AIMTo explore the effects of gentamicin ototoxicity on the expression of heat shock protein 70 in guinea pigs cochlea.

METHODSWe used immunohistochemistry staining and image quantitative analysis system, combined with auditory brainstem response (ABR) measurement to investigate the change on the expression of HSP70 in guinea pigs cochlea of gentamicin ototoxicity.

RESULTSThe levels of HSP70 immunoreactivity in guinea pigs cochlea of experimental animals were high including Corti's organ, stria vascularis, medial spiral limbus, spiral ganglion cells and the threshold of ABR was in high correlation with the expression of HSP70 ([ r] > 0.8, P < 0.01).

CONCLUSIONGentamicin can induce expression of HSP 70 in guinea pigs cochlea and protect hearing function.

Animals ; Cochlea ; drug effects ; physiopathology ; Gentamicins ; toxicity ; Guinea Pigs ; HSP70 Heat-Shock Proteins ; metabolism ; Heat-Shock Response ; drug effects

OBJECTIVETo study the expression and the possible role of JWA protein in oxidative stress-induced damage of MCF-7 cells, especially the relationship between JWA and heat shock proteins (HSPs).

METHODSMCF-7 cells were exposed to different concentration of H(2)O(2) (0.01,0.10, 1.00 mmol/L) for different time (10, 30, 60 and 180 min) respectively. DNA damage was detected by using DNA gel electrophoresis. The MTT assay was used to analyze the effect of H(2)O(2) on the cytotoxicity and relative cell proliferation ratio of the cells. The expressions of JWA, HSP70, HSP27 and HSF1 were determined by Western-blot.

RESULTSThe inhibitory effect on MCF-7 cells viability induced by H(2)O(2) was shown a dose-and time-dependent manner and MCF-7 cells proliferation, and was almost completely inhibited by the exposure of H(2)O(2) at 1.00 mmol/L for 180 min. Hydrogen peroxide treatment of MCF-7 cells caused oxidative stress which up-regulated the expressions of JWA, HSP70 and heat shock factor 1 (HSF1) in a dose-dependent manner, and the expression pattern of JWA was very similar to those of HSP70 and HSF1 but not to HSP27.

CONCLUSIONJWA might enhance intracellular defenses against H(2)O(2)-induced oxidative damage in human breast carcinoma cells. JWA is determined functioning as an effective environmental responsive protein and as a parallel molecule of HSP70 actively participates in the signal pathways of oxidative damage which might be regulated by HSF1.

Cell Line, Tumor ; DNA Damage ; drug effects ; DNA-Binding Proteins ; biosynthesis ; Dose-Response Relationship, Drug ; Female ; HSP27 Heat-Shock Proteins ; HSP70 Heat-Shock Proteins ; biosynthesis ; Heat Shock Transcription Factors ; Heat-Shock Proteins ; biosynthesis ; Humans ; Hydrogen Peroxide ; adverse effects ; Intracellular Signaling Peptides and Proteins ; Neoplasm Proteins ; biosynthesis ; Oxidative Stress ; drug effects ; Transcription Factors ; biosynthesis ; Up-Regulation

OBJECTIVETo examine the inhibition of nitric oxide (NO) synthesis during ischemic preconditioning (IP) upon the induction of heat-shock protein 72 (HSP72) and the size-limiting effect of the second window of protection on infarction.

METHODSRabbits were subjected to either 4 cycles of 5-min long coronary artery occlusion separated by 10 min of reperfusion, or a sham operation. During this procedure, we administered 10 mg/kg of N(G)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) intravenously 5 min before IP followed by its continuous infusion (1.5 mg/kg/min). Twenty-four hours after IP or the sham operation, the hearts were rapidly excised for assay of HSP72 expression or were subjected to 30 min of coronary artery occlusion followed by 120 min of reperfusion, at which point infarct size (IS) was measured.

RESULTSTwenty-four hours after IP or the sham operation, there was no difference in heart rate or mean arterial pressure between the groups. Immunoblotting revealed an increase in HSP72 protein levels in the IP group, which was blocked by L-NAME. IS in the IP rabbits was reduced compared with controls (29.8 +/- 3.7% vs. 50.8 +/- 4.3%, P < 0.01). IS in the IP rabbits was elevated as a result of L-NAME treatment (46.0 +/- 5.1%). Administration of L-arginine reversed the effects of L-NAME on the induction of HSP72 and IS (33.5 +/- 4.0%). The intravenous administration of S-nitroso-N-acetylpenicillamine (an NO donor, 15 microg/kg/min) over 20 min increased the induction of HSP72 and reduced IS (31.3 +/- 5.7%, P < 0.01 vs. control) 24 h later.

CONCLUSIONThese findings suggest that NO may be involved in the induction of HSP72 and the opening of the second window of protection of IP.

Animals ; HSP72 Heat-Shock Proteins ; Heat-Shock Proteins ; biosynthesis ; Hemodynamics ; drug effects ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Infarction ; pathology ; Nitric Oxide ; physiology ; Penicillamine ; analogs & derivatives ; pharmacology ; Rabbits

OBJECTIVE: To observe the effect of heat shock factor 1 (HSF1) on heat stress-induced apoptosis in Raw264.7 macrophages. METHODS: Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-HSF1 were exposed to heat stress (42.5 degrees C +/- 0.5 degrees C) for 1 h and recovered at 37 degrees C for 6, 9, 12, and 24 h respectively. Flow cytometry (FCM), Hoechst 33258 staining and DNA ladder assays were performed to assess the apoptosis. RESULTS: After heat stress, FCM showed that apoptotic cells were increased significantly and reached the peak at 9 h in Raw 264.7 cells transfected with pcDNA3.1, and were characterized with classical morphologic changes including apoptotic body and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly at 6, 9, and 12 h after the heat stress. But the overexpression of HSF1 could reduce the number of apoptotic cells and inhibit DNA fragmentation. CONCLUSION HSF1 can inhibit heat stress-induced apoptosis in Raw264.7 macrophages.
Animals ; Apoptosis ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; pharmacology ; Heat Shock Transcription Factors ; Heat-Shock Response ; Macrophages ; cytology ; Mice ; Rats ; Transcription Factors ; pharmacology ; Transfection

OBJECTIVE: To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells. METHODS: We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay. RESULTS: Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis. CONCLUSION Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis.
Cell Line ; Collagen Type I ; metabolism ; HSP47 Heat-Shock Proteins ; metabolism ; Heat-Shock Response ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo explore the relationship between the expression of heat shock protein 90, 60 and 27 (HSP90, HSP60 and HSP27) and genetic damage in peripheral blood of workers exposed to coke oven emissions.

METHODS288 coke oven workers in a steel factory were divided into the high-dose group and the low-dose group on the basis of environment monitoring result and work place. There were 172 men in high-dose group (workers who worked at the oven top and oven side) and 116 men in low-dose group (workers who worked at the oven bottom and others who were engaged to aided work). 38 workers unexposed occupationally to carcinogenic substances were selected as the control group, who were employed in medical therapy unit nearby 2 kilometers from the steel factory. Their general information, history of personal and occupational exposure, and the work environment were investigated. Blood samples were collected immediately after a shift at the end of a working day from 288 coke oven workers and 38 control workers. Levels of HSP90, HSP60 and HSP27 in peripheral blood lymphocytes were measured by Western blot, and the degree of DNA damage was detected by the comet assay.

RESULTSLevels of HSP90 in peripheral blood lymphocytes in three groups were 0.24 +/- 0.32, 0.12 +/- 0.30 and 0.06 +/- 0.33 respectively. They increased significantly compared with that of the control. But levels of HSP60 and HSP27 were not significantly different among those groups. Compared with the control group, there was significant difference in tail length, olive tail moment et al of SCGE (G +/- s(G)) of occupational exposure workers. High-dose group > low-dose group > control group (P < 0.05). The degree of DNA damage increased with the rise of exposure BaP dose (Spearman r = -0.345, P < 0.01).

CONCLUSIONLevels of HSP90 in peripheral blood lymphocytes and the degree of DNA damage increase with the rise of exposure polycyclic aromatic hydrocarbons (PAHs) dose.

Adult ; Chaperonin 60 ; blood ; Coke ; adverse effects ; Comet Assay ; DNA Damage ; drug effects ; HSP27 Heat-Shock Proteins ; blood ; HSP90 Heat-Shock Proteins ; blood ; Humans ; Lymphocytes ; metabolism ; Male ; Occupational Exposure ; adverse effects ; Polycyclic Aromatic Hydrocarbons ; adverse effects

MDR1 promoter has been shown to contain heat shock elements (HSE), and it has been reported that FM3A/M and P388/M MDR cells show a constitutively activated heat shock factor (HSF), suggesting that HSF might be an important target for reversing the multidrug resistance. Therefore, it was examined whether quercetin, which has been shown to interfere with the formation of the complex between HSE and HSF, and to downregulate the level of HSF1, can sensitize MDR cells against anticancer drugs by inhibition of HSF DNA-binding activity. In this study, quercetin appeared to inhibit the constitutive HSF DNA-binding activity and the sodium arsenite-induced HSF DNA-binding activity in the MDR cells. The basal and sodium arsenite-induced MDRCAT activities were remarkably suppressed by the treatment of quercetin. These results were well consistent with the finding that the treatment of quercetin decreased the expression level of P-gp, MDR1 gene product, in dose-dependent manner, and markedly increased the sensitivity of MDR cells to vincristine or vinblastine. These results suggest that quercetin can decrease the expression of P-gp via inhibition of HSF DNA-binding activity, and might be useful as a chemosensitizer in MDR cells.
Animal ; Antineoplastic Agents/pharmacology ; Arsenites/pharmacology ; Carcinoma/drug therapy ; Drug Resistance, Multiple/physiology* ; Drug Resistance, Neoplasm/physiology ; Heat-Shock Proteins/metabolism ; Heat-Shock Proteins/drug effects* ; Heat-Shock Proteins/antagonists & inhibitors ; Leukemia, Experimental/drug therapy ; Mice ; P-Glycoprotein/genetics ; P-Glycoprotein/drug effects ; Quercetin/pharmacology* ; Sodium Compounds/pharmacology ; Tumor Cells, Cultured ; Vinblastine/pharmacology ; Vincristine/pharmacology

Cell Line ; Glucocorticoids ; pharmacology ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Mucin 5AC ; metabolism ; Respiratory System ; drug effects ; metabolism

OBJECTIVETo investigate the protective role of inducible heat shock protein 70 (Hsp70) against damage induced by formaldehyde.

METHODSHuman bronchial epithelium (HBE) cells were transfected with plasmid harboring hsp70 gene to increase the protein expression level. HBE cells transfected with pcDNA3.1 plasmid were used as transfection control and HBE cells cultured at normal condition served as control. Three groups were marked as HBE/hsp70, HBE/pcDNA and HBE. Hsp70 expression levels of 3 groups were detected. The cells of HBE/hsp70 and HBE groups were exposed to different concentrations of formaldehyde (0,0.39,1.56,6.25 mmol/L) for 4 h. The contents of GSH and MDA were measured, and KCl-SDS method was applied to measure DNA-protein crosslink (DPC).

RESULTSHsp70 level in HBE/hsp70 group increased by 80% compared with HBE group. GSH contents in HBE/hsp70 group significantly increased and were 141.0, 119.6 mg/gpro at 0.39, 1.56 mmol/L, respectively (P<0.01), as compared with HBE group. However, it decreased when formaldehyde concentration increased to 6.25 mmol/L. While GSH content in HBE group remained decreasing. MDA contents in HBE/hsp70 and HBE group increased with formaldehyde. MDA content in HBE/hsp70 was 0.088 micromol/gpro and significantly lower than that (0.138 micromol/gpro) in HBE group (P<0.05) when formaldehyde concentration was 1.56 mmol/L, At the formaldehyde dose of 6.25 mmol/L MDA content in HBE/hsp70 was 0.140 micromol/gpro which was significantly lower than that (0.289 micromol/gpro) in HBE group (P<0.01). DPC% in two groups increased with formaldehyde. At the formaldehyde dose of 0.39 mmol/L, DPC% in HBE/hsp70 group was 3.94% which was significantly lower than that (6.25%) in HBE group (P< 0.01). At the formaldehyde dose of 1.56 mmol/L, DPC% in HBE/hsp70 group was 11.86% which was significantly lower than that (20.89%) in HBE group (P<0.05).

CONCLUSIONHsp70 can reduce formaldehyde-induced damages in human bronchial epithelium cells in vitro.

Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; Formaldehyde ; toxicity ; Glutathione ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Malondialdehyde ; metabolism ; Transfection

OBJECTIVETo investigate the effects of metformin on the proliferation and apoptosis of human oral cancer cell line KB in vitro.

METHODSHuman oral cancer cell line KB was exposed to different doses of metformin (0, 1.25, 2.5, 5, 10, and 20 mmol/L), and the changes in cell viability were detected using MTT assay. Colony formation of the cells was observed following an 8-day metformin exposure. The changes in mitochondrial membrane potential were measured by JC-1 assay, and PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the changes in the protein expressions of GRP78 and activated caspase-3.

RESULTSMetformin exposure caused time- and dose-dependent suppression of KB cell proliferation, and exposure to 5 mmol/L metformin for 24, 48 and 72 h resulted in cell survival rates of 68.0%, 36.9%, and 14.5%, respectively. Metformin significantly inhibited KB cell colony formation. Exposure of the cells to increased concentrations of metformin gradually increased the apoptotic rate and decreased mitochondrial membrane potential. Metformin caused an initial up-regulation followed by a down-regulation of GRP78 expression in KB cells and increased the expression of activated caspase-3.

CONCLUSIONMetformin can inhibit the proliferation and induce apoptosis of KB cells, the mechanism of which may involve the activation of the mitochondrial apoptotic pathway and endoplasmic reticulum stress.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Heat-Shock Proteins ; metabolism ; Humans ; KB Cells ; Membrane Potential, Mitochondrial ; drug effects ; Metformin ; pharmacology