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Heat shock proteins (HSPs) are available and/or induced for the survival of all organisms, including eukaryotic, prokaryotic, and plants, from higher temperature stresses. They are the chaperone proteins that protect all cells against heat, as the name implies. In addition to thermal stress, they also protect them from chemical, physical, and other stresses, including exposure to oxidative stress, nutritional deficiencies, ultraviolet radiation, ethanol, viral infection, ischemia-reperfusion injury, and cancer-related stresses. They are classified based on their molecular weights in kDa, such as HSP90 and HSP70. In our label-free, high-throughput, quantitative LC-MS/MS-based proteomic studies of MDA-MB-231, human, triple-negative breast cancer cells, treated with electrical pulses (EP) and cisplatin (CsP), we identified a number of HSPs, such as HSP90AA1, and others to be significantly downregulated in EP + CsP, compared to CsP alone. This indicates that cells will undergo apoptotic cell death and hence could cause effective cancer cure/treatment. Considering that over 2 million new cases and over 600,000 deaths in 2020, of which ~ 15% are TNBC, heat shock proteins could be the untapped resources, available for the next biomarkers and/or inhibitors for new/additional therapies., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Type 2 DCs (DC2s) comprise the majority of conventional DCs within most tumors; however, little is known about their ability to initiate and sustain antitumor immunity, as most studies have focused on antigen cross-presenting DC1s. Here, we report that DC2 infiltration identified by analysis of multiple human cancer data sets showed a significant correlation with survival across multiple human cancers, with the benefit being seen in tumors resistant to cytotoxic T cell control. Characterization of DC subtype infiltration into an immunotherapy-resistant model of breast cancer revealed that impairment of DC1s through 2 unique models resulted in enhanced DC2 functionality and improved tumor control. BATF3 deficiency depleted intratumoral DC1s, which led to increased DC2 lymph node migration and CD4+ T cell activation. Enhancing DC2 stimulatory potential by genetic deletion of Hsp90b1 (encoding molecular chaperon GP96) led to a similar enhancement of T cell immunity and improved survival in a spontaneous breast cancer model. These data highlight the therapeutic and prognostic potential of DC2s within checkpoint blockade-resistant tumors.

In spite of the abundance of antifungal therapies, 75% of women in the world suffer from the second most common cause of vaginal infection named vulvovaginal candidiasis. This complication is characterized with overgrowth of Candida albicans. The low efficacy and side effects of current antifungal therapies have convinced the researchers to look for a non-antibiotic based treatment such as cold atmospheric plasmas (CAP). The aim of this research was to evaluate the effects of CAP on C. albicans growth, ergosterol and biofilm formation. In addition, antibiotic resistance, phospholipase and proteinase activity, and structural properties were examined with different exposure duration. Putative critical effect of CAP on the expression of HSP90 as a target of anti-fungal therapy was investigated. ROS production in C. albicans exposed to CAP was assessed. For this purpose, C. albicans subjected to 0, 90, 120, 150, 180 and 210 s of He/O2 (2%), and non-treated cells as control were examined in terms of the mentioned virulence factors. The results showed that CAP had a significant effect on inhibition of C. albicans growth, Inhibition of biofilm formation, ergosterol content, and fluconazole and amphotericin B antibiotic sensitivity were significant in 210 s treatment group. This effect was validated based on changes of the cell architecture and morphology given the microscopy imaging results. The expression of HSP90 in both C. albicans ATCC 10231 and C. albicans PFCC 9362 was inhibited in 210 s of exposition. CAP exposition induced intracellular ROS, which may cause membrane damage and cell death in C. albicans. Taken together, the potential of CAP for therapeutic purposes in C. albicans-induced fungal infections is supported., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Untranslated regions (UTRs) of the transcripts play significant roles in translation regulation and continue to raise many intriguing questions in our understanding of cellular stress physiology. Internal ribosome entry site (IRES) mediated alternative translation initiations are emerging as unique mechanisms. Present study is aimed to indentify a functional short 92 base pair length putative sequence located at the 5' untranslated region of bovine heat shock protein 90 AA1 (Hsp90AA1) may interact with ribosomal as well as eukaryotic initiation factor binding site. Here we have predicted both the two and three dimensional structures of bovine Hsp90AA1 IRES (MF400854) element with their respective free energy. Molecular interactions between bovine RPS5 and IRES have been determined after the preparation of docking complex of IRES bound RPS5. Structure of bovine ribosomal translational initiation factor (TIF) has also been determined and docked with IRES. Molecular interaction between bovine TIF and IRES was analyzed from the complex structure. We further detected the relative expression efficiency of the viral (original) in relation with Hsp90AA1 IRES-driven GFP expression, which revealed that efficiency under the control of identified bovine Hsp90AA1 IRES was slightly lower than viral origin. It was also noted that identified bovine HSP90 IRES may increase the expression level of GFP under in vitro heat stressed condition.

Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH 2 -terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5-1 U) and urea (0-3 M). Each HSP90 construct was highly purified and approximately 0.1-1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection - Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a, murf1b, murf2a, murf2b, murf3 and a murf2-like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A tight quality control system over protein folding, turnover and synthesis, involving molecular chaperones and co-chaperones, maintains the balance of cardiac proteins. Various cardiac pathologies, including myocardial infarction, increase stresses and post-translational modifications favoring misfolding due to an overwhelmed quality control system. The toxic nature of accumulated misfolded proteins further worsens the condition. The important molecular chaperones which act as quality control proteins are involved in protecting the heart, these include heat shock protein70 (HSP70) and HSP90. Here, we review the emerging roles of heat shock proteins in the maintenance of cardiac cell populations in experimental models of ischemia/reperfusion (I/R) injury. Furthermore, we discuss the expression of HSP70 and HSP90 with therapeutic and diagnostic considerations. Although there is only a partial understanding of these important HSPs in I/R injury, there is an immense therapeutic potential of modulating these HSPs to counteract the imbalance between misfolding and endogenous protein quality control systems.

Aims: HSP90, as a molecular chaperone, has numerous substrate proteins, including HIF-1α and p-AKT, but the relationships among HSP90, HIF-1α and p-AKT have not been investigated in NPC. We examined and analysed the correlation between expression of HSP90, HIF-1α and p-AKT and clinicopathological features of NPC., Methods: We collected 445 cases of NPC and 54 cases of non-cancerous nasopharyngeal epithelia tissues, detected expression of HSP90, HIF-1α and p-AKT proteins in these tissues by immunohistochemistry., Results: The results indicated that overexpression of HSP90, HIF-1α and p-AKT in NPC was significantly higher than that in non-cancerous nasopharyngeal epithelia (P < 0.05). The overexpression of HIF-1α in primary NPC was significantly lower than that in matched lymph node metastatic NPC (P = 0.024) or recurrent NPC (P = 0.039). The overexpression of HSP90 (P < 0.001) and HIF-1α (P = 0.031) was evidently higher in late stage NPC. NPC patients with lymph node metastasis (LNM) had a higher overexpression rate of HSP90 (P < 0.001) than those without LNM. Increased HSP90 expression was positively associated with HIF-1α expression (r = 0.367, P < 0.001) and p-AKT (r = 0.142, P = 0.003) expression in NPC. Furthermore, HIF-1α was also related to p-AKT expression (r = 0.114, P = 0.017). The overall survival rate for NPC patients with up-regulated HSP90 was significantly lower than those with down-regulated HSP90 (P < 0.001), as was found with raised HIF-1α (P = 0.036) and increased p-AKT (P = 0.044). Multivariate Cox regression analysis further identified that HSP90 and HIF-1α were independent poor prognostic factors for NPC., Conclusions: Taken together, elevated HSP90 was associated with expression of HIF-1α and p-AKT in NPC. Furthermore, high expression of HSP90 and HIF-1α could be used as a novel independent poor prognostic biomarker for patients with NPC., (© 2019 John Wiley & Sons Ltd.)

Objectives: Heat shock protein 90 (HSP90) contributes to cutaneous vasodilatation during exercise in the heat through nitric oxide (NO) synthase (NOS)-dependent mechanisms in young adults. We hypothesized that similar responses would be observed in older middle-aged adults., Methods: In nineteen habitually active older middle-aged (56 ± 5 years) men (n = 9) and women (n = 10), cutaneous vascular conductance (CVC) was measured at four forearm skin sites continuously treated with (a) lactated Ringers solution (Control), (b) 10 mmol/L L-NAME (NOS inhibitor), (c) 178 μmol/L geldanamycin (HSP90 inhibitor), or (d) 10 mmol/L L-NAME and 178 μmol/L geldanamycin combined. Participants rested in an upright semi-recumbent position in the heat (35°C) for 70 minutes, followed by a 50-minute bout of moderate-intensity cycling (~55% peak oxygen uptake) and a 30-minute recovery period in the heat., Results: In both men and women, we observed no significant effects of HSP90 inhibition on CVC throughout rest, exercise, and recovery in the heat (all P > 0.27). Conversely, NOS inhibition and dual NOS and HSP90 inhibition attenuated CVC relative to Control throughout the protocol (all P ≤ 0.05)., Conclusions: While NOS mediates cutaneous vasodilatation during rest, exercise, and recovery in the heat, HSP90 does not measurably influence this response in habitually active older middle-aged men or women under these conditions., (© 2019 John Wiley & Sons Ltd.)

Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 cells., (Copyright © 2019. Published by Elsevier B.V.)

Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae., Competing Interests: The authors have declared that no competing interests exist.

Up-regulation of heat shock protein 90 (HSP90) gene takes place in lung cancer cells. Therefore, targeting HSP90 in lung cancer may be promising step in lung cancer therapy. The present study aimed to evaluate the efficiency of implantable 17-dimethylaminoethylamino-17-demethoxy geldanamycin (17-DMAG)-loaded Poly(caprolactone)-poly(ethylene glycol) (PCL/PEG) nanofibers to increase the anti-cancer effects via inhibition of HSP90 expression and telomerase activity. For this purpose, 17-DMAG-loaded PCL/PEG nanofibers were successfully fabricated via electrospinning and characterized using FE-SEM and FTIR. Colorimetric MTT assay was used to determine the drug cytotoxicity. Also, the expression levels of HSP90 mRNA in the A549 cells treated with the nanofibers were assessed using Quantitative Real-Time PCR. The effect of free 17-DMAG and 17-DMAG-loaded PCL/PEG nanofiber treatment on telomerase activity was monitored by TRAP assay. MTT assay confirmed that loading of 17-DMAG into PCL/PEG nanofiber enhanced dramatically cytotoxicity in the lung cancer cells. This finding was associated with reduction of HSP90 mRNA expression and telomerase activity in the cells seeded on 17-DMAG-loaded PCL/PEG nanofibers in relative to free 17-DMAG. In conclusion, the findings demonstrated that 17-DMAG-loaded PCL/PEG nanofibers are more effectual than free 17-DMAG against A549 lung cancer cells via modulation of Hsp90 expression and inhibition of telomerase activity. Hence, the implantable 17-DMAG-loaded nanofibrous scaffolds might be an excellent tool for efficiently killing of the lung residual cancer cells and avoid the local cancer recurrence., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Heat shock protein 90 (Hsp90) plays a vital role in the progress of malignant disease and elevated Hsp90 expression has been reported in pancreatic cancer. In this study, we radiolabeled a dimeric Sansalvamide A derivative (Di-San A1) with 64 Cu, and evaluated the feasibility of using 64 Cu-Di-San A1 for PET imaging of Hsp90 expression in a mouse model of pancreatic cancer. A macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-trisacetic acid) was conjugated to Di-San A1. 64 Cu-Di-San A1 was successfully prepared in a radiochemical yield > 97% with a radiochemical purity > 98%. 64 Cu-Di-San A1 is stable in PBS and mouse serum with > 92% of parent probe intact after 4 h incubation. The cell binding and uptake revealed that 64 Cu-Di-San A1 binds to Hsp90-positive PL45 pancreatic cancer cells, and the binding can be effectively blocked by an Hsp90 inhibitor (17AAG). For microPET study, 64 Cu-Di-San A1 shows good in vivo performance in terms of tumor uptake in nude mice bearing PL45 tumors. The Hsp90-specific tumor activity accumulation of 64 Cu-Di-San A1 was further demonstrated by significant reduction of PL45 tumor uptake with a pre-injected blocking dose of 17AAG. The ex vivo PET imaging and biodistribution results were consistent with the quantitative analysis of PET imaging, demonstrating good tumor-to-muscle ratio (5.35 ± 0.46) of 64 Cu-Di-San A1 at 4 h post-injection in PL45 tumor mouse xenografts. 64 Cu-Di-San A1 allows PET imaging of Hsp90 expression in PL45 tumors, which may provide a non-invasive method to quantitatively characterize Hsp90 expression in pancreatic cancer.

Background/aims: Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice., Methods: MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting., Results: URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein., Conclusions: URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The molecular chaperone Hsp90 is an essential member of the cellular proteostasis system. It plays an important role in the stabilisation and activation of a large number of client proteins and is involved in fatal disease processes, e.g. Alzheimer disease, cancer and cystic fibrosis. This makes Hsp90 a crucial protein to study. Mechanistic studies require large amounts of protein but the production and purification of recombinant human Hsp90 in Escherichia coli is challenging and laborious. Here we identified conditions that influence Hsp90 production, and optimised a fast and efficient purification protocol. We found that the nutrient value of the culturing medium and the length of induction had significant effect on Hsp90 production in Escherichia coli. Our fast, single-day purification protocol resulted in a stable, well-folded and pure sample that was resistant to degradation in a reproducible manner. We anticipate that our results provide a useful tool to produce higher amount of pure, well-folded and stable recombinant human Hsp90β in Escherichia coli in an efficient way.

The expression of Mx1 in EPC cells after treatment with poly(I:C) or infection with viral hemorrhagic septicemia virus (VHSV) was significantly suppressed by treatment with dexamethasone. However, the titer of VHSV did not increase but instead decreased after dexamethasone treatment. This suggests that dexamethasone not only downregulates type I IFN but also affects certain factors that are necessary for VHSV replication. An important effect of HSP90 on replication of RNA viruses and downregulation of HSP90 by glucocorticoids have been reported. In this study, dexamethasone downregulated HSP90α expression in EPC cells that were stimulated with poly(I:C) or infected with VHSV. Furthermore, cells treated with an HSP90 inhibitor, geldanamycin, showed significantly decreased titers of VHSV, suggesting that HSP90 may be an important host component involved in VHSV replication, and HSP90 inhibition might be one of the causes for the observed reduction in viral titer caused by dexamethasone treatment.

Recently, orexin (ORX)ergic system has gained great attention for its major neuroregulatory role on fish motor, circadian, feeding activities and above all during water toxic conditions. Fish are particularly sensitive to acute sublethal copper (Cu) concentrations while little is known about neurobehavioral data after chronic Cu exposure. For this work, the marine teleost ornate wrasse (Thalassoma pavo) was monitored for 21 days during exposure to a relatively low CuCl 2 concentration (0.25 mg/l). In particular, at 1 day fish displayed moderate reductions (-32%) of feeding behaviors versus controls that decreased up to -94% at 21 days. Swimming activities were also moderately (-44%) reduced at 1 day, which diminished even further (~ -75%) from 7 to 21 days. The lethargic attitudes seemed to be frequently interrupted by aquatic surface respiration and abnormal motor behaviors such as rapid and abrupt changes of direction. Contextually, an upregulatory trend of HSP90 mRNAs occurred especially in the lateral part of the dorsal telencephalon (Dl; +72%), medial preglomerular nucleus (NPGm; +87%), and torus longitudinalis (TLo; +108%). Moreover, some of these nuclei displayed extensive argyrophilic signals that are typical of notable neurodegenerative events. Interestingly, a single intraperitoneal administration of ORX-A (10 ng/g of body weight) at 21 days attenuated not only some behavioral impairments but also HSP90 mRNA levels and neurodegenerative events. Behavioral indications of the present results tend to underlie the valuable role of HSP90 transcript together with the ORXergic system as probable rescuing factors operating in marine fish during persistent adverse environmental states with eventual bearings on human health issues.

This study evaluated the effect of the antiretroviral ritonavir on protease secretion in different strains of Cryptococcus neoformans isolated from the environment and investigated the expression of heat shock protein (Hsp90), classically described virulence factors in other yeast in the presence of the same antiretroviral. The presence of the enzyme was detected by the formation of a degradation of the halo around the colonies. The results were classified as follows: level 1 (without proteases), level 2 (positive for proteases), and level 3 (strongly positive for proteases). Total protein extract isolated from the cell walls of the 12 strains incubated in the absence and presence of ritonavir (0.3125 mg mL -1 ) were resolved by SDS-PAGE and analyzed by Western blot assays using an antiserum against Hsp90 from Blastocladiella emersonii. All strains tested showed inhibition of proteinase activity in the presence of ritonavir at 0.3125 to 1.25 mg mL -1 . High levels of Hsp90 were observed in the absence of ritonavir (0.3125 mg mL -1 ), except for the non-virulent control cells. In contrast, in the presence of the antiretroviral, a drastic reduction in the expression of the chaperone was observed. The data suggest that ritonavir, in addition to containing viral replication, could inhibit the expression of virulence factors in opportunistic yeast, as proteases and Hsp90. According to our current knowledge, this is the first time that the inhibition of Hsp90 by an antiretroviral was reported for environmental isolates of C. neoformans.

Heat shock protein 90 (HSP90), which is expressed in cancer cells, profoundly affects progression, invasion, and metastasis. However, to our knowledge, in East Asia, the correlation between the expression of HSP90 and clinicopathological variables has seldom been discussed. We therefore investigated this and its prognostic value in 36 patients newly diagnosed with oral squamous cell carcinoma (SCC) in northern Taiwan. Samples of tumour and normal samples from the patients were compared immunohistochemically. HSP90 was expressed mainly in the samples of tumour, and was significantly higher in these than in the normal epithelium (p<0.001). Metastases to the lymph nodes in the 36 patients also correlated with expression of HSP90. Correlation between expression of HSP90 and the size of the tumour or pathological staging was not significant, but strong expression correlated with poor survival. In general, expression was low among our samples (30/36). It was significantly higher in the tumour samples than in normal samples, and correlated with metastases to lymph nodes in the neck., (Copyright © 2017. Published by Elsevier Ltd.)

TRAP1 is a HSP90 molecular chaperone upregulated in colorectal carcinomas and involved in control of intracellular signaling, cell cycle, apoptosis and drug resistance, stemness and bioenergetics through co-traslational regulation of a network of client proteins. Thus, the clinical significance of TRAP1 protein network was analyzed in human colorectal cancers. TRAP1 and/or its client proteins were quantified, by immunoblot analysis, in 60 surgical specimens of colorectal carcinomas at different stages and, by immunohistochemistry, in 9 colorectal adenomatous polyps, 11 in situ carcinomas and 55 metastatic colorectal tumors. TRAP1 is upregulated at the transition between low- and high-grade adenomas, in in situ carcinomas and in about 60% of human colorectal carcinomas, being downregulated only in a small cohort of tumors. The analysis of TCGA database showed that a subgroup of colorectal tumors is characterized by gain/loss of TRAP1 copy number, this correlating with its mRNA and protein expression. Interestingly, TRAP1 is co-expressed with the majority of its client proteins and hierarchical cluster analysis showed that the upregulation of TRAP1 and associated 6-protein signature (i.e., IF2α, eF1A, TBP7, MAD2, CDK1 and βCatenin) identifies a cohort of metastatic colorectal carcinomas with a significantly shorter overall survival (HR 5.4; 95% C.I. 1.1-26.6; p=0.037). Consistently, the prognostic relevance of TRAP1 was confirmed in a cohort of 55 metastatic colorectal tumors. Finally, TRAP1 positive expression and its prognostic value are more evident in left colon cancers. These data suggest that TRAP1 protein network may provide a prognostic signature in human metastatic colorectal carcinomas.

Background: Colorectal cancer is the major cause of cancer mortality, despite development of therapeutic strategies. The novel marker tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial heat shock protein that has been related to drug resistance and protection from apoptosis in colorectal cancer. This study aims to delineate the clinicopathologic significance of TRAP1 expression in colorectal cancer., Methods: Seven-hundred and fourteen FFPE tissues were collected from colorectal cancer patients who underwent surgery from February 2002 to July 2011 at Dong-A University Medical Center, Busan, South Korea. We performed TRAP1 immunohistochemistry using tissue microarray, and divided into two groups, TRAP1 high expression group and low expression group. Statistical analysis was utilized to evaluate the association of TRAP1 with clinicopathologic characteristics and disease-specific survival of patients., Results: High TRAP1 expression was observed in 564 cases (79%) and low expression was 150 cases (21%). TRAP1 expression was significantly increased in colorectal cancer with advanced pathologic T-stage compared with that in early T-stage (p = 0.008). By univariate survival analysis, high TRAP1 expression was significantly associated with worse disease-specific survival (p = 0.01). But, TRAP1 expression was marginally associated with lymph node involvement and tumor differentiation (p = 0.085, p = 0.082, respectively). Multivariate analysis indicated that TRAP1 expression (hazard ratio, 1.947; 95% CI, 1.270 to 2.984; p = 0.002), and pathologic T stage (hazard ratio, 3.190; 95% CI, 1.275 to 7.983; p = 0.013) were independent prognostic factors for colorectal adenocarcinomas., Conclusions: Here, we found that overexpression of TRAP1 might contribute to tumor cell local invasion of colorectal cancer. The association between TRAP1 overexpression and worse disease-specific survival also suggested that TRAP1 protein expression might have oncogenic role. Consequently, our data demonstrated that TRAP1 expression was a good prognostic biomarker for depth of invasion and disease-specific survival in colorectal cancer.

Heat shock protein 90 is a chaperone molecule that aids in proper folding of target proteins. Recently, heat shock protein 90 was found to play a role in would healing through regulation of fibroblast functions. The aim of the present study was to investigate the role of heat shock protein 90 in collagen synthesis in human dermal fibroblasts. The effects of transforming growth factor-β, 17-N-allylamino-17-demethoxygeldanamycin, and transfection of heat shock protein 90 were evaluated by real-time PCR, western blot, and immunofluorescence assays. The Smad 2/3 and Akt pathways were evaluated to identify the signaling pathways involved in collagen synthesis. Heat shock protein 90 and collagen levels were compared in keloid and control tissues by immunohistochemical analysis. The expression of collagen was significantly increased after treatment with transforming growth factor-β, while 17-N-allylamino-17-demethoxygeldanamycin inhibited transforming growth factor-β-induced collagen synthesis. Overexpression of heat shock protein 90 itself with or without transforming growth factor-β increased collagen synthesis. These effects were dependent on Smad 2/3 pathway signaling. Finally, expression of heat shock protein 90 was increased in keloid tissue compared with control tissues. Taken together, these results demonstrate that modulation of heat shock protein 90 influences transforming growth factor-β-induced collagen synthesis via regulation of Smad 2/3 phosphorylation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Antibiotics are among the most frequently prescribed drugs in human and animal medicine. With antibiotic resistance being a serious threat to veterinary and public health, the prudent use of antibiotics receives much attention. Less well known is that incorrect use of antimicrobial agents may also lead to increased bacterial virulence with the potential of a more severe clinical course of infection. Therefore, the aim of this study was to investigate the effect of subtherapeutic doses of tetracyclines on htpG virulence gene expression in Salmonella Typhimurium and on the course of salmonellosis., Methods: Salmonella strains containing an htpG-luxCDABE transcriptional fusion were constructed. Phenotype microarrays and tetracycline treatment were used to investigate their htpG expression. A Salmonella transposon mutant bank was used to identify genes involved in the induction of htpG gene expression. Finally, the in vitro results were linked to the in vivo situation using a Salmonella mouse model., Results: We demonstrate that subtherapeutic antimicrobial concentrations can exacerbate bacterial infections through direct up-regulation of bacterial virulence factors using Salmonella Typhimurium 112910a phage type 120/ad as a model organism. Phenotype microarrays showed that expression of the Salmonella Typhimurium virulence gene htpG is increased by several tetracycline antimicrobials at values below their MIC, a process that requires intact Salmonella LPS genes. Exposure of experimentally infected DBA/2J mice to subtherapeutic doxycycline concentrations resulted in htpG-mediated exacerbation of Salmonella Typhimurium infection., Conclusions: These findings show that the Salmonella isolate used in this study can respond to subtherapeutic tetracycline pressure by increasing its virulence and disease severity., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

Introduction: Prediction of response to preoperative breast cancer chemotherapy may offer a substantial optimization of medical management of this disease. The most efficient prediction would be done a priori, before the start of chemotherapy and based on the biological features of patient and tumor. Numerous markers have been proposed but none of them has been applied as a routine. The role of MKI67 and HSP90 expression has been recently suggested to predict treatment sensitivity in HER2-positive breast cancer. The aim of this study was to validate the utility of proliferation based markers (MKI67 and CDK1) and heat shock proteins (namely HSP90) to predict response to chemotherapy in cohort of breast cancer patients treated preoperatively., Material and Methods: Ninety-three patients with breast cancer, all females, mean age 42.2 years, among them 32% T1-T2 patients, 49% T3 patients and 13% with T4 tumor stage, 27% N0, 42% N1, 16% N2, 15% N3 were subjected to initial chemotherapy. The majority of patients (86%) received anthracycline and taxane chemotherapy. Among the patients there were 9 individuals with metastatic disease (M1) at initial presentation, and 11 patients were not treated surgically after initial chemotherapy (no sufficient disease response). From 82 patients operated on, 20 patients (24%) showed pathological complete response (pCR), while in 62 patients there was no pCR. 42% of patients were hormone-sensitive HER2-negative, 20% hormone-sensitive HER2-positive, 9% only HER-positive and 29% with triple negative breast cancer. Four gene transcripts (MKI67, cyclin-dependent kinase 1 [CDK1], heat shock proteins HSP90AA1 and HSP- 90AB1) were analyzed in total RNA isolated from single core obtained during preoperative core needle biopsy by quantitative real-time PCR with fluorescent probes (Universal Probe Library, Roche). Results were normalized to the panel of reference genes., Results: There were no statistically significant differences in MKI67 and CDK1 expression between pCR and no pCR groups (p = 0.099 and 0.35, respectively), although the median expression of both genes was slightly higher in pCR group. In contrast, both HSP90AA1 and HSP90AB1 transcripts showed decreased expression in pCR group (medians 0.77 and 0.55) when compared to no p CR group (median 0.86 and 0.73), statistically significant for HSP90AA1 (p = 0.031) and of borderline significance for HSP90AB1 (p = 0.054). The most significant predictor of pCR was the ratio of CDK1 transcript to HSP90AA transcript. This ratio was significantly higher in CR group (median 0.99) than in no CR group (median 0.68, p = 0.0023), and showed a potential diagnostic utility (area under receiver operating characteristic [ROC] curve 0.72)., Conclusions: HSP90AA1 and AB1 genes exhibit low expression in breast cancers highly sensitive to chemotherapy and may indicate the patients with higher probability of pathological complete response. The ratio of HSP90AA1 to proliferation-related markers (CDK1 or MKI67) may be even better predictor of pCR chance, with higher expression of proliferation genes and lower stress response in patients sensitive to chemotherapy.

Background: Mutations in myosin chaperones Unc45b and Hsp90aa1.1 as well as in the Unc45b-binding protein Smyd1b impair formation of myofibrils in skeletal muscle and lead to the accumulation of misfolded myosin. The concomitant transcriptional response involves up-regulation of the three genes encoding these proteins, as well as genes involved in muscle development. The transcriptional up-regulation of unc45b, hsp90aa1.1 and smyd1b is specific to zebrafish mutants with myosin folding defects, and is not triggered in other zebrafish myopathy models., Results: By dissecting the promoter of unc45b, we identify a Heat shock factor 1 (Hsf1) binding element as a mediator of unc45b up-regulation in myofibers lacking myosin folding proteins. Loss-of-function of Hsf1 abolishes unc45b up-regulation in mutants with defects in myosin folding., Conclusions: Taken together, our data show that skeletal muscle cells respond to defective myosin chaperones with a complex gene program and suggest that this response is mediated by Hsf1 activation.

Low-intensity pulsed ultrasound (LIPUS) has demonstrated its positive effects on osteogenic differentiation of mesenchymal stem cells and the proliferation and differentiation of osteoblasts, negative effects on osteoclast growth, and promotion of angiogenesis, leading to improvement of the tissue perfusion. Heat-shock proteins (HSPs) are initially identified as molecules encouraged and expressed by heat stress or chemical stress to cells and involved in the balance between differentiation and apoptosis of osteoblasts. However, it remains unclear if the effect of LIPUS on osteoblast differentiation could involve HSP expression and contribution. In this study, mouse calvarial osteoblasts were exposed to LIPUS at a frequency of 3.0 MHz by 30 mW/cm(2) for 15 min or to 42°C heat shock for 20 min at day 3 of cell culture and examined for osteogenesis with pursuing induction of HSP27, HSP70, and HSP90. LIPUS as well as heat shock initially upregulated HSP90 and phosphorylation of Smad1 and Smad5, encouraging cell viability and proliferation at 24 h, enhancing mineralized nodule formation stronger by LIPUS after 10 days. However, HSP27, associated with BMP2-stimulated p38 mitogen-activated protein kinase during osteoblast differentiation, was downregulated by both stimulations at this early time point. Notably, these two stimuli maintained Smad1 phosphorylation with mineralized nodule formation even under BMP2 signal blockage. Therefore, LIPUS might be a novel inducer of osteoblastic differentiation through a noncanonical signal pathway. In conclusion, LIPUS stimulation enhanced cell viability and proliferation as early as 24 h after treatment, and HSP90 was upregulated, leading to dense mineralization in the osteoblast cell culture after 10 days.

Background: Undifferentiated pleomorphic sarcoma (UPS) is a heterogeneous tumor group, and little is known about molecular target therapy for UPS. Heat shock protein 90 (HSP90) is an expressed chaperone that refolds certain denatured proteins under stress conditions. One of these proteins is Akt. The disruption of Akt signaling plays an important role in tumor progression. The present study's purpose was to analyze the HSP90 expression, Akt/mTOR pathway activation and the correlation between HSP90 expression and its pathway activation in UPS., Methods: The status of HSP90 and the profiles of the Akt/ mTOR pathway were assessed by immunohistochemistry in 79 samples of UPS, and these data were compared with clinicopathological and histopathological findings. The expressions of indicated proteins were assessed by Western blotting in five frozen samples. After treating UPS cells with the HSP90 inhibitor, we assessed the antitumor effect of the inhibitor., Results: Immunohistochemically, phosphorylated Akt (p-Akt), p-mTOR, p-S6RP and p-4EBP were positive in 57.3, 51.9, 54.5 and 57.1% of the UPS samples, respectively. The expressions of those phosphorylated proteins were correlated with each other. HSP90 expression was elevated in 56.4% of the samples and was correlated with p-Akt, p-mTOR and p-S6RP. The immunohistochemical results were confirmed by Western blotting. The HSP90 inhibitor led to decreased viability and invasiveness of the cells and inactivated the AKT/mTOR pathway in vitro., Conclusion: Elevated expression of HSP90 is a poor-prognosis factor and is involved in the activation of the Akt/mTOR pathway in UPS. HSP90 inhibition is a potential treatment option for UPS.

Objective: To investigate the expression and clinical significance of TRAP1 (tumor necrosis factor receptor-associated protein 1) in kidney cancer., Methods: TRAP1 expression was detected in kidney cancer and normal kidney tissues by qRT-PCR and immunohistochemistry (IHC), respectively. Then, the correlation of TRAP1 expression with clinicopathological characters and patients' prognosis was evaluated in kidney cancer., Results: IHC results revealed that the high-expression rates of TRAP1 in kidney cancer tissues and normal kidney tissues were 51.3% (41/80), 23.3% (7/30), and the difference was statistically significant (P=0.01). Also, TRAP1 mRNA level in kidney cancer was found to be significantly greater compared with those in normal kidney by qRT-PCR. In addition, TRAP1 expression in kidney cancer significantly correlated with lymph node metastasis and clinical stage (P<0.05). Kaplan-Meier survival analysis indicated that the mean survival time of patients with TRAP1 low-expression was significantly higher (56 months) than those patients with TRAP1 high-expression (47 months). Meanwhile, Kaplan-Meier and Cox survival analysis indicated that TRAP1, lymph node metastasis and clinical stage were correlated with patients' prognosis., Conclusion: TRAP1 is highly expressed in kidney cancer and correlates with patients prognosis, which may be served as a potential marker for the diagnosis and treatment of kidney cancer.

Aims: The aim of this study was to investigate the expression of Hsp90β and GRP94, and elucidate the clinical significance of their expression, in patients with resectable non-small-cell lung cancer (NSCLC)., Methods and Results: Surgical tissue specimens were obtained from 208 patients with NSCLC who underwent surgical resection. The expression levels of Hsp90β and GRP94 were assessed with tissue microarrays and immunohistochemistry. No correlations were observed between Hsp90β or GRP94 expression and several clinicopathological factors. The high-Hsp90β group [median overall survival (OS) 20.4 months; 95% confidence interval (CI) 0.000-40.864] showed a significant decrease in OS as compared with the low-Hsp90β group (median OS not reached; P = 0.003). In contrast to the Hsp90β analysis, the GRP94 analysis did not show a difference in OS. Moreover, in subgroup analyses of patients with squamous cell carcinoma histology, OS (P = 0.012) and relapse-free survival (P = 0.044) were significantly worse in the high-Hsp90β group than in the low-Hsp90β group. Multivariate analysis suggested that old age [hazard ratio (HR) 1.568; 95% CI 1.019-2.412; P = 0.041], advanced disease (HR 2.066; 95% CI 1.218-3.502; P = 0.007) and high Hsp90β expression (HR 1.802; 95% CI 1.061-3.060; P = 0.029) were independent poor prognostic factors for OS., Conclusions: Hsp90β expression might be a useful marker of poor OS, although further large prospective studies are warranted to validate our findings., (© 2015 John Wiley & Sons Ltd.)

Heat shock protein 90α (Hsp90α), encoded by the HSP90AA1 gene, is the stress inducible isoform of the molecular chaperone Hsp90. Hsp90α is regulated differently and has different functions when compared to the constitutively expressed Hsp90β isoform, despite high amino acid sequence identity between the two proteins. These differences are likely due to variations in nucleotide sequence within non-coding regions, which allows for specific regulation through interaction with particular transcription factors, and to subtle changes in amino acid sequence that allow for unique post-translational modifications. This article will specifically focus on the expression, function and regulation of Hsp90α., (Published by Elsevier B.V.)

Radiotherapy is an essential part of multi-modal treatment for soft tissue sarcomas. Treatment failure is commonly attributed to radioresistance, but comprehensive analyses of radiosensitivity are not available, and suitable biomarkers or candidates for targeted radiosensitization are scarce. Here, we systematically analyzed the intrinsic radioresistance of a panel of soft tissue sarcoma cell lines, and extracted scores of radioresistance by principal component analysis (PCA). To identify molecular markers of radioresistance, transcriptomic profiling of DNA damage response regulators was performed. The expression levels of HSP90 and its clients ATR, ATM, and NBS1 revealed strong, positive correlations with the PCA-derived radioresistance scores. Their functional involvement was addressed by HSP90 inhibition, which preferentially sensitized radioresistant sarcoma cells and was accompanied by delayed γ-H2AX foci clearance and HSP90 client protein degradation. The induction of apoptosis and necrosis was not significantly enhanced, but increased levels of basal and irradiation-induced senescence upon HSP90 inhibition were detected. Finally, evaluation of our findings in the TCGA soft tissue sarcoma cohort revealed elevated expression levels of HSP90, ATR, ATM, and NBS1 in a relevant subset of cases with particularly poor prognosis, which might preferentially benefit from HSP90 inhibition in combination with radiotherapy in the future., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study aims to investigate the effect of heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in the malignant pheochromocytoma using a xenograft mouse model. Treatment with 17-AAG induced a marked reduction in the volume and weight of PC12 pheochromocytoma cell tumor xenografts in mice. Furthermore, 17-AAG also significantly inhibited the expression of HSP90 and its client proteins. Our results validated HSP90 as an important target in pheochromocytoma and provided rationale for the testing of HSP90 inhibitors as a promising therapeutic agent in the antitumor therapy of pheochromocytoma.

Heat shock protein 90 (HSP90), a highly conserved and multi-functional molecular chaperone, plays an essential role in cellular metabolism and stress response. In this study, HSP90 cDNA named MaHSP90 was cloned from Wuchang bream (Megalobrama amblycephala) gills by using rapid amplification of cDNA ends. The full-length MaHSP90 cDNA is 2674 bp and consists of a 3',5'-untranslated region and a 2250-bp open reading frame encoding a 750-amino acid long protein. Identity analysis revealed that the amino acid sequence of MaHSP90 is highly conserved. Homology analysis and structure comparison further indicated that MaHSP90 should be the β isoform member of the HSP90 family. MaHSP90 mRNA was ubiquitously expressed in the liver, heart, muscle, gill, intestine, kidney, and brain. The MaHSP90 mRNA levels under nitrite stress were analyzed using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR); the mRNA levels significantly increased at 3, 6, and 12 h after nitrite exposure in the gills and then stabilized between 24 and 48 h. Furthermore, a similar relationship between mRNA expression (qRT-PCR) and HSP90 protein levels (densitometric band analysis) was found. Transcriptional analysis of caspase-8 and caspase-9 expression in the gills of juvenile M. amblycephala after a 48-h exposure to nitrite suggested that MaHSP90 expression is related positively with nitrite-induced apoptosis. Fish exposed to nitrite also showed gill damage. Our results suggest that MaHSP90 mRNA is constitutively expressed in various tissues and inducible in the gills under nitrite stress, suggesting its important role in nitrite stress response.

Humic acid (HA) has been implicated as a contributory factor for blackfoot disease, which is an endemic peripheral vascular disease. We investigated the effect of HA on the regulation of endothelial nitric oxide (NO) synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) to evaluate the involvement of eNOS and related factors in peripheral vascular impairment with HA exposure. Treatment of HUVECs with HA induced upregulation of eNOS. This result coincides with those of previous studies. Furthermore this is the first study to report that HA induces upregulation of heat shock protein (Hsp)90α, Hsp90β, eNOS phosphorylation at Ser1177, and eNOS phosphorylation at Thr495, as compared to that in the control. In contrast, treatment with BAPTA, an intracellular Ca(2+) chelator, inhibited upregulation of these proteins induced by HA. This study demonstrates that HA treatment leads to increases in both Hsp90α and Hsp90β proteins and indicates that Hsp90α leads to eNOS phosphorylation at Ser1177 and that Hsp90β leads to eNOS phosphorylation at Thr495, respectively. Upregulation of eNOS, Hsp90α, and Hsp90β in HUVECs is regulated by intracellular Ca(2+) accumulation induced by HA. These results suggest that upregulation of eNOS phosphorylation at Ser1177 and eNOS phosphorylation at Thr495 produce NO and superoxide anions, respectively, resulting in generation of peroxynitrite, which causes impairment of vascular endothelial cells., (© 2013 Wiley Periodicals, Inc.)

Iron is an essential cellular nutrient, being a critical cofactor of several proteins involved in cell growth and replication. Compared with normal cells, neoplastic cells have been shown to require a greater amount of iron, thus laying the basis for the promising anticancer activity of iron chelators. In this work, we evaluated the effects of molecules with iron chelation activity on neuroblastoma (NB) cell lines. Of the 17 iron chelators tested, six reduced cell viability of two NB cell lines with an inhibition of growth of 50% below 10 µM; four of the six molecules-ciclopirox olamine (CPX), piroctone, 8-hydroxyquinoline, and deferasirox-were also shown to efficiently chelate intracellular iron within minutes after addition. Effects on cell viability of one of the compounds, CPX, were indeed dependent on chelation of intracellular iron and mediated by both G0/G1 cell cycle block and induction of apoptosis. By combined transcriptome and translatome profiling we identified early translational downregulation of several members of the heat shock protein group as a specific effect of CPX treatment. We functionally confirmed iron-dependent depletion of HSP90 and its client proteins at pharmacologically achievable concentrations of CPX, and we extended this effect to piroctone, 8-hydroxyquinoline, and deferasirox. Given the documented sensitivity of NB cells to HSP90 inhibition, we propose CPX and other iron chelators as investigational antitumor agents in NB therapy., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Tumour cell-derived heat shock proteins (HSPs) are used as vaccines for immunotherapy of cancer patients. However, it is proposed that the peptide chaperoned on HSPs, not HSPs themselves, elicited a potent immune response. Given that HSPs are highly expressed by most myeloma cells and vital to myeloma cell survival, we reasoned that HSPs themselves might be an ideal myeloma antigen. In the present study, we explored the feasibility of targeting HSPs themselves for treating multiple myeloma. We identified and chose HLA-A*0201-binding peptides from human HSPB1 (HSP27) and HSP90AA1 (HSP90), and confirmed their immunogenicity in HLA-A*0201 transgenic mice. Dendritic cells pulsed with HSPB1 and HSP90AA1 peptides were used to stimulate peripheral blood mononuclear cells from healthy volunteers and myeloma patients to generate HSP peptide-specific cytotoxic T lymphocytes (CTLs). HSP peptide-specific CTLs efficiently lysed HLA-A*0201(+) myeloma cells (established cell lines and primary plasma cells) but not HLA-A*0201(-) myeloma cells in vitro, indicating that myeloma cells naturally express HSP peptides in the context of major histocompatibility complex class I molecules. More importantly, HSP peptide-specific CTLs effectively reduced tumour burden in the xenograft mouse model of myeloma. Our study clearly demonstrated that HSPs might be novel tumour antigens for immunotherapy of myeloma., (© 2014 John Wiley & Sons Ltd.)

Background: Heat shock protein (Hsp) 90 is a key regulator of various oncogene products and cell-signaling molecules, while Hsp70 protects against heat-induced apoptosis. We previously described a system in which hyperthermia was produced using thermosensitive ferromagnetic particles (FMPs) with a Curie temperature (T c) of 43 °C to mediate automatic temperature control, and demonstrated its antitumor effect in a mouse melanoma model. In the present study, the antitumor effects of combining Hsp90 inhibitor (17DMAG) and Hsp70 inhibitor (quercetin) with FMP-mediated hyperthermia were examined., Methods: Expressions of Hsp90/70 and Akt were evaluated using Western blotting in vitro. In an in vivo study, melanoma cells were subcutaneously injected into the backs of C57BL/6 mice. FMPs were then injected into the resultant tumors, and the mice were divided into groups treated with quercetin and/or 17DMAG with/without hyperthermia. When exposed to a magnetic field, the temperature of tissues containing FMPs increased and stabilized at the T c. The TUNEL method was used to determine whether hyperthermia induced apoptosis within tumors., Results: In the group pretreated with hyperthermia + quercetin + 17DMAG, Akt expression was reduced in vitro, the incidence of apoptosis within tumors was greater, and tumor growth was significantly suppressed 20 days after FMP injection in vivo, compared with other treatment groups. The survival rates among tumor-bearing mice observed for a period of 40 days were significantly higher in the hyperthermia + quercetin + 17DMAG group., Conclusion: Combining Hsp90/70 inhibition with hyperthermia appears to increase their antitumor effects. Thus, the combination of FMP-mediated, self-regulating hyperthermia with Hsp90/70 inhibition has important implications for cancer treatment.

Heat shock protein 90 (Hsp90) is a molecular chaperone for numerous client proteins, many of which are crucial for the pathogenesis of non-small cell lung cancers (NSCLCs). To date, therapeutic approaches using epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib or erlotinib for the treatment of NSCLCs have been limited due to the emergence of acquired drug resistance mainly mediated by a secondary T790M mutation in EGFR. Considering this, Hsp90 inhibition seems promising as it leads to overall degradation of the oncogenic EGFR family proteins. In this regard, the present study provides the preclinical basis for a new Hsp90 inhibitor, WK88-1, for the treatment of NSCLCs harboring the T790M mutation in EGFR. Our data revealed that inhibition of Hsp90 by WK88-1 induced overall degradation of multiple oncogenic signaling molecules including EGFR, ErbB2 and ErbB3, leading to subsequent growth arrest and apoptosis in the gefitinib-resistant H1975 cell line. In addition, treatment with WK88-1 markedly inhibited proliferation, migration and invasion in H1975 cells. Moreover, an in vivo xenograft assay indicated that WK88-1 markedly suppressed tumor growth in the H1975 xenografts, highlighting the potential efficacy of WK88-1 for overcoming gefitinib resistance in NSCLCs harboring the T790M mutation in EGFR.

Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-μ (PFT-μ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-μ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-μ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-μ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer.

Pancreatic cancer (PC) is an aggressive and devastating disease with a dismal prognosis. The study aimed to investigate the role of HSP90α and PDIA3 in patients with PC. Immunohistochemistry was performed on tissue microarrays containing 186 pairs of PC and normal pancreatic tissues to assess the expression levels of HSP90α and PDIA3. The expression levels of cytoplasmic HSP90α (P = 0.032) and PDIA3 (P = 0.043) in PCs were significantly higher than those in normal pancreas tissues, but nuclear HSP90α showed lower expression in PC tissues (P = 0.002). In addition, cytoplasmic expression of HSP90α and PDIA3 was significantly associated with perineural invasion (PNI) (P = 0.004) and sex (P = 0.014), respectively. These results indicate that cytoplasmic HSP90α may serve as a biomarker for PNI in PCs.

Unlabelled: The TNF receptor-associated protein 1 (TRAP1) is a mitochondrial HSP that has been related to drug resistance and protection from apoptosis in colorectal and prostate cancer. Here, the effect of TRAP1 ablation on cell proliferation, survival, apoptosis, and mitochondrial function was determined in non-small cell lung cancer (NSCLC). In addition, the prognostic value of TRAP1 was evaluated in patients with NSCLC. These results demonstrate that TRAP1 knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1 downregulation impairs mitochondrial functions such as ATP production and mitochondrial membrane potential as measured by TMRM (tetramethylrhodamine methylester) uptake, but it does not affect mitochondrial density or mitochondrial morphology. The effect of TRAP1 silencing on apoptosis, analyzed by flow cytometry and immunoblot expression (cleaved PARP, caspase-9, and caspase-3) was cell line and context dependent. Finally, the prognostic potential of TRAP1 expression in NSCLC was ascertained via immunohistochemical analysis which revealed that high TRAP1 expression was associated with increased risk of disease recurrence (univariate analysis, P = 0.008; multivariate analysis, HR: 2.554; 95% confidence interval, 1.085-6.012; P = 0.03). In conclusion, these results demonstrate that TRAP1 impacts the viability of NSCLC cells, and that its expression is prognostic in NSCLC., Implications: TRAP1 controls NSCLC proliferation, apoptosis, and mitochondrial function, and its status has prognostic potential in NSCLC., Competing Interests: of potential conflicts of interest: No potential conflicts of interest are disclosed, (©2014 AACR.)

The objective of this study was to investigate the mechanism of heat shock protein 90 alpha (Hsp90α) protection against heart damage resulting from heat stress by detecting Hsp90α mRNA, Hsp90α protein, protein localization, and cell damage in primary myocardial cells of neonatal rats in response to heat stress in vitro. The cells were heat-stressed at 42°C in an incubator with 95% air and 5% CO2 for different periods. Levels of Hsp90α, protein localization, enzymes, and cytopathological lesions were detected using Western blot, immunocytochemistry enzymatic assays, and cytopathological techniques. Aspartate aminotransferase, lactate dehydrogenase, and creatine kinase enzyme levels were elevated during heat stress, and acute cellular lesions that were characterized by vacuolar degeneration and necrosis were observed. Hsp90α levels decreased between 10 and 60 min of heat stress and increased after 360 and 480 min, while Hsp90α mRNA decreased after 360 min. These results indicate that heat stress might induce irreversible damage in certain myocardial cells. The elevated Hsp90α level at the end of heat stress and its positive signal in the cytoplasm of myocardial cells after heat stress could be associated with its protective role. Additionally, the consumption of Hsp90α exceeded its production in the first period of treatment.

Multiple human proteins have been shown to both support and restrict viral replication, and confirmation of virus-associated changes in the expression of these genes is relevant for future therapeutic efforts. In this study a well-characterized panel of 49 individuals either infected with HIV-1 or uninfected was compiled and analyzed for the effect of HIV infection status, viral load, and antiretroviral treatment on specific gene expression. mRNA was extracted and reverse transcribed from purified CD4+ cells, and quantitative real-time PCR was utilized to scrutinize differences in the expression of four host genes that have been demonstrated to either stimulate (HSP90 and LEDGF/p75) or restrict (p21/WAF1 and APOBEC3G) proviral integration. HIV infection status was associated with slight to moderate alterations in the expression of all four genes. After adjusting for age, mRNA expression levels of HSP90, LEDGF/p75 and APOBEC3G were found to all be decreased in infected patients compared to healthy controls by 1.43-, 1.26-, and 4.71-fold, respectively, while p21/WAF1 expression was increased 2.35-fold. Furthermore, individuals receiving raltegravir exhibited a 1.28-fold reduction in LEDGF/p75 compared to those on non-raltegravir antiretroviral treatment. Identification of these and similar HIV-induced changes in gene expression may be valuable for delineating the extent of host cell molecular mechanisms stimulating viral replication., (© 2014 Wiley Periodicals, Inc.)

Invasive aspergillosis is a deadly infection for which new antifungal therapies are needed. Heat shock protein 90 (Hsp90) is an essential chaperone in Aspergillus fumigatus representing an attractive antifungal target. Using a thiamine-repressible promoter (pthiA), we showed that genetic repression of Hsp90 significantly reduced virulence in a murine model of invasive aspergillosis. Moreover, substituting the A. fumigatus hsp90 promoter with 2 artificial promoters (potef, pthiA) and the Candida albicans hsp90 promoter resulted in hypersensitivity to caspofungin and abolition of the paradoxical effect (resistance at high caspofungin concentrations). By inducing truncations in the hsp90 promoter, we identified a 100-base pair proximal sequence that triggers a significant increase of hsp90 expression (≥1.5-fold) and is essential for the paradoxical effect. Preventing this increase of hsp90 expression was sufficient to abolish the paradoxical effect and therefore optimize the antifungal activity of caspofungin.