RESUMEN
BACKGROUND: Intratumor subsets with tumor-initiating features in glioblastoma are likely to survive treatment. Our goal is to identify the key factor in the process by which cells develop temozolomide (TMZ) resistance. METHODS: Resistant cell lines derived from U87MG and A172 were established through long-term co-incubation of TMZ. Primary tumors obtained from patients were maintained as patient-derived xenograft for studies of tumor-initating cell (TIC) features. The cell manifestations were assessed in the gene modulated cells for relevance to drug resistance. RESULTS: Among the mitochondria-related genes in the gene expression databases, superoxide dismutase 2 (SOD2) was a significant factor in resistance and patient survival. SOD2 in the resistant cells functionally determined the cell fate by limiting TMZ-stimulated superoxide reaction and cleavage of caspase-3. Genetic inhibition of the protein led to retrieval of drug effect in mouse study. SOD2 was also associated with the TIC features, which enriched in the resistant cells. The CD133+ specific subsets in the resistant cells exhibited superior superoxide regulation and the SOD2-related caspase-3 reaction. Experiments applying SOD2 modulation showed a positive correlation between the TIC features and the protein expression. Finally, co-treatment with TMZ and the SOD inhibitor sodium diethyldithiocarbamate trihydrate in xenograft mouse models with the TMZ-resistant primary tumor resulted in lower tumor proliferation, longer survival, and less CD133, Bmi-1, and SOD2 expression. CONCLUSION: SOD2 plays crucial roles in the tumor-initiating features that are related to TMZ resistance. Inhibition of the protein is a potential therapeutic strategy that can be used to enhance the effects of chemotherapy.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Resistencia a Antineoplásicos/genética , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Superóxido Dismutasa/administración & dosificación , Temozolomida/farmacología , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos/fisiopatología , Humanos , Ratones , Células Madre Neoplásicas/fisiologíaRESUMEN
Klebsiella pneumoniae (KP) is the most common pathogen of pyogenic liver abscess in East and Southeast Asia and diabetes mellitus (DM) is a major risk factor. The effect and mechanism of diabetes on KP liver abscess was examined in streptozotocin-induced diabetic mice and Akita mice (C57BL/6J-Ins2Akita). KP translocation to liver and plasma alaine transaminase levels were increased and liver clearance of KP was decreased in DM mice. Diabetic mice exhibited overgrowth of Enterococcus as well as E.coli and decreased lactobacilli/bifidas growth in intestine, increased intestinal iNOS protein and nitrite levels in portal vein, and increased IL-1ß and TNF-α expression of Kupffer cells. Fructooligosaccharides (FOS) or dead L. salivarius (dLac) supplementation reversed diabetes-induced enteric dysbiosis, NO levels in portal vein, and KP translocation to liver. L-NAME treatment decreased intestinal iNOS protein expression as well as Kupffer cell activation and increased liver clearance of KP in DM mice. Dead E.coli (2×108 CFU/ml) feeding for one week induced iNOS and TLR4 expression of intestine in germ-free (GF) mice. Dead bacteria feeding induced IL-1ß and TNF-α expression of Kupffer cells in GF mice but not in GF TLR4-/- mice. In conclusion, balance of intestinal microflora is important for preventing intestinal iNOS expression, Kupffer cell activation, and KP liver translocation in diabetes. Reversal of diabetes-induced enteric dysbiosis with FOS or dead L. salivarius decreases diabetes-induced intestinal iNOS expression and KP liver translocation. Diabetes induces Kupffer cell activation and KP liver translocation through enteric dysbiosis and nitric oxide production.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Infecciones por Klebsiella/etiología , Infecciones por Klebsiella/fisiopatología , Macrófagos del Hígado/patología , Hígado/microbiología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Alanina Transaminasa/genética , Alanina Transaminasa/metabolismo , Animales , Western Blotting , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/terapia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Infecciones por Klebsiella/prevención & control , Klebsiella pneumoniae/fisiología , Ligilactobacillus salivarius/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Oligosacáridos/uso terapéutico , ARN Ribosómico 16S/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Altered intestinal microbiota and subsequent endotoxemia play pathogenic roles in diabetes. We aimed to study the mechanisms of intestinal defense impairment in type 1 diabetes and the effects of Lactobacillus salivarius as well as fructooligosaccharides (FOS) supplementation on diabetes-induced bacterial translocation. Alterations in the enteric microbiome, expression of mucosal antibacterial proteins and bacteria-killing activity of the intestinal mucosa in streptozotocin (STZ)-induced diabetic mice and Ins2(Akita) mice were investigated. The effects of dead L. salivarius (2×10(8)CFU/ml) and FOS (250 mg per day) supplementation for 1 week on endotoxin levels and Klebsiella pneumoniae translocation were also examined. Finally, germ-free mice were cohoused with wild-type or Ins2(Akita) mice for 2 weeks to examine the contribution of microbiota on the antibacterial protein expression. STZ-induced diabetic mice developed intestinal defense impairment as demonstrated by decreased mucosal bacteria-killing activity; reduction of non-defensin family proteins, such as Reg3ß, Reg3γ, CRP-ductin and RELMß, but not the defensin family proteins; and increased bacterial translocation. Intestinal bacteria overgrowth, enteric dysbiosis and increased intestinal bacterial translocation, particularly pathogenic K. pneumoniae in STZ-induced diabetic mice and Ins2(Akita) mice, were noted. Treating diabetic mice with dead L. salivarius or FOS reversed enteric dysbiosis, restored mucosal antibacterial protein and lessened endotoxin levels as well as K. pneumoniae translocation. Moreover, germ-free mice cohoused with wild-type mice demonstrated more intestinal Reg3ß and RELMß expression than those cohoused with Ins2(Akita) mice. These results indicate that hyperglycemia induces enteric dysbiosis, reduction of non-defensin proteins as well as bacteria-killing activity of the intestinal mucosa and intestinal defense impairment. Reversal of enteric dysbiosis with dead L. salivarius or FOS supplementation decreases diabetes-induced K. pneumoniae translocation and endotoxin levels through the induction of non-defensin proteins.
Asunto(s)
Diabetes Mellitus Tipo 1/dietoterapia , Suplementos Dietéticos , Disbiosis/dietoterapia , Inmunidad Mucosa , Mucosa Intestinal/microbiología , Ligilactobacillus salivarius/inmunología , Animales , Traslocación Bacteriana , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/microbiología , Disbiosis/inmunología , Disbiosis/metabolismo , Disbiosis/microbiología , Endotoxinas/antagonistas & inhibidores , Endotoxinas/sangre , Endotoxinas/metabolismo , Regulación de la Expresión Génica , Vida Libre de Gérmenes , Hormonas Ectópicas/agonistas , Hormonas Ectópicas/genética , Hormonas Ectópicas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Klebsiella pneumoniae/inmunología , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/fisiología , Ligilactobacillus salivarius/química , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Oligosacáridos/uso terapéutico , Proteínas Asociadas a Pancreatitis , Prebióticos , Proteínas/agonistas , Proteínas/genética , Proteínas/metabolismo , Distribución AleatoriaRESUMEN
Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoid Staphylococcus aureus mastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goat ß-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn(125) but not Asn(232) is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn(232) to Gln(232) substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn(125) to Gln(125) substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn(125) of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus.