Proteomic identification of heat shock protein 70 as a candidate target for enhancing apoptosis induced by farnesyl transferase inhibitor

Farnesyl transferase inhibitors (FTIs) are novel antitumor drugs with clinical activity. FTIs inhibit cell growth not only by preventing direct Ras farnesylation but also through a Ras-independent pathway. Proteomics has been shown to be a powerful tool to monitor and analyze molecular networks and fluxes within the living cells and to identify the proteins that participate in these networks upon perturbation of the cellular environment. To observe early and dynamic protein changes in the cellular response to FTI in ovarian cancer cells, total proteins were extracted from 2774 cells treated or not with 10 microM manumycin, an FTI, for 3, 6 and 16 h. The proteins in the cells that were differentially expressed following treatment with manumycin for 3, 6 and 16 h were noted by two-dimensional electrophoresis and further identified by peptide mass fingerprinting as stress proteins. Both heat shock protein 70 (HSP70) and altered HSP70 were significantly up-regulated as early as 16 h in 2774 cells after exposure to manumycin. Since HSP70 plays an important role in protecting cells under stress, we treated the 2774 cells with the HSP inhibitor quercetin in combination with FTI. Quercetin dramatically enhanced the manumycin-mediated apoptosis in 2774 cells. Inducible HSP70 by manumycin in surviving ovarian cancer cells was also inhibited by quercetin as demonstrated by enzyme-linked immunosorbent assay. The inhibition of HSP70 by quercetin was correlated with enhancement of manumycin-induced mediated apoptosis in 2774 cells. The inhibition of HSP70 by 50 microM quercetin was also correlated with a decreased expression of procaspase-3 and enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragment in 2774 cells treated with manumycin. The interaction between the HSP70 inhibitor and FTI confirms the functional significance of the up-regulation of HSP70 as a protective mechanism against FTI-induced apoptosis and provides the framework for combination treatment of ovarian cancer.     

Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure

Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF/TOF spectrometer, MS/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in both breeds, while 4 protein spots were expressed specifically in the bivoltine breed and 12 protein spots were expressed specifically in the multivoltine breed. In the present proteomics approach, 5 separate spots of sHSP proteins (HSP19.9, HSP20.1, HSP20.4, HSP20.8, and HSP21.4) were identified. Protein spot intensity of sHSPs was lower in the multivoltine breed than in the bivoltine breed after the 45°C heat shock treatment, while the difference between two breeds was not significant after the 41°C heat shock treatment. These results indicated that some other mechanisms might be engaged in thermal tolerance of multivotine breed except for the expression of sHSP and HSP70. There were visible differences in the intensity of heat shock protein expression between male and female, however, differences were not statistically significant.     

HSP70 binds to SHP2 and has effects on the SHP2-related EGFR/GAB1 signaling pathway

The Src homology phosphotyrosyl phosphatase, SHP2, is a positive effector of EGFR signaling. However, the molecular mechanism and biological functions of SHP2 regulation are still not completely known. To better understand the cellular processes in which SHP2 participates, we carried out mass spectrometry to find SHP2 binding proteins. FLAG-SHP2 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by LC/MS/MS mass spectrometry. Among the identified proteins, we focus in this report on the heat shock protein 70 (HSP70). Physical interactions of SHP2 with HSP70 were confirmed in vivo. Further experiments demonstrate that EGF does not activate binding of SHP2 with HSP70 rather the binding appears to be constitutive. However, the formation of an HSP70/SHP2 complex affected the binding of SHP2 with EGFR and (or) GAB1. These data suggest that binding of HSP70 with SHP2 regulates to some extent the EGF signaling pathway. In addition, immunostaining experiments indicated that SHP2 and HSP70 co-localized in the cell membrane region after EGF treatment. Our findings propose a possible involvement of HSP70 in the regulation of EGF signaling pathway by SHP2.     

Inhibition of heat shock proteins (HSP) expression by quercetin and differential doxorubicin sensitization in neuroblastoma and Ewing's sarcoma cell lines

Neuroblastoma (NB) and Ewing's sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over-expression was correlated to drug-resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin-stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti-proliferative effect in NB (IC₅₀: 6.9 ± 5.8 μmol/L) than in ES cells (IC₅₀: 85.5 ± 53.1 μmol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC₅₀ decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro-apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB.     

Prevention and treatment of alopecia areata with quercetin in the C3H/HeJ mouse model

Alopecia areata (AA) is an autoimmune non-scarring hair loss disorder. AA can be acute, recurrent, or chronic. Current therapeutic options for AA are limited, and there is no effective prevention for recurrent AA. We have previously shown a correlation between the expression of HSP70 (HSPA1A/B), a heat shock protein involved in the inflammatory response, and the onset of AA in the C3H/HeJ mouse model. In this study, we tested the effects of quercetin, a bioflavonoid with anti-inflammatory properties, on AA development and HSP70 expression in the C3H/HeJ model. Mice with spontaneous AA were treated with subcutaneous quercetin or sham injections. Hair regrowth was observed in lesional areas in all the quercetin-treated mice, but in none of the sham-treated mice. In addition, non-alopecic C3H/HeJ mice were heat-treated to induce alopecia, along with quercetin or sham injections. Whereas 24% of the heat-treated mice with sham injections developed alopecia, none of the mice receiving quercetin injections did. As expected, the level of HSP70 expression in quercetin-treated areas was comparable to control. Furthermore, we showed that systemic delivery of quercetin by intraperitoneal injections prevented/reduced spontaneous onset of AA. Our results demonstrated that quercetin provided effective treatment for AA as well as prevention of onset of AA in the C3H/HeJ model, and warrant further clinical studies to determine whether quercetin may provide both treatment for preexisting AA and prevention of recurrent AA. The ready availability of quercetin as a dietary supplement may lead to increased patient compliance and positive outcomes for AA.     

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.     

The use of stress‐70 proteins in physiology: a re‐appraisal

There are few factors more important to the mechanisms of evolution than stress. The stress response has formed as a result of natural selection, improving the capacity of organisms to withstand situations that require action. The ubiquity of the cellular stress response suggests that effective mechanisms to counteract stress emerged early in the history of life, and their commonality proves how vital such mechanisms are to operative evolution. The cellular stress response (CSR) has been identified as a characteristic of cells in all three domains of life and consists of a core 44 proteins that are structurally highly conserved and that have been termed the ‘minimal stress proteome’ (MSP). Within the MSP, the most intensely researched proteins are a family of heat‐shock proteins known as HSP70. Superficially, correlations between the induction of stress and HSP70 differential expression support the use of HSP70 expression as a nonspecific biomarker of stress. However, we argue that too often authors have failed to question exactly what HSP70 differential expression signifies. Herein, we argue that HSP70 up‐regulation in response to stressors has been shown to be far more complex than the commonly accepted quasi‐linear relationship. In addition, in many instances, the uncertain identity and function of heat‐shock proteins and heat‐shock cognates has led to difficulties in interpretation of reports of inducible heat‐shock proteins and constitutive heat‐shock cognates. We caution against the broad application of HSP70 as a biomarker of stress in isolation and conclude that the application of HSP70 as a meaningful index of stress requires a higher degree of validation than the majority of research currently undertakes.     

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.     

Thermotolerance and heat acclimation may share a common mechanism in humans

Thermotolerance and heat acclimation are key adaptation processes that have been hitherto viewed as separate phenomena. Here, we provide evidence that these processes may share a common basis, as both may potentially be governed by the heat shock response. We evaluated the effects of a heat shock response-inhibitor (quercetin; 2,000 mg/day) on established markers of thermotolerance [gastrointestinal barrier permeability, plasma TNF-α, IL-6, and IL-10 concentrations, and leukocyte heat shock protein 70 (HSP70) content]. Heat acclimation reduced body temperatures, heart rate, and physiological strain during exercise/heat stress) in male subjects (n = 8) completing a 7-day heat acclimation protocol. These same subjects completed an identical protocol under placebo supplementation (placebo). Gastrointestinal barrier permeability and TNF-α were increased on the 1st day of exercise/heat stress in quercetin; no differences in these variables were reported in placebo. Exercise HSP70 responses were increased, and plasma cytokines (IL-6, IL-10) were decreased on the 7th day of heat acclimation in placebo; with concomitant reductions in exercise body temperatures, heart rate, and physiological strain. In contrast, gastrointestinal barrier permeability remained elevated, HSP70 was not increased, and IL-6, IL-10, and exercise body temperatures were not reduced on the 7th day of heat acclimation in quercetin. While exercise heart rate and physiological strain were reduced in quercetin, this occurred later in exercise than with placebo. Consistent with the concept that thermotolerance and heat acclimation are related through the heat shock response, repeated exercise/heat stress increases cytoprotective HSP70 and reduces circulating cytokines, contributing to reductions in cellular and systemic markers of heat strain. Exercising under a heat shock response-inhibitor prevents both cellular and systemic heat adaptations.     

Characterization of Schinus lentiscifolius Marchand (Anacardiaceae) Bark Extract and Its Effects on Lymphocyte Oxidative Stress and Heat Shock Response

Schinus lentiscifolius Marchand has been used in folk medicine to treat immunoinflammatory related diseases, which are marked by OS and altered HSR. Our study aimed to evaluate OS and HSR in lymphocytes treated with S. lentiscifolius bark extracts. S. lentiscifolius barks were partitioned with solvents to obtain hexane (SL‐HEX), ethyl acetate (SL‐ACOET) and methanol (SL‐MEOH) extracts, and the presence of bioactive compounds was evaluated by thin layer chromatography. Total phenols were measured by the Folin–Ciocalteu method and flavonoids were identified by HPLC‐DAD‐ESI‐MS/MS. Antioxidant capacity was verified by DPPH method, cell viability by Trypan Blue method, lipid peroxidation by TBARS and HSP70 by immunoblotting. The SL‐ACOET extract presented higher content of phenolic compounds and antioxidant activity in vitro. It was able to reduce lipid peroxidation levels in lymphocytes induced by H₂O₂ and improved cell viability. The SL‐ACOET extract inhibited HSR by a decrease in both intracellular content and release of 70 kDa heat shock proteins (HSP70) and also by decrease extra‐to‐intracellular HSP70 ratio in lymphocytes submitted to heat shock (2 h, 41 °C). S. lentiscifolius bark extract has antioxidant activity and inhibitory effect on HSR probably due to the presence of polyphenols as the flavonoids quercetin and kaempferol.     

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurrence of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-crystallin was observed. HSP32 represents heme oxygenase 1 (HO-1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress-induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO-1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase-1 has been linked to inflammatory processes and oxidative stress, its specific up-regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.     

Expression of heat shock proteins during development of barley

Barley heat shock proteins have been cloned, characterized by hybrid release translation and sequenced. Clones coding for proteins of 17, 18, 30, 32 and 70 kDa have been obtained. Out of these the 32 and 30 kDa proteins have been characterized as precursors to plastidic proteins of 26 kDa by posttranslational transport and by cDNA sequencing. The coding regions of these two transcribed genes are highly homologous. Accumulation of the plastid HSP as well as of HSP 70 as well as their corresponding mRNAs has been studied in 2- to 6-day old seedlings and in the 7-day old barley leaf. The mRNA for all investigated proteins were only found after a heat shock; the mRNA levels increase towards the tip of the leaf and with development. Furthermore, under the conditions used the mRNAs for all investigated heat shock proteins accumulate in parallel. Unexpectedly, both proteins, HSP 70 and HSP 26, are found by western blotting in the 2-day old control plants in the absence of any inducing heat shock. At later stages of development and in the leaf gradient only immunoreactivity with HSP 70 was observed. In contrast to the levels of their mRNAs the highest levels of HSP 30–26 and 70 have been observed in the basal segments indicating that translational control plays a role during HSP expression. Under severe heat shock a protein of 30 kDa is induced whose identity is not known but which reacts with the antibody to HSP 30–26 and might represent the accumulating precursors of the plastidic proteins.     

Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain

Heat shock protein 70 (HSP70) is an intracellular stress protein that confers cytoprotection to a variety of cellular stressors. Several lines of evidence have suggested that augmentation of the heat shock response by increasing the expression of HSP70 represents a potential therapeutic strategy for the treatment of critically ill patients. The Tat protein of human immunodeficiency virus 1 (HIV-1) has been used previously to deliver functional cargo proteins intracellularly when added exogenously to cultured cells. We generated a Tat-HSP70 fusion protein using recombinant methods and treated HSF -/- cells with either Tat-HSP70 or recombinant HSP70 prior to exposure to hyperoxia or lethal heat shock. We showed that biologically active, exogenous HSP70 can be delivered into cells using the HIV-1 Tat protein, and that the Tat-mediated delivery of HSP70 confers cytoprotection against thermal stress and hyperoxia and may represent a novel approach to augmenting intracellular HSP70 levels.     

The human oesophageal squamous epithelium exhibits a novel type of heat shock protein response.

The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.     

Targeting HSP70: The second potentially druggable heat shock protein and molecular chaperone?

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.     

HSP70 is part of the synaptonemal complex in Eimeria tenella

In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction.