OBJECTIVETo investigate the relationship between sensitivity to cisplatin (DDP) and the expression of HSP70 in cervical cancer cells in vitro.

METHODSCervical cancer Hela229 cells treated with different concentrations of DDP and the HSP70 inhibitor (PFT-µ) were examined for cell viability using MTT assay and colony forming ability. The cell apoptosis was analyzed by flow cytometry with propidium iodide staining and DAPI staining, and JC-1 staining was used to determine mitochondrial membrane potential. The expressions of HSP70, Bcl-2, Bax and caspase-3 were measured with Western blotting. A nude mouse model bearing Hela229 cell xenograft was used to evaluate the effect of DDP and PFT-µ on tumor growth.

RESULTSHela229 cells expressed a higher level of HSP70 than normal cervical cells. The combined use of PFT-µ significantly enhanced the inhibitory effect of DDP (P<0.01) and increased the cell apoptosis in Hela229 cells. JC-1 staining demonstrated that DDP combined with PFT-µ more obviously reduced mitochondrial membrane potential. DDP combined with PFT-µ more strongly lowered Bcl-2 expression and increased the expressions of casepase-3 and Bax than DDP alone. In the nude mouse model, PFT-µ significantly enhanced DDP sensitivity of Hela229 cell xenografts (P<0.01).

CONCLUSIONSInhibition of HSP70 expression can enhance the sensitivity of cervical cancer cell to DDP both in vivo and in vitro possibly by promoting cell apoptosis, suggesting the potential of HSP70 as a new target for gene therapy of cervical cancer.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Caspase 3 ; metabolism ; Cell Proliferation ; Cell Survival ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Female ; HSP70 Heat-Shock Proteins ; antagonists & inhibitors ; HeLa Cells ; Humans ; Membrane Potential, Mitochondrial ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Sulfonamides ; pharmacology ; Uterine Cervical Neoplasms ; drug therapy ; pathology ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the effects of 5-Aza-deoxycytidine (5-Aza-CdR) on the amount of exosomes and immuno-associated proteins produced in hepatoma HepG2 and Hep3B cells.

METHODSExosomes derived from HepG2 and Hep3B cells with or without treatment by 5-Aza-CdR were isolated and purified by combination of ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under the electron microscope. The concentration of proteins in exosomes was detected by BCA. The expression of HSP70, HLA-I and NY-ESO-1 proteins in exosomes was assayed by Western blot and immuno-electron microscopy. The mRNA expression of p53 gene was observed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were significantly increased after treatment by 5-Aza-CdR (P < 0.05). The immuno-electron microscopy and Western blotting showed that after treatment with 5-Aza-CdR, the contents of HSP70, HLA-I and NY-ESO-1 proteins were increased in exosomes in both HepG2 and Hep3B hepatoma cells.

CONCLUSION5-Aza-CdR, an inhibitor of DNA methyltransferase, can increase the amount of exosomes and exosome-containning immuno-associated proteins secreted by hepatoma cells. It may be contributed by up-regulation of p53 gene and demethylation mechanism of 5-Aza-CdR.

Antigens, Neoplasm ; metabolism ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; secretion ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Exosomes ; secretion ; Gene Expression Regulation, Neoplastic ; HSP70 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Liver Neoplasms ; pathology ; secretion ; Membrane Proteins ; metabolism ; RNA, Messenger ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Up-Regulation

OBJECTIVESTo investigate role of hypoxia inducible factor 1 (HIF-1) in the transcriptional activation of heat shock protein 70-2 (HSP70-2) in hepatocellular carcinoma (HCC) cells under hypoxic conditions.

METHODSHCC cells were exposed to reduced oxygen atmosphere (1% O2), or treated with YC-1 or HIF-1 alpha siRNA, the expression of HIF-1 alpha and HSP70-2 were detected by Western blot analysis. Serial deletions of the HSPA2 promoter were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with HIF-1 alpha siRNA, and the promoter activities were detected with the dual luciferase assay.

RESULTSWestern blot analysis showed that both HIF-1 alpha and HSP70-2 proteins were strongly increased after HCC cells were exposed to hypoxic conditions (1% O2) for 6 h, and the expression level of HSP70-2 was increased in a time-dependent manner. Treatment of HepG2 cells with YC-1 or HIF-1 alpha siRNA significantly inhibited the expression of HIF-1 alpha and HSP70-2. In silico analysis of the HSP70-2 promoter using the Gene2 Promoter software revealed the presence of two putative hypoxic response element (HRE) consensus at -446bp (HRE1) and -238bp (HRE2). Depletion of promoter sequence between -653 and -385 led to a dramatic reduction of promoter activity, whereas further deletion to position -201 did not reduce the activity further. These data suggested that HRE1 plays an important role in hypoxia-induced activation of the HSPA2 promoter. Site-directed mutagenesis further confirmed these results. Mutation of HRE1 but not of HRE2 abrogated the sensitivity of the HSP70-2 promoter to hypoxia.

CONCLUSIONSHSP70-2 expression is up-regulated in response to hypoxia and a HIF-1 binding site (HRE1) in the HSP70-2 promoter is involved in this response.

Base Sequence ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Hypoxia ; Gene Expression Regulation, Neoplastic ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; antagonists & inhibitors ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Molecular Sequence Data ; Plasmids ; genetics ; Promoter Regions, Genetic ; RNA, Small Interfering ; genetics ; Transfection ; Up-Regulation

Heat shock proteins (HSPs) form the most ancient defense system in all living organisms. These proteins act as molecular chaperones by helping the refolding of misfolded proteins and assisting their elimination if they become irreversibly damaged. HSPs interact with a number of cellular systems and form efficient cytoprotective mechanisms. HSPs allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked, and HSPs induced in response to stress appear to function at key regulatory points in the control of apoptosis. HSPs include antiapoptotic and proapoptotic proteins that interact with a variety of cellular proteins. Their expression levels can determine the fate of the cell in response to death stimulus. On the other hand, HSPs are overexpressed in tumor cells, and the inhibition of HSP90 has recently been regarded as a very promising tool to combat various cancers. HSPs can be secreted to circulatory system from a variety of cell types in response to stress. The secreted exogenous proteins act as cytokines and have potential modulatory functions in immune system. Cell surface-bound HSP70 can render tumor cell more sensitive to natural killer cell-mediated cytolytic attack. Therefore, modulator of chaperone activities is becoming a new target of drug development, such as in apoptosis and tumor immunity fields.
Animals ; Apoptosis ; drug effects ; Cytoprotection ; drug effects ; Drug Delivery Systems ; economics ; methods ; trends ; Drug Screening Assays, Antitumor ; HSP70 Heat-Shock Proteins ; antagonists & inhibitors ; metabolism ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; metabolism ; Heat-Shock Proteins ; antagonists & inhibitors ; metabolism ; Humans ; Molecular Chaperones ; Pharmaceutical Preparations ; administration & dosage ; chemical synthesis

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.
Antigens, CD36/*physiology ; Cell Line, Tumor ; Chromans/pharmacology ; Cycloheximide/pharmacology ; HSP70 Heat-Shock Proteins/*biosynthesis ; Humans ; Lipoproteins, LDL/pharmacology/*physiology ; Monocytes/drug effects/metabolism ; PPAR gamma/agonists/antagonists & inhibitors/*physiology ; Protein Synthesis Inhibitors/pharmacology ; Signal Transduction ; Thiazolidinediones/pharmacology

Heat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Go6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKC micron, we then evaluated the role of PKC micron in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKC micron with siRNA or the inhibition of PKC micron activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKC micron evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKC micron performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification.
Animals ; Carbazoles/pharmacology ; Cell Line ; Flavonoids/pharmacology ; HSP70 Heat-Shock Proteins/*biosynthesis ; Humans ; Indoles/pharmacology ; Isoquinolines/pharmacology ; MAP Kinase Signaling System/physiology ; Maleimides/pharmacology ; Mice ; NFATC Transcription Factors/metabolism ; Phosphorylation ; Promoter Regions, Genetic ; Protein Kinase C/antagonists & inhibitors/*physiology ; Protein Transport ; Saline Solution, Hypertonic/*pharmacology ; Signal Transduction ; Sulfonamides/pharmacology

BACKGROUNDA number of studies suggest that the expression of heat shock protein 70 (HSP(70)) induced by heat stress are associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated by other factors in the same stress. This study was conducted to explore the protective role of HSP(70) expression in acute myocardial anoxia/reoxygeneration (A/R) injury with a liposome-mediated gene transfer technique for the introduction of pCDNA HSP(70) into the neonatal rat myocardial cells. In addition, heat shock stress cytoprotection was also investigated for comparison.

METHODSThe cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups (n = 8): control group, A/R group, HS + A/R group and pCDNA HSP(70) + A/R group. A liposome-coated HSP(70) pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP(70) mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell viability was assayed by monotetrazolium (MTT) and the lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity of cells during incubation and the changes in cells ultrastructure were examined. NF-kappaB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry.

RESULTSCompared with viability in the A/R group ((35.4 +/- 6.9)%) the cell viability in the HS + A/R group ((72.8 +/- 11.6)%) and the pCDNA HSP(70) + A/R group ((76.3 +/- 12.2)%) was improved significantly (P < 0.05). The activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS + A/R group and pCDNA HSP(70) + A/R group, significant decreases in activity were observed. The cell ultrastructure of the A/R group cells was abnormal, whereas nearly normal ultrastructure was observed in HS + A/R group and pCDNA HSP(70) + A/R group. HSP(70) mRNA and protein were slightly expressed in the myocardiocytes of the A/R group. However, obvious overexpression was observed in the HS + A/R group and in the pCDNA HSP(70) + A/R group (P < 0.01). And there was a significant difference between the HS + A/R group and the pCDNA HSP(70) + A/R group in the expression of HSP(70) mRNA and protein (P < 0.01). A high activity of NF-kappaB (5.76 +/- 0.64) was detected in the A/R group. But in the HS + A/R group there was a statistically significant decrease in the activity of NF-kappaB compared with the A/R group (3.11 +/- 0.52 vs 5.76 +/- 0.64, P < 0.01). The same statistically significant difference was also observed in the pCDNA HSP(70) + A/R group and A/R group (2.83 +/- 0.49 vs 5.76 +/- 0.64, P < 0.01).

CONCLUSIONSOverexpression of HSP(70) alone by gene transfection leads to protection for cardiac myocyte against anoxia-reoxygeneration. These cardioprotective effects were related to the reduction in activation of NF-kappaB.

Animals ; Cell Hypoxia ; Cell Survival ; Cells, Cultured ; Cytoprotection ; HSP70 Heat-Shock Proteins ; genetics ; physiology ; Myocytes, Cardiac ; pathology ; ultrastructure ; NF-kappa B ; antagonists & inhibitors ; Oxygen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVEAnimal trials have demonstrated that memantine has neuroprotective effects on hypoxic-ischemic (HI) brain damage. Whether memantine can improve the long-term prognosis of rats with HI brain damage has not been reported. This study was designed to investigate the long-term effect of memantine therapy on neonatal rats with HI brain damage.

METHODSSixty postnatal 7-day-old newborn rats were randomly assigned into Normal control, HI and Memantine treated groups. Memantine (20 mg/kg) was administered immediately after HI in the Memantine-treated group. All subjects received a 5-day training of Morris water maze test from 23 days old. The escape latency (EL) was recorded at 28 and 35 days old.

RESULTSThe EL values of the Normal control, HI and Memantine-treated groups at 28 days old were 23.1 +/- 21.8, 35.1 +/- 5.3, and 20.6 +/- 3.4 seconds, respectively. There was a significant difference in the EL value between the HI and the Normal control groups (P < 0.05). The EL value of the Normal control, HI and Memantine-treated groups at 35 days old were 19.7 +/- 16.7, 35.6 +/- 32.3, and 16.3 +/- 13.2 seconds, respectively. A prolonged EL induced by HI still existed (P < 0.05 vs Normal controls) but memantine treatment shortened the EL (P < 0.01 vs HI group) at 35 days old.

CONCLUSIONSAdministering memantine immediately after HI can markedly increase the abilities of spatial discrimination, learning and memory and improve the long-term prognosis in rats with HI brain damage.

Animals ; Animals, Newborn ; Avoidance Learning ; drug effects ; Brain ; metabolism ; Excitatory Amino Acid Antagonists ; therapeutic use ; Female ; HSP70 Heat-Shock Proteins ; genetics ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; psychology ; Male ; Maze Learning ; drug effects ; Memantine ; therapeutic use ; Rats ; Rats, Sprague-Dawley

Angiotensin II (Ang II), which is an important mediator of both vascular responsiveness and growth, has been shown to induce vascular smooth muscle cell (VSMC) hypertrophy via the activation of a complex series of intracellular signaling events. Heat shock protein 70 (Hsp70) has recently been shown to protect against Ang II-induced hypertension. In this study, we tested the hypothesis that Hsp70 can protect VSMC from Ang II-induced hypertrophy. We treated VSMCs with Ang II to induce hypertrophy and to activate MAPK signaling pathway. We observed that the augmentation of Hsp70 expression inhibited Ang II-stimulated VSMC hypertrophy. This inhibitory effect of Hsp70 appears to be partly due to extracellular signal-regulated kinase (ERK1/2) inactivation, which in turn, may possibly result from the accumulation of MAP kinase phosphatase-1 (MKP-1).
Rats, Sprague-Dawley ; Rats ; RNA, Small Interfering/pharmacology ; Protein-Tyrosine-Phosphatase/metabolism/physiology ; Phosphoprotein Phosphatase/metabolism/physiology ; Muscle, Smooth, Vascular/*cytology/*drug effects ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors ; Male ; MAP Kinase Kinase 2/metabolism ; MAP Kinase Kinase 1/metabolism ; Immediate-Early Proteins/metabolism/physiology ; Hypertrophy ; HSP70 Heat-Shock Proteins/antagonists & inhibitors/*pharmacology ; Flavonoids/pharmacology ; Enzyme Stability/drug effects ; Cells, Cultured ; Cell Cycle Proteins/metabolism/physiology ; Aorta/drug effects/pathology ; Animals ; Angiotensin II/*pharmacology

AIMTo investigate the inhibitory effects and mechanism of action of isoliensinine (IL) on the proliferation of porcine coronary arterial smooth muscle cells (CASMCs) induced by phenylephrine (Phen) and its mechanisms of action.

METHODSMTT assay, immunohistochemical method and Western blotting were adopted.

RESULTSIL (0.03 - 3 micromol x L(-1)) could inhibit the CASMCs proliferation induced by Phen (0.1 micromol x L(-1)) in a concentration-dependent manner. IL (0.1 micromol x L(-1)) antagonized Phen-induced overexpression of PDGF-beta and bFGF from 0.545 +/- 0.026 and 0.47 +/- 0.03 to 0.458 +/- 0.019 and 0.376 +/- 0.017 (P < 0.01 , P < 0.01). IL (0.1 micromol x L(-1)) also decreased c-fos, c-myc and hsp70 overexpression induced by Phen from 0.57 +/- 0.04, 0.44 +/- 0.04 and (173 +/- 36)% to 0.46 +/- 0.05, 0.372 +/- 0.021 and (115 +/- 35)% respectively (P < 0.01, P < 0.01, P < 0.01).

CONCLUSIONIL exerted antiproliferative effect on CASMCs induced by phenylephrine, and its mechanisms were related to decrease the overexpression of growth factors (PDGF-beta, bFGF), protooncogene (c-fos, c-myc) and hsp70.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coronary Vessels ; cytology ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Isoquinolines ; administration & dosage ; isolation & purification ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; Nelumbo ; chemistry ; Phenylephrine ; antagonists & inhibitors ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-fos ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Proto-Oncogene Proteins c-sis ; metabolism ; Swine