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Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and in vivo assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The 70 kDa heat shock protein (HSP70) is a stress-inducible protein that has been shown to protect the brain from various nervous system injuries. It allows cells to withstand potentially lethal insults through its chaperone functions. Its chaperone properties can assist in protein folding and prevent protein aggregation following several of these insults. Although its neuroprotective properties have been largely attributed to its chaperone functions, HSP70 may interact directly with proteins involved in cell death and inflammatory pathways following injury. Through the use of mutant animal models, gene transfer, or heat stress, a number of studies have now reported positive outcomes of HSP70 induction. However, these approaches are not practical for clinical translation. Thus, pharmaceutical compounds that can induce HSP70, mostly by inhibiting HSP90, have been investigated as potential therapies to mitigate neurological disease and lead to neuroprotection. This review summarizes the neuroprotective mechanisms of HSP70 and discusses potential ways in which this endogenous therapeutic molecule could be practically induced by pharmacological means to ultimately improve neurological outcomes in acute neurological disease.

Aims: HSP70, a molecular chaperone, helps to maintain proteostasis. In muscle biology, however, evidence suggests HSP70 to have a more versatile range of functions, as genetic deletion of its inducible genes impairs Ca 2+ handling, and consequently, cardiac and skeletal muscle contractility. Still, it is unknown whether HSP70 is involved in vascular reactivity, an intrinsic physiological mechanism of blood vessels. Therefore, we designed this study to test the hypothesis that proper vascular reactivity requires the assistance of HSP70., Main Methods: We performed functional studies in a wire-myograph using thoracic aorta isolated from male Sprague Dawley rats. Experiments were conducted with and without an HSP70 inhibitor as well as in heat-stressed vessels. The expression levels of HSP70 were evaluated with Western blotting. NO and ROS levels were assessed with fluorescence microscopy., Key Findings: We report that blockade of HSP70 weakens contraction in response to phenylephrine (dose-response) in the aorta. Additionally, we demonstrated that inhibition of HSP70 affects the amplitude of the fast and of the slow components of the time-force curve. Corroborating these findings, we found that inhibition of HSP70, in vessels over-expressing this protein, partly rescues the contractile phenotype of aortic rings. Furthermore, we show that blockade of HSP70 facilitates relaxation in response to acetylcholine and clonidine without affecting the basal levels of NO and ROS., Significance: Our work introduces an additional physiological role for HSP70, the assistance of vascular reactivity, which highlights this protein as a new player in vascular physiology, and therefore, uncovers a promising research avenue for vascular diseases., Competing Interests: Declaration of competing interest Both authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cancer metastasis represents a multistep process, including alteration of cell adhesion/motility in the microenvironment and sustained angiogenesis, which is essential for supporting cancer growth in tissues that are distant from the primary tumor. There is growing evidence suggesting that heat shock proteins (HSPs) (also known as heat stress proteins), which constitute a family of stress-inducible proteins, may be involved in the pathogenesis of cancer. Curcumin (diferuloylmethane) is a potent anti-inflammatory, antioxidant, antimicrobial, and antitumor agent. Curcumin has been shown to regulate different members of HSPs including HSP27, HSP40, HSP60, HSP70, and HSP90 in cancer. Here, we present extent findings suggesting that curcumin may act as a potential therapeutic agent for the treatment of cancer through its regulation of HSPs., (© 2019 International Union of Biochemistry and Molecular Biology.)

Acute myocardial infarction is a major cause of death worldwide. The most important therapy for limiting ischemic injury and infarct size is timely and efficient myocardial reperfusion treatment, which may instead induce cardiomyocyte necrosis due to myocardial ischemia-reperfusion (I/R) injury. Heat shock protein 70 (HSP70), a stress-inducible protein, is overexpressed during myocardial I/R. The induced HSP70 is shown to regulate several intracellular proteins (e.g., transcription factors, enzymes, and apoptosis-related proteins) and signaling pathways (e.g., c-Jun N-terminal kinase pathway and extracellular-signal-regulated kinase 1/2 pathway), forming a complicated network that contributes to reducing reactive oxygen species accumulation, improving calcium homeostasis, inhibiting cellular apoptosis, thereby enhancing the stress adaption of myocardium to I/R injury. In addition, the extracellular HSP70, which is released from injured cardiomyocytes during I/R, acts as a proinflammatory mediator that results in cell death, while the intracellular HSP70 exerts antiinflammatory effects by suppressing proinflammatory signaling pathways. Notably, HSP70 is induced and contributes to the cardioprotection in several types of preconditioning and postconditioning. Meanwhile, it is shown that the cardioprotective effectiveness of preconditioning-induced HSP70 (e.g., hyperthermia preconditioning-induced HSP70) can be impaired by certain pathological conditions, such as hyperlipidemia and hyperglycemia. Thus, we highlight the widespread cardioprotective involvement of HSP70 in preconditioning and postconditioning and elucidate how HSP70-mediated cardioprotection is impaired in these pathological conditions. Furthermore, several therapeutic potentials of HSP70 against myocardial I/R injury and potential directions for future studies are also provided in this review., (© 2018 Wiley Periodicals, Inc.)

Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson's Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70. Next, twelve MAL1-271 derivatives were synthesized and examined in a refined α-synuclein aggregation model as well as in an assay that monitors maturation of a disease-causing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant, which is also linked to Hsp70 function. Compared to the control, MAL1-271 significantly increased the number of cells lacking α-synuclein inclusions and increased the steady-state levels of the CFTR mutant. We also found that a nitrile-containing MAL1-271 analog exhibited similar effects in both assays. None of the derivatives exhibited cellular toxicity at concentrations up to 100 μm, nor were cellular stress response pathways induced. These data serve as a gateway for the continued development of a new class of Hsp70 agonists with efficacy in these and potentially other disease models., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Trichlorfon is a moderately toxic organophosphate pesticide that is widely used in aquaculture. This study investigated the effects of trichlorfon on hematological parameters, biochemical factors, and stress reaction in Cyprinus carpio L. The fish were exposed to acute concentrations of trichlorfon (0, 0.5, 1.0, 2.0, and 4.0 mg L -1 ) at 25 °C and 15 °C for 1 and 2 weeks, after which several parameters were evaluated to assess the effects of the pesticide. Significant decreases were observed in red blood cell (RBC) Count, hemoglobin (Hb) level, hematocrit (Ht), and plasma protein levels after each exposure period. In contrast, notable increases in mean corpuscular volume (MCV), mean cell hemoglobin (MCH), calcium, and glucose levels were observed in the trichlorfon-treated groups. Additionally, there were significant increases in the plasma levels of glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and alkaline phosphatase (ALP) following the exposure to trichlorfon. Furthermore, the results showed a relationship between toxic stress and increment in HSP70 and cytochrome P450 1A (CYP1A) expression over time. Ht, MCV, MCH, and the value of other biochemical parameters were quite lower at 15 °C than their corresponding values were at 25 °C, which indicated the decreased physical activity at 15 °C. The results of the present work indicate that acute exposure to trichlorfon and thermal stimulus could damage erythropoietic tissue. Additionally, hepatocytes function and physiological mechanisms could be impaired. Ht, glucose, GOT, GPT, HSP70, and CYP1A levels might be useful biomarkers of trichlorfon toxicity in contaminated aquatic ecosystems., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To investigate oxidative stress responses to cadmium and lead, the freshwater water flea Daphnia magna was exposed to Cd and Pb for 48 h. Following treatment with sub-lethal concentrations, intracellular reactive oxygen species (ROS) levels, as well as modulation of multiple biomarker, such as superoxide dismutase (SOD) activity, glutathione (GSH) contents, glutathione S-transferase (GST) activity, antioxidant enzyme - coding genes (three GST isoforms, glutaredoxin [GRx], glutathione peroxidase [GPx], and thioredoxin [TRx]), and stress-response proteins (heat shock protein 70 [Hsp70] and Hsp90) were examined. The results showed that intracellular ROS level was not changed at 24 h, but reduced at 48 h. Levels of total GSH content were reduced by Cd, but highly induced by Pb. SOD and GST activities were stimulated 48 h after exposure to Cd and Pb. A significant modulation of oxidative stress marker genes was observed after exposure to each element with different expression patterns depending on the metal and developmental stages. In particular, the expression levels of GST-sigma, HSP70, and HSP90 genes were enhanced in Cd - and Pb - exposed neonates. These findings imply that oxidative stress markers appear to be actively involved in cellular protection against metal-induced oxidative stress in D. magna. This study would facilitate the understanding of the molecular response to Cd and Pb exposure in water fleas., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Heat shock protein 70 (Hsp70) is widely involved in immune disorders, making it as an attractive drug target for inflammation diseases. Nonselective induction of Hsp70 upregulation for inflammation therapy could cause extensive interference in inflammation-unrelated protein functions, potentially resulting in side effects. Nevertheless, direct pharmacological activation of Hsp70 via targeting specific functional amino acid residue may provide an insight into precise Hsp70 function regulation and a more satisfactory treatment effect for inflammation, which has not been extensively focused. Here we show a cysteine residue (Cys306) for selective Hsp70 activation using natural small-molecule handelin. Covalent modification of Cys306 significantly elevates Hsp70 activity and shows more satisfactory anti-neuroinflammation effects. Mechanism study reveals Cys306 modification by handelin induces an allosteric regulation to facilitate adenosine triphosphate hydrolysis capacity of Hsp70, which leads to the effective blockage of subsequent inflammation signaling pathway. Collectively, our study offers some insights into direct pharmacological activation of Hsp70 by specially targeting functional cysteine residue, thus providing a powerful tool for accurately modulating neuroinflammation pathogenesis in human with fewer undesirable adverse effects., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Schistosomiasis is a debilitating disease that affects over 240 million people worldwide and is considered the most important neglected tropical disease following malaria. Free-swimming freshwater cercariae, one of the six morphologically distinct schistosome life stages, infect humans by directly penetrating through the skin. Cercariae identify and seek the host by sensing chemicals released from human skin. When they reach the host, they burrow into the skin with the help of proteases and other contents released from their acetabular glands and transform into schistosomula, the subsequent larval worm stage upon skin infection. Relative to host invasion, studies have primarily focused on the nature of the acetabular gland secretions, immune response of the host upon exposure to cercariae, and cercaria-schistosomulum transformation methods. However, the molecular signaling pathways involved from host-seeking through the decision to penetrate skin are not well understood. We recently observed that heat shock factor 1 (Hsf1) is localized to the acetabular glands of infectious schistosome cercariae, prompting us to investigate a potential role for heat shock proteins (HSPs) in cercarial invasion. In this study, we report that cercarial invasion behavior, similar to the behavior of cercariae exposed to human skin lipid, is regulated through an Hsp70-dependent process, which we show by using chemical agents that target Hsp70. The observation that biologically active protein activity modulators can elicit a direct and clear behavioral change in parasitic schistosome larvae is itself interesting and has not been previously observed. This finding suggests a novel role for Hsp70 to act as a switch in the cercaria-schistosomulum transformation, and it allows us to begin elucidating the pathways associated with cercarial host invasion. In addition, because the Hsp70 protein and its structure/function is highly conserved, the model that Hsp70 acts as a behavior transitional switch could be relevant to other parasites that also undergo an invasion process and can apply more broadly to other organisms during morphological transitions. Finally, it points to a new function for HSPs in parasite/host interactions., Competing Interests: The authors have declared that no competing interests exist.

The hsp70 and hsp90 expression patterns and catalase (CAT) activity in the freshwater planaria Dugesia japonica exposed to cadmium (Cd) under laboratory conditions were investigated. Planaria were exposed to a range of Cd concentrations (0-150 μg Cd/L) for 24 h. The expression levels of hsp70 and hsp90 were determined by relative quantitative real-time polymerase chain reaction. Within the overall dose range in the experiment, the expression level of hsp70 and the activity of CAT in D. japonica were altered significantly. Hsp70 was induced in D. japonica upon Cd exposure concentrations as low as 9.375 μg Cd/L. No significant effect on the expression level of hsp90 was observed. Our findings demonstrated that stress gene hsp70, but not hsp90, was responsive to Cd contamination in D. japonica CAT activity was significantly induced at concentrations of 18.75, 37.5, and 75 μg Cd/L after 24-h exposure. We recommend that the use of hsp70 as a biomarker should be complemented by evidence of changes in other parameters, such as CAT activity, in D. japonica., (© The Author(s) 2014.)

Skeletal muscle atrophy can be defined as a decrease of muscle volume caused by injury or lack of use. This condition is associated with reactive oxygen species (ROS), resulting in various muscular disorders. We acquired 2D and 3D images using micro-computed tomography in gastrocnemius and soleus muscles of sciatic-denervated mice. We confirmed that sciatic denervation-small animal model reduced muscle volume. However, the intraperitoneal injection of Oenothera odorata root extract (EVP) delayed muscle atrophy compared to a control group. We also investigated the mechanism of muscle atrophy's relationship with ROS. EVP suppressed expression of SOD1, and increased expression of HSP70, in both H2O2-treated C2C12 myoblasts and sciatic-denervated mice. Moreover, EVP regulated apoptotic signals, including caspase-3, Bax, Bcl-2, and ceramide. These results indicate that EVP has a positive effect on reducing the effect of ROS on muscle atrophy.

Zinc oxide nanoparticles (ZnONPs) are widely used in several commercial products due to their unique physicochemical properties. However, their release into the aquatic environments through various anthropogenic activities will lead to toxic effect in aquatic organisms. Although several investigations have been reported on the effect of ZnONPs in aquatic organisms using traditional end points such as survival, growth, and reproduction, the molecular level end points are faster and sensitive. In this study, the expression of different genes involved in oxidative stress response, detoxification, and cellular defense was studied in an ecotoxicologically important bio-monitoring organism Chironomus riparius in order to understand the subcellular effects of ZnONPs. The fourth instar larvae were exposed to 0, 0.2, 2, 10, and 20 mg/L of ZnONPs and Zn ions (in the form of ZnSO4.7H2O) for 24 and 48 h period. The expression of CuZn superoxide dismutase, manganese superoxide dismutase, catalase, phospholipid hydroperoxide glutathione peroxidase, thioredoxin reductase 1 and delta-3, sigma-4 and epsilon-1 classes of glutathione S-transferases, cytochrome p4509AT2, and heat shock protein 70 were studied using real-time polymerase chain reaction method. Gene expression results showed that the expression of genes related to oxidative stress response was more pronounced as a result of ZnONPs exposure as compared to Zn ions. The mRNA expression of genes involved in detoxification and cellular protection was also modulated. Significantly higher expression levels of oxidative stress-related genes shows that oxidative stress is an important mechanism of toxicity as a result of ZnONPs exposure in C. riparius., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Heat shock protein 70 (Hsp70) is one of the most widely used biomarker for monitoring environment perturbations in biological systems. To facilitate the analysis of hsp70 expression as a biomarker, we generated a Tg(hsp70:gfp) transgenic medaka line in which green fluorescence protein (GFP) reporter gene was driven by the medaka hsp70 promoter. Here, we characterized Tg(hsp70:gfp) medaka for inducible GFP expression by seven environment-relevant heavy metals, including mercury, arsenic, lead, cadmium, copper, chromium, and zinc. We found that four of them (mercury, arsenic, lead, and cadmium) induced GFP expression in multiple and different organs. In general, the liver, kidney, gut, and skin are among the most frequent organs to show induced GFP expression. In contrast, no detectable GFP induction was observed to copper, chromium, or zinc, indicating that the transgenic line was not responsive to all heavy metals. RT-qPCR determination of hsp70 mRNA showed similar induction and non-induction by these metals, which also correlated with the levels of metal uptake in medaka exposed to these metals. Our observations suggested that these heavy metals have different mechanisms of toxicity and/or differential bioaccumulation in various organs; different patterns of GFP expression induced by different metals may be used to determine or exclude metals in water samples tested. Furthermore, we also tested several non-metal toxicants such as bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 4-introphenol, and lindane; none of them induced significant GFP expression in Tg(hsp70:gfp) medaka, further suggesting that the inducibility of Tg(hsp70:gfp) for GFP expression is specific to a subset of heavy metals.

Heat shock proteins (HSPs) represent an important element in the body's defense against various damaging factors. The probably also play an important role in the pathogenesis and treatment of several diseases, including autoimmune pathology and neoplasms. Recently, several investigators have focused their attention on the involvement of the HSP70 protein family in the morbid process of inflammatory bowel diseases (IBD). The HSP70 family of is represented by two distinct forms of protein, the HSP72 protein (also known as the HSP70.1 protein), the expression of which is clearly increased in conditions of stress; and the HSP73 (or HSC73) protein, which manifests stable expression. HSP70 proteins are present in the colorectal epithelium. In patients with inflammatory bowel diseases, their expression in significantly increased during the active stage of the disease. In experimental studies, overexpression of HSP70 was found to prevent the development of inflammatory process in the large intestinal mucosa provoked by various damaging factors. In physiological conditions, various mechanisms are considered to be responsible for an increased expression of HSP70. One of them involves lymphocyte activity and the production of cytokines (mainly IL-2). Another suggested mechanism involves the presence of bacteria in the large intestine, including both physiological flora (Lactobacillus GG, Bacteroides fragilis) and pathogenic bacteria (Salmonella, Escherichia coli). HSP70 expression is probably also increased by physical activity. There is also a potential for pharmacological stimulation of HSP70 expression, linked (for example) to geranylgeranylacetone, polaprezinc and mesalazine. Thus, augmentation of HSP70 expression may become a new element in IBD therapy.

Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief but do not modify disease progression. Here we explore a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polyglutamine androgen receptor in spinal and bulbar muscular atrophy, huntingtin in Huntington's disease, α-synuclein in Parkinson's disease, and tau in Alzheimer's disease, are client proteins of heat shock protein 90 (Hsp90), and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. Hsp90 and Hsp70 have opposing effects on client protein stability in protein quality control; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes ubiquitination dependent on CHIP (C terminus of Hsc70-interacting protein) and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models.

Allicin, the main biologically active compound derived from garlic, exerts a broad spectrum of pharmacological activities and is considered to have therapeutic potential in many neurological disorders. Using an in vitro spinal cord injury model induced by glutamate treatment, we sought to investigate the neuroprotective effects of allicin in primary cultured spinal cord neurons. We found that allicin treatment significantly attenuated glutamate-induced lactate dehydrogenase (LDH) release, loss of cell viability and apoptotic neuronal death. This protection was associated with reduced oxidative stress, as evidenced by decreased reactive oxygen species (ROS) generation, reduced lipid peroxidation and preservation of antioxidant enzyme activities. The results of western blot analysis showed that allicin decreased the expression of inducible nitric oxide synthase (iNOS), but had no effects on the expression of neuronal NOS (nNOS) following glutamate exposure. Moreover, allicin treatment significantly increased the expression of heat shock protein 70 (HSP70) at both mRNA and protein levels. Knockdown of HSP70 by specific targeted small interfere RNA (siRNA) not only mitigated allicin-induced protective activity, but also partially nullified its effects on the regulation of iNOS. Collectively, these data demonstrate that allicin treatment may be an effective therapeutic strategy for spinal cord injury, and that the potential underlying mechanism involves HSP70/iNOS pathway-mediated inhibition of oxidative stress.

Heat shock proteins (hsps) are induced by a wide range of environmental stressors including heavy metals in aquatic organisms. However, the effect of heavy metals on zooplankton at the molecular level remains still unclear. In this study, we measured the intracellular reactive oxygen species (ROS) level and the antioxidant enzyme activities for 96 h after exposure to five heavy metals: arsenic (As), cadmium (Cd), copper (Cu), silver (Ag), and zinc (Zn) in the intertidal copepod Tigriopus japonicus. Activities of the antioxidant enzymes were highly elevated in metal-exposed copepods, indicating that heavy metals can induce oxidative stress by generating ROS, and stimulate the involvement of antioxidant enzymes as cellular defense mechanisms. Subsequently, transcriptional changes in hsp gene families were further investigated in the metal-exposed groups for 96 h. The ROS level and glutathione (GSH) content were significantly increased in Ag-, As-, and Cu-exposed copepods, while they were only slightly elevated in Cd- and Zn-exposed groups. Based on the numbers of significantly modulated hsp genes and their expression levels for 96 h, we measured the effect of heavy metals to stress genes of T. japonicus in the following order: Cu > Zn > Ag > As > Cd, implying that Cu acts as a stronger oxidative stress inducer than other heavy metals. Of them, the expression of hsp20 and hsp70 genes was substantially modulated by exposure to heavy metals, indicating that these genes would provide a sensitive molecular biomarker for aquatic monitoring of heavy metal pollution., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sulfamethoxazole (SMZ) is one of the most widely employed sulfonamides. Because of the widespread use of SMZ, a considerable amount is indeed expected to be introduced into the environment. The cytotoxicity of SMZ relies mainly on arylhydroxylamine metabolites (S-NOH) of SMZ and it is associated with the production of reactive oxygen species (ROS). There is limited information about the toxic potential of SMZ at the cellular and molecular levels, especially in aquatic and/or non-target organisms. In the present study, the red swamp crayfish (Procambarus clarkii), being tolerant to extreme environmental conditions and resistant to disease, was used as a model organism to profile the molecular and physiological response to SMZ. Haemolymphatic-immunological parameters such as glucose serum levels and total haemocyte counts were altered; moreover, a significant increase in Hsp70 plasma levels was detected for the first time. Variations at the transcriptional level of proinflammatory genes (cyclooxygenase-1, COX 1, and cyclooxygenase-2, COX 2), antioxidant enzymes (glutathione-S-transferase, GST and manganese superoxide dismutase MnSOD), stress response and Fenton reaction inhibitor genes (heat-shock protein 70 HSP70, metallothionein, MT and ferritin, FT) were evaluated, and alterations in the canonical gene expression patterns emerged. Considering these results, specific mechanisms involved in maintaining physiological homeostasis and adaptation in response to perturbations are suggested., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although selective striatal cell death is a characteristic hallmark in the pathogenesis of Huntington's disease (HD), the underlying mechanism of striatal susceptibility remains to be clarified. Heat shock proteins (HSPs) have been reported to suppress the aggregate formation of mutant huntingtin and concurrent striatal cell death. In a previous study, we observed that heat shock transcription factor 1 (HSF1), a major transcription factor of HSPs, significantly attenuated 3‑nitropropionic acid (3NP)‑induced reactive oxygen species (ROS) production and apoptosis through the expression of HSP 70 in striatal cells. To investigate the differential roles of HSPs in 3NP‑induced striatal cell death, the effect of geldanamycin (GA), an HSP 90 inhibitor, was examined in 3NP‑stimulated striatal cells. GA significantly attenuated 3NP‑induced striatal apoptosis and ROS production with an increased expression of HSP 70. Triptolide (TL), an HSP 70 inhibitor, abolished GA‑mediated protective effects in 3NP‑stimulated striatal cells. To understand the underlying mechanism by which GA‑mediated HSP 70 protects striatal cells against 3NP stimulation, the involvement of various signaling pathways was examined. GA significantly attenuated 3NP‑induced c‑Jun N‑terminal kinase (JNK) phosphorylation and subsequent c‑Jun phosphorylation in striatal cells. Taken together, the present study demonstrated that GA exhibits protective properties against 3NP‑induced apoptosis and JNK activation via the induction of HSP 70 in striatal cells, suggesting that expression of HSP 70 may be a valuable therapeutic target in the treatment of HD.

Background and Aim: Apoptosis is a major cause of myocyte death, and taurine is anti-apoptotic. Heat shock protein 70 (HSP70) (which is regulated by heat shock factor-HSF-1) is also anti-apoptotic, and caspase 3 stimulates the apoptotic pathway. This study investigated whether taurine affects atherogenic diet-induced myocardial apoptosis, and whether HSP70, HSF-1 and caspase 3 are involved., Methods: New Zealand white rabbits were divided into 3 groups for 4 weeks according to their diet. Group 1 (control) was fed a normal rabbit diet; Group 2 (MC) received a normal rabbit diet with 1% methionine plus 0.5% cholesterol. Group 3 received MC diet + 2.5% taurine (MCT)., Results: The atherogenic diet did not affect myocardial HSP70 or HSF-1 protein, but increased myocardial apoptotic nuclei to 40% (p < 0.01) versus 7% in con and 12% in MCT (p < 0.01). However, in MCT, myocardial HSP70 expression increased by 42.7% versus con and MC (p = 0.016), HSF-1 by 12% versus con and MC (p < 0.05), and total nuclei count increased by 37% versus MC (p < 0.05). Caspase 3 subunits remained unchanged in all groups, and HSP70 was increased approximately twofold in endothelial layer of arterioles (p = 0.01)., Conclusion: This study shows that taurine could reduce myocardial apoptotic nuclei and thus confer myocardial cytoprotection via stimulating myocardial HSP70 via HSF-1 and caspase 3-independent mechanisms.

In this article we show that long-term cultivation of Chinese hamster fibroblasts of line V-79 RJK at elevated temperature resulted in the selection of variants with genetic changes at the level of karyotype. From the first steps of resistance selection to elevated temperature we identified population of cells with changes in karyotype (polyploidy cells, deletions, inversions, translocations of chromosomes, and some cells with DM-chromosomes). Further cultivation was accompanied with selection of cells with paracentrical chromosome breakages and HSR's on chromosomes. Nonspecific destabilization of the karyotype (on first steps of selection) was associated with increased expression of hsc70 and pgp. After long-term incubation at an elevated temperature, the cells with karyotypic changes had the basal level of hsc70 and pgp expression.

Actinomycin D (Act D) is a general transcriptional inhibitor that is approved for the treatment of sarcomas, and Wilms, germ cell and trophoblastic tumors. Little is known about the molecular mechanisms that dictate the sensitivity of cancer cells to Act D. In this study, we investigated the effects of Act D on heat shock proteins (HSPs) and the expression and roles of HSP27 in Act D-induced cancer cell apoptosis. We show that Act D upregulates HSP27 and HSP70 expression in cancer cells, whereas it inhibits HSP90 expression. The upregulation of HSP27 by Act D is not attributable to changes in HSP27 transcription or HSP27 synthesis. HSP27 knockdown leads to an increase in Act D-induced caspase 3 and caspase 7 cleavage, and sensitizes rhabdosarcoma cells and breast cancer cells to Act D-induced apoptosis. We conclude that upregulation of HSP27 represents an adaptive response that compromises the anticancer activity of Act D., (© 2013 FEBS.)

Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.

Aims: We previously found that paeoniflorin, a major constituent of Paeonia lactiflora Pall, could induce heat shock proteins (HSPs) in cultured mammalian cells without apparent toxicity (Yan et al. 2004). We here investigated the induction of HSPs by paeoniflorin in mouse stomach and the effect of paeoniflorin on the HCl- and ethanol-triggered gastric mucosal injury in mouse., Main Methods: Paeoniflorin and quercetin were intraperitoneally administered in mouse and Hsp70 and other proteins in mouse tissues were detected by western blotting., Key Findings: The intraperitoneal administration of paeoniflorin clearly induced Hsp70 in mouse stomach, and paeoniflorin had a protective effect on the HCl- and ethanol-triggered gastric mucosal injury. When quercetin was injected before paeoniflorin administration, the induction of Hsp70 was suppressed and the protective effect of paeoniflorin was also diminished. Thus, the expression level of Hsp70 was well correlated with the extent of protection against irritant-induced gastric mucosal injury. Oral injection of HCl activated nuclear factor kappa B (NF-κB) and elicited the expression of cyclooxygenase-2 (COX-2) in gastric mucosa. Prior administration of paeoniflorin, however, suppressed these effects. No apparent systemic side effect of paeoniflorin has been observed so far. Hsp70 was also induced in the liver, heart, and brain by paeoniflorin., Significance: From these results, it is suggested that paeoniflorin and paeoniflorin-containing herbal medicines might be used clinically as HSP inducers for the prevention and treatment of diseases associated with protein conformation and of various other pathological states, such as stress ulcers and irritant- or ischemia-induced injuries., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Sunscreens have long been used to protect against the acute effects of UV radiation. They can also have protective effects on chronic UV-induced changes, such as photoaging and skin cancer. Recent studies have focused on marine organisms as a source of natural bioactive molecules and some UV-absorbing algal compounds are under investigation as candidates for new natural sunscreens., Objective: The cutaneous photoprotective ability of the mycosporine-like aminoacids (MAAs) Porphyra-334 and shinorine (P-334+SH), high UV-absorbing compounds isolated from the red alga Porphyra rosengurttii, was evaluated by in vivo procedures in mouse skin. The expression of the heat shock protein HSP70 as a potential biomarker for acute UV damage was also investigated., Methods: A galenic formulation containing the MAA combination of P-334+SH was applied topically to the dorsal skin of SkhR-1 H hairless mice, which were irradiated with a single UV radiation dose of 3.87Jcm(-2) and compared with a combination of UVB- and UVA-absorbing reference filters. Clinical signs of sunburn, such as erythema and edema, as well as other quantifiable histological and biochemical parameters, such as the expression of the heat shock protein 70 and antioxidant enzyme activities, were measured from skin biopsies at 6, 24, 48 and 72h post-radiation., Results: The formulation containing MAA prevented sunburn cell formation, as well as corneum stratum, malphigian, dermal and hypodermal thickening and other structural and morphological alterations observed in biopsies of non-photoprotected skin. A significant increase in Hsp70 was observed in the epidermis of non-photoprotected mouse skin, besides a de novo expression in deeper layers. P-334+SH protected against the significant decrease in superoxide dismutase and catalase activities observed in non-photoprotected mice., Conclusion: The topical application of P-334+SH protected against UV-induced skin damage in mice and contributed to maintaining the antioxidant defence system of the skin as well as Hsp70 expression.

Exploring the expression of Toll-like receptor (TLR) in cyclosporine (CsA)-induced renal injury in humans, we evaluated the expression of TLR4 in both biopsied renal tissue and cultured tubular cells. Immunohistochemical stains for TLR4, heat shock protein (HSP) 47, and HSP70 were performed in both pre- and post-treatment biopsies obtained from 18 patients of minimal change nephrotic syndrome or IgA nephropathy treated with CsA, and the percentage of positive tubules was compared in each case. For in vitro experiments, HK-2 cells were treated with CsA (2, 5, and 10 microg/ml) for 24, 48, and 72 h. TLR4 mRNA and protein were measured using real-time RT-PCR and Western blot. In addition, hypoxic effect was added by GasPak System. The tubular expressions of TLR4 (2.2 +/- 1.2% vs 4.4 +/- 2.0%, p < 0.001) and HSP70 (2.6 +/- 2.8% vs 6.1 +/- 4.2%, p = 0.002) were increased after CsA treatment. TLR4 mRNA and protein expression were also increased in a dose-dependent manner. Hypoxia enormously increased TLR4 expression. In summary, CsA increased tubular expression of TLR4 and its ligand HSP70. As hypoxia was shown to be a strong stimulus for TLR4 expression, it can be said that TLR4 is influenced by both direct toxicity and impediment of renal microcirculation in human CsA nephrotoxicity.

Natural killer (NK) cells are important effectors in the immune response to tumors. A number of cell-surface inhibitory and activating receptors on NK cells tightly regulate their interaction with target cell ligands. In particular, the strength of an anti-tumor immune response appears to depend critically on surface levels of one activating receptor, NKG2D. Correspondingly, expression of NKG2D ligands on target cells is a requirement for effective tumor immunosurveillance and the elimination of pathogen-infected cells. Sodium butyrate, a potent repressor of histone deacetylase (HDAC), has recently been proposed as a potential agent in cancer treatment based on its ability to modify, in several cancer cell types, the expression of a variety of genes related to cell cycle regulation and apoptosis. Here we report that, in the HeLa and HepG2 tumor cell lines, sodium butyrate upregulated the expression of the MHC class I-related chain molecules A and B (MICA and MICB) at both the mRNA and protein levels, resulting in an enhanced susceptibility of cells in both lines to NK lysis. It also led to an elevated expression of heat shock protein 70 (HSP70) and transcription factor Sp1, and increased the binding of transcription factors Sp1 and heat shock transcription factor 1 (HSF1) to the MICA/B promoter, resulting in increased expression of MICA and MICB. siRNA targeting Sp1 significantly attenuate the enhancement of MICA expression by sodium butyrate. These results suggest that sodium butyrate and other HDAC inhibitors may have therapeutic potential by enhancing the immune response to cancer.

Background: Glutamine (GLN) improves outcome in experimental and clinical states of illness and injury. The authors hypothesized GLN-mediated enhancement of O-glycosylation and subsequent phosphorylation of key transcription factors in the HSP70 pathway would lead to increased HSP70 expression following experimental sepsis., Methods: Mice underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 30 minutes after CLP. A separate group of mice was treated with mithramycin, an Sp1 inhibitor. Lung tissue was harvested at 1, 2, 6, and 24 hours after CLP and was analyzed for HSF-1 and Sp1 O-GlcNAc modification, alpha-p-threonine modification, and HSP70., Results: GLN increased O-GlcNAc modification of HSF-1 and Sp1 at 1 and 2 hours after sepsis (P < .001 vs saline). Samples immunoprecipitated for Sp1 and probed for subsequent phosphorylation showed a significant increase in nuclear alpha-p-threonine-modified Sp1 at 2 and 6 hours after sepsis (P < .001 vs saline). GLN increased phosphorylated nuclear HSF-1 at 1 and 2 hours after CLP (P < .001). Finally, GLN treatment increased HSP70 4-fold (P < .01), but when treated with mithramycin, this increase was attenuated at 2, 6, and 24 hours (P < .001 vs no mithramycin treatment)., Conclusions: These results indicate that GLN induces HSF-1 and Sp1, which is known to lead to their nuclear translocation. The molecular mechanism of GLN-mediated HSP70 expression appears to be dependent on O-GlcNAc pathway activation and subsequent O-glycosylation and phosphorylation of key transcription factors required for HSP70 induction.

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid beta (Abeta) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Abeta aggregation. These observations led to the hypothesis that chaperones might interact with Abeta and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Abeta has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Abeta aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Abeta self-assembly and that these chaperones are effective at substoichiometric concentrations (approximately 1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Abeta is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.

Our prior work demonstrated that geldanamycin (GA) reduced injury due to oxygen-glucose deprivation (OGD) in primary astrocyte cultures. Using medium with an ionic composition similar to that observed during in vivo global ischemia, the selectivity and temporal profile of CA1 neuronal damage seen in vivo was mimicked with OGD in mouse hippocampal organotypic slice cultures. The present study tested the ability of GA to reduce delayed neuronal death in such cultures. Treating organotypic cultures with 100 nM GA for 24 h prior to OGD induced Hsp70 and significantly reduced CA1 neuronal damage. Staining with ubiquitin to identify protein aggregates revealed reduced redistribution of ubiquitin, consistent with reduced protein aggregation likely due at least in part to induction of Hsp70 by GA.

Objective: Heat shock protein (HSP) expression is vital to cellular and tissue protection after stress or injury. However, application of this powerful tool in human disease has been limited, as known enhancers of HSPs are toxic and not clinically relevant. Glutamine (GLN) can enhance HSP expression in non-clinically relevant animal injury models. The aim of this study was to assess the ability of GLN to enhance pulmonary HSP expression, attenuate lung injury, and improve survival after sepsis in the rat., Design: Prospective, randomized, controlled animal trial., Setting: University research laboratory., Subjects: Male Sprague-Dawley rats., Interventions: We utilized a rat model of cecal ligation and puncture to induce sepsis. GLN or saline was administered 1 hr after initiation of sepsis via single tail-vein injection. We analyzed heat shock factor-1 phosphorylation, HSP-70, and HSP-25 via Western blot. Tissue metabolism was assayed by magnetic resonance spectroscopy. Occurrence of lung injury was determined via histopathologic examination. An inhibitor of HSP expression, quercetin, was utilized to assess role of HSP expression in prevention of sepsis-related mortality., Measurements and Main Results: GLN, given after initiation of sepsis, enhanced pulmonary heat shock factor-1 phosphorylation, HSP-70, HSP-25, and attenuated lung injury after sepsis. Further, GLN improved indices of lung tissue metabolic function (adenosine 5-triphosphate/adenosine 5-diphosphate ratio, nicotinamide adenine dinucleotide) after sepsis. No significant effect of GLN on lung tissue-reduced glutathione was observed. GLN treatment led to a significant decrease in mortality (33% [6 of 18] GLN-treated rats vs. 78% [14 of 17] saline-treated rats). Administration of the HSP inhibitor quercetin blocked GLN-mediated enhancement of HSP expression and abrogated GLN's survival benefit., Conclusions: GLN has been safely administered to critically ill patients and shown to improve outcome without clear understanding of the protective mechanism. Our results indicate GLN may prevent the occurrence of lung injury, lung tissue metabolic dysfunction, and mortality after sepsis via enhancement of deficient lung heat shock factor-1 phosphorylation/activation and HSP expression.

Unlabelled: Nitric oxide (NO) may be a necessary but not sufficient mediator of cytokine-mediated, selective beta-cell destruction. Previously, we have described a difference in NO-dependent IL-1beta sensitivity in vivo and in vitro of pancreatic islets from two rat strains, Brown Norway (BN) and Wistar Kyoto (WK), the latter being the more sensitive strain. Here we investigated whether strain-dependent, differential islet iNOS expression was associated with differences in islet expression of the IL-1 receptor type 1(IL-1RI) or interferon regulating factor 1 (IRF-1), and/or caused differences in HSP70 expression, a marker of cell defence against oxidative stress., Methods: isolated islets from both rat strains were exposed to increasing concentrations of IL-1beta (0-150 pg/ml) for 24 hours or for varying culture periods (4-48 hours) to 15 pg/ml of IL-1beta., Measurements: accumulated insulin and nitrite release into incubation medium, and islet mRNA and protein expression of iNOS, IL-1RI, IRF-1 and HSP70 by semi-quantitative RT-PCR and Western blotting., Results: Higher insulin and lower nitrite release into the incubation medium were seen for BN compared to WK rats islets in both dose- and time-response experiments. IRF-1 expression preceded iNOS expression and was more pronounced in WK than in BN islets. No strain differences were observed for islet expression of IL-1RI. A strain-dependent HSP70 expression in response to IL-1beta with the highest levels in WK rat islets following iNOS expression was seen., Conclusion: There was a strain-dependent difference in iNOS expression which was associated with IRF-1 and HSP70 expression.