoma; Neurofilament Proteins; Neurons; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Zinc

The aim of this study was to evaluate the synergistic antiangiogenic effect of low dose of vinblastine (VBL) and rapamycin (RAP) in neuroblastoma (NB). Both in vitro (endothelial cells proliferation assay; TUNEL assay; phosphatidylserine exposure and cell cycle analysis) and in vivo (chick embryo chorioallantoic membrane, CAM) assays were used. Each compound alone was able to induce a significant dose- and time-response inhibition of in vitro endothelial cells (EC) growth. Interaction index evaluation indicates that a synergistic effect was found when both drugs were combined at very low doses. Comparable effects were obtained when EC were preincubated with conditioned medium (CM) derived from the human NB cell line HTLA-230. Morphological changes were induced by each drug, and their combination resulted in a clear and stronger effect. Apoptosis was demonstrated by the TUNEL assay and confirmed by Annexin V-FITC staining of EC treated with VBL, showing an increase in the percentage of cells with a G2-M and sub-G1 DNA content, whereas in those treated with RAP a block in the G1 cell fraction and inhibition of progression to the S phase were observed. Here too, the combination resulted in a synergistic cell cycle arrest and induction of apoptosis. Similar results were obtained in vivo with the CAM assay. The angiogenic responses induced by HTLA-230-derived CM, NB tumor xenografts, and human NB biopsy specimens were inhibited by each drug and more significantly by their combination. The observation that these well-known drugs display synergistic effects as antiangiogenics when administered frequently at very low dose may be of significance in the designing of new ways of treating NB.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Culture Media, Conditioned; Drug Synergism; Endothelium, Vascular; Female; Humans; In Situ Nick-End Labeling; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Neuroblastoma; Sirolimus; Vinblastine

MYCN and insulin-like growth factor (IGF) system are important for the pathogenesis and development of neuroblastoma. We previously reported evidence of a direct linkage between MycN and the IGF system in KP-N-RT human neuroblastoma cells, where IGF-I induced both MycN expression at the RNA level and G1-S cell cycle progression through the IGF-I receptor (IGF-IR)/ MEK/ mitogen-activated protein kinase (MAPK) pathway (A. Misawa et al., Cancer Res, 2000; 60:64-9). Our data also showed the possibility of a potent IGF-IR downstream signal cascade that accelerates progression into the S-phase, other than the MAPK pathway. In this study, we further investigated the role of this alternative pathway in the growth of neuroblastoma cells. A phosphoinositide 3-kinase (PI3K) inhibitor wortmannin blocked IGF-I-mediated induction of MycN. Our data suggest that the inhibition of MycN by wortmannin was transmitted through the MAPK pathway. Progression of the cell cycle from G1 to S phase was inhibited up to 90% by wortmannin or rapamycin, an inhibitor of mTOR, which acts downstream of PI3K. Despite its effects on induction of MycN and on progression through S phase, wortmannin did not block proliferation of neuroblastoma cells. On the other hand, rapamycin inhibited both IGF-I-induced cell cycle progression and cell proliferation in complete medium, although it had no effect on IGF-I-mediated MycN induction. Our study indicates maintenance of cell proliferation requires mTOR function, which is independent of MycN induction in human neuroblastoma cells.

Topics: Androstadienes; Cell Cycle; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinases; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Sirolimus; Time Factors; Tumor Cells, Cultured; Wortmannin

Glial cell line-derived neurotrophic factor (GDNF) activates c-Ret tyrosine kinase and several downstream intracellular pathways; the biological effects caused by the activation of each of these pathways, however, remain to be elucidated. Here we report the ability of GDNF to induce proliferation, rather than differentiation, of neuroblastoma cells (SH-SY5Y) by targeting the signaling pathway responsible for mediating this proliferative effect. GDNF induces the phosphorylation of Akt and p70S6 kinase (p70S6K) in SH-SY5Y cells in which Ret protein expression is relatively low. Interestingly, treating SH-SY5Y cells with retinoic acid greatly increases Ret protein levels and GDNF-induced Ret tyrosine phosphorylation, but does not affect the mitogenic action of GDNF and the activation of the Akt/p70S6K pathway. In contrast, the activation of the ERK pathway and the resulting induction of immediate-early genes parallel the increases in Ret protein levels. Rapamycin, a specific inhibitor of p70S6K activation by the mammalian target of rapamycin, completely prevents GDNF-induced proliferation and activation of p70S6K. These results suggest that GDNF promotes cell proliferation via the activation of p70S6K, independent of the ERK signaling pathway, and that GDNF activates the Akt/p70S6K pathway more efficiently than the ERK pathway in the cells in which Ret expression is low.

Topics: Blotting, Western; Brain Neoplasms; Cell Division; Cell Line; Enzyme Activation; Glial Cell Line-Derived Neurotrophic Factor; Humans; Immunosuppressive Agents; Mitogen-Activated Protein Kinases; Mitogens; Nerve Growth Factors; Neuroblastoma; Precipitin Tests; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Tumor Cells, Cultured; Up-Regulation

The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+ release and Ca2+ uptake in permeabilized cell types. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+ fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells. Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to approximately 30 % of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This FK506-induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-Mg2+-ATPase (SERCA) Ca2+ pump. Oxalate-facilitated 45Ca2+ uptake in SH-SY5Y microsomes was inhibited by FK506 with an IC50 of 19 microM. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. FK506 (10 microM) did not affect IP3-induced Ca2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC3H1 cells. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3-induced Ca2+ release.

Topics: Animals; Antiprotozoal Agents; Aorta; B-Lymphocytes; Biological Transport; Caffeine; Calcimycin; Calcium; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Chickens; Enzyme Inhibitors; Humans; Immunosuppressive Agents; Inositol 1,4,5-Trisphosphate Receptors; Ionophores; Ivermectin; Mice; Microsomes; Muscle, Smooth, Vascular; Neuroblastoma; Oxalates; Phosphodiesterase Inhibitors; Rats; Receptors, Cytoplasmic and Nuclear; Sirolimus; Spermine; Tacrolimus; Thapsigargin; Tumor Cells, Cultured

In human neuroblastoma SH-SY5Y cells, treatment with immunosuppressants such as FK506, cyclosporin A or rapamycin for 4 days induced the enhancement of the 27-kDa Bcl-2alpha protein level. Among immunosuppressants, rapamycin has most potency. Treatment with herbimycin A or wortmannin also enhanced Bcl-2 expression, but the BB type of platelet-derived growth factor decreased the level. These results suggest that Bcl-2 expression is probably regulated by the cascade of tyrosine kinase, phosphatidylinositol 3-kinase and rapamycin-sensitive p70 S6-kinase in human neuroblastoma SH-SY5Y cells.

Topics: bcl-X Protein; Gene Expression; Humans; Immunosuppressive Agents; Neuroblastoma; Polyenes; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Sirolimus; Tumor Cells, Cultured