Expression of Heat Shock Protein 70 and 27 in Non-small Cell Lung Cancer and Its Clinical Significance

The heat shock proteins （HSPs） 70 and HSP 27 expression in patients with non-small cell lung cancer （NSCLC） was studied and the relation.ship between HSP 70 and HSP 27 with the clinicopathological features of NSCLC was investigated. The expression of HSP 70 and HSP 27 was detected in tumor tissues from 60 patients with NSCLC by S-P immunohistochemistry. The findings were analyzed in combination with the histological types, histopathological differentiation, lymph node metastasis, patients＇ clinical stages, smoking history and gender. The results showed that of the 60 NSCLC tissue specimens studied, the immunoreactivity of HSP 70 and HSP 27 was detected in 47 （78.3 %） and 43 （71.7 %） specimens, respectively. A positive correlation was found between the overexpression of HSP 70 and HSP 27. The histopathological differentiation, lymph node metastasis, clinical stages and smoking history were correlated to the expression of HSP 70, but not to the expression of HSP 27. No statistical significance was observed in histological types and gender with respect to both HSP 70 and HSP 27 expression. It is suggested that the HSP 70 expression is a powerful and significant prognostic indicator and is related to histopathological differentiation, lymph node metastasis, patients＇ clinical stages, smoking history, whereas HSP 27 expression is not.

Molecular cloning of human heat shock protein 27 and study of its protective effects on oxidative damage in rat cardiomyocte H9c2

Objective： To clone human cardiac heat shock protein 27 （HSP27） gene and to determine the effects of HSP27 on the oxidative stress in rat cardiomyocyte cell line H9c2. Methods： Full length of HSP27 cDNA which got by RT-PCR was constructed into pCDNA3.1^＋ . The recombinant was transfected into rat cardiomyocyte cell line H9c2 and the stable trahsfection cell line was selected by G418. Then we observe the effects of HSP27 over-expression on LDH release and apoptosis induced H2O2 in H9c2. Results： ①pCDNA3.1^＋/HSP27 provided a sound expression of HSP27 in both 293T and H9c2. ②LDH releasing induced by 0, 100,250,500, 1000 μmol/L H2O2 in HSP27 over-expression group and wild type group were 0.396±0.017 vs. 0.390±0.01）9 （p 〉0.05）, 0.437±0. 014 vs. 0.416±0.015 （P〈0.05）, 0.471±0.018 vs. 0.417±0.009 （P 〈0.001）, 0.505±0.030 vs. 0.657± 0.022（P 〈0.001）, 0.547 ±0.027 and 0.661 ± 0.011（ P 〈 0. 001 ）, respectively. ③Apoptosis induced by 150 μmol/L H2O2 in HSP27 over-expression group and wild type group were （10.693± 1.122）% vs. （4.027 ± 1.628）%（ P 〈0.01）. Conclusion： We cloned and constructed human cardiac HSP27 gene successfully, and over-expression of human HSP27 could inhibit oxidative damage significantly in H9c2.

Effects of Sodium Salicylate on the Expression of HSP27 Protein during Oxidative Stress in Tissue-cultured Human Lens Epithelial Cells

The effects of sodium salicylate on the expression of heat shock protein 27 （HSP27） during oxidative stress in tissue-cultured human lens epithelial cells were investigated. Cultured human lens epithelial cells （HLB-3） were divided into 3 groups： control group （group A）, oxidation injury group （group B） and sodium salicylate group （group C）. Apoptosis of human lens epithelial cells cultured in vitro was induced in the presence of 150 μmol/L H2O2. Cells viability and the expression of HSP27 were analyzed. Viability of the cells was measured by methyl thiazole tetrazolium （MTT） chromatometry. The expression of HSP27 in HLB-3 cells was detected by using immunohistochemistry and image analysis system, Sodium salicylate could induce the expression of HSP27, and the cells viability in group C was significantly higher than in group B （0.2667±0.01414 vs 0.2150±0.01080, P=0.012〈0.05）. The average gray value of HSP27 in group B was less than that in group C （P=0.000〈0.05）. The increased expression of HSP27 by sodium salicylate might play an important role in the protection of hydrogen peroxide-induced injury of human lens epithelial cells, suggesting that sodium salicylate could suppress, at least in part, the apoptosis of human lens epithelial cells.

HSP27:a candidate differentially expressed protein between left-and right-sided colon carcinomas

Objective:The aim of the study was to screen differentially expressed proteins between left-and right-sided colon cancers by proteomics techniques and provide molecular genetic basis for oncobiological difference between left-and rightsided colon cancers.Methods:Tissue samples including left-and right-sided colon cancers were collected and preserved in the-80 ℃ refrigeratory.In the first part of our experiment,protein separating was performed by using two-dimensional gel electrophoresis(2-DE) and the images of the gels were acquired by the scanner and then analyzed to find the differentially expression protein-spots in different groups.The peptide mass fingerprintings(PMF) was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and the proteins were identified by data searching in the Mascot-database.Differentially expression proteins were assayed by RT-PCR,Western blot,and immunohistochemical methods.Results:The 55 differentially expressed protein spots were screened and 23 spots of them were identified.Compared to right-sided colon cancer,15 proteins up-regulated and 8 proteins down-regulated including HSP27 in left-sided colon cancer.HSP27 expressed higher in right-sided than in left-sided colon cancers by RT-PCR,Western blot and immunohistochemical methods.Conclusion:There were differentially expressed proteins between left-and right-sided colon cancers,especially differences in HSP27 expression in mRNA and protein level,which were molecular genetic basis for oncobiological difference between left-and right-sided colon cancers.

Heat shock protein 27 regulates oxidative stress-induced apoptosis in cardiomyocytes:mechanisms via reactive oxygen species generation and Akt activation

Background Increased reactive oxygen species （ROS） formation, which in turn promotes cardiomyocytes apoptosis, is associated with the pathogenesis and progression of various cardiac diseases such as ischemia and heart failure. Recent studies have shown that over expression of heat shock protein 27 （Hsp27） confers resistance to cardiac ischemia/reperfusion injury. However, not much is known about the regulation of myocyte survival by Hsp27. Methods The rat cardiac cell line H9c2, with a stable overexpression of Hsp27, was established, with empty vector transfected H9c2 cells as controls. Following the cells challenged by Hydrogen Peroxide （H2O2）, lactate dehydrogenase （LDH） release, apoptosis, intracellular ROS, cell morphology, mitochondrial transmembrane potential and the activation of serine/threonine kinase Akt were determined. Results Along with marked suppression of H2O2-induced injury by Hsp27 overexpression in H9c2 cells, ROS generation and the loss of mitochondrial membrane potential were also significantly depressed. Furthermore, augmented Akt activation was observed in Hsp27 overexpressed H9c2 cells following H2O2 exposure. Conclusions Hsp27 inhibits oxidative stress-induced H9c2 damage and inhibition of ROS generation and the augmentation of Akt activation may be involved in the protective signaling.

Paclitaxel-doxorubicin sequence is more effective in breast cancer cells with heat shock protein 27 overexpression

Background Cancer cells with overexpression of heat shock protein 27 （HSP27） are resistant to chemotherapeutic drug doxorubicin （Dox）. Paclitaxel （Pacl） was reported to suppress HSP27 expression in ovarian and uterine cancer cells. The purposes of this study were to investigate whether Pacl inhibits the expression of HSP27 in breast cancer cells, whether Pacl can sensitize breast cancer cells with HSP27 overexpression to Dox, and to define a more effective schedule for the combination of Dox with Pacl. Methods The HSP27 high-expressing human breast cancer cell lines, MCF-7 and MDA-MB-435, and the HSP27 low-expressing cell line, MDA-MB-231, were used in this study. The level of HSP27, topoisomerase （Topo） IIα and β expression were assessed by Western blotting. The cytotoxic activities of Dox, Pacl and combination of these two drugs were evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay and flow cytometric assays. Results Pacl （0.1 μmol/L） inhibited HSP27 expression by approximately 2-fold in MCF-7 and MDA-MB-435 cells, while up-regulating the level of topo IIα and β. In contrast, expression of HSP27 in MDA-MB-231 did not change significantly following Pacl treatment. There were synergistic effects in both treatment sequences （Pacl-Dox and Dox-Pacl） when Pacl was combined with Dox. Compared with those treated with the Dox-Pacl sequence, the Pacl-Dox sequence had a stronger effect in cancer cells with HSP27 overexpression, as MCF-7 and MDA-MB-435 treated with the Pacl-Dox sequence had lower viabilities and a higher apoptotic rate. Conclusions Paclitaxel significantly decreases the level of HSP27 in breast cancer cells overexpressing HSP27. In combination therapies, the Pacl-Dox sequence is more effective in clearing breast cancer cells with high HSP27 expression compared with the Dox-Pacl sequence.

A novel intronic circular RNA,circGNG7,inhibits head and neck squamous cell carcinoma progression by blocking the phosphorylation of heat shock protein 27 at Ser78 and Ser82

Background:There is increasing evidence that circular RNAs(circRNAs)play a significant role in pathological processes including tumorigenesis.In contrast to exonic circRNAs,which are the most frequently reported circRNAs in cancer so far,the studies of intronic circRNAs have been greatly lagged behind.Here,we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma(HNSCC).Methods:We conducted whole-transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients.Then,we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients.We also identified interactions between circGNG7 and functional proteins,which alter downstream signaling that regulate HNSCC progression.Results:In this study,we identified a new intronic circRNA,circGNG7,and validated its functional roles in HNSCC progression.CircGNG7 was predominately localized to the cytoplasm,and its expression was downregulated in both HNSCC tissues andCAL27,CAL33,SCC4,SCC9,HN6,and HN30 cells.Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients.Consistent with this finding,overexpression of circGNG7 strongly inhibited tumor cell proliferation,colony formation,in vitro migration,and in vivo tumor growth.Mechanistically,the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4(SMAD4).Importantly,we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27(HSP27),occupying its phosphorylation sites and hindering its phosphorylation,which reduced HSP27-JNK/P38 mitogen-activated protein kinase(MAPK)oncogenic signaling.Downregulation of circGNG7 expression in HNSCC increased HSP27-JNK/P38 MAPK signaling and promoted tumor progression.Conclusions:Our results revealed that a new intronic circRNA,circGNG7,functions as a strong tumor suppressor and that circGNG7/HSP27-JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

Expression of heat shock protein 27 in the esophageal tissue of rats with reflux esophagitis

Background Little attention has been paid to the expression of heat shock protein 27 （HSP27） in patients with reflux esophagitis （RE）, and few studies of the importance of HSP27 in esophagitis have been carried out in animal models. This study aimed to explore the expression of HSP27 in the esophageal tissue of rats with RE. Methods Eighty female Wistar rats were randomly divided into experimental groups A and B and control groups C and D （n=20 in each group）. To establish RE, rats in the two experimental groups received pylorus and forestomach ligations, while rats in the control group received gastrostomy and gastric perforation repair. The rats in groups A and C were sacrificed 7 days after surgery, and the rats in groups B and D were sacrificed 14 days after surgery. In groups A and B, 10 and 8 rats were diagnosed with RE by pathological examination, respectively （they were included in groups A＇ and B＇, respectively）. The histopathological diagnosis of all the lower esophageal tissues in groups C and D was normal and 20 normal specimens were randomly selected for groups C＇ and D＇ with 10 specimens in each group. Macroscopic and microscopic esophagitis scores were assessed for the specimens in groups A＇ and B＇. Lower esophageal tissues were collected from groups A＇, B＇, C＇, and D＇, and paraffin-embedded slices were made using part of the tissues. The expression of HSP27 in the tissues was detected using the two-step streptavidin-peroxidase immunohistochemical method. Some collected tissues were frozen, and expressions of HSP27 mRNA were detected using fluorescence quantitative polymerase chain reaction （FQ-PCR）. Results Median macroscopic and microscopic esophagitis scores in groups A＇ （n=10） and B＇ （n=8） were 1.0 and 1.5, and 2.0 and 2.5, respectively. There were no significant differences in the macroscopic or microscopic esophagitis scores between the two groups （Z=-0.330, P=-0.741; Z=-0.142, P=-0.887, respectively）. Immunohistochemical staining showed that HSP27 was expressed in all layers of the esophageal epithelia in RE and control rats. FQ-PCR showed that HSP27 mRNA levels in the lower esophageal tissue in RE group （groups A＇ and B＇） were higher than those in control group （groups C＇ and D＇） （Z=-0.249, P=0.001）. HSP27 mRNA expression in the lower esophageal tissue was significantly different in groups B＇ and D＇ （Z=-3.027, P=-0.002）. And the levels of HSP27 mRNA expression in severe RE group （microscopic esophagitis score： 3） were higher than in mild RE group （microscopic esophagitis score： 1-2） and control group （Z=-3.396, ,P=-0.001; Z=--3.855, P 〈0.001）. Conclusions HSP27 mRNA expression in the lower esophageal tissue of rats with RE is significantly higher than in the normal controls. Although reflux is a persistent stimulating factor, increased expression of HSP27 in the lower esophageal tissue of rats with RE requires aggravated esophageal injury.

Comparison of Hsps Expression after Radio-frequency Field Exposure in Three Human Glioma Cell Lines

Objective To investigate and compare the effect of radio-frequency （RF） field exposure on expression of heat shock proteins （Hsps） in three human glioma cell lines （MO54, A172, and T98）. Methods Cells were exposed to sham or 1950 MHz continuous-wave for 1 h. Specific absorption rates （SARs） were 1 and 10 W/kg. Localization and expression of Hsp27 and phosphorylated Hsp27 （（78） Ser） （p-Hsp27） were examined by immunocytochemistry. Expression levels of Hsp27, p-Hs27, and Hsp70 were determined by Western blotting. Results The Hsp27 was primarily located within the cytoplasm, p-Hsp27 in both cytoplasm and nuclei of MO54, A172, and T98 cells. RF field exposure did not affect the distribution or expression of Hsp27. In addition, Western blotting showed no significant differences in protein expression of Hsp27 or HspT0 between sham- and RF field-exposed cells at a SAR of 1 W/kg and 10 W/kg for 1 h in three cells lines. Exposure to RF field at a SAR of 10 W/kg for 1 h slightly decreased the protein level of phosphorylated Hsp27 in MO54 cells. Conclusion The 1950 MHz RF field has only little or no apparent effect on Hsp70 and Hsp27 expression in MO54, A172, and T98 cells.

Preconditioning crush increases the survival rate of motor neurons after spinal root avulsion

In a previous study, heat shock protein 27 was persistently upregulated in ventral motor neurons following nerve root avulsion or crush. Here, we examined whether the upregulation of heat shock protein 27 would increase the survival rate of motor neurons. Rats were divided into two groups： an avulsion-only group （avtflsion of the L4 lumbar nerve root only） and a crush-avulsion group （the L4 lumbar nerve root was crushed 1 week prior to the avulsion）. Immunofluores- cent staining revealed that the survival rate of motor neurons was significantly greater in the crush-avulsion group than in the avulsion-only group, and this difference remained for at least 5 weeks after avulsion. The higher neuronal survival rate may be explained by the upregulation of heat shock protein 27 expression in motor neurons in the crush-avulsion group. Further- more, preconditioning crush greatly attenuated the expression of nitric oxide synthase in the motor neurons. Our findings indicate that the neuroprotective action of preconditioning crush is mediated through the upregulation of heat shock protein 27 expression and the attenuation of neuronal nitric oxide synthase upregulation following avulsion.