Inducible Hsp70 Research Articles

Oxidative stress, immunological response, and heat shock proteins induction in the Chinese Mitten Crab, Eriocheir sinensis following avermectin exposure.

In this study, the Chinese mitten crabs, Eriocheir sinensis were exposed to avermectin at 0.03, 0.06, 0.12, 0.24, and 0.48 mg/L respectively for 96 hours. The results showed that levels of superoxide dismutase, catalase, and glutathione peroxidase in hepatopancreas were slightly induced at concentration of 0.03 and 0.06 mg/L, but significantly (P < .05) decreased at higher concentrations, meanwhile similar trend of the activities of acid phosphatase, alkaline phosphatase and lysozyme were observed. Significant induction of HSP70 and HSP90 mRNA expression was detected at 24 hours whereas no significant change was found in HSP60. In addition, levels of reactive oxygen species in hepatocytes increased in dose- and time- dependent manners, and cell viabilities of hepatocytes and haemocytes decreased. These results indicated that sublethal concentration exposure of avermectin had a prominent oxidative stress effect on E. sinensis based on the antioxidative and immunological activity inhibition, and HSP60, 70, and 90 perform a protective response during the exposure.

HSP90 as a novel therapeutic target for posterior capsule opacification

Posterior capsule opacification (PCO) is a common complication of cataract surgery, resulting from a combination of proliferation, migration, epithelial-mesenchymal transition (EMT) of residual capsular epithelial cells and fibrosis of myofibroblasts. HSP90 is known to regulate the proteostasis of cells under pathophysiological conditions. The role of HSP90 in PCO formation, however, is not clear. To do this, the lens epithelial cell lines and an ex vivo cultured rat capsular bag model were used to study the role of HSP90 in PCO formation. The expression of protein and mRNA was measured by immunoblotting and quantitative RT-PCR, and cell apoptosis was measured by TUNEL(TdT-mediated dUTP nick-end labeling). The cell proliferation was measured by cell viability assays. The results showed that 17-AAG (Tanespimycin), an inhibitor of HSP90, suppresses the proliferation of immortalized lens epithelial cell lines HLE-B3, SRA01/04, and mLEC, with IC50 values of 0.27, 0.27, and 0.49 μM, respectively. In an ex vivo cultured rat capsular model, the capsular residual epithelial cells resisted the stress of the capsulorhexis surgery and took 3–6 days to completely overlay the capsular posterior wall. During this process, heat shock factor 1 and its downstream targets HSP90, HSP25, αB-crystallin, and HSP40 were upregulated. Treatment with 17-AAG inhibited the viability of capsular residual epithelial cells and induced the cells apoptosis, characterized by increases in ROS levels, apoptotic DNA injury, and the activation of caspases 9 and 3. HSP90 participated in regulating both EGF receptor (EGFR) and TGF receptor (TGFR) signaling pathways. HSP90 was found to interact with the EGFR, such that inhibition of HSP90 by 17-AAG destabilized the EGFR protein and suppressed p-ERK1/2 and p-AKT levels. 17-AAG also inhibited the TGF-β-induced phosphorylation of SMAD2/3 and ERK1/2 and the decrease in E-cadherin and ZO-1 expression. Accordingly, these data suggest that the induction of HSP90 protects capsular residual epithelial cells against capsulorhexis-induced stress and participates in regulating the processes of proliferation, EMT and migration of rat capsular residual epithelial cells, at least partly, through the EGFR and TGFR signaling pathways. Treatment with 17-AAG suppresses PCO formation and is therefore a potential therapeutic candidate for PCO prevention.

4-HNE induces proinflammatory cytokines of human retinal pigment epithelial cells by promoting extracellular efflux of HSP70

Oxidative stress and subsequent chronic inflammation result in dysfunction of the retinal pigment epithelium (RPE) and represent therapeutic targets in the context of age-related macular degeneration (AMD). However, molecular mechanisms that linked oxidative stress and inflammation still unclear. As an important byproduct of oxidative stress, 4-hydroxynonenal (4-HNE) induces apoptosis and lysosome dysregulation of RPE cells. In the present study, we evaluated cytokines production of RPE cells induced by 4-HNE by using cytokine array and confirmed that 4-HNE induced IL-6, IL-1β and TNF-α production in a concentration dependent manner. Specifically, 4-HNE also induced IL-10 and TGF-β production in low concentration. Molecular analysis revealed that intracellular HSP70 inhibited 4-HNE-induced production of pro-inflammatory cytokines, and 4-HNE exerted proinflammatory effects in RPE cells by enhancing extracellular release of HSP70, as efflux inhibitor Methyl-β-cyclodextrin (MBC) treatment significantly blocked the release of HSP70 and decreased IL-6 production of RPE cells induced by 4-HNE. Meanwhile, HSP70 inducer arimoclomol increased intracellular HSP70 production, but showed no influence on its extracellular level, also performed anti-inflammatory effects in 4-HNE-stimulated RPE cells. Whereas the anti-inflammatory effects of paeoniflorin, an HSP70 inducer simultaneously promoted its extracellular efflux, was lower than arimoclomol. In addition, we further confirmed that MBC exhibited synergetic effect with both paeoniflorin and arimoclomol to inhibit the production of proinflammatory cytokines induced by 4-HNE. Taken together, these results indicate that HSP70 plays a vital role in regulating inflammation of RPE cells induced by oxidative stress and might be a potential novel target for clinical treatment of AMD.

Protective Effects of Propolis Extract in a Rat Model of Traumatic Brain Injury via Hsp70 Induction.

BACKGROUND:Traumatic brain injury (TBI) is one of the major global health problems. Secondary brain injury is a complex inflammation cascades process that causes brain cell apoptosis. Propolis is a natural product that has neuroprotective property.AIM:This study aimed to investigate the effect of propolis toward Hsp70 expression with apoptosis marker in brain tissue after TBI.METHODS:Thirty-three Sprague Dawley rats were randomised into three treatments group, i.e. sham-operated controls, closed head injury (CHI), and CHI with propolis extract (treatment group). In the treatment group, propolis was given 200 mg/kg per oral for 7 days then harvested brain tissues after sacrificed by cervical dislocation at day 8. We investigated Hsp70, Caspase 3, apoptosis-inducing factor (AIF), and TUNEL assay expression using immunohistochemistry staining. Statistical test using one-way ANOVA test and Tukey HSD as post hoc test.RESULTS:Mean of positive Hsp70 stained cells in group 1 was 6.82 ± 2.14, group 2 was 3.91 ± 2.26, and group 3 was 9.64 ± 3.53 with a significant difference of Hsp70 expression distribution within groups (p = 0.0001). Mean of positive caspase 3 stained cells in group 1 was 5.45 ± 2.30, group 2 was 13.82 ± 2.44, and group 3 was 7.03 ± 1.54 with a significant difference of caspase3 expression distribution within groups (p=0.0001). Mean of positive AIF stained cells in group 1 was 5.36 ± 2.11, group 2 was 12.82 ± 1.40, and group 3 was 8.09 ± 1.81 with a significant difference of AIF expression distribution within groups (p = 0.0001). Mean of positive TUNEL assay stained cells in group 1 was 4.82 ± 2.04, group 2 was 11.55 ± 1.51, and group 3 was 7.64 ± 1.96 with a significant difference of TUNEL test expression distribution within groups (p = 0.0001).CONCLUSION:Propolis may protect brain cell from apoptosis after injury by maintaining Hsp70 expression in addition to antioxidant and anti-inflammatory.

Hsp70 inhibitors: Implications for the treatment of colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies in the world. Despite intensive advances in diagnosis and treatment of CRC, it is yet one of the leading cause of cancer related morbidity and mortality. Therefore, there is an urgent medical need for alternative therapeutic approaches to treat CRC. The 70 kDa heat shock proteins (Hsp70s) are a family of evolutionary conserved heat shock proteins, which play an important role in cell homeostasis and survival. They overexpress in various types of malignancy including CRC and are typically accompanied with poor prognosis. Hence, inhibition of Hsp70 may be considered as a striking chemotherapeutic avenue. This review summarizes the current knowledge on the progress made so far to discover compounds, which target the Hsp70 family, with particular emphasis on their efficacy in treatment of CRC. We also briefly explain the induction of Hsp70 as a strategy to prevent CRC.

Amide-tethered quinoline-resorcinol conjugates as a new class of HSP90 inhibitors suppressing the growth of prostate cancer cells

The study is focused on the design and synthesis of amide tethered quinoline-resorcinol hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led to the identification of compound 11, which possesses potent cell growth inhibitory effects against HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid residues of the HSP90 chaperone protein were determined.

Effect of Feeding Strategies on Molecular Responses of Biotransformation Genes in Crassostrea gigas Exposed to Cadmium

Heavy metals, including cadmium (Cd), are widespread pollutants of great environmental concern because of their accumulation and toxicity. Environmental conditions can affect the accumulation and elimination of Cd in organisms. The aim of this study is to understand the respective responses of metallothioneins (MTs), P-glycoprotein (P-gp), and heat shock protein 70 (hsp70) genes in the mantle and digestive gland of the oyster Crassostrea gigas exposed to 10 µg L−1 Cd for 28 days, followed by a depuration period of 35 days under different feeding strategies. The MT expression in the digestive gland was higher than in mantle, and was induced in a fluctuating pattern throughout the time of the whole experiment when the oyster was exposed to Cd. The results indicated an enhanced MT activity is required for Cd detoxification. Expression levels of P-gp in the mantle of Cd-exposed oysters fed with algae increased in the first two weeks of the accumulation phase. Then the induction weakened, but the expression still increased significantly compared with the control group fed without algae. The P-gp levels in the digestive gland were under-regulated during both the contamination and decontamination periods except in the group fed with Dicrateria inornata at day 0.083. The induction of P-gp is possibly related to the important role of pumping Cd out of the cell, while a major implication of P-gp expression inhibition would be a disruption of the protein synthesis. Hsp70 expression exhibited an overall decreasing trend. The highest relative hsp70 inhibition occurred during day 42 to day 63, with approximately 4-fold and 10-fold lower hsp70 levels as compared to the C.gigas feeding with no algae in the mantle and digestive gland, respectively. The induction of hsp70 may be explained by the refolding of protein in presence of Cd, followed by the inhibition of hsp70 when the protein synthesis was disrupted.

Expression of thermal tolerance genes in two Drosophila species with different acclimation capacities.

Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. We predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, we compared basal levels of gene expression at different developmental temperatures within each of the two species. Furthermore, we explored the heat hardening dynamics by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster we found an indication of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively.

Description of strongly heat-inducible heat shock protein 70 transcripts from Baikal endemic amphipods

Heat shock proteins/cognates 70 are chaperones essential for proper protein folding. This protein family comprises inducible members (Hsp70s) with expression triggered by the increased concentration of misfolded proteins due to protein-destabilizing conditions, as well as constitutively expressed cognate members (Hsc70s). Previous works on non-model amphipod species Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, both endemic to Lake Baikal in Eastern Siberia, have only revealed a constitutively expressed form, expression of which was moderately further induced by protein-destabilizing conditions. Here we describe heat-inducible hsp70s in these species. Contrary to the common approach of using sequence similarity with hsp/hsc70 of a wide spectrum of organisms and some characteristic features, such as absence of introns within genes and presence of heat shock elements in their promoter areas, the present study is based on next-generation sequencing for the studied or related species followed by differential expression analysis, quantitative PCR validation and detailed investigation of the predicted polypeptide sequences. This approach allowed us to describe a novel type of hsp70 transcripts that overexpress in response to heat shock. Moreover, we propose diagnostic sequence features of this Hsp70 type for amphipods. Phylogenetic comparisons with different types of Hsp/Hsc70s allowed us to suggest that the hsp/hsc70 gene family in Amphipoda diversified into cognate and heat-inducible paralogs independently from other crustaceans. Thus, the cognate and inducible hsp70 types in distant taxa may not be recognized by sequence similarity.

Biologically sound formal model of Hsp70 heat induction

A proper response to rapid environmental changes is essential for cell survival and requires efficient modifications in the pattern of gene expression. In this respect, a prominent example is Hsp70, a chaperone protein whose synthesis is dynamically regulated in stress conditions. In this paper, we expand a formal model of Hsp70 heat induction originally proposed in previous articles. To accurately capture various modes of heat shock effects, we not only introduce temperature dependencies in transcription to Hsp70 mRNA and in dissociation of transcriptional complexes, but we also derive a new formal expression for the temperature dependence in protein denaturation. We calibrate our model using comprehensive sets of both previously published experimental data and also biologically justified constraints. Interestingly, we obtain a biologically plausible temperature dependence of the transcriptional complex dissociation, despite the lack of biological constraints imposed in the calibration process. Finally, based on a sensitivity analysis of the model carried out in both deterministic and stochastic settings, we suggest that the regulation of the binding of transcriptional complexes plays a key role in Hsp70 induction upon heat shock. In conclusion, we provide a model that is able to capture the essential dynamics of the Hsp70 heat induction whilst being biologically sound in terms of temperature dependencies, description of protein denaturation and imposed calibration constraints.

Regional differences in the inflammatory and heat shock response in glia: implications for ALS

Preferential neuronal vulnerability is characteristic of several neurodegenerative diseases including the motor neuron disease amyotrophic lateral sclerosis (ALS). It is well established that glia play a critical role in ALS, but it is unknown whether regional differences in the ability of glia to support motor neurons contribute to the specific pattern of neuronal degeneration. In this study, using primary mixed glial cultures from different mouse CNS regions (spinal cord and cortex), we examined whether regional differences exist in key glial pathways that contribute to, or protect against, motor neuron degeneration. Specifically, we examined the NF-κB-mediated inflammatory pathway and the cytoprotective heat shock response (HSR). Glial cultures were treated with pro-inflammatory stimuli, tumour necrosis factor-ɑ/lipopolysaccharide or heat stressed to stimulate the inflammatory and HSR respectively. We found that spinal cord glia expressed more iNOS and produced more NO compared to cortical glia in response to inflammatory stimuli. Intriguingly, we found that expression of ALS-causing SOD1G93A did not elevate the levels of NO in spinal cord glia. However, activation of the stress-responsive HSR was attenuated in SOD1G93A cultures, with a reduced Hsp70 induction in response to stressful stimuli. Exposure of spinal cord glia to heat shock in combination with inflammatory stimuli reduced the activation of the inflammatory response. The results of this study suggest that impaired heat shock response in SOD1G93A glia may contribute to the exacerbated inflammatory reactions observed in ALS mice.Graphical abstractMixed primary glial cultures were established from cortical and spinal cord regions of wild-type mice and mice expressing ALS–causing mutant human SOD1 and the inflammatory and heat shock responses were investigated in these cultures. In the absence of stress, all cultures appeared to have similar cellular composition, levels of inflammatory mediators and similar expression level of heat shock proteins. When stimulated, spinal cord glia were more reactive and activated the inflammatory pathway more readily than cortical glia; this response was similar in wild-type and SOD1G93A glial cultures. Although the heat shock response was similar in spinal cord and cortical glial, in SOD1G93A expressing glia from both the spinal cord and cortex, the induction of heat shock response was diminished. This impaired heat shock response in SOD1G93A glia may therefore contribute to the exacerbated inflammatory reactions observed in ALS mice.

1863-P: The Hepatic Role of Heat Shock Protein 72 in the Pathogenesis of Type 2 Diabetes

Cell stresses, such as ER stress or oxidative stress are considered to be key mediators in pathophysiology of type 2 diabetes. Heat shock protein (HSP) 72, a major inducible HSP against heat, serves to protect cells from those cellular stresses and works in protein quality control. Induction of HSP72 improves glucose intolerance in type 2 diabetic model mice. In this study, we investigated glucose metabolism in whole body HSP72 deficient (KO) mice. The effects of hepatic HSP72 induction in KO mice were also investigated. KO mice and control mice were subjected to a high-fat diet regimen. Metabolic parameters and pathophysiological examination were performed. HSP72 was overexpressed in mice by lenti-virus to investigate the role of HSP72 in liver. There were no significant differences in body weight and food intake between two groups. Fasting and random-fed blood glucose levels were significantly higher in KO mice than those in control mice. The fasting insulin level was significantly lower in KO mice. Upon glucose challenge, blood glucose levels were higher in KO mice. On insulin tolerance test, KO mice exhibited insulin resistant phenotype. Visceral fat mass was increased and hepatic steatosis was obvious in KO mice with increased SREBP-1c mRNA expression. Upon insulin stimulation, phosphorylation of Akt was decreased in KO liver, with increased activation of JNK. Hepatic gluconeogenesis was not suppressed in KO accompanied by increased gluconeogenic enzymes. When restoring HSP72 expression in liver by lenti-virus on KO mice, blood glucose, hepatic steatosis and gluconeogenesis were ameliorated. Interestingly, visceral fat size and macrophage infiltration were both reversed by hepatic HSP72 induction. Deficiency of HSP72 leads to type 2 diabetic phenotype. As induction of HSP72 in liver is beneficial to improve glucose homeostasis in KO, the abundance of HSP72 in liver is critical in diabetic pathophysiology, and appropriate HSP72 induction may serve as a target in the treatment of type 2 diabetes. Disclosure T. Kondo: None. S. Kitano: None. N. Miyakawa: None. T. Watanabe: None. R. Goto: None. K. Ono: None. M. Igata: None. J. Kawashima: None. H. Motoshima: None. T. Matsumura: None. E. Araki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Merck &amp; Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi. Funding Japan Society for the Promotion of Science

Heat Shock Protein 70 Messenger RNA in Rat Leukocytes Elevates After Severe Intestinal Ischemia–Reperfusion

BackgroundHeat shock protein 70 (HSP70) confers protection against heat shock, oxidative stress, infection, and inflammation in many cell types. A recent study reported that the induction of HSP70 was associated with morphologic protection against ischemia–reperfusion injury (IRI) in the rat small intestine. This study investigated the dynamics of HSP70 in leukocytes during intestinal IRI in a rat model. Materials and methodsSerial blood samples were collected at 60-minute intervals up to 240 min from male Wistar rats (n = 15). The rats were divided into three groups of five each: the control group, the nonlethal IRI group, and the lethal IRI group. Rats belonging to the control group underwent a sham operation, and laparotomy was performed on rats in the lethal and nonlethal IRI groups. The nonlethal group experienced a 30-minute clamping of the superior mesenteric artery, and the lethal group experienced a 75-minute clamping of the superior mesenteric artery. The expression of HSP70 messenger RNA (mRNA) in leukocytes was measured by real-time quantitative polymerase chain reaction. Mixed-effects modeling of repeated measures was used to carry out the statistical analysis. The Bonferroni correction was applied to multiple comparisons. A P value < 0.0167 was considered to indicate statistical significance. ResultsThe expression of HSP70 mRNA in leukocytes increased 60 min after reperfusion in both IRI groups, and it was 12.8 times higher in the lethal group and 3.6 times higher in the nonlethal group compared with the control group. The expression of mRNA in the lethal group was significantly increased compared with the nonlethal group and the control group at 120 and 180 min after reperfusion. At 120 min after reperfusion, the expression of HSP70 mRNA was 6.1 times higher in the lethal group than in the nonlethal group (P = 0.0075) and 17.7 times higher than in the control group (P = 0.0011). At 180 min after reperfusion, the expression of HSP70 mRNA was 6.8 times higher in the lethal group than in the nonlethal group (P = 0.0007) and 4.3 times higher than in the control group (P = 0.0032). Although the expression of HSP70 mRNA in the nonlethal group was elevated in the early stages of reperfusion, there was no difference between the nonlethal group and the control group (P = 0.0212 at 60 min). ConclusionsThe expression of HSP70 mRNA in leukocytes may be a clinically useful indicator for evaluating pathologic conditions in intestinal IRI.

Phase I trial of the combination of the heat shock protein-90 inhibitor onalespib (AT13387) and the cyclin-dependent kinase inhibitor AT7519M in patients with advanced solid tumors.

2619 Background: The 90kDa heat shock protein (HSP90) participates in the folding, stabilization, activation, and proteolytic turnover of aberrant proteins that contribute to the growth and survival of cancer cells. HSP90 inhibition leads to degradation of these aberrant proteins through the ubiquitin-proteosome pathway, allowing for simultaneous targeting of multiple pathways. Inhibition of HSP90 alone stimulates a compensatory upregulation of HSP70. The transcriptional induction of HSP70 has been linked to the activity of CDK9. Combined inhibition of onalespib-mediated HSP90 inhibition and AT7519-mediated CDK9 inhibition has demonstrated synergistic anti-tumor activity in preclinical models at NOAEL doses, justifying a Phase 1 study. Methods: We conducted an open-label phase 1 study following a 3+3 trial design. Patients received a 1-week lead-in of onalespib alone (C0), followed by onalespib/AT7519M on days 1,4,8, and 11 of a 21-day cycle. Pharmacokinetic samples were obtained on C0D1 after onalespib alone, and on C1D1 and D11 with the combination. HSP70 protein levels were analyzed in PBMC and plasma samples collected at baseline, after onalespib alone, and after the combination, in order to demonstrate AT7519-mediated suppression of HSP70 expression. Patients enrolled to the expansion phase underwent optional paired tumor biopsies for assessment of proof-of-mechanism demonstration of modulation of client proteins. Results: Twenty-eight patients have been treated, 10 of whom were enrolled to the expansion cohort with optional tumor biopsies. The MTD is DL2: onalespib 80 mg/m2 IV + AT7519M 21 mg/m2 IV on days 1,4,8, and 11 of a 21-day cycle. At DL3, DLTs included Grade 3 troponin elevation and mucositis. Drug-related adverse events occurring in ≥ 30% of patients include diarrhea, fatigue, mucositis, nausea, and vomiting, consistent with known toxicities of these agents. Two patients with colorectal and endometrial cancer, respectively, remained on study for 10 cycles with SD as the best response. Modulation of HSP70 were demonstrated in patient plasma samples. Conclusions: The combination of onalespib and AT7519 is tolerable, although the doses of both agents were below the monotherapy MTDs. Prolonged disease stabilizations were observed. Pharmacokinetic and pharmacodynamic analyses are ongoing, including assessment of HSP70 expression in plasma and tumor. Clinical trial information: NCT02503709.

Cell line-specific efficacy of thermoradiotherapy in human and canine cancer cells in vitro.

ObjectiveAims were to investigate sensitivity of various human and canine cancer cell lines to hyperthermia and the influence of particular treatment conditions, and to analyze the DNA-damage response and mode of cell death in cell line radiosensitized by hyperthermia. Additionally, we were interested in the involvement of HSP70 in radiosensitization.MethodsRadiosensitization by hyperthermia was determined in a panel of human and canine cancer cell lines using clonogenic cell survival assay, as well as levels of heat shock proteins (HSPs) using immunoblotting. The influence of the hyperthermia-radiotherapy time gap, different temperatures and the order of treatments on clonogenicity of hyperthermia-sensitive A549 cells was investigated. Additionally, DNA damage and cell death were assessed by Comet assay and an apoptosis/necrosis assay. Further we induced transient knockdown in A549 cells to test HSP70’s involvement in radiosensitization.ResultsOut of eight cell lines tested, only two (A549 and Abrams) showed significant decrease in clonogenic cell survival when pre-treated with hyperthermia at 42°C. Strong induction of HSP70 upon thermoradiotherapy (HT-RT) treatment was found in all cell lines. Transient knockdown of HSP70 in A549 cells did not result in decrease of clonogenic cell survival in response to HT-RT.ConclusionTumor cell-type, temperature and order of treatment play an important role in radiosensitization by hyperthermia. However, hyperthermia has limited potency to radiosensitize canine cancer cells grown in a 2D cell culture setting presented here. DNA damage and apoptosis/necrosis did not increase upon combined treatment and cytosolic levels of HSP70 appear not to play critical role in the radiosensitization of A549 cells.

HSF1 phosphorylation by cyclosporin A confers hyperthermia sensitivity through suppression of HSP expression

Heat shock factor 1 (HSF1) is a transcription factor essential for tumorigenesis, and targeting HSF1 may be effective in combined therapeutics for cervical cancer. Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation. However, the roles and regulatory mechanisms by which CsA modulates HSP expression remain largely unknown. In this study, we found that CsA pretreatment prevented induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 on HSF1 and thus inhibiting its transcriptional activity. Suppression of ERK1/2, GSK3β and CK2 activities attenuated CsA-induced down-regulation of HSP expression and up-regulation of HSF1 phosphorylation. CsA interfered with HSF1-SSBP1 complex formation and HSF1 nuclear translocation and recruitment to the HSP70 promoter. CsA clearly caused HeLa cell death during proteotoxic stress through reduced expression of HSPs. These results indicate that CsA suppresses HSP induction during heat shock by regulating the phosphorylation and nuclear translocation of HSF1. Our results provide a conceptual framework for the development of novel therapeutic strategies for cervical cancer through application of CsA during hyperthermia or chemotherapy.

Carbenoxolone Could Deteriorate Streptozotocin-induced Diabetes through Induction of Heat Shock Protein 70 and IFN-γ in C57BL/6 Mice

Type 1 diabetes (T1D), a spontaneous autoimmune disease, is associated with destruction of insulin-producing β-cells in the pancreas. Since some heat shock proteins (HSP), such as HSP70 exert a protective effect in both tissues and cells, the present study was conducted to elucidate the effects of carbenoxolone (CBX) as an HSP70 inducer on T1D. The disease was induced in male C57BL/6 mice using streptozotocin (STZ) and subjects were allocated to therapeutic 1 and therapeutic 2 groups, as well as negative and positive control groups. The treated mice (therapeutic 1 and therapeutic 2 groups) received 50 mg/kg CBX intraperitoneally every 24 hours, in the therapeutic 1 group the drug was injected before and after disease induction whereas in the therapeutic 2 group the drug was injected only after disease induction. Serum fasting blood sugar (FBS) level, cytokines production (Interferon-gamma (IFN-γ), Interleukin 10 (IL-10), and IL-17), serum HSP70 level and CD4+CD25+Foxp3+ regulatory T cell (Treg) frequency measurements were outperformed 14 days after the last STZ injection. Our results showed that in the treated groups, serum HSP70, IFN-γ, and IL-17 levels were increased in contrast to the untreated groups. The IL-10 level was markedly decreased in comparison to untreated diabetic mice (p<0.05). Moreover, it was found that the frequency of Tregs in treated mice was lower in comparison to the untreated mice but the difference was not significant (p>0.05). Our results confirm that CBX might through HSP70 induction, followed by increasing IFN-γ level leads to suppression of IL-10 production in diabetic mice resulted in toxic effects on pancreatic islet beta cells and deteriorating of disease.

Enhancement by HSP90 inhibitor of PGD2-stimulated HSP27 induction in osteoblasts: Suppression of SAPK/JNK and p38 MAP kinase

Heat shock protein (HSP) 90 that is ubiquitously expressed in various tissues is a major molecular chaperone. We have previously demonstrated that prostaglandin D2 (PGD2), a bone remodeling factor, elicits the expression of HSP27, a small HSP, through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of HSP90 in the PGD2-stimulated HSP27 induction and the underlying mechanism in MC3T3-E1 cells. Onalespib, an inhibitor of HSP90, significantly enhanced the PGD2-stimulated HSP27 induction. In addition, geldanamycin, another HSP90 inhibitor, potentiated the HSP27 induction. Both onalespib and geldanamycin markedly amplified the PGD2-induced phosphorylation of SAPK/JNK and p38 MAP kinase. SP600125, an inhibitor of SAPK/JNK, and SB203580, an inhibitor of p38 MAP kinase, suppressed the amplification by onalespib of the PGD2-stimulated HSP27 induction. These results strongly suggest that HSP90 plays a negative role in the HSP27 induction stimulated by PGD2 in osteoblasts, and that the inhibitory effect of HSP90 is mediated through the regulation of SAPK/JNK and p38 MAP kinase.

Neuroprotective Effects of HSF1 in Retinal Ischemia-Reperfusion Injury.

PurposeRetinal ischemia, a common cause of several vision-threatening diseases, contributes to the death of retinal neurons, particularly retinal ganglion cells (RGCs). Heat shock transcription factor 1 (HSF1), a stress-responsive protein, has been shown to be important in response to cellular stress stimuli, including ischemia. This study is to investigate whether HSF1 has a role in retinal neuronal injury in a mouse model of retinal ischemia-reperfusion (IR).MethodsIR was induced by inserting an infusion needle into the anterior chamber of the right eye and elevating a saline reservoir connected to the needle to raise the intraocular pressure to 110 mm Hg for 45 minutes. HSF1, Hsp70, molecules in the endoplasmic reticulum (ER) stress branches, tau phosphorylation, inflammatory molecules, and RGC injury were determined by immunohistochemistry, Western blot, or quantitative PCR.ResultsHSF1 expression was significantly increased in the retina 6 hours after IR. Using our novel transgenic mice carrying full-length human HSF gene, we demonstrated that IR-induced retinal neuronal apoptosis and necroptosis were abrogated 12 hours after IR. RGCs and their function were preserved in the HSF1 transgenic mice 7 days after IR. Mechanistically, the beneficial effects of HSF1 may be mediated by its induction of chaperone protein Hsp70 and alleviation of ER stress, leading to decreased tau phosphorylation and attenuated inflammatory response 12 to 24 hours after IR.ConclusionsThese data provide compelling evidence that HSF1 is neuroprotective against retinal IR injury, and boosting HSF1 expression may be a beneficial strategy to limit neuronal degeneration in retinal diseases.

Induction of HSP27 and HSP70 by constitutive overexpression of Redd1 confers resistance of lung cancer cells to ionizing radiation

Redd1 is a stress response protein that functions as a repressor of mTORC1, a central regulator of protein translation, resulting in the inhibition of cell growth and metabolism. However, paradoxically, high Redd1 expression favors cancer progression and generates resistance to cancer therapy. Herein, we revealed that constitutive overexpression of Redd1 induced HSP27 and HSP70 expression in lung cancer cells. The expression of Redd1, HSP27 and HSP70 was highly increased in lung cancer tissues compared with that in normal lung tissues. Inhibition of HSP27 or HSP70 suppressed AKT phosphorylation, which was induced by constitutive overexpression of Redd1 and enhanced the inhibitory effects on viability of Redd1‑overexpressing cells. Inhibition of AKT phosphorylation resulted in a decrease of HSP27 and HSP70 expression in Redd1‑overexpressing cells. These data indicated that HSPs and AKT in Redd1‑overexpressing cells positively regulated the function and expression of each other and were involved in lung cancer cell survival. Knockdown of HSP27, HSP70 or AKT enhanced ionizing radiation (IR) sensitivity, particularly in lung cancer cells in which Redd1 was stably overexpressed. Collectively, constitutive overexpression of Redd1 led to HSP27 and HSP70 induction and AKT activation, which were involved in lung cancer cell survival and resistance to IR, suggesting that Redd1 may be used as a therapeutic target for lung cancer.