Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.
Animals ; Berberine ; pharmacology ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Heat Stress Disorders ; drug therapy ; genetics ; metabolism ; Hot Temperature ; Humans ; Male ; Mice ; Mice, Inbred ICR ; TATA Box ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

PURPOSE: There are currently no consistently effective treatments for the excessive collagen produced by keloid fibroblasts. Previously, we reported that heat shock protein 70 (Hsp70) is up-regulated in keloid fibroblasts and keloid tissue. We, therefore, investigated whether Hsp70 is related to excessive collagen production in keloid fibroblasts. MATERIALS AND METHODS: We inhibited Hsp70 in keloid fibroblasts by RNA interference and examined the resulting collagen expression. Thus, we selected small interfering RNAs (siRNAs) specific for human Hsp70, transfected them into keloid fibroblasts, and evaluated the resulting phenotypes and protein production using real-time polymerase chain reaction (PCR), Western blot, and a collagen assay. RESULTS: The siRNAs dramatically suppressed Hsp70 mRNA expression, resulting in a decrease in collagen production in the keloid fibroblasts compared with controls. The siRNAs did not influence the viability of the keloid fibroblasts. CONCLUSION: Hsp70 overexpression likely plays an important role in the excessive collagen production by keloid fibroblasts. RNA interference has therapeutic potential for the treatment of keloids.
Adolescent ; Adult ; Blotting, Western ; Collagen/*drug effects/metabolism ; Female ; Fibroblasts/metabolism ; Gene Expression Regulation ; HSP70 Heat-Shock Proteins/genetics/metabolism/*pharmacology ; Humans ; Keloid/*drug therapy/genetics/metabolism ; Male ; RNA, Messenger/*genetics ; RNA, Small Interfering/*genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; Up-Regulation

The purpose of this study was to verify that a combination of mild hyperthermia and docetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach. The effects of docetaxel on the proliferation of cells from the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and effective experimental concentrations of docetaxel were determined. The effects of mild hyperthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The effects of these combined treatments on cell cycle progression in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cytometry. The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase (MAPK) pathways were analyzed by using Western blotting. The effects of these combined treatments on the expression of the heat shock protein 70 (HSP70) and the multi-drug resistance (MDR) gene product P-glycoprotein (Pgp) were examined by using Western blotting. The results showed that the half-maximal inhibitory concentration (IC50) of docetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31 μmol/L respectively. Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453 cells. Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from (23.66±3.59)% and (18.51±3.17)% in docetaxel treatment group to (47.12±6.73)% and (55.16±7.42)% in mild hyperthermia plus docetaxel group, indicating that the mild hyperthermia and docetaxel therapeutic approaches exhibited significant synergistic antitumor effects. Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells. Western blotting demonstrated that proteins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone. As compared with blank control group, cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lymphoma 2 (Bcl-2) protein expression but slightly increased Bcl-2-associated X protein (Bax) expression. Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia. It was concluded that treatments of mild hyperthermia plus docetaxel inhibited the proliferation of human breast cancer cells, promoted apoptosis of breast cancer cells, and produced synergistic antitumor effects.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; Cell Cycle ; drug effects ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Hot Temperature ; Humans ; MAP Kinase Signaling System ; MCF-7 Cells ; Receptors, Estrogen ; genetics ; metabolism ; Taxoids ; pharmacology

BACKGROUNDIt is important to identify the multiple sites of leptin activity in obese women with breast cancer. In this study, we examined the effect of exogenous human leptin on heat shock protein 70 (HSP70) expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice.

METHODSWe cultured MCF-7 human breast cancer cells and established nude mice bearing xenografts of these cells, and randomly divided them into experimental and control groups. The experimental group was treated with human leptin, while the control group was treated with the same volume of normal saline. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues. Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells. Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues.

RESULTSLeptin activated HSP70 in a dose-dependent manner in vitro: leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells (P < 0.001). There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group (P > 0.05). Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group (P > 0.05).

CONCLUSIONSA nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor. HSP70 may be target of leptin in breast cancer. Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.

Animals ; Blotting, Western ; Breast Neoplasms ; drug therapy ; metabolism ; Cell Line, Tumor ; Female ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Leptin ; pharmacology ; therapeutic use ; Mice ; Mice, Nude ; Real-Time Polymerase Chain Reaction ; Xenograft Model Antitumor Assays

The purpose of the present study is to investigate whether glucose-regulated protein 75 (GRP75) overexpression inhibits apoptosis induced by glucose deprivation through Raf/Mek/Erk1/2 signaling pathway. After pretreatment with Mek-specific inhibitor U0126, GRP75 overexpressing PC12 cells were incubated in glucose-free DMEM medium for indicated time (6, 12 and 24 h). And DMSO-treated GRP75 overexpressing PC12 cells were applied as control. Western blot was used to determine the expression and phosphorylation level of Erk1/2. MTT assay was used to measure cell viability. Hoechst 33258 staining and flow cytometry using propidium iodide (PI) staining was used to analysis apoptosis. Immunofluorescence with antibody against cytochrome c (Cyt c) was used to detect Cyt c release from mitochondrion. The results showed U0126 prevented the activation of Erk1/2 maintained by GRP75, but the total Erk1/2 expression was not affected. U0126-treated group showed lower cell viability and higher apoptotic rate compared with control group. Immunofluorescence indicated the delay in release of Cyt c was blocked by U0126. These results suggest U0126 prevents protective effect of GRP75 on PC12 cells by inhibiting Erk1/2 phosphorylation, which certifies that GRP75 can inhibit the mitochondria-dependent apoptotic pathway through Raf/Mek/Erk1/2 signaling cascade.
Animals ; Apoptosis ; physiology ; Butadienes ; pharmacology ; Cells, Cultured ; Culture Media ; Glucose ; pharmacology ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; MAP Kinase Signaling System ; physiology ; Membrane Proteins ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitriles ; pharmacology ; PC12 Cells ; Phosphorylation ; Rats ; raf Kinases ; metabolism

The probable mechanism of the reduction of rat cerebral ischemic-reperfusion injury by propyl gallate was studied. Intraluminal suture middle cerebral artery occlusion model of rat was employed. Propyl gallate was injected immediately after the ischemia was happened. The activity of NF-kappaB, and the expression of COX-2 and HSP70 on the peripheral ischemia were determined by Western blotting. The expression of TNF-alpha was determined by ELISA assay. RT-PCR and immunofluorescence staining were employed to detect the transcription and expression of TLR-4. Results showed that propyl gallate could inhibit the activity of NF-kappaB in the peripheral ischemia, and reduce the expression of COX-2 and TNF-alpha. As the upstream of NF-kappaB, the transcription and expression of TLR-4 decreased, as well as HSP70, the endogenic ligand of TLR-4. As an antioxidant, propyl gallate could reduce the cerebral ischemic-reperfusion injury through inhibiting the activity of NF-kappaB and decreasing the COX-2 and TNF-alpha in the peripheral ischemia. It also could influence HSP70 and TLR-4.
Animals ; Cyclooxygenase 2 ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Infarction, Middle Cerebral Artery ; complications ; Male ; Propyl Gallate ; pharmacology ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; etiology ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).

METHODSWistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).

RESULTS(1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P<0.01, P<0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P<0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P<0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P<0.05 and P<0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P<0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P<0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P<0.05). The concentration of HSP70 did not show any 0.05).

CONCLUSIONSThe TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.

Animals ; Animals, Newborn ; Benzofurans ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; NF-kappa B ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; enzymology ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription, Genetic ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate whether the administration of the ultra-filtration extract from Danggui Buxue Decoction (EDBD) was able to protect cardiomyocytes from oxidative injury of rats induced by hydrogen peroxide (H(2)O(2)) and its potential mechanism.

METHODSMyocardial cells from 1- to 2-day-old neonatal rats were cultured in Dulbecco's modified Eagle's medium low-glucose and Ham's F12 medium (1:1), and the cellular injury was induced by H(2)O(2). The ultra-filtration extract mixture from Angelica sinensis and Hedysarum polybotrys was given in three doses of 3.75, 7.5, and 15 mg/mL. Morphological changes of cardiomyocytes were observed by microscope. Survival rate of myocardial cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cardiomyocyte damages were estimated by detecting lactate dehydrogenase (LDH) and creatine kinase (CK) releases in the medium, superoxide dismutase (SOD) activities, and intracellular malondialdehyde (MDA) and myeloperoxidase (MPO) contents. The levels of caspase-3 and heat shock protein 70 (hsp70) mRNA expression in cardiomyocytes were measured by reverse transcription polymerase chain reaction.

RESULTSThe EDBD could protect the cardiomyocytes from H(2)O(2) injury in a dosedependent manner (3.75, 7.50, and 15.00 mg/mL). The EDBD could significantly decrease LDH and CK leakages and intracellular MDA and MPO contents, increase SOD activity, up-regulate hsp70 expression, and down-regulate caspase-3 expression.

CONCLUSIONThe EDBD has protection on cardiomyocytes injured by H(2)O(2) through improving cell antioxidant ability, up-regulating hsp70 expression, and inhibiting caspase-3 activity.

Animals ; Animals, Newborn ; Caspase 3 ; genetics ; metabolism ; Cell Survival ; drug effects ; Creatine Kinase ; metabolism ; Cytoprotection ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Mice ; Myocardium ; pathology ; ultrastructure ; Myocytes, Cardiac ; drug effects ; enzymology ; pathology ; Oxidative Stress ; drug effects ; Peroxidase ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Superoxide Dismutase ; metabolism ; Ultrafiltration

OBJECTIVETo observe the expression levels of heat shock protein 70 (hsp70) and NF-kappaB p65 mRNA in lung tissue of acute paraquat (PQ) poisoning rats, and intervention effects of ulinastatin (UTI).

METHODSSeventy-two Sprague-Dawley (SD) rats were randomly divided into three groups: PQ poisoning group, UTI group and control group. The rats were exposed intragastrically to PQ at the dose of 80 mg/kg to establish a model of the rat acute lung injury. The UTI group was intervened by peritoneal injection with 10000 U/kg UTI in 30 minutes. On the 12, 24, 48, 72 h after exposure, myeloperoxidase (MPO) activity in lung tissue were detected. The expression of the NF-kappaB p65 mRNA and hsp70 mRNA in lung tissue was detected by the reverse transcription-PCR (RT-PCR). The lung pathological changes of rats were observed.

RESULTSThe degree of lung injury in PQ group and UTI group was higher than that in control group. But in UTI group the degree of lung injury was lower than PQ group. MPO activity in the lung tissues in PQ group was (31.72 +/- 6.42), (56.23 +/- 8.63), (87.21 +/- 10.02) and (107.21 +/- 13.52) micro/g in 12, 24, 48 and 72 h, respectively which was significantly higher than that [(11.38 +/- 1.25) micro/g] in control group (P < 0.01). MPO activity in the lung tissues in UTI group was (15.65 +/- 3.21), (35.98 +/- 5.74), (59.33 +/- 9.65) and (71.25 +/- 10.58) micro/g in 12, 24, 48 and 72 h, respectively which was significantly lower than those in PQ group (P < 0.01). The expression levels of NF-kappaB p65 mRNA of lung tissues in UTI group in 12, 24, 48 and 72 h were 0.3288 +/- 0.0147, 0.5337 +/- 0.0328, 0.7357 +/- 0.0424 and 0.7547 +/- 0.0905, respectively, which were significantly lower that those (0.4185 +/- 0.0294, 0.8532 +/- 0.0841, 0.9554 +/- 0.0975 and 1.0094 +/- 0.0703) in PQ group (P < 0.01). hsp70 mRNA expression levels in 12, 24, 48 and 72 h of the UTI group were 0.5193 +/- 0.0254, 0.8289 +/- 0.0606, 0.7566 +/- 0.0277 and 0.4873 +/- 0.0105, respectively, which were significantly higher than those (0.3897 +/- 0.0125, 0.5904 +/- 0.0186, 0.4007 +/- 0.0237 and 0.2293 +/- 0.0137) in PQ group (P < 0.01).

CONCLUSIONThe expression levels of hsp70 mRNA and NF-kappaB p65 mRNA of rats after intoxication increased significantly. UTI can protect the lung tissues by elevating the expression of hsp70 and reducing the expression of NF-kappaB in the lung tissues of rats with acute paraquat poisoning.

Animals ; Glycoproteins ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Male ; Paraquat ; poisoning ; Peroxidase ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; metabolism

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.
ADP Ribose Transferases/*pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Bacterial Toxins/*pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell/drug therapy/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Chromatography, Liquid ; Cyclin B/metabolism ; Cyclin-Dependent Kinase 2/metabolism ; Drug Resistance, Neoplasm/*drug effects ; E2F1 Transcription Factor/metabolism ; Electrophoresis ; Exotoxins/*pharmacology ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Mouth Neoplasms/drug therapy/*metabolism ; Tandem Mass Spectrometry ; Tumor Suppressor Protein p53/metabolism ; Virulence Factors/*pharmacology