This study is to investigate the antitumor activity of ophiopogonin B (OP-B). MTT assay, flow cytometric analysis, acridine orange staining, Lyso-Tracker Red staining and HeLa-GFP-LC3 transfect cells assay were used to detect the proliferation activity, apoptosis and autophagy of HeLa cells. The results showed that OP-B exerted potent antiproliferative activity on HeLa cells, the cell growth inhibition effect of OP-B was not due to apoptosis and OP-B could induce autophagy of HeLa cells. OP-B also induced the protein expression up-regulation of Beclin-1 and promoted LC3 I transformation LC3 II, which were representative proteins of autophagy. Furthermore, 3-MA, an inhibitor of autophagy, not only inhibited OP-B-mediated autophagy but also almost completely reversed the antiproliferative effect of OP-B, suggesting that the growth inhibition effect of OP-B was autophagy dependent. Western blotting demonstrated that OP-B inhibited the phosphorylation of Akt and its' downstream vital protein, such as mTOR and p70S6K. In addition, OP-B also induced the protein expression up-regulation of PTEN, which is a negative regulation protein for Akt/mTOR signaling pathway. However, OP-B did not affect the protein expression of total Akt. Collectively, the antitumor effects of OP-B were autophagy-dependent via repression Akt/mTOR signaling pathway. Therefore, OP-B is a prospective inhibitor of Akt/mTOR and may be used as an alternative compound to treat cervical carcinoma.
Adenine ; analogs & derivatives ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; Beclin-1 ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; HeLa Cells ; Humans ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Ophiopogon ; chemistry ; PTEN Phosphohydrolase ; metabolism ; Phosphorylation ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-akt ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Spirostans ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; Up-Regulation

Treatment with the combination of Chinese herbs and cytotoxic chemotherapies showed a higher survival rate in clinical trials. In this report, the results demonstrated that the tanshinone II A, a key component of Salvia miltiorrhiza bunge, when it is combined with the cytotoxic drug cisplatin showed synergistic antitumor effects on human prostate cancer PC3 cells and LNCaP cells in vitro. Antiproliferative effects were detected with MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometer. Protein expression was detected by Western blotting. The intracellular concentration of cisplatin was detected by high performance liquid chromatography. The results demonstrated that tanshinone II A significantly enhanced the antiproliferative effects of cisplatin on human prostate cancer PC3 cells and LNCaP cells with the increase of the intracellular concentration of cisplatin. These effects were correlated with cell cycle arrested at S phase and cell apoptosis. The apoptosis might be achieved through death receptor pathway and mitochondrial pathway. Furthermore, the Bcl-2 family members were also involved in this apoptotic process. Collectively, these results indicated that the combination of tanshinone II A and cisplatin had a better treatment effect in vitro not only on androgen-dependent LNCaP cells but also on androgen-independent PC3 cells.
Androgens ; metabolism ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Diterpenes, Abietane ; isolation & purification ; pharmacology ; Drug Synergism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Male ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Prostatic Neoplasms ; metabolism ; pathology ; Salvia miltiorrhiza ; chemistry

Objective To explore the effect and mechanism of salvianolic acid B on bronchial epithelial cell apoptosis induced by cigarette smoke extract.Methods Human bronchial epithelial cells 16HBE were divided into control group,model group(induced by cigarette smoke extract),experimental low-dose group(induced by cigarette smoke extract+11 μmol·L-1 salvianolic acid B treatment),and experimental medium-dose group(cigarette smoke extract Induction+22 μmol·L-1 salvianolic acid B treatment),experimental high-dose group(cigarette smoke extract induction+44 μmol·L-1 salvianolic acid B treatment),experimental high-dose+Compound C group(cigarette smoke extract induction+44 μmol·L-1 Salvianolic acid B+AMPK signaling pathway inhibitor Compound C treatment).Cell counting kit-8(CCK-8)assay was used to detect proliferation;Western blot was used to detect the phosphorylated adenosine monophosphate activates protein kinase(p-AMPK),CCAAT/enhancer-binding protein C/EBP(CHOP),activating transcription factor 4(ATF4)protein expression;PI single staining was used to detect cell cycle;Annexin V-FITC/PI double staining was used to detect apoptosis,and spectrophotometry was used to detect caspase-3 activity.Results The cell proliferation activity(OD value)in the bronchial epithelial cells of the control group,model group,experimental low-dose group,experimental medium-dose group,experimental high-dose group,and experimental high-dose+Compound C group were 0.86±0.07,0.38±0.03,0.45±0.03,0.54±0.04,0.68±0.03 and 0.42±0.04;the expression levels of p-AMPK/AMPK protein were 0.41±0.03,0.13±0.03,0.20±0.02,0.28±0.04,0.36±0.04 and 0.22±0.02;G0/G1 phase were(54.40±5.84)％,(82.93±4.50)％,(75.45±4.73)％,(67.41±2.70)％,(59.15±3.73)％and(69.80±6.59)％;apoptosis rate were(3.21±0.49)％,(24.90±3.35)％,(20.56±1.73)％,(13.55±1.68)％,(9.20±1.07)％and(18.04±1.79)％.Compared experimental low-dose group,experimental medium-dose group,experimental high-dose group with model group,the difference of above indicators were all statistically significant(all P＜0.05);compared the experimental high-dose+Compound C group with the experimental high-dose group,the difference of above indicators were all statistically significant(all P＜0.05).Conclusion Salvianolic acid B affects endoplasmic reticulum stress by activating AMPK signaling to reduce bronchial epithelial cell apoptosis induced by cigarette smoke extract.

OBJECTIVETo map the gene locus in a Chinese pedigree with autosomal dominant nonsyndromic hearing loss.

METHODSA genome wide screening was performed with 394 microsatellite markers distributed with an average spacing of 10 cM (ABI Prism Linkage Mapping Set 2, Applied Biosystems, Foster City, CA, U.S.A.).

RESULTSAffected family members showed a bilateral, symmetrical, progressive neurosensory deafness. Significant linkage was found to marker D1 S937 (maximum two point LOD score of 5. 71 at theta = 0.05) on chromosome 11q. The position of the novel deafness locus, DFNA11, was delimited by analysis of the recombinant haplotypes (D11S165-D11S1874). This analysis placed DFNA11 between the proximal marker D11S1314 and the distal marker D11S898, which define a critical interval of 25.34 cM.

CONCLUSIONSMapping of the DFNA11 locus further confirms the great genetic heterogeneity underlying the autosomal dominant forms of hereditary deafness. Reports of more families with hearing impairment linked to this locus should contribute to the identification of the responsible gene, providing insights into the auditory function and the molecular pathophysiology of age related hearing loss.

Adult ; Aged ; Chromosome Mapping ; Deafness ; congenital ; genetics ; Female ; Genes, Dominant ; Haplotypes ; Humans ; Male ; Microsatellite Repeats ; Middle Aged ; Myosins ; genetics ; Pedigree ; Young Adult

AIMNucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, beta-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2. 2. 15 cells derived from HepG2 cells transfected with HBV genome.

METHODSbeta-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, 1H NMR and MS. 2. 2. 15 Cells were placed at a density of 5 x 10(4) per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2. 2. 15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated.

RESULTSThe synthesized compound structure conformed with beta-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 0.2 micromol x L(-1). The experiment of cytotoxicity gained IC50 200 micromol x L(-10.

CONCLUSIONbeta-L-D4A has been synthesized successfully. beta-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.

Antiviral Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Replication ; drug effects ; DNA, Viral ; drug effects ; Dideoxyadenosine ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Genome, Viral ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Humans ; Liver Neoplasms ; pathology ; Transfection ; Virus Replication ; drug effects

AIMTo study on synthesis and antibacterial activity evaluation of polyheterocycles.

METHODSThe condensation of 4-amino-3-pyridin-3-yl-4H-[1,2,4] triazole-5-thiol with 2-chloromethyl-5-substituted phenyl-[1,3,4] oxadiazoles gave the corresponding title heterocycle amines, and the in vitro antibacterial activity was primarily evaluated by the method of cup-plate diffusion solution.

RESULTSTwelve novel compounds were synthesized, and their structures were confirmed by IR, 1H NMR, MS and element analysis. Biological screening results demonstrated that most of the compounds prepared showed good antibacterial activity.

CONCLUSIONOxadiazoles incorporting pyridyl triazole ring may be a pharmacophor structure in the molecule for developing antibacterial candidate drugs.

Anti-Bacterial Agents ; chemical synthesis ; chemistry ; pharmacology ; Escherichia coli ; drug effects ; Oxadiazoles ; chemical synthesis ; chemistry ; pharmacology ; Proteus vulgaris ; drug effects ; Staphylococcus aureus ; drug effects ; Triazoles ; chemical synthesis ; chemistry ; pharmacology

OBJECTIVETo investigate the clinical classification and relationship with conductive deafness of congenital middle ear malformations.

METHODSFrom 1995 to 2004, 64 patients (82 ears) with single congenital middle ear malformations were operated in the ENT department of the General Hospital of Chinese People's Liberation Army. According to the histology and embryology of middle ear and the findings of surgical exploration, the clinical classification was performed. Statistical analysis was used to judge the differences of hearing loss in different type of congenital middle ear malformations.

RESULTSAccording to the embryologic development of the structures in middle ear, congenital middle ear malformations were classified 4 types. Type A: congenital ossicular chain anomalies; type B: congenital fusion of stapes; type C: congenital hypoplasia or atresia of oval/round widows. Hearing loss of three types on language frequency have no obvious difference (P = 0.1617), but there were statistical difference on high frequency ( > 2 kHz) between type A with type B and type C (P <0.05). Furthermore, descension of bone conduction and mixed deafness were familiar in type B and C.

CONCLUSIONSAccording to embryologic development, it was rational that congenital middle ear malformations were classed 3 types mentioned above. Hearing loss due to middle ear malformations could be distinguished by descension of bone conduction and air conduction on high frequency ( >2 kHz).

Adolescent ; Adult ; Child ; Child, Preschool ; Congenital Abnormalities ; classification ; diagnosis ; Ear Ossicles ; abnormalities ; Ear, Middle ; abnormalities ; Female ; Hearing Loss, Conductive ; classification ; congenital ; diagnosis ; Humans ; Male ; Retrospective Studies ; Young Adult

OBJECTIVETo identify the activity of hammerhead ribozyme against transforming growth factor beta1 (TGFbeta1) in a cell-free system and in activated hepatic stellate cells (HSCs).

METHODSThe ribozyme against TGFb1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX large scale RNA production system. The in vitro cleavage reactions were performed through incubation of 32P-labeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were constructed, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFbeta1 expression.

RESULTSThe ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFbeta1 expression in activated HSCs, while the disabled ribozyme displayed only a slight effect on TGFbeta1 expression.

CONCLUSIONThe ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFbeta1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.

Animals ; Cell-Free System ; Cells, Cultured ; Genetic Vectors ; Hepatocytes ; cytology ; RNA ; genetics ; metabolism ; RNA, Catalytic ; RNA, Messenger ; genetics ; metabolism ; Rats ; Transcriptional Activation ; Transfection ; Transforming Growth Factor beta ; genetics ; metabolism

AIMStudies on synthesis and antibacterial activity of new heterocycles.

METHODSThe cyclocondensation of [(3-pyridyl)-1,3,4-oxadiazol-2-yl] thio acetic acid with various aroyl hydrazines in the presence of POCl3 and xylene gave the corresponding titled compounds, and the in vitro antibacterial activity was primarily evaluated by the method of cupplate diffusion solution.

RESULTSSixteen novel titled compounds were synthesized, their structures were confirmed by IR, 1HNMR, MS and elemental analysis. Biological screening results demonstrated that most of the compounds prepared displayed potential antibacterial activity.

CONCLUSIONOxadiazoles incorporting pyridyl oxadiazole ring may be usefully antibacterial candidate drugs.

Anti-Bacterial Agents ; chemical synthesis ; chemistry ; pharmacology ; Escherichia coli ; drug effects ; Oxadiazoles ; chemical synthesis ; chemistry ; pharmacology ; Proteus vulgaris ; drug effects ; Staphylococcus aureus ; drug effects