RÉSUMÉ
Facioscapulohumeral muscular dystrophy (FSHD), one of the most common muscular dystrophies, is caused by an abnormal expression of the DUX4 gene in skeletal muscles, resulting in muscle weakness. In this study, we investigated MT-DUX4-ASO, a novel gapmer antisense oligonucleotide (ASO). MT-DUX4-ASO decreased the expression of DUX4 and its target genes in FSHD patient-derived myoblasts. For the first time, we demonstrated that a systemically administered ASO, even without a ligand for drug delivery, could significantly improve muscle injury and motor function in the ACTA1-MCM/FLExDUX4 (DUX4-TG) mouse model of FSHD. Tamoxifen (TMX) injection transiently induces skeletal-muscle-specific DUX4 expression in DUX4-TG mice, while the skeletal muscles of TMX-untreated DUX4-TG mice have leaky DUX4 expression in a small subset of myofibers similar to those of FSHD patients. Subcutaneous 10 mg/kg of MT-DUX4-ASO at two-week intervals significantly suppressed muscular DUX4 target gene expression, histological muscle injury, and blood muscle injury marker elevation in TMX-untreated DUX4-TG mice. Notably, MT-DUX4-ASO at 10 mg/kg every other week significantly prevented the TMX-induced declines in treadmill test running speed and muscle force in DUX4-TG mice. Thus, the systemically administered unconjugated MT-DUX4-ASO suppressed disease progression in DUX4-TG mice, extending the potential of unconjugated ASOs as a promising FSHD treatment strategy.
RÉSUMÉ
2'-Amino-locked nucleic acid has a functionalizable nitrogen atom at the 2'-position of its furanose ring that can provide desired properties to a nucleic acid as a scaffold. In this study, we synthesized a novel nucleic acid, 2'-N-methanesulfonyl-2'-amino-locked nucleic acid (ALNA[Ms]) and conducted comparative studies on the physical and pharmacological properties of the ALNA[Ms] and on conventional nucleic acids, such as 2'-methylamino-LNA (ALNA[Me]), which is a classical 2'-amino-LNA derivative, and also on 2',4'-BNA/LNA (LNA). ALNA[Ms] oligomers exhibited binding affinities for the complementary RNA strand that are similar to those of conventional nucleic acids. Four types of ALNA[Ms] nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. The knockdown abilities of Malat1 RNA using the Matat1 antisense oligonucleotide (ASO) containing ALNA[Ms] were higher than those of ALNA[Me] and were closer to those of LNA. Furthermore, the ASO containing ALNA[Ms] showed different tissue tropism from that containing LNA. ALNA[Ms] exhibited biological activities that were distinct from conventional constrained nucleic acids, suggesting the possibility that ALNA[Ms] can serve as novel modified nucleic acids in oligonucleotide therapeutics.
Sujet(s)
Acides nucléiques , Acides nucléiques/composition chimique , Oligonucléotides/pharmacologie , Oligonucléotides/composition chimique , Oligonucléotides antisens/pharmacologie , Oligonucléotides antisens/composition chimique , ARN/composition chimique , ARN complémentaireRÉSUMÉ
Guanidine-bridged nucleic acid (GuNA) is a novel 2',4'-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) analog containing cations that exhibit strong affinity for target RNA and superior nuclease resistance. In this study, Malat1 antisense oligonucleotide (ASO) bearing GuNA was evaluated for target knockdown (KD) activity and tolerability. The GuNA ASO did not interfere with RNase H recruitment on the target RNA/ASO heteroduplex and did show potent target KD activity in a skeletal muscle-derived cell line equivalent to that of the LNA ASO under gymnotic conditions, whereas almost no KD activity was observed in a hepatocyte-derived cell line. The GuNA ASO exhibited potent KD activity in various tissues; the KD activity in the skeletal muscle was equivalent with that of the LNA ASO, but the KD activities in the liver and kidney were clearly lower compared with the LNA ASO. In addition, despite the higher accumulation of the GuNA ASO in the liver, levels of aspartate aminotransferase and alanine aminotransferase with the GuNA ASO administration were not elevated compared with those induced by the LNA ASO. Our data indicate that the GuNA ASO is tolerable and exhibits unique altered pharmacological activities in comparison with the LNA ASO in terms of the relative effect between liver and skeletal muscle.
Sujet(s)
Acides nucléiques , Oligonucléotides antisens , Guanidine/métabolisme , Guanidines/métabolisme , Foie/métabolisme , Oligonucléotides antisens/pharmacologie , ARN/métabolisme , Distribution tissulaireRÉSUMÉ
We recently designed guanidine-bridged nucleic acids (GuNA), and GuNA bearing a thymine (T) nucleobase was synthesized and successfully incorporated into oligonucleotides. The GuNA-T-modified oligonucleotides possessed high duplex-forming ability towards their complementary single-stranded RNAs and were highly stable against 3'-exonuclease. Therefore, GuNA is a promissing artificial nucleic acid for therapeutic antisense oligonucleotides. We herein report the facile synthesis of GuNA phosphoramidites bearing adenine (A), guanine (G), and 5-methylcytosine (mC) nucleobases and a robust method for the preparation of GuNA-modified oligonucleotides, even with sequences having acid-sensitive purine nucleobases. Oligonucleotides modified with GuNA-A, -G, or -mC possessed high duplex-forming ability, similar to those modified with GuNA-T. Moreover, some of the GuNA-modified oligonucleotides were revealed to have high base discriminating ability compared with that of their natural counterparts. GuNA nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. Thus, all GuNAs (GuNA-T, -A, -G, and -mC) are now available to be examined in therapeutic applications.
Sujet(s)
OligonucléotidesRÉSUMÉ
BACKGROUND: Finding an abdominal mass or hematuria is the initial step in diagnosing Wilms tumor. As the first manifestation of Wilms tumor, it is exceedingly rare for pulmonary tumor embolism to present with cardiac arrest. A case of a patient whose sudden cardiac arrest due to massive pulmonary tumor embolism of Wilms tumor was not responsive to resuscitation is presented. CASE PRESENTATION: The patient was a five-year-old girl who collapsed suddenly during activity in nursery school and went into cardiac arrest in the ambulance. Unfortunately, she was not responsive to conventional resuscitation. A judicial autopsy conducted at the local police department showed the main cause of her sudden cardiac arrest was attributed to multiple pulmonary tumor embolisms of stage IV Wilms tumor. CONCLUSIONS: Except for one reported case, treatments were not successful in all eight cardiac arrest cases with pulmonary tumor embolism of Wilms tumor. These results indicate that it is challenging not only to make an accurate diagnosis, but also to provide proper specific treatment in the cardiac arrest setting. We propose that flexible triage and prompt transfer to a tertiary hospital are necessary as an oncologic emergency to get such patients to bridging therapy combined with extracorporeal membrane oxygenation or immediate surgical intervention under cardiopulmonary bypass.
Sujet(s)
Arrêt cardiaque/étiologie , Tumeurs du rein/complications , Embolie pulmonaire/complications , Tumeur de Wilms/complications , Enfant d'âge préscolaire , Femelle , Humains , Tumeurs du rein/diagnostic , Tumeur de Wilms/diagnosticRÉSUMÉ
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) experiments require a suitable match of the matrix and target compounds to achieve a selective and sensitive analysis. However, it is still difficult to predict which metabolites are ionizable with a given matrix and which factors lead to an efficient ionization. In the present study, we extracted structural properties of metabolites that contribute to their ionization in MALDI-MS analyses exploiting our experimental data set. The MALDI-MS experiment was performed for 200 standard metabolites using 9-aminoacridine (9-AA) as the matrix. We then developed a prediction model for the ionization profiles (both the ionizability and ionization efficiency) of metabolites using a quantitative structure-property relationship (QSPR) approach. The classification model for the ionizability achieved a 91% accuracy, and the regression model for the ionization efficiency reached a rank correlation coefficient of 0.77. An analysis of the descriptors contributing to such model construction suggested that the proton affinity is a major determinant of the ionization, whereas some substructures hinder efficient ionization. This study will lead to the development of more rational and predictable MALDI-MS analyses.
Sujet(s)
Composés chimiques organiques/analyse , Relation quantitative structure-activité , Spectrométrie de masse MALDI/méthodes , Métabolomique , Composés chimiques organiques/composition chimique , Analyse de régressionRÉSUMÉ
Type 2 diabetes mellitus is frequently accompanied by fatty liver/nonalcoholic fatty liver disease. Hence, accumulation of lipids in the liver is considered to be one of the risk factors for insulin resistance and metabolic syndrome. Ursodeoxycholic acid (UDCA) is widely used for the treatment of liver dysfunction. We investigated the therapeutic effects of UDCA on type 2 diabetes mellitus exacerbating hepatic steatosis and the underlying mechanisms of its action using KK-A(y) mice fed a high-fat diet. KK-A(y) mice were prefed a high-fat diet; and 50, 150, and 450 mg/kg of UDCA was orally administered for 2 or 3 weeks. Administration of UDCA decreased fasting hyperglycemia and hyperinsulinemia. Hyperinsulinemic-euglycemic clamp analyses showed that UDCA improved hepatic (but not peripheral) insulin resistance. Hepatic triglyceride and cholesterol contents were significantly reduced by treatment with UDCA, although the genes involved in the synthesis of fatty acids and cholesterol, including fatty acid synthase and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, were upregulated. Fecal levels of bile acids, neutral sterols, fatty acids, and phospholipids were significantly increased by UDCA treatment. The gene expression levels and protein phosphorylation levels of endoplasmic reticulum stress markers were not changed by UDCA treatment. These results indicate that UDCA ameliorates hyperglycemia and hyperinsulinemia by improving hepatic insulin resistance and steatosis in high-fat diet-fed KK-A(y) mice. Reduction of hepatic lipids might be due to their excretion in feces, followed by enhanced utilization of glucose for the synthesis of fatty acids and cholesterol. Ursodeoxycholic acid should be effective for the treatment of type 2 diabetes mellitus accompanying hepatic steatosis.
Sujet(s)
Cholagogues et cholérétiques/usage thérapeutique , Alimentation riche en graisse/effets indésirables , Stéatose hépatique/traitement médicamenteux , Insulinorésistance , Acide ursodésoxycholique/usage thérapeutique , Animaux , Cholestérol/analyse , Cholestérol/biosynthèse , Cholestérol/génétique , Diabète de type 2/traitement médicamenteux , Stress du réticulum endoplasmique/effets des médicaments et des substances chimiques , Acides gras/analyse , Acides gras/biosynthèse , Acides gras/génétique , Stéatose hépatique/métabolisme , Analyse de profil d'expression de gènes , Hyperglycémie/traitement médicamenteux , Hyperinsulinisme/traitement médicamenteux , Métabolisme lipidique/effets des médicaments et des substances chimiques , Mâle , Souris , Phosphorylation/effets des médicaments et des substances chimiques , Régulation positiveRÉSUMÉ
Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists such as fenofibrate are used to treat dyslipidemia. Although fenofibrate is considered safe in humans, it is known to cause hepatocarcinogenesis in rodents. To evaluate untargeted metabolic profiling as a tool for gaining insight into the underlying pharmacology and hepatotoxicology, Fischer 344 male rats were dosed with 300 mg/kg/day of fenofibrate for 14 days and the urine and plasma were analyzed on days 2 and 14. A combination of liquid and gas chromatography mass spectrometry returned the profiles of 486 plasma and 932 urinary metabolites. Aside from known pharmacological effects, such as accelerated fatty acid beta-oxidation and reduced plasma cholesterol, new observations on the drug's impact on cellular metabolism were generated. Reductions in TCA cycle intermediates and biochemical evidence of lactic acidosis demonstrated that energy metabolism homeostasis was altered. Perturbation of the glutathione biosynthesis and elevation of oxidative stress markers were observed. Furthermore, tryptophan metabolism was up-regulated, resulting in accumulation of tryptophan metabolites associated with reactive oxygen species generation, suggesting the possibility of oxidative stress as a mechanism of nongenotoxic carcinogenesis. Finally, several metabolites related to liver function, kidney function, cell damage, and cell proliferation were altered by fenofibrate-induced toxicity at this dose.
Sujet(s)
Fénofibrate/toxicité , Hypolipémiants/toxicité , Foie/anatomopathologie , Métabolomique/méthodes , Acidose lactique/métabolisme , Animaux , Marqueurs biologiques/sang , Marqueurs biologiques/urine , Chromatographie en phase liquide , Cycle citrique/effets des médicaments et des substances chimiques , Acides gras/métabolisme , Fénofibrate/administration et posologie , Chromatographie gazeuse-spectrométrie de masse , Hypolipémiants/administration et posologie , Métabolisme lipidique/effets des médicaments et des substances chimiques , Mâle , Stress oxydatif/effets des médicaments et des substances chimiques , Récepteur PPAR alpha/métabolisme , Rats , Rats de lignée F344 , Tests de toxicité chronique , Tryptophane/métabolismeRÉSUMÉ
We report a 52-year-old patient with a small hepatic mass which was ultrasonographically anechoic with scattered high echoic spots, and appearing slightly hyperattenuating relative to the surrounding parenchyma on unenhanced CT scans. Laparotomy revealed that the lesion was a unilocular cyst containing a mucinous fluid. The histologic diagnosis was ciliated hepatic foregut cyst (CHFC). The CHFC is a rare congenital cystic tumor which derives from the embryologic foregut. Up to 2006, only 24 cases, including our case, had been reported in Japan. The patients were 13 men and 11 women, aged between 41 years and 79 years. All of the lesions were solitary and unilocular. In 22 cases, the CHFC was located in the medial segment of the left lobe, mostly just beneath the hepatic surface. In all 24 Japanese cases, the cystic wall was benign histologically. However, reports of 3 malignant cases overseas indicates we should treat this disease cautiously.
Sujet(s)
Kystes/congénital , Maladies du foie/congénital , Kystes/anatomopathologie , Humains , Maladies du foie/anatomopathologie , Mâle , Adulte d'âge moyenRÉSUMÉ
High expression of epidermal growth factor receptor (EGFR) is thought to be correlated with cell proliferation, invasion, metastasis, resistance to chemoradiotherapy, and poor prognosis in various kinds of human cancers. Blockade of EGFR signal transduction can be a promising strategy for cancer therapy. Approximately 40-70% of esophageal squamous cell carcinomas (ESCCs) show high expression of EGFR. In this study, we examined the antitumor effect of gefitinib, an EGFR tyrosine kinase inhibitor, against ESCC cells in vitro and in vivo. In three ESCC cell lines (TE8, T.T and T.Tn), cell proliferation had been inhibited in a dose-dependent manner and IC(50) values (respectively, 8.49, 18.9 and 17.3muM). Gefitinib inhibited EGF-induced autophosphorylation of EGFR and its downstream signaling pathways, Ras/Raf/MAPK and PI3K/Akt, and caused G(1) arrest of cell cycle and apoptosis confirmed with flow cytometry. We examined the effect of gefitinib on nude mice bearing established TE8 and T.T xenografts. Gefitinib (100 or 200mg/kg once-daily, p.o.) showed antitumor activity in a dose-dependent manner, resulting in a significantly improved survival of treated mice as compared with untreated mice. Immunohistochemical examination of the harvested tumor was performed to examine the status of phosphorylated EGFR, PCNA, Factor VIII and apoptosis. We found inhibition of EGFR phosphorylation, cell cycle arrest (by PCNA staining), decrease of microvessel density (Factor VIII) and induction of apoptosis by TUNEL staining. In conclusion, our findings demonstrate that gefitinib is effective for growth inhibition of ESCC cell lines in vitro and in vivo and suggest that gefitinib may be one of the new therapeutic options for ESSC.