OBJECTIVETo investigate the effects of cycle-dependent kinase (CDK) inhibitor SNS-032 on apoptosis in human acute myeloid leukemia (AML) HL-60 cells and its molecular mechanisms.

METHODSCultured AML HL-60 cells were treated with various concentrations of SNS-032. Cell apoptosis was determined with flow cytometry;cell viability was measured by MTT assay; the profiles of microRNA expression of HL-60 cells were analyzed by microRNA microarray;the protein expressions of JAK2/STAT3 pathway were detected by Western blotting.

RESULTSApoptosis of AML HL-60 cells was induced by SNS-032; the rate of apoptosis was (5.9±1.7)%, (12.1±3.1)% and (59.4±3.6)% when HL-60 cells were treated with 0,100 and 200 nmol/L SNS-032. MicroRNA microarray analysis revealed that the levels of miR-30a, miR-183, miR-20b, miR-26b, miR-20a, miR-589, miR-107, miR-181a, miR-106a, miR-17 and miR-378c were down-regulated by SNS-032,whereas the levels of miR-320a and miR-H7* were up-regulated. Western blotting showed that SNS-032 strongly inhibited phosphorylation of STAT3 and protein expression of JAK2,C-MYC and MCL-1.

CONCLUSIONCDK inhibitor SNS-032 can induce apoptosis of AML HL-60 cells, which is associated with the inhibition of MCL-1,C-MYC and JAK2/STAT3, and down-regulation of miR-17-92 family.

Apoptosis ; Cell Survival ; Down-Regulation ; Flow Cytometry ; HL-60 Cells ; Humans ; Janus Kinase 2 ; metabolism ; MicroRNAs ; metabolism ; Oxazoles ; pharmacology ; Phosphorylation ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Thiazoles ; pharmacology

OBJECTIVETo investigate the correlation between atorvastatin inhibiting the expression level of matrix metalloproteinase 9 (MMP-9) and peroxisome proliferator activated receptor alpha (PPARalpha) signal channel in myocyte of aging rat.

METHODSPrimary cultures of myocyte were got ten from aging rats. Myocyte were divided into control group, DMSO group, atorvastatin group, atorvastatin plus GW6471 group, which treated respectively by cell culture medium, DMSO, atorvastatin, atorvastatin plus GW6471. The expression of MMP-9 mRNA was evaluated by RT-PCR, and content of MMP-9 protein was detected by Western blot.

RESULTS(1) There was no difference between control group and DMSO group in level of MMP-9 mRNA and protein expression (P > 0.05); (2) The level of MMP-9 mRNA and MMP-9 protein expression in atorvastatin group were significantly lower than those in control group (P < 0.01); (3) Both level of MMP-9 mRNA and protein expression in atorvastatin plus GW6471 group were significantly higher than those in atorvastatin group (P < 0.05), but were still lower than those in control group (P < 0.05).

CONCLUSIONAtorvastatin inhibit MMP-9 expression of aging myocytes by PPARalpha signal channel.

Aging ; Animals ; Atorvastatin Calcium ; Cells, Cultured ; Heptanoic Acids ; pharmacology ; Matrix Metalloproteinase 9 ; metabolism ; Muscle Cells ; drug effects ; metabolism ; Oxazoles ; pharmacology ; PPAR alpha ; metabolism ; Pyrroles ; pharmacology ; Rats ; Rats, Wistar ; Signal Transduction ; Tyrosine ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate the effect of peroxisome proliferator-activated receptor gamma (PPARgamma) activation on transforming growth factor beta1 (TGF-beta1)-induced connective tissue growth factor (CTGF) expression in rat hepatic stellate cells (HSCs).

METHODSCultured HSCs with or without PPARgamma-specific antagonist GW9662 treatment prior to the addition of an increasing amount of PPARgamma natural ligand (15-d-PGJ2) or synthetic ligand (GW7845) were stimulated with TGF-beta1. The mRNA and protein levels of CTGF expression were detected by semi-quantitative RT-PCR and Western blotting, respectively. The morphological changes of the HSC were observed by electron microscope.

RESULTS15-d-PGJ2 and GW7845 significantly inhibited TGF-beta1-induced CTGF expression at both mRNA and protein levels in HSCs, and the inhibitory effect was dramatically, if not completely, abolished by pretreatment with GW9662, suggesting that the inhibition was mediated by PPARgamma. Morphological observation revealed that PPARgamma activation caused obvious changes of HSCs from activated to quiescent phenotypes.

CONCLUSIONPPARgamma ligand shows potent inhibitory effect on TGF-beta1-induced CTGF expression in rat HSCs, suggesting its potential as a candidate agent for treatment and prevention of hepatic fibrosis.

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Oxazoles ; pharmacology ; PPAR gamma ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Signal Transduction ; Transforming Growth Factor beta1 ; pharmacology ; Tyrosine ; analogs & derivatives ; pharmacology

Diabetes mellitus is a common metabolic disease with a high and growing prevalence affecting 4% of the population worldwide, the development of safe and effective therapeutic drug is the major thrust for chemists and pharmacists. To search for active antidiabetic lead compound, we designed and synthesized some novel beta-amino ketone derivatives containing sulfamethoxazole moiety directly through Mannich reaction of sulfamethoxazole, 4-bromoacetophenone and some aromatic aldehydes catalyzed by concentrated hydogen chloride or iodine in the solution of ethanol at 24-40 degrees C with convenient operation, mild reaction condition and satisfactory yield (32%-90%). Their chemical structures were characterized by 1H NMR, 13C NMR, MS and HR-MS. Biological activity tests showed that, in the range of low concentration (5-10 microg x mL(-1)), these title compounds to a certain degree possess protein tyrosine phosphatase 1B (PTP1B) inhibitory activity and a-glucosidase inhibitory activity, moreover, some could activate peroxisome proliferator-activated receptor response element (PPRE) moderately. The PPRE agonist activities of seven compounds are almost 40% of that of Pioglitazone (the positive control), compound 12 shows the strongest activity (66.35%) among them. Thus, it was found that some of 4-(3-(4-bromophenyl)-3-oxo-1-arylpropylamino)-N-(5-methyl-isoxazol-3-yl) benzenesulfonamide containing sulfamethoxazole moiety exhibited antidiabetic activity for the first time.
Glycoside Hydrolase Inhibitors ; Humans ; Hypoglycemic Agents ; chemical synthesis ; chemistry ; pharmacology ; Molecular Structure ; Oxazoles ; chemistry ; Peroxisome Proliferator-Activated Receptors ; agonists ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; Response Elements ; Structure-Activity Relationship ; Sulfonamides ; chemistry ; Thiazolidinediones ; pharmacology

This study was aimed to investigate the effect of SNS-032 (C17 H24 N4O2S2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice. The self-renewal capability of bone marrow cells was measured by cobblestone forming cell test. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were detected by flow cytometry. The results showed that there was no significant difference of the frequency of HSC between SNS-032 and control group. The expressions of CDK1, CDK2, CDK7 and p27 decreased in HSC (P < 0.05) while the expressions of CDK4, CDK6, p21, p18, p19, Bcl-2, Bax, Puma, p53, Bim1, Sall4 and Notch1 showed no difference between SNS-032 group and control group (P > 0.05). The fraction of viable HSC in each phase of cell cycle remained unchanged after the treatment of SNS-032 (P > 0.05). Furthermore, there was no statistical difference in the apoptotic fractions between control and drug-treated groups (P > 0.05). It is concluded that SNS-032 induce apoptosis of cancer cells. Interestingly, SNS-032 has no significant inhibitory effect on self-renewal and differentiation of normal HSC, as well as no obvious effect inducing apoptosis of normal HSC and HPC.
Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Oxazoles ; pharmacology ; Thiazoles ; pharmacology

OBJECTIVETo explore the dose-effect relationship between premature chromosome condensation induced by Calyculin A and irradiation dose.

METHODSThe human peripheral blood was irradiated by (137)Cs gamma radial. The irradiation dose included 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 Gy. The premature chromosome condensation induced by Calyculin A was observed, and dyed by centromeric banding.

RESULTSThere was the quadratic relation between the total aberration, fragment, dicentric+centric ring (dic+r) ration and irradiation dose.

CONCLUSIONPremature chromosome condensation induced by Calyculin A can be used as a biodosimetry.

Cell Line ; Chromosome Aberrations ; radiation effects ; Chromosome Banding ; Chromosomes ; radiation effects ; Dose-Response Relationship, Radiation ; Female ; Gamma Rays ; adverse effects ; Humans ; Lymphocytes ; radiation effects ; Male ; Oxazoles ; pharmacology ; Young Adult

OBJECTIVETo investigate the antitumor efficacy and mechanism of HSP90 inhibitor FW-04-806 against Bcr/Abl(+) leukemia K562 and HL60 cells and their mechanisms of action.

METHODSMTT assay was used to assess the proliferation-inhibiting effect of FW-04-806. Cell cycle was analyzed with propidium iodide by flow cytometry. Cell apoptosis was determined using the FITC mV apoptosis detection kit. Western blot was applied to reveal the protein expression of related proliferative and apoptotic signaling pathways. The changes of mitochondrial membrane potential were detected by flow cytometry. Protein-protein interactions was shown by co-immunoprecipitation. The level of mRNA was assessed by real-time RT-PCR.

RESULTSFW-04-806 obviously inhibited cell proliferation in the HL60, K562 and HL60/Bcr-Abl cell lines, with an IC50 of (30.89 ± 0.12) µmol/L, (9.76 ± 0.19) µmol/L and (8.03 ± 0.26) µmol/L, respectively (P<0.001). Compared with the vehicle group, the two increasing doses of FW-04-806 showed inhibition of tumor growth at a rate of (17.40 ± 0.34)% and (34.33 ± 5.00)%, respectively, in the K562 cell line groups (P=0.003), and (18.90 ± 1.45)% and (35.60 ± 3.55)% (P=0.001) in the HL60/Bcr-Abl cell line groups. FW-04-806 dissociated Hsp90/Cdc37 chaperon/co-chaperon complex, followed by degradation of the Hsp90 proteins through proteasome pathway without affecting mRNA expression. FW-04-806 induced apoptosis and led to G2/M arrest.

CONCLUSIONOur findings indicate that FW-04-806 displays potential antitumor effect by suppressing the proliferation and apoptosis in Bcr/Abl(+) leukemia cells in vivo.

Apoptosis ; drug effects ; Cell Cycle ; Cell Proliferation ; drug effects ; Fusion Proteins, bcr-abl ; HL-60 Cells ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; K562 Cells ; Leukemia ; drug therapy ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Oxazoles ; pharmacology ; RNA, Messenger ; metabolism ; Signal Transduction

Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.
Arachidonic Acid/metabolism ; Ca(2+)-Calmodulin Dependent Protein Kinase/metabolism ; Cell Differentiation ; Dimethyl Sulfoxide/pharmacology* ; Enzyme Inhibitors/pharmacology* ; HL-60 Cells ; Human ; N-Formylmethionine Leucyl-Phenylalanine/pharmacology ; NADPH Oxidase/metabolism* ; Neutrophils/metabolism* ; Neutrophils/drug effects ; Oxazoles/pharmacology* ; Oxygen/metabolism ; Phosphoprotein Phosphatase/antagonists & inhibitors ; Phosphoproteins/immunology ; Signal Transduction ; Tetradecanoylphorbol Acetate/pharmacology ; Time Factors

Antitumor activity and the mechanism of CPUY013, a novel Topo I inhibitor, on gastric adenocarcinoma BGC823 cells were studied in vitro and in vivo. The proliferation was investigated by MTT assay and colony formation assay. Apoptosis was determined by both dual fluorescence staining with AO and EB and DNA agarose gel electrophoresis analysis methods. Nude mice model of BGC823 xenograft tumor was established by subcutaneous inoculation. The suppression activity of the CPUY013 by intragastric administration on xenograft mice model was detected. The change of cell cycle was studied by flow cytometry assay. The expressions of Topo I, widetype p53, active caspase-3, bcl-2 and bax proteins were analyzed by Western blotting assay. Results showed that CPUY013 could inhibit BGC823 cell proliferation at a certain range of dose. The flow cytometry analysis showed that CPUY013 and topoecan (TPT) led to a decrease in the proportion of G1 phase cells and an increase in the proportion of S phase cells, suggesting that they arrested the transition of tumor cells from S phase to G2 phase. The sub-G1 group was analyzed by flow cytometry. Compared with control, after 48 h treatment with CPUY013 or TPT, the sub-G1 group significantly increased in a dose-dependent manner. CPUY013 and TPT induced apoptosis in tumor cells. Cells treated with CPUY013 for 48 h were stained with AO/EB mixture. Then the cells were observed under fluorescence microscope. And it was found that early and late apoptosis cells were identified by perinuclear condensation of chromatin stained by AO/EB, respectively. Necrotic cells were identified by uniform labeling with EB. With the increase of concentration of CPUY013 and TPT, these morphological changes under the fluorescence microscope become clearer, indicating that the proportion of apoptosis cells increased gradually. By using JC-1 kit, loss of deltapsim was also detected in BGC823 cells treated with CPUY013 and TPT, which represent mitochondria function. And characteristic DNA ladder was observed apparently in BGC823 cells treated with CPUY013. When the xenograft tumor mice were treated with 150 mg x kg(-1) CPUY013, the tumor growth inhibition rate was 62.1%. The expression of bax and p53 proteins increased significantly and bcl-2 and bcl-2/bax decreased after the treatment of the CPUY013. The CPUY013 down-regulated Topo I protein expression and up-regulated active caspase-3 protein expression. The novel Topo I inhibitor CPUY013 can significantly suppress the growth of BGC823 xenograft tumor in vivo and inhibit the proliferation by inducing apoptosis of BGC823 cells in vitro.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Fluoroquinolones ; pharmacology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oxazoles ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Topoisomerase I Inhibitors ; Topotecan ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the molecular elements responsible for the enhanced apoptotic susceptibility in U937 cells mediated by silencing atactic telangiectasis mutation (ATM) gene.

METHODSTwo U937 cell mutants, U937-ASPI3K (ATM gene negative) and U937-pZEOSV2 (+) (ATM gene positive) were used as a cell model system. Apoptosis was examined by measuring free nucleosome concentrations in U937 cells. Western blotting was employed to measure nuclear protein abundances of cdc25A, cdc25B, cdc25C, total p34cdc2, p34cdc2 (Thr161) or p34cdc2 (Thr14, Tyr15). RT-PCR was used to estimate cdc25 transcript levels.

RESULTSU937-ASPI3K exhibited an enhanced apoptotic susceptibility to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-theronine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors could abolish the enhancement. Upon irradiation, p34cdc2 in U937-pZEOSV2 (+) was in an inactive state owing to phosphorylation of Thr 14 and Tyr15, which was associated with a dramatic decrease of nuclear cdc25A, cdc25B and cdc25C poteins. In contrast, p34cdc2 in U937-ASPI3K was in an active state owing to the low phosphorylation of Thr14 and Tyr15, which was associated with constant nuclear cdc25A, cdc25B and cdc25C protein abundance before and after irradiation. The responsive decrease of nuclear cdc25 proteins occurred at the post-transcription level.

CONCLUSIONSilencing ATM gene blocks the irradiation induced responsive decrease of nuclear cdc25 proteins, resulting in an abnormal activation p34cdc2 is the critical molecular mechanism for the enhanced apoptotic responses.

Apoptosis ; drug effects ; genetics ; radiation effects ; Ataxia Telangiectasia Mutated Proteins ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; Cycloheximide ; pharmacology ; DNA-Binding Proteins ; Enzyme Inhibitors ; pharmacology ; Gene Silencing ; drug effects ; radiation effects ; Humans ; Nuclear Proteins ; genetics ; metabolism ; Oxazoles ; pharmacology ; Phosphoprotein Phosphatases ; antagonists & inhibitors ; Protein Synthesis Inhibitors ; pharmacology ; Protein-Serine-Threonine Kinases ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Proteins ; U937 Cells ; cdc25 Phosphatases ; genetics ; metabolism