Abrogation of heat-shock protein (HSP)70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m

Aim: To investigate the effect of abrogating heat shock protein (HSP) 70 expression by antisense HSP70 oligonucleotides treatment on human androgen-independent prostate cancer cell line PC-3m growth. Methods: PC-3m cells were treated with 0-16 μmol/L antisense HSP70 oligomers for 0-100 hr. Cell growth inhibition was analyzed using a trypan blue dye exclusion test. Apoptotic cells were detected and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression of HSP70 and bcl-2 affected by antisense HSP70 oligomers were determined using Western blot. Results: Antisense HSP70 oligomer induced apoptosis and then inhibited proliferation of PC-3m cells in a dose- and time-dependent manner. Ladder-like patterns of DNA fragments were observed in PC-3m cells treated with 10 μmol/L antisense HSP70 oligomer for 48 hr or 8 μmol/L for 72 hr on agarose gel electrophoresis. Antisense HSP70 oligomer pretreatment enhanced the subsequent induction of apoptosis by heat shock in PC-3m cells. In addition, undetectable HSP70 expression was observed at a concentration of 10 μmol/L antisense HSP70 oligomer treatment for 48 hr or 8 μmol/L for 72 hr in Western blot, which was paralleled by decreased expression levels of anti-apoptotic protein bcl-2. Conclusion: HSP70 antisense oligomer treatment abrogates the expression of HSP70, which may disrupt HSP70-bcl-2-interactions and further down-regulate bcl-2 expression, in turn inducing apoptosis and inhibiting cell growth in PC-3m cells.

HSP110 aggravates ischemia-reperfusion injury after liver transplantation by promoting NF-κB pathway

Background:Ischemia-reperfusion injury(IRI)poses a significant challenge to liver transplantation(LT).The underlying mechanism primarily involves overactivation of the immune system.Heat shock protein 110(HSP110)functions as a molecular chaperone that helps stabilize protein structures.Methods:An IRI model was established by performing LT on Sprague-Dawley rats,and HSP110 was silenced using siRNA.Hematoxylin-eosin staining,TUNEL,immunohistochemistry,ELISA and liver enzyme analysis were performed to assess IRI following LT.Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes.Results:Our findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT(P<0.05).However,when rats were injected with siRNAHSP110,IRI subsequent to LT was notably reduced(P<0.05).Additionally,the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced(P<0.05).Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver(P<0.05).Conclusions:HSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells.Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI.

Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.

Effect of Ultraviolet Radiation on Hsp70 Protein Expression in HaCaT Cells

Ultraviolet radiation by its wavelength is divided into: UVA, UVB and UVC. Only UVA and UVB manage to penetrate the ozone layer, but due to anthropological activities, all of them are capable of interacting with humans to a greater or lesser extent, and can generate adverse effects such as cellular stress when interacting with intra-and extracellular biomolecules. The skin is the first organ in contact with UV radiation, and the stress it generates can be analyzed by the expression of a bioindicator of cellular damage such as Hsp70. Therefore, the objective of the project was: to determine the effect of UVA, UVB and UVC radiation on HaCaT epithelial cells, by analyzing the expression of Hsp70. Materials and methods: HaCaT cells were cultured in vitro, which were irradiated with UVA, UVB and UVC light at different doses, to subsequently determine the degree of Hsp70 expression by Immunodetection by PAGE-SDS and Western Blot. Results: Basal expression of Hsp70 was observed in no irradiated HaCaT cells. When HaCaT cells were irradiated with UVA, UVB, UVC, an increase in this Hsp70 protein was observed. With UVA, a higher degree of expression was observed at a time of 30 minutes of irradiation. With UVB the highest expression shifted to a time of 20 minutes. With UVC, overexpression was observed after 10 minutes. Conclusion: UV radiation generates cellular stress on HaCaT cells, evaluated by the stress bioindicator Hsp70. According to the wavelength of UV radiation, those that have a shorter wavelength have a greater potential for cellular damage, such as UVC.

THE ROLE OF INTRONIC HSE IN THE HEAT SHOCK INDUCED EXPRESSION OF HUMAN hsp 90β GENE

The first intron of human hsp90β gene is not only essential in maintaining high constitutive expression but also critical for heat shock inducibility of the hsp90β gene. Typical HSEs in the first intron play a vital role in the heat induced expression of human hsp90β gene. Slot blot analysis shows that hsp90β gene mRNA transcripts initiated from the 3’ of the first intron dominates over that of the first exon. The intronic HSEs of the hsp90β gene show much higher binding affinity toward the recombinant heat shock factor HSF1 than that of the recombinant heat shock factor HSF2.

cDNA Cloning of Heat Shock Protein Genes and Their Expression in an Indigenous Cryptic Species of the Whitefly Bemisia tabaci Complex from China

Thermal adaptation plays a fundamental role in shaping the distribution and abundance of insects,and heat shock proteins（Hsps）play important roles in the temperature adaptation of various organisms.To better understand the temperature tolerance of the indigenous ZHJ2-biotype of whitefly Bemisia tabaci species complex,we obtained complete cDNA sequences for hsp90,hsp70,and hsp20 and analyzed their expression profiles under different high temperature treatments by real-time quantitative polymerase chain reaction.The high temperature tolerance of B.tabaci ZHJ2-biotype was determined by survival rate after exposure to different high temperatures for 1 h.The results showed that after 41°C heat-shock treatment for 1 h,the survival rates of ZHJ2 adults declined significantly and the estimated temperature required to cause 50% mortality（LT50）is 42.85°C for 1 h.Temperatures for onset（Ton）or maximal（Tmax）induction of hsps expression in B.tabaci ZHJ2-biotype were 35 and 39°C（or 41°C）.Compared with previous studies,indigenous ZHJ2-biotype exhibits lower heat temperature stress tolerance and Ton（or Tmax）than the invasive B-biotype.

Heat shock protein inhibitor, quercetin, as a novel adjuvant agent to improve radiofrequency ablation-induced tumor destruction and its molecular mechanism

Background： We investigated the effect of a small molecular inhibitor of heat shock protein （HSP）, qnercetin, on tumor radiofrequency （RF） ablation, and explored the underlying molecular mechanisms. Methods： In in vivo study, rats with R3230 breast adenocarcinoma were sacrificed 24 h post-treatment and gross coagulation areas were compared, and next, randomized into four treatment arms （control, quercetin alone, RF alone, and combination） for Kaplan-Meier analysis of defined endpoint survival. Then the distribution and expression levels of heat shock protein 70 （HSP70）, cleaved caspase-3 and heat shock factor 1 （HSF1） were analyzed after different treatments. In in vitro study, we used quercetin to promote SK- HEP-I （hepatic） and MCF-7 （breast） cancer cell apoptosis in heat shock cell model, and siRNA was used to block c-Jun and to explore the role of activating protein-1 （AP-1） signaling pathways. Results： We found the effects of quercetin plus RFA resulted in increase on the tumor destruction/ endpoint survival （26.5±3.4 d） in vivo, compared with RF alone （17.6±2.5 d） and quercetin alone （15.7±3.1 d）. Most importantly, quercetin-induced cancer cell death required the presence of HSF1 in animal model. Furthermore, quercetin directly down-regulated expression of HSF1 in vitro, which our findings have revealed, required the activation of AP-1 signaling pathways by loss-of-function analysis using siRNA mediated targeting of c-Jun. Conclusions： These results indicated a protective role of quercetin in tumor ablation and highlighted a novel mechanism involving HSP70 with HSF1 pathway in thermal ablation of solid tumors.

Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR （FQ RT-PCR）. The results showed that the activities of creatine kinase （CK） and glutamic-pyruvic transaminase （GPT） were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 （HSC70） mRNA was changeable. The transcription of heat shock protein 70 （HSPT0） mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

Advance in Research on Biological Function and TranscriptionalControl of Heat Shock Proteins

The regulation of heat shock transcription factor to heat shock protein expression and the newest knowledge about the effect of heat shock protein on aging,immune response and the balance of cell survival and apoptosis are summarized in the paper.

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 （Hsp70） is one important member of heat shock protein （Hsp） family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone （Haliotis discus hannai Ino）. For the chronic exposure, abalones were maintained at 8, 12, 20, and 30∩ for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30~C-acclimated group, followed by the 8~C-acclimated group and then the 12~C- and 20~C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8~C and 38~C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

What we know about ST13, a co-factor of heat shock protein, or a tumor suppressor?

This article is to summarize the molecular and functional analysis of the gene “suppression of tumorigenicity 13” (ST13). ST13 is in fact the gene encoding Hsp70 interacting protein (Hip), a co-factor (co-chaperone) of the 70-kDa heat shock proteins (Hsc/Hsp70). By collaborating with other positive co-factors such as Hsp40 and the Hsp70-Hsp90 organizing protein (Hop), or competing with negative co-factors such as Bcl2-associated athanogen 1 (Bag1), Hip facilitates may facilitate the chaperone function of Hsc/Hsp70 in protein folding and repair, and in controlling the activity of regulatory proteins such as steroid receptors and regulators of proliferation or apoptosis. Although the nomenclature of ST13 implies a role in the suppression of tumorigenicity (ST), to date available experimental data are not sufficient to support its role in cancer development, except for the possible down-regulation of ST13 in gastric and colorectal cancers. Further investigation of this gene at the physiological level would benefit our understanding of diseases such as endocrinological disorders, cancer, and neurodegeneration commonly associated with protein misfolding.

STUDY ON THE ANTI-TUMOR EFFICACY INDUCED BY HEAT SHOCK PROTEIN 70-PEPTIDE COMPLEXES DERIVED FROM TUMOR CELLS

OBJECTIVE: To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. METHODS: Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used. RESULTS: HSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P

Polymorphism of heat shock protein 70-2 and enterocutaneous fistula in Chinese population

AIM: To investigate whether the heat shock protein 70-2 (HSP70-2) polymorphism is associated with enterocutaneous fistulas in a Chinese population.

Cytotoxic T-lymphocytes response in vitro activated by dendritic cells pulsed with heat shock protein 70 derived from human bladder tumor cell lines of EJ

Objective： To investigate whether human dendritic cells （DC） derived from peripheral blood mononuclear cells （PBMC）, which were pulsed by heat shock protein 70 （HSP70） isolated from human bladder tumor cell lines of E J, were able to induce peptide specific cytotoxic T-lymphocytes （CTL） response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods： The E J-derived HSP70 co-cultured with DC from the healthy volunteers＇ PBMC, along with the crude lysate （the supematant before HSP70 purification） from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes （SI） and interferon-y （IFN-γ） were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51^Cr releasing test. Results： Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate （the supernatant before HSP70 purification） （P 〈 0.05）. DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells （P 〈 0.05）. Conclusion： The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.

The Heat Shock Protein Story—From Taking mTORC1,2 and Heat Shock Protein Inhibitors as Therapeutic Measures for Treating Cancers to Development of Cancer Vaccines

Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.

ACUPUNCTURE EFFECTS ON INDUCIBLE NITRIC OXIDE SYNTHASE mRNA, iNOS PRODUCT AND HEAT SHOCK PROTEIN IN PERITONEAL MACROPHAGES OF THE MOUSE

To study the acupuncture effects on the inducible nitric oxide synthase (iNOS) mRNA,iNOS product and heat shock protein(hsp) 70 in the mouse macrophages, after peritoneal stimulation with steriled paraffin oil for 48 h, 24 Kunming mice were randomly divided into 3 groups: a, electroacupuncture (EA) groupl treated with EA; b, control 1 (C 1) group, peritoneal macrophages treated with culture; and c, control 2 (C 2) group, treated with neither EA nor culture. The macrophages of mice in 3 groups collected from respective peritoneal cavities were prepared into two kinds of specimens, including slide and nitrocellulose membrane (NCM). The iNOS mRNA, iNOS and hsp 70 were detected respectively with in situ hybridization, cytochemistry, immunohistochemistry and RNA and protein dot blots. The results showed that the signals of iNOS mRNA and iNOS product were localized in the macrophage cytoplasm; the immunoreactivity(IR) of hsp 70 localized in both cytoplasm and nucleus. The dot blot signal scanning value of those in 3 groups in comparison with each other showed as follows: EA group>C 2 group>C 1 group, (P<0.01).

Genome-Wide Identification of heat shock protein 10/60 Genes in Japanese Flounder (Paralichthys olivaceus) and Their Regulated Expression After Bacterial Infection

Heat shock proteins 10/60(hsp10/60)are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions.Besides the chaperone and housekeeping functions,they are also known to be involved in immune response during bacterial infection.In this study,we identified and annotated 10 hsp10/60 genes through bioinformatic analysis in Japanese flounder(Paralichthys olivaceus).Among them one member of hsp10(hspe)family and nine members of hsp60(hspd)family were identified.Phylogenetic and selection pressure analysis showed that the hsp10/60 genes were evolutionarily constrained and their function was conserved.Besides,hsp10/60 genes were involved in different embryonic and larval stages and acted as the sentinel role in an unchallenged organism.In addition,we also observed the expression patterns of hsp10/60 genes after Edwardsiella tarda infection,for the first time in Japanese flounder.Eight out of 10 genes were differentially expressed after bacterial challenges,the significantly regulated expressions of flounder hsp10/60 genes after bacterial infections suggested their involvement in immune response in flounder.Our results provide valuable information for clarifying the evolutionary relationship,and early insights of the immune functions of hsp10/60 genes in Japanese flounder.

Enhancing the treatment effects of tumor cell purified autogenous heat shock protein 70-peptide complexes on HER-3-overexpressing breast cancer

Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes(HSP70-PCs)on HER-3-overexpressing breast cancer.Methods In this study,we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics.We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells(DCs)and CD8+T cells induced by these complexes.Third,recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3-overexpressing breast cancer cells,and the resulting immunological response was examined.Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+T cell killing of HER-3-positive breast cancer cells.Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.

Heat shock proteins and immunotherapy

Being one of the most abundant intracellular proteins, heat shock proteins (HSPs) have many housekeeping functions which are crucial for the survival of organisms. In addition, some HSPs are new immunoactive molecules which play important roles in both adaptive and innate immunity. They could activate CD8 + and CD4 + lymphocytes, induce innate immune response including natural killer (NK) cell activation and cytokine secretion, and induce maturation of dendritic cells (DCs). These characteristics have been used for immunotherapy of various types of cancers and infectious diseases. This review focuses on the main HSP families——HSP70 and 90 families. The mechanism of HSPs′ function in eliciting immune response are elucidated and various forms of HSPs used in immunotherapy are discussed in details. At the end of this review, authors summarize clinical trials related to HSPs and evaluate their clinical efficacy.

苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析

世界检疫性害虫苹果蠹蛾Cydia pomonella是一种温度耐受可塑性很高的物种。本研究针对温度波动可能导致其耐热性增强的科学问题,采用生测法鉴定了苹果蠹蛾实验种群的高温耐受阈值,采用同源克隆、RACE和实时荧光定量PCR(RT-qPCR)等方法研究了苹果蠹蛾热激蛋白Hsp90基因的应激表达对耐热性的重要作用。高温耐受阈值研究结果表明,苹果蠹蛾实验种群的死亡率随温度的升高和时间的延长显著性升高,1-5龄幼虫分别经50℃和52℃高温处理2,5和10min后,3龄幼虫耐热性最差,5龄幼虫最强。50℃和52℃分别处理10min和5min均可导致1-4龄幼虫全部死亡,而5龄幼虫在这两种处理下仍有25.0%和11.1%的存活率。以35℃处理的5龄雌幼虫为材料克隆苹果蠹蛾Hsp90基因全长cDNA,结果显示该基因全长为2470bp,完整开放阅读框为2148bp,共编码716个氨基酸,预测分子量为82.07kDa,命名为Cphsp90(GenBank登录号JN624775)。该基因编码的氨基酸序列与亚洲玉米螟Ostrinia furnacalis和甘蓝夜蛾Mamestra brassicae等昆虫的Hsp90的氨基酸序列一致性高达96%,表明了Hsp90家族的保守特性。Cphsp90mRNA的相对表达量在32～44℃高温胁迫下随温度的升高而显著增高,证实Cphsp90是诱导型热激基因,且mRNA相对表达量与胁迫程度正相关。Cphsp90基因的表达还具有组织特异性,35℃处理幼虫的表皮中Cphsp90相对表达量显著高于血淋巴、脂肪体和中肠,应激响应最为活跃。与未经温热预处理的昆虫相比,35℃温热预处理3h后的5龄幼虫在40,45和50℃更高的温度胁迫下,Cphsp90mRNA达到最高表达量所需要的胁迫温度有所提升,由未经预热处理的40℃处理10min提高到45℃处理10min,这与温热预处理会增强5龄幼虫耐热性的现象相符,表明Cphsp90基因的响应表达在苹果蠹蛾耐热性及其可塑性过程中发挥重要的作用。