Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.
Biological Products/metabolism* ; Lactams, Macrocyclic/metabolism* ; Multigene Family ; Organisms, Genetically Modified ; Streptomyces/metabolism*

OBJECTIVETo investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism.

METHODSCCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays.

RESULTSThe inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment.

CONCLUSIONThe 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Apoptosis ; Benzoquinones ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; HL-60 Cells ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Leukemia ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptome

OBJECTIVETo investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism.

METHODSThe multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively.

RESULTSThe 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P < 0.05 and r = -0.473, P < 0.05), while the culture medium without 17-AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P < 0.05); At same time of culture, the more high of 17-AAG concentration, the more high of cell ratio in G1 phase (P < 0.05), at same concentration of 17-AAG, the more long time of culture, the more high of cell ratio in G1 phase (P < 0.05).

CONCLUSIONThe HSP90 inhibitory 17-AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.

Apoptosis ; Benzoquinones ; pharmacology ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Multiple Myeloma ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo explore apoptosis of multiple myeloma (MM) cells and its mechanism by the combined inhibition of mTORC2 signaling pathway and heat shock protein 90.

METHODSThe effects of Rapamycin, 17-AAG and the combination on proliferation of MM cell lines U266 and KM3 were assessed using MTT at different time points (0, 8, 24, 48 hour). Cell apoptosis and cell cycle distribution were measured by flow cytometry. The specific proteins p-AKT (ser473), p-AKT (thr450), p-S6 (S235/236) and AKT were detected by Western blotting.

RESULTSRapamycin, 17- AAG and the combination suppressed the proliferation of MM cell lines U266 and KM3, especially the combination of Rapamycin and 17-AAG synergistically inhibited the proliferation (P<0.05); Rapamycin induced G1 arrest both at 24 and 48 hours, 17-AAG also induced G1 arrest, especially at 48 hours (P<0.01); Rapamycin, 17-AAG alone decreased the expression of AKT and induced MM cell apoptosis to some extent (P<0.01); Chronic rapamycin treatment inhibited mTORC2; Inhibition of both mTORC2 and chaper on pathways degraded AKT and induced MM cell apoptosis, which was significantly higher than that of any single agent (P<0.01).

CONCLUSIONInhibition of both mTORC2 and chaper on pathways decreased the expression of AKT to induce apoptosis of MM cells in vitro.

Apoptosis ; Benzoquinones ; pharmacology ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; drug effects ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Mechanistic Target of Rapamycin Complex 2 ; Multiple Myeloma ; pathology ; Multiprotein Complexes ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo study the effect of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on cell proliferation and apoptosis of human cancer SGC-7901 cells and explore the mechanisms.

METHODSThe inhibitory effect of 17-AAG on the proliferation and morphology of SGC-7901 cells was assessed with MTT assay and DNA-PI staining, respectively. Flow cytometry was employed to analyze the changes in cell cycle and apoptosis of the cells following 17-AAG exposure. The cellular expression of Fas protein was detected by immunohistochemistry.

RESULTS17-AAG significantly suppressed the proliferation of SGC-7901 cells in a time- and dose-dependent manner. After treatment with 17-AAG for 48 h, SGC-7901 cells showed cell cycle arrested at G(2)/M stage, and the cell apoptosis rate increased with the 17-AAG concentration. The expression of Fas protein in the cytoplasm of SGC-7901 cells increased gradually with the increase of 17-AAG concentration.

CONCLUSION17-AAG can induce apoptosis, alters the cell cycle distribution and up-regulates the expression of Fas protein in SGC-7901 cells to suppress the cell proliferation.

Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Stomach Neoplasms ; pathology ; fas Receptor ; metabolism

In the present study, a new compound named 17-(6-cinnamamido-hexylamino-)-17-demethoxygeldanamycin (CDG) was obtained by introducing the cinnamic acid (CA) group into the 17-site of geldanamycin (GDM). The anti-cancer effects of CDG in vitro and in vivo were evaluated. MTT assay was used to examine the inhibitory effect of CDG on the proliferation of MCF-7, HepG2, H460 and SW1990 cells. Immunofluorescent staining flow cytometry combined with Annexin V-FITC/PI staining were used to detect apoptotic cells. Transwell assay was used to analyze the effect of CDG on cell invasion and migration ability. Western blotting was used to detect the expression levels of RAF-1, EGFR, AKT, CDK4 and HER-2 of MCF-7, HepG2 and H460 cells. The toxicities of CDG and GDM were evaluated in mice. Using the subcutaneously transplanted MCF-7 xenograft in nude mice, inhibitory effect was evaluated in vivo. The results showed that CDG inhibited the proliferation of cancer cells (IC50: 13.6-67.4 microg.mL-1). After exposure to CDG for 48 h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus. The rates of apoptosis of MCF-7, HepG2, H460 and SW1990 cells incubated with 10 microg.mL-1 CDG were 23.16%, 27.55%, 22.21%, 20.47%, respectively. A dose-dependent reduction of migration of four cell lines was found after exposure to CDG. The decreased levels of RAF-1, EGFR, AKT, CDK4 and HER-2 showed that CDG possessed HSP90 inhibitory effect. The result of animal toxicity test on the mice suggested that CDG had lower toxicity than GDM. Meanwhile, CDG inhibited the growth of MCF-7 xenografts of athymic mice.
Animals ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Benzoquinones ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase 4 ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; chemical synthesis ; chemistry ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Proto-Oncogene Proteins A-raf ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; metabolism ; pathology ; Drug Resistance, Neoplasm ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Mutation ; Oncogene Proteins, Fusion ; antagonists & inhibitors ; genetics ; metabolism ; Protein Kinase Inhibitors ; therapeutic use ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Triazoles ; pharmacology

OBJECTIVETo explore correlation of seven apoptosis-related proteins (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with clinical outcomes of ALK+ anaplastic large-cell lymphoma (ALCL).

METHODSUsing immunohistochemistry and immunofluorescence double staining methods, the expressions of these seven apoptosis-associated proteins were studied to clarify their relationship with clinical outcomes of 36 ALK+ and 25 ALK-systemic ALCL patients enrolled between 1996 and 2006. The relationship of these apoptosis-regulating proteins with NPM-ALK status was also evaluated with the tyrosine inhibitor herbimycin A (HA) in vitro by immunocytochemistry, Western blotting and flow cytometric assays.

RESULTSThe presence of Hsp90α-, MDM2-, Bax-, Cytochrome C, and Cleaved caspase3-positive tumor cells was found significantly different in ALK+ and ALK-ALCLs, which was correlated with highly favorable clinical outcome. The Bcl-2- and p53-positive tumor cells were found in groups of patients with unfavorable prognosis. Inhibition of NPM-ALK by HA could reactivate the p53 protein and subsequent apoptosis-related proteins and therefore induced apoptosis in ALK+ ALCL cells.

CONCLUSIONOur results suggest that these seven proteins might be involved in apoptosis regulation and associated with clinical outcome of ALK+ systemic ALCLs. We also reveal a dynamic chain relation that NPM-ALK regulates p53 expression and subsequent apoptosis cascade in ALK+ ALCLs.

Adolescent ; Adult ; Aged ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Benzoquinones ; pharmacology ; Biomarkers, Tumor ; metabolism ; Blotting, Western ; Cell Culture Techniques ; Cell Survival ; drug effects ; Child ; Child, Preschool ; Disease-Free Survival ; Enzyme Inhibitors ; pharmacology ; Female ; Flow Cytometry ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Lactams, Macrocyclic ; pharmacology ; Lymphoma, Large-Cell, Anaplastic ; enzymology ; metabolism ; pathology ; Male ; Microscopy, Fluorescence ; Middle Aged ; Neoplasm Staging ; Prognosis ; Protein-Tyrosine Kinases ; metabolism ; Receptor Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Retrospective Studies ; Rifabutin ; analogs & derivatives ; Young Adult

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.
Benzoquinones/*pharmacology ; Cell Line, Tumor ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/enzymology/metabolism/microbiology ; GTP-Binding Protein alpha Subunits, Gi-Go/metabolism ; Gastric Mucosa/enzymology/metabolism/*microbiology ; *Helicobacter pylori ; Humans ; Lactams, Macrocyclic/*pharmacology ; Oligonucleotides, Antisense ; Pertussis Toxin/*pharmacology ; RNA, Messenger/metabolism ; Receptor, PAR-2/*physiology ; src-Family Kinases/metabolism

To identify the anti-bacterial compound(s) from Streptomyces hygroscopicus 17997, a geldanamycin producer, silica gel thin layer chromatography (TLC) TLC was used to separate the secondary metabolites of S. hygroscopicus 17997. Compound(s) from the silica gel TLC with anti-Gram positive bacteria activity and becoming red upon color reaction by 2.0 mol/L NaOH was analyzed by HPLC. The UV absorption profile and the retention time of a peak of HPLC were identical to those of authentic elaiophylin. A conserved region of dTDP-glucose-4,6-dehydratase (Tgd) gene was amplified by PCR from the genomic DNA of Streptomyces hygroscopicus 17997. DNA sequence analysis of the amplified DNA fragment indicated that it should be the tgd gene of elaiophylin biosynthetic gene cluster. These results implied that the compound in the peak of HPLC was elaiophylin, a macrodiolide antibiotic. The compound was then confirmed to be elaiophylin by LC-(+)-ESI-MS, which revealed that Streptomyces hygroscopicus 17997 was an elaiophylin producer. At the same time, a fast procedure, which consisted of silica gel TLC, color reaction, HPLC, PCR detection and DNA sequence analysis of tgd gene, and LC-(+)-ESI-MS, was established for rapid identification of elaiophylin and its producer.
Benzoquinones ; metabolism ; Chromatography, Liquid ; methods ; DNA, Bacterial ; genetics ; Hydro-Lyases ; genetics ; Lactams, Macrocyclic ; metabolism ; Macrolides ; analysis ; isolation & purification ; metabolism ; Mass Spectrometry ; methods ; Sequence Analysis, DNA ; Streptomyces ; genetics ; isolation & purification ; metabolism