OBJECTIVE: To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells. METHODS: Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist. RESULTS: VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells. CONCLUSIONS VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.
Autophagy ; drug effects ; Autophagy-Related Proteins ; genetics ; metabolism ; Cell Line ; Cysteine Endopeptidases ; genetics ; metabolism ; Dactinomycin ; pharmacology ; Down-Regulation ; Genes, Reporter ; Humans ; MicroRNAs ; antagonists & inhibitors ; metabolism ; Microtubule-Associated Proteins ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; Transfection ; Valproic Acid ; administration & dosage ; antagonists & inhibitors ; pharmacology

We report an unusual case of 9.5-cm-sized embryonal rhabdomyosarcoma arose from a mediastinal mature teratoma in a 46-yr-old man. A man presented with chest trauma as a result of an accident at 10 September 2011. On chest X-ray, an anterior mediastinal mass was detected. To obtain further information, chest computed tomography (CT) with contrast enhancement was performed, revealing an anterior mediastinal mass. Complete surgical excision was performed and entire specimen was evaluated. Pathologic diagnosis was embryonal rhabdomyosarcoma arising in mature cystic teratoma. After surgical excision, two cycles of dactinomycin-based chemotherapy were performed. Lung metastasis was detected on follow up CT in September 2012, and wedge resection was performed. Pathological finding of the lung lesion showed same feature with that of primary rhabdomyosarcoma.
Antibiotics, Antineoplastic/therapeutic use ; Dactinomycin/therapeutic use ; Desmin/metabolism ; Humans ; Immunohistochemistry ; Lung Neoplasms/radiography/secondary/surgery ; Male ; Mediastinal Neoplasms/*diagnosis/pathology ; Middle Aged ; Neoplasms, Germ Cell and Embryonal/drug therapy/*radiography/surgery ; Rhabdomyosarcoma, Embryonal/drug therapy/*radiography/surgery ; Teratoma/*diagnosis/pathology ; Tomography, X-Ray Computed

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child, Preschool ; Dactinomycin ; administration & dosage ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Humans ; Infant ; Kidney Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rhabdoid Tumor ; drug therapy ; metabolism ; pathology ; surgery ; Rhabdomyosarcoma ; pathology ; Sarcoma, Clear Cell ; metabolism ; pathology ; Synaptophysin ; metabolism ; Vimentin ; metabolism ; Vincristine ; administration & dosage ; Wilms Tumor ; pathology

Nucleophosmin/B23 (NPM) is a universally expressed nucleolar phosphoprotein that participates in proliferation, apoptosis, ribosome assembly, and centrosome duplication; however, the role of NPM in cell cycle regulation is not well characterized. We investigated the mechanism by which NPM is involved in cell cycle regulation. NPM was knocked down using siRNA in HepG2 hepatoblastoma cells. NPM translocation following actinomycin D (ActD) treatment was investigated using immunofluorescent staining. Expression of NPM and other factors involved in cell cycle regulation was examined by Western blotting. Cell cycle distribution was measured using flow cytometry to detect 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Cell proliferation was quantified by the MTT assay. Knockdown of NPM increased the percentage of HepG2 cells in S phase and led to decreased expression of P53 and P21Cip1/WAF1. S-phase arrest in HepG2 cells was significantly enhanced by ActD treatment. Furthermore, knockdown of NPM abrogated ActD-induced G2/M phase cell cycle arrest. Taken together, these data demonstrate that inhibition of NPM has a significant effect on the cell cycle.
Antibiotics, Antineoplastic ; pharmacology ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Dactinomycin ; pharmacology ; Gene Knockdown Techniques ; Hep G2 Cells ; Humans ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; S Phase ; Tumor Suppressor Protein p53 ; metabolism

Adult ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cyclophosphamide ; therapeutic use ; Cystadenocarcinoma, Papillary ; pathology ; Dactinomycin ; therapeutic use ; Diagnosis, Differential ; Ependymoma ; drug therapy ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Hysterectomy ; Ovarian Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Ovariectomy ; Teratoma ; pathology ; Vimentin ; metabolism ; Vincristine ; therapeutic use

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
Alpha-Amanitin/pharmacology ; Animals ; Antibodies, Monoclonal/analysis/metabolism ; Antibodies, Protozoan/analysis/metabolism ; Dactinomycin/pharmacology ; Fluorescent Antibody Technique, Direct ; Gene Expression/*physiology ; Green Fluorescent Proteins/genetics ; Hela Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Nucleolus Organizer Region/drug effects/*metabolism ; Pol1 Transcription Initiation Complex Proteins/metabolism ; Protein Sorting Signals/physiology ; Protozoan Proteins/*biosynthesis/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Toxoplasma/*physiology ; Transfection

Akabane, Aino and Chuzan virus are arthropod-borne (arbo)viruses mainly associated with reproductive failures in cattle. We investigated apoptosis in Vero cells (C-1586) infected with Akabane, Aino and Chuzan virus. The fragmentation of chromosomal DNA was simultaneously detected with the progress of cytopathic effect from 48 hr to 72 hr post infection, depending on viruses. Although the treatment of cycloheximide blocked apoptosis in Vero cells infected with three viruses, actinomycin D did not prevent DNA oligomerization, thus indicating that de novo viral protein synthesis is critical for viral apoptosis. In addition, the activation of caspase-3 was also detected in Vero cells by indirect fluorescent assay. From the present results, it is of future interest whether apoptotic characteristics of these viruses are related to pathogenecity in vivo.
Animals ; Apoptosis/*physiology ; Bunyaviridae/*physiology ; Caspase 3 ; Caspases/metabolism ; Cercopithecus aethiops ; Cytopathogenic Effect, Viral/*physiology ; DNA Fragmentation/physiology ; Dactinomycin ; Enzyme Activation ; Orbivirus/*physiology ; Vero Cells

OBJECTIVETo study the inhibitory effect of antisense oligonucleotides against telomerase RNA on the growth of human choriocarcinoma transplant in nude mice.

METHODSChoriocarcinoma xenografts were established by transplanting JAR cells subcutaneously to female nude mice, and were treated with high and low doses of antisense oligonucleotides. Control groups were treated with NS, random sequence and actinomycin D (Act-D). Tumor growth was monitored once every other day. Telomerase relative activity was assayed by TRAP-ELISA. Western blotting was used to detect expression of hTERT.

RESULTSLow and high doses antisense oligonucleotides, and Act-D inhibited tumor growth by 76.6%, 93.8% and 85.4% respectively, which were significantly different when compared with random sequence and NS groups. Expression of telomerase relative activity and hTERT were decreased as well. But the differences among the first three groups had no significance.

CONCLUSIONTelomerase RNA antisense oligonucleotide inhibits growth of human choriocarcinoma xenografts in nude mice. It may be a novel approach to the treatment of choriocarcinoma.

Animals ; Antibiotics, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Choriocarcinoma ; enzymology ; pathology ; DNA-Binding Proteins ; metabolism ; Dactinomycin ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oligonucleotides, Antisense ; administration & dosage ; pharmacology ; Pregnancy ; Telomerase ; genetics ; metabolism ; Uterine Neoplasms ; enzymology ; pathology

15-deoxy-delta12,14-PGJ2(15d-PGJ2) is a natural ligand that activates the peroxisome proliferators-activated receptor (PPAR) gamma, a member of nuclear receptor family implicated in regulation of lipid metabolism and adipocyte differentiation. Recent studies have shown that 15d-PGJ2 is the potent anti-inflammatory agent functioning via PPARgamma-dependent and -independent mechanisms. Most postulated mechanisms for anti-inflammatory action of PPARgamma agonists are involved in inhibiting NF-kappaB signaling pathway. We examined the possibility that IL-6 signaling via the Jak-Stat pathway is modulated by 15d-PGJ2 in lymphocytes and also examined whether the inhibition of IL-6 signaling is dependent of PPARgamma. 15d-PGJ2 blocked IL-6 induced Stat1 and Stat3 activation in primary human lymphocytes, Jurkat cells and immortalized rheumatoid arthritis B cells. Inhibition of IL-6 signaling was induced rapidly within 15 min after treatment of 15d-PGJ2. Other PPARgamma-agonists, such as troglitazone and ciglitazone, did not inhibit IL-6 signaling, indicating that 15d-PGJ2 affect the IL-6-induced Jak-Stat signaling pathway via PPARgamma-independent mechanism. Although cycloheximide reversed 15d-PGJ2-mediated inhibition of Stat3 activation, actinomycin D had no effect on 15d-PGJ2-mediated inhibition of IL-6 signaling, indicating that inhibition of IL-6 signaling occur independent of de novo gene expression. These results show that 15d-PGJ2 specifically inhibit Jak-Stat signaling pathway in lymphocytes, and suggest that 15d-PGJ2 may regulate inflammatory reactions through the modulation of different signaling pathway other than NF-kappaB in lymphocytes.
Arthritis, Rheumatoid/metabolism/pathology ; Chromans/pharmacology ; Cycloheximide/pharmacology ; DNA-Binding Proteins/*metabolism ; Dactinomycin/pharmacology ; Gene Expression Regulation ; Humans ; Hypoglycemic Agents/pharmacology ; Interleukin-6/*pharmacology ; Jurkat Cells/metabolism/pathology ; Lymphocytes/cytology/*drug effects/*metabolism ; NF-kappa B/metabolism ; PPAR gamma/metabolism ; Phosphorylation ; Prostaglandin D2/*analogs & derivatives/pharmacology ; Protein Synthesis Inhibitors/pharmacology ; Research Support, Non-U.S. Gov't ; *Signal Transduction ; Thiazolidinediones/pharmacology ; Trans-Activators/*metabolism

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 mM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.
Antineoplastic Agents/*metabolism ; Bleomycin/metabolism/pharmacology ; Circular Dichroism ; Comparative Study ; DNA/chemistry/drug effects/*metabolism ; DNA Damage ; Dactinomycin/metabolism ; Doxorubicin/*analogs & derivatives/metabolism ; Human ; Nogalamycin/metabolism ; Nucleic Acid Conformation ; *Repetitive Sequences, Nucleic Acid ; Telomere/drug effects/*genetics