Oxidative stress and methylglyoxal (MG), a reactive dicarbonyl metabolites produced by enzymatic and non-enzymatic reaction of normal metabolism, induced aldose reductase (AR) expression in rat aortic smooth muscle cells (SMC). AR expression was induced in a time-dependent manner and reached at a maximum of 4.5-fold in 12 h of MG treatment. This effect of MG was completely abolished by cyclohemide and actinomycin D treatment suggesting AR was synthesized by de novo pathway. Pretreatment of the SMC with N-acetyl-L-cysteine significantly down-regulated the MG-induced AR mRNA. Furthermore, DL-Buthionine-(S,R)-sulfoximine, a reagent which depletes intracellular glutathione levels, increased the levels of MG-induced AR mRNA. These results indicated that MG induces AR mRNA by increasing the intracellular peroxide levels. Aminoguanidine, a scanvenger of dicarbonyl, significantly down-regulated the MG-induced AR mRNA. In addition, the inhibition of AR activities with statil, an AR inhibitor, enhanced the cytotoxic effect of MG on SMC under normal glucose, suggesting a protective role of AR against MG-induced cell damages. These results imply that the induction of AR by MG may contribute to an important cellular detoxification of reactive aldehyde compounds generated under oxidative stress in extrahepatic tissues.
Acetylcysteine ; Aldehyde Reductase* ; Animals ; Dactinomycin ; Glucose ; Glutathione ; Metabolism ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Oxidative Stress* ; Pyruvaldehyde ; Rats ; RNA, Messenger

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

Development of drug resistance in tumors during treatment is a major factor limiting the clinical use of anticancer agents. When tumor cells acquire resistance to anticancer drug, they show cross-resistance to other antitumor agents. In the present study, SNU-1 cell was induced adriamycin 10-7 drug resistance, SNU-1/ADR, in vitro culture system. We got the doubling time and number for viability test during 96 hours by MTT assay. To investigate the cross resistance of various anticancer drugs in human stomach cancer cell SNU-1 and SNU-1/ADR, We compared IC50 (drug concentration of 50% reduction) and the relative resistance (RR). SNU-1/ADR was expressed multidrug resistant with vinblastine (RR;>31.62), vincristine (RR;29.50), dactinomycin (RR;21.37), epirubicin (RR;17.78), daunorubicin (RR;14.12), adriamycin (RR;7.76), and etoposide (RR;4.46), and other drugs, 5-fluorouracil, cisplatin, cyclophosphamide, methotrexate, and calarubicin, have not cross resistant with adriamycin. There was double minute chromosome in SNU-1/ADR by karyotyping although this change was not seen in SUN-1.
Antineoplastic Agents ; Cisplatin ; Cyclophosphamide ; Dactinomycin ; Daunorubicin ; Doxorubicin ; Drug Resistance ; Epirubicin ; Etoposide ; Fluorouracil ; Humans ; In Vitro Techniques ; Inhibitory Concentration 50 ; Karyotyping ; Metabolism* ; Methotrexate ; Stomach Neoplasms* ; Stomach* ; Vinblastine ; Vincristine

PURPOSE: Growth hormone(GH) produces a variety of effects in adipose tissue via GHRs on the cell membrane. In mouse, alternative splicing of the nascent transcript from the GHR gene produces two major transcripts:GHR mRNA and GHR binding protein(GHBP) mRNA. These two transcripts share the common extracellular ligand-binding domain, but differ in the C-terminal sequence. Since GHR plays an important role in mediating the actions of GH in adipose metabolism, I initiated these studies to examine GHR gene expression during mouse 3T3-Ll preadipocyte-adipocyte differentiation. METHODS: GHR and GHBP transcripts were detected by RNase protection assay (RPA) using the antisense riboprobes complementary either to the specific sequence of the GHR or to the sequence shared by both GHR and GHBP mRNAs. RNA prepared from 3T3-L1 cells at day 0(preadipocytes) and day 7(adipocytes) after treatment with actinomycin D was analyzed by RPA. RESULTS: After stimulation of differentiation, mRNA abundance increased 25-fold and reached a maximal level by day 7 of adipogenesis. The GHR mRNA:GHBP mRNA ratio was 1.3+/-.15 and remained unchanged during differentiation. The decay rate for both mRNAs, estimated by treating the cells with actinomycin D, was approximately 24 hours and showed no significant difference between preadipocytes and adipocytes. CONCLUSION: GHR gene expression is upregulated during preadipocyte-adipocyte differentiation.
3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Adipose Tissue ; Alternative Splicing ; Animals ; Cell Membrane ; Dactinomycin ; Gene Expression ; Growth Hormone* ; Metabolism ; Mice ; Negotiating ; Receptors, Somatotropin* ; Ribonucleases ; RNA ; RNA, Messenger

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

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

BACKGROUND: All-trans-retinoic acid metabolism by cytochrome P450 is one of the major mechanisms that can regulate the level of retinoids in cells. Therefore, enhanced metabolism of all-trans retinoic acid by all-trans retinoic acid induced cytochrome P450 would probably decrease the therapeutic effects of active retinoids. We previously reported that the tail of all-trans retinoic acid (the carboxyl-terminus) may bind to a binding site of cytochrome P450 in part by electrostatic forces, and the head of RA (the beta-cyclogeranylidene ring) may bind to an active site of cytochrome P450 in part by hydrophobic forces. It is very interesting to study the interactions between the RA binding site of cytochrome P450 induced by all-trans retinoic acid and the structural analogs of all-trans retinoic acid and its effects of RA metabolism. OBJECTIVE: The purpose of this study is to examine the effects of sorbic acid, that has a similar structure with the tail of all-trans retinoic acid, on RA metabolism in head-neck squamous cell carcinoma cell line AMC-HN-6 which showed a much increased induction of cytochrome P450 by all-trans retinoic acid. METHODS: We examined the effects of sorbic acid on RA metabolism in all-trans retinoic acid specific cytochrome P450-inducible AMC-HN-6 cell line using cytochrome P450 enzyme assay with total cell lysates and microsomal proteins. Radioisotope-labeled polar metabolites of all-trans retinoic acid were separated by thin layer chromatography and the radioactivity was measured by beta-counter. Metabolic activity was expressed as the percentage of total radioactivity of polar metabolites. RESULTS: The results are summurized as follows: 1. RA metabolism of AMC-HN-6 cell line was inhibited by actinomycin D and cyclohexamide and was also inhibited by ketoconazole, the cytochrome P450 inhibitor, in a concentration-dependent manner. 2. Cytochrome P450-mediated oxidation was induced by all-trans retinoic acid, 13-cis-RA, 9-cis-RA, and retinal, but not by retinol in AMC-HN-6 cell line.3. Sorbic acid inhibited RA metabolism of AMC-HN-6 cell line in a concentration-dependent manner when the enzyme assay was performed on microsomal protein but could not inhibit RA metabolism in total cell lysate enzyme assay. CONCLUSION: The conversion of all-trans retinoic acid to polar metabolites is inhibited by sorbic acid in microsomal enzyme assay of AMC-HN-6 cell line, but not in total cell assay. These results suggest that sorbic acid can bind to the active binding site of cytochrome P450 but binding affinity of sorbic acid to RA binding molecules such as CRABP-I,-II, RARs, RXRs may be stronger than that of sorbic acid to cytochrome P450.
Binding Sites ; Carcinoma, Squamous Cell ; Catalytic Domain ; Cell Line* ; Chromatography, Thin Layer ; Cytochrome P-450 Enzyme System ; Cytochromes ; Dactinomycin ; Enzyme Assays ; Head ; Ketoconazole ; Metabolism* ; Radioactivity ; Retinaldehyde ; Retinoids* ; Sorbic Acid* ; Tretinoin* ; Vitamin A

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

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

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