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

OBJECTIVETo investigate whether the heat shock protein (HSP) 70 antisense oligomers can enhance the sensitivity of bladder cancer cell EJ to mitomycin C.

METHODSThe HSP70 mRNA of EJ cells was blocked by the 10 micromol/L HSP70 antisense oligomers, while its effect on cell growth was evaluated by methyl thiazolyl tetrazolium (MTT) and colony forming ability test.

RESULTSThe HSP70 expressions in HSP70 antisense treated group were lower than the corresponding sense and nonsense treated groups (P < 0.01). While, the increased sensitivity of EJ to mitomycin C was found in antisense treated group, compared with the corresponding sense and nonsense treated groups (P < 0.01).

CONCLUSIONThe sensitivity of bladder cancer cell EJ to mitomycin C was enhanced by the blockage of the HSP70 expression.

Cell Division ; drug effects ; Cell Line, Tumor ; HSP70 Heat-Shock Proteins ; antagonists & inhibitors ; biosynthesis ; genetics ; Humans ; Mitomycin ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology

OBJECTIVETo compare the effects of Buyang Huanwu Decoction (BYHWD) and its active regions combination on brain heat shock protein 70 (HSP70) expression.

METHODSCerebral ischemia model gerbils produced by ligating bilateral common carotid artery were injected intraperitoneally by BYHWD and its active regions combination separately. HSP70 mRNA expression was determined by Northern blot test and protein expression determined by Western blot test 48 hrs after ischemia for 15 min and reperfusion.

RESULTSThe level of HSP70 mRNA and protein expression in ischemic/reperfusion brain tissue increased significantly. BYHWD and its active regions combination could decrease the HSP70 mRNA expression obviously, the inhibition effect of BYHWD was more significant than that of its active regions combination, but neither of them showed any effect on HSP70 protein expression.

CONCLUSIONBYHWD and its active regions combination might down-regulate HSP70 mRNA expression in ischemic brain tissue through its anti-ischemic effect.

Animals ; Brain ; metabolism ; Brain Ischemia ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gerbillinae ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Reperfusion Injury ; metabolism

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

OBJECTIVETo evaluate the neuroprotective effect of memantine, a non-competitive antagonist at the N-methyl-D-aspartate receptor, against hypoxic ischemia (HI) by exploring its regulation on the expression and synthesis of heat shock protein 70 (HSP70) gene in neonatal rat models with cerebral HI.

METHODSMemantine was intraperitoneally injected at a dose of 20 mg/kg in neonatal rat models either before (PRE group) or after (POST group) induction of HI. The expression and synthesis of the HSP70 gene and its corresponding product were determined by rapid competitive PCR and immunohistochemistry, respectively.

RESULTSThere was an increase in the expression of HSP70 mRNA two hours after induction of HI, which reached its peak at 48 hours, then decreased gradually. The same expression occurred at relatively low levels in the control group. Also, HSP70 synthesis was detected as early as 2h after HI, reached its peak between 48 and 72 hours, then declined over time. After memantine administration, the expression of the gene and its synthesis of the corresponding product decreased significantly during the time intervals 24 - 72 h for the gene and 48 - 72 h for the product compared to the HI group.

CONCLUSIONIt was shown that HI is very sensitive to the expression of the HSP70 gene and synthesis of its corresponding product, which could be regulated by memantine. The latter may have the ability to reduce brain damage; thus decreased HSP70 mRNA expression could be a marker for HI. It is suggested that memantine can be a promising agent for neuroprotection against HI, although an overall and objective assessment of memantine is required to see if it can be used on neonates clinically later on.

Animals ; Female ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; Male ; Memantine ; pharmacology ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDBladder cancer is a relatively common tumor in the urinary system, in which mitomycin C (MMC)-based chemotherapy or combination chemotherapy has been mainly used to treat patients with advanced bladder cancer. The prognosis of patients with advanced bladder cancer is still extremely poor in spite of recent therapeutic advances. To improve the prognosis, the sensitivity of tumor cells to mitomycin C by the induction of apoptosis with the abating heat shock protein 70 (HSP70) expression in human bladder cancer cell lines of BIU-87 was investigated.

METHODSHSP70 expression was abated in BIU-87 cells by HSP mRNA antisense oligomers. MTT assay and the clone-forming test were used for evaluating the sensitivity of cells to MMC. Apoptosis was assessed using both fluorescent microscopy after staining the cells with Hoechst 33258 and DNA fragment ladder agarose electrophoresis. Thirty-two male six-week-old BALB/c nude mice, at the beginning of the experiment, were used to evaluate the effect of antisense oligomers (ASO) on the tumor formation in vivo.

RESULTSHSP70 expression in BIU-87 was effectively abated by HSP70 mRNA antisense oligomers. The percentage of apoptotic cells in ASO group was greater than in sense oligomers (SO) [P < 0.05, (18.31 +/- 2.89)% vs (1.89 +/- 0.74)%], nonsense oligomers (NO) [P < 0.05, (18.31 +/- 2.89)% vs (1.78 +/- 0.92)%] and blank groups [P < 0.05, (18.31 +/- 2.89)% vs (1.87 +/- 0.84)%], while the sensitivity of tumor cells to mitomycin C was enhanced. The in vivo tumor inhibition rate of ASO plus MMC (> 50%) was more than that of ASO or MMC group alone (all P < 0.05).

CONCLUSIONSThe abating level of HSP70 expression can strengthen the sensitivity of BIU-87 to MMC. One of this effect might be related to the induction of apoptosis by abating HSP70 expression.

Apoptosis ; drug effects ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; HSP70 Heat-Shock Proteins ; antagonists & inhibitors ; genetics ; physiology ; Humans ; Mitomycin ; pharmacology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Urinary Bladder Neoplasms ; drug therapy ; metabolism ; pathology

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

To obtain the expression of Mycobacterium tuberculosis heat shock protein 70 in methylotropic yeast. The expression vector pPIC9K-hsp70 was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The result protein was secreted into the supernatant induced by 0.5% methanol at 30 degrees C and purified by centrifugation, ultrafiltration and ATP-agarose. The recombinant Hsp70 was identified by SDS-PAGE, Western blot, mice experiment and effect on the immature DC. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed Hsp70 was about 70 kD and the expressed protein could specifically react with anti-Mt. Hsp70 IgG. And mice immunization indicated the expressed hsp70 had immunogenicity. Hsp70 could induce DC maturation and release Th1 cytokine. The secreted 70 kD protein was about 120 mg/L which accounted for more than 30% of the total supernatant protein and was purified to electrophoretic purity. The Hsp70, which had the biological activity, is successfully secretorily expressed in the Pichia pastoris GS115.
Animals ; Bacterial Proteins ; genetics ; immunology ; metabolism ; pharmacology ; Blotting, Western ; Cells, Cultured ; Dendritic Cells ; drug effects ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Vectors ; genetics ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; metabolism ; pharmacology ; Humans ; Interleukin-12 ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Tumor Necrosis Factor-alpha ; metabolism

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

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