OBJECTIVETo investigate the mechanism by which heat shock protein 90 (HSP90) regulates 26S proteasome in hyperthermia.

METHODSHyperthermic HepG2 cell models established by exposure of the cells to 42 degrees celsius; for 3, 6, 12, and 24 h were examined for production of reactive oxygen species (ROS) and cell proliferation, and the changes in Hsp90α and 26S proteasome were analyzed.

RESULTSROS production in the cells increased significantly after hyperthermia (F=28.958, P<0.001), and the cell proliferation was suppressed progressively as the heat exposure time extended (F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level (F=164.174, P<0.001) and activity (F=133.043, P<0.001) of 26S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26S proteasome (F=180.231, P<0.001).

CONCLUSIONThe intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.

HSP90 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Hot Temperature ; Humans ; Proteasome Endopeptidase Complex ; metabolism ; RNA, Small Interfering ; genetics ; Reactive Oxygen Species ; metabolism ; Up-Regulation

The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Aspirin/*pharmacology ; Cell Nucleus/genetics ; Chickens ; Gene Expression Regulation/*drug effects ; HSP90 Heat-Shock Proteins/*genetics ; Hot Temperature ; Myocytes, Cardiac/*drug effects/enzymology/pathology ; Stress, Physiological/*drug effects

Animals ; Cadherins ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Early Growth Response Protein 1 ; genetics ; metabolism ; Estradiol ; physiology ; Eukaryotic Initiation Factor-3 ; metabolism ; Genes, Tumor Suppressor ; physiology ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Iron ; metabolism ; Neoplasms ; metabolism ; pathology

Along with the increase in fungal infections, Candida albicans prevention and control become the focus of anti-fungal infection at present. This study aims to discuss the effect monomer andrographolide (AG) on C. albicans biofilm dispersion. In the experiment, micro-well plates and medical catheter pieces were used to establish the C. albicans biofilm model. It was discovered by XTT assay and flat band method that 1 000, 500, 250 mg x L(-1) AG could impact the activity of C. albicans biofilm dispersion cells. The morphological structures of residual biofilms on catheter pieces were observed with scanning electron microscopy, which showed that 1 000, 500, 250 mg x L(-1) AG could induce C. albicans biofilm dispersion in a dose-dependent manner, and the dispersed cells were dominated by the yeast phase. According to the real-time fluorescence quantification PCR (qRT-PCR) test, AG could up-regulate HSP90 expression and down-regulate UME6 and PES1 expressions. This study demonstrates that AG could induce C. albicans biofilm dispersion to some extent.
Anti-Inflammatory Agents ; pharmacology ; Biofilms ; drug effects ; Candida albicans ; genetics ; physiology ; ultrastructure ; Diterpenes ; pharmacology ; Dose-Response Relationship, Drug ; Fungal Proteins ; genetics ; Gene Expression Regulation, Fungal ; drug effects ; HSP90 Heat-Shock Proteins ; genetics ; Microscopy, Electron, Scanning ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Survival ; drug effects ; ErbB Receptors ; antagonists & inhibitors ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; MCF-7 Cells ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Minor Histocompatibility Antigens ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Transplantation, Heterologous ; Tyrphostins ; pharmacology

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 evaluate the effect of heat shock protein 90 (HSP90) on hepatitis B virus (HBV) replication in hepatocytes and to investigate the related molecular mechanism.

METHODSA eukaryotic plasmid expressing human HSP90 was constructed (designated as HA-HSP90). HepG2 cells were co-transfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen (HBsAg) was determined by enzyme-linked immunosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering (si)RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1b and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR.

RESULTSThe HA-HSP90 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSP90 transfection, but neither IL-1b nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication.

CONCLUSIONHSP90 can inhibit HBV replication and TBK1 is not involved in this process.

HSP90 Heat-Shock Proteins ; genetics ; Hep G2 Cells ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Protein-Serine-Threonine Kinases ; genetics ; Transfection ; Virus Replication

OBJECTIVETo investigate the expressions of GRα, GRβ, GRγ, GRp and heat shock protein 90 (HSP90) in primary immune thrombocytopenia (ITP) patients and their correlations with glucocorticoid resistance.

METHODSThe expressions of glucocorticoid receptors (GRα, GRβ, GRγ, GRp) mRNA and HSP90 in peripheral blood mononuclear cells of 40 newly diagnosed ITP patients and 29 healthy volunteers were examined by real time PCR. Of them, 28 patients received glucocorticoid (GC) therapy divided into GC sensitive group (GCS) and GC resistant group (GCR) according to GC response. GRα, GRβ, GRγ, GRp, HSP90 mRNA and HSP90/GRα were analyzed in paired groups.

RESULTSThe expression of HSP90 mRNA was significantly decreased in ITP patients \[0.91(0.48 - 2.21)\] than in normal subjects \[1.41(0.83 - 2.61)\] (P < 0.05). There were no significant differences in mRNA expressions of GRα, GRβ, GRγ, GRp and HSP90/GRα between ITP patients and normal controls. The expression of GRα mRNA in GCS patients was significant higher than in GCR patients (P < 0.05). Moreover, no significant differences in mRNA expressions of GRβ, GRγ, GRp and HSP90 and the ratio of HSP90 to GRα were observed between GCS and GCR patients.

CONCLUSIONThe expression of HSP90 mRNA decreased in adult ITP patients. GC resistance in adult ITP patients was associated with reduced expression of GRα. The very low expression of GRβ mRNA may be not involved in GC resistance in adult ITP.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Female ; Glucocorticoids ; metabolism ; HSP90 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Receptors, Glucocorticoid ; metabolism ; Thrombocytopenia ; metabolism ; Young Adult

OBJECTIVETo investigate the expression of heat shock protein 90 (HSP90) on the cell surface of highly invasive human prostate cancer cells PC3 and its possible molecular mechanisms of its effect on cell invasion through analyzing FAK/Src signaling pathway.

METHODSThe expression of cell surface HSP90 on PC3 cells was studied by immunofluorescence staining and surface biotinylation assay respectively. A specific HSP90 antibody was used to inhibit the cell surface HSP90. In vitro cell invasion was assessed by modified Boyden chambers. Phosphorylated FAK on tyr 397, 576, 577 and 925, and phosphorylated c-Src on tyr 416 were examined by Western blot assay. The association between FAK and c-Src was analyzed by immunoprecipitation. The effects of FAK knockdown by siRNA or Src kinases inhibitor PP2, with or without anti-HSP90 antibody, on PC3 cell invasion were also evaluated.

RESULTSA pool of HSP90 was detected on the cell surface of PC3 cells. A specific HSP90 antibody significantly retarded tumor cell invasion. Concomitant with this finding, targeting cell surface HSP90 significantly inhibited the phosphorylations of FAK and c-Src, and also the interactions between FAK and c-Src. FAK knockdown or PP2 dramatically suppressed cell invasion, however, anti-HSP90 antibody didn't further inhibit cell invasion.

CONCLUSIONSCell surface HSP90 promotes human prostate cancer cell invasion through a FAK/c-Src signaling, with may be a novel therapeutic target against metastatic tumors.

Antibodies ; pharmacology ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Gene Knockdown Techniques ; HSP90 Heat-Shock Proteins ; immunology ; metabolism ; Humans ; Male ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Pyrimidines ; pharmacology ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; src-Family Kinases ; antagonists & inhibitors ; metabolism

This study was aimed to investigate the correlation of heat shock protein 90 (HSP90) expression with migration ability of human multiple myeloma cells. The HSP90 mRNA expression and migration change of human multiple myeloma cell line (U266) were detected by RT-PCR and Transwell chamber respectively after treatment of U266 cells with final concentration 50, 100, 150, 200 nmol/L of bortezomib (proteosome inhibitor) for 4 hours. The results indicated that along with the increasing of bortezomib concentration, the expression level of HSP90alpha mRNA in U266 cells was enhanced, while no obvious increase of HSP90beta mRNA expression was observed in spite of statistical difference as a whole (p<0.05), but with the increasing of drug concentration in cells, their migration ability gradually decreased (p<0.05). It is concluded that the correlation of HSP90 expression with migration ability of human multiple myeloma cells exists.
Cell Line, Tumor ; Cell Movement ; HSP90 Heat-Shock Proteins ; genetics ; Humans ; Multiple Myeloma ; genetics ; pathology ; RNA, Messenger ; genetics