HSP90β Expression Research Articles

HSP90β regulates EAAT2 expression and participates in ischemia‑reperfusion injury in rats.

Cerebrovascular diseases (CVDs) have become a global public health problem and ischemia‑reperfusion injury, the major cause of neurological impairment exacerbation, is closely related to excitotoxicity. The present study aimed to investigate the effects of changes in heat shock protein (HSP)90β expression and verify whether HSP90β regulates EAAT2 expression in a cerebral ischemia‑reperfusion injury model. Healthy adult Sprague‑Dawley (SD) male rats were used to establish a control group, sham‑operated group, middle cerebral artery occlusion (MCAO) group, empty virus group and lentivirus group. A model of cerebral ischemia‑reperfusion was established using the MCAO method. Lentivirus construction and injection were used to interfere with the expression of HSP90β. The modified neurological severity score was used to assess neurological deficits. Triphenyltetrazolium chloride staining was used to detect infarct areas. Immunofluorescence was used to detect HSP90β expression localization and the expression levels of HSP90β and EAAT2 were determined using western blotting and reverse transcription‑quantitative PCR. An MCAO model was successfully established and it was found that HSP90β, but not HSP90α, was upregulated after MCAO. HSP90β expression coincided with astrocyte markers in the ischemic penumbra area, while no expression was observed in microglia. Inhibition of HSP90β expression improved neurological deficits and alleviated brain injury by increasing EAAT2 expression. These results suggested that HSP90β is involved in the process of cerebral ischemia‑reperfusion injury in rats and that inhibition of HSP90β expression increases EAAT2 levels, conferring a neuroprotective effect in MCAO model rats.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90β expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

Identification, characterization, and function of GRP94 and HSP90β in cold stress response in cold water fish Phoxinus lagowskii

The glucose-regulated protein (GRP94) and HSP90β are highly conserved and diverse molecular chaperones that could prevent protein aggregation and denaturation while also playing an important role in cellular stress responses. We show through our study the cloning and characterization of GRP94 and HSP90β genes and analyze their mRNA expression in the brain, heart, liver, and muscle tissues of Phoxinus lagowskii after exposure to cold stress. The strong stress resistance to the temperature of this cold-water fish in high latitudes makes it a good model for this study. GRP94 and HSP90β contain a KDEL and a MEEVD motif at the C-terminus and a histidine kinase-like ATPases (HATPases-c domain) at the N-terminus. These genes were found to be highly expressed in the liver, gills, ovary, testis, and other tissues. In the brain, liver, and muscle tissue, there was an increase in GRP94 mRNA expression during early acute cold stress compared to a control fish. However, the mRNA expression levels of HSP90β increase in the brain and heart in response to exposure to acute cold stress. Relative long-term stress showed no significant difference in the GRP94 and HSP90β expression levels in the liver, heart, and muscle tissue. Significantly high expression of GRP94 was shown in the brain during 14 days, suggesting that these two genes may protect brain tissue from damage during relative long-term cold stress. Our work suggests that HSP90 enhances AKT signaling and may participate in the glucose mechanism. Furthermore, HSP90 may activate IKK signaling via AKT, resulting in a variety of immune responses. This concludes the first study to focus on the GRP94 and HSP90β functions in P. lagowskii during cold stress and provides important tools for future research into HSP90 and GRP94 genes in cold-water fish.

HSP90AA1 promotes the inflammation in human gingival fibroblasts induced by Porphyromonas gingivalis lipopolysaccharide via regulating of autophagy

BackgroundPeri-implantitis of tooth seriously affects the life quality of patients. This study aimed to investigate the role of HSP90AA1 in the inflammatory of human gingival fibroblasts (HGFs) induced by porphyromonas gingivalis lipopolysaccharide (Pg-LPS), and to provide a potential therapeutic target for clinical treatment of peri-implantitis.MethodsPg-LPS (0.1, 1, 10 μg/mL) was used to construct the inflammatory model of HGFs to evaluate the effect of Pg-LPS on HGFs. Then HSP90AA1-siRNA was transfected to construct HSP90AA1 low expression HGFs cell line, and 3-MA was also added. After that, cell viability, apoptosis, the contents of inflammatory cytokines were detected by CCK-8, flow cytometry and ELISA assay, respectively. Intracellular ROS, the expressions of HSP90α, HSP90β were detected by immunofluorescence. The levels of HSP90AA1, p-NF-κB p65/NF-κB p65, LC3 II/I, ATG5, Beclin-1 and TLR protein were detected by western blot.ResultsPg-LPS treatment didn’t affect the viability of HGFs cells, but induced the cell apoptosis and ROS generation, increased the contents of IL-1β, IL-6, TNF-α, and the protein expressions of HSP90AA1, p-NF-κBp65/NF-κBp65, LC3II/I, ATG5, and Beclin-1 in HGFs. While HSP90AA1-siRNA transfected into Pg-LPS induced HGFs significantly reduced the HSP90AA1, HSP90α, HSP90β expression, decreased the inflammatory factors, ROS generation, cell apoptosis rate, and autophagy-related proteins and TLR2/4 protein levels. What’s more, the addition of autophagy inhibitor 3-MA further promote the effect of HSP90AA1-siRNA on Pg-LPS treated HGFs.ConclusionsThis study showed that HSP90AA1 promoted the inflammatory response of Pg-LPS induced HGFs by regulating autophagy. The addition of 3-MA further confirmed that autophagy may mediate siHSP90AA1 to enhance the inflammatory response.

Glycocholic acid and butyrate synergistically increase vitamin D-induced calcium uptake in Caco-2 intestinal epithelial cell monolayers

BackgroundRoux-en-Y gastric bypass (RYGB) substantially decreases intestinal calcium absorption and may eventually lead to bone resorption. This is likely a consequence of bile diversion from the alimentary limb, as the presence of bile seems necessary for vitamin D-mediated calcium uptake. We recently suggested that the mediating mechanism may be a down-regulation of the vitamin D co-activator heat-shock protein (Hsp)90β. Recent evidence suggests that vitamin D may have effects on both active and passive calcium absorption. AimTo identify mechanisms in vitro that may be responsible for the decreased calcium absorption after RYGB. We hypothesized that bile, alone or in concert with nutritional compounds, could be of importance. Material & methodsCaco-2 cells were grown confluent on semi-permeable membranes in a double-chamber setup to mimic small intestinal mucosa. The effect of bile acids chenodeoxycholic, lithocholic, glycocholic and taurocholic acid, with and without the addition of the fatty-acid butyrate, were tested for their effects on Hsp90β expression and active and passive calcium-flux monitored using radioactive 45Ca. ResultsWe initially found that whole human bile, but only together with the fatty acid butyrate, potently induced Hsp90β expression. In line with this, a single bile acid, e.g. glycocholic acid (GCA), in combination with butyrate, increased Hsp90β expression (40 ± 13% vs. GCA, butyrate or vehicle alone; p < 0,001; n = 14–25). Further, this combination together with vitamin D increased the passive gradient-driven flux of calcium, compared to stimulation with vitamin D alone or in combination with either GCA or butyrate (880 ± 217% vs. vitamin D and GCA or butyrate, or vitamin D only; p = 0,01–0.006; n = 5–11). Surprisingly, this combination had no effect on active calcium transport in the absence of calcium gradient. ConclusionThe combination of GCA and butyrate increased gradient-driven calcium uptake up to 9-fold in Caco-2 intestinal epithelial cells, but had no effect on active calcium absorption. This effect was mediated via the vitamin D receptor co-activator Hsp90β.

Osteoking improves OP rat by enhancing HSP90‑β expression.

Osteoporosis (OP) is a chronic bone disease that affects individuals worldwide. Osteoporosis is primarily asymptomatic, and patients with OP suffer from pain, inconvenience, economic pressure and osteoporotic fracture (OPF). Osteoking, a Traditional Chinese Medicine compound that originates from the Yi ethnic group, has been used for a number of years to treat fractures. In our previous study, osteoking exhibited therapeutic effects on rats with OPF by promoting calcium deposition. Based on bioinformatics and network pharmacology analyses of a component-target-disease database, heat shock protein HSP 90-β (HSP90-β), also known as HSP90-β, was identified to be a key target of osteoking in OP. High HSP90-β expression levels were observed in osteoporotic rats and rat bone mesenchymal stem cells (rBMSCs) following osteoking treatment. After 12 weeks of administration in vivo, there was increased bone mineral density (BMD) (P<0.05), increased bone alkaline phosphatase (P<0.05), and improved bone microstructure in the osteoking group compared with those of the negative control group. In vitro, increased calcium deposition in rBMSCs was observed after 4 weeks of osteoking treatment. These results suggest that the mechanisms of osteoking are closely associated with HSP90-β and activate the bone morphogenetic protein (BMP) signalling pathway, primarily through BMP-2. Osteoking treatment improves OP in rats by enhancing HSP90-β expression.

토종 종계의 암수 합사가 개체의 스트레스 반응 정도에 미치는 영향

This study was performed to investigate the effect of mixed rearing of male and female chickens on the stress response in Korean native chickens. To identify the degree of the stress response, heterophil-lymphocyte ratio (H/L ratio), heat shock protein genes (HSPs) expression, and intracellular nuclear DNA damage rate were analyzed before and after the mixed rearing of male and female chickens. The results showed that the H/L ratio of chickens after mixing males and females was more than thrice as higher than before mixing (<i>P</i>&lt;0.001), but the differences between males and females were not significant. HSP-70, HSP90-α, and HSP90-β expression levels were 2.5 to 3.4 times higher after mixing male and female chickens, compared to before mixing (<i>P</i>&lt;0.01). In the mixed rearing of male and female chickens, the increase in HSPs expression in females was higher than in males. Comet indicators in intracellular DNA damage rate analysis showed a significant increase after mixing male and female chickens compared to before mixing (<i>P</i>&lt;0.001). However, there was no significant difference between males and females with respect to DNA damage rate. Taken together, these results suggest that male and female mixed rearing acts as a strong external stressor in both male and female chickens.

Heat Shock Protein 90 as a Prognostic Marker and Therapeutic Target for Adrenocortical Carcinoma.

Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC.Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines.Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90β. Within a cohort of ACC patients, HSP90β expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2—and AKT—pathways by luminespib and ganetespib treatment.Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.

Differential Expression of HSP90β in MDA-MB-231 and MCF-7 Cell Lines after Treatment with Doxorubicin.

BackgroundBreast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including HSP90α and HSP90β in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed.Material and MethodsThe cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. Immunofluorescence and western blotting techniques were used to determine the expression of HSP90β in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of HSP90α.ResultsThe MTT assay results showed that the MDA-MB- 231 cells (IC50=14.521 μM) were more sensitive than the MCF-7 cells (IC50=16.3315 μM) to DOX. The immunofluorescence results indicated that the expression of HSP90α in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that HSP90β expression decreased in the MCF-7 cells but increased in the MDA-MB- 231 cells after DOX treatment. Conclusion: The obtained results suggested that HSP90α and HSP90β expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, HSP90α expression was reduced while HSP90β was found to be overexpressed following DOX treatment.

Clinical significance of heat shock protein 90α expression as a biomarker of prognosis in patients with gastric cancer.

Heat shock protein 90 (HSP90) possesses two major isoforms - HSP90α and HSP90β. They have essential roles in the protection against stressful conditions. They are also important for the re-establishment of cellular homeostasis. We investigated the clinical significance of HSP90α and HSP90β expression in patients with gastric cancer (GC). HSP90α and HSP90β expression levels were examined immunohistochemically in surgical specimens obtained from 186 GC patients. The correlations between their expression levels and clinicopathological parameters including patient survival were analyzed. The frequencies of larger tumor size (maximum diameter ≥4 cm) and more prominent tumor invasion (≥pT3) in the high intensity HSP90α expression group were 73.4% and 68.8% higher, respectively, than those in the low intensity group (both P = 0.001). High HSP90α expression level was also significantly associated with lymphatic invasion, lymph node metastasis, and advanced stage (TNM stage ≥III) disease (P = 0.047, P = 0.046, and P = 0.004, respectively). Patients with high HSP90α expression levels demonstrated significantly worse survival than those with low HSP90α expression levels (P = 0.047). In contrast, survival did not differ significantly according to the intensity of HSP90β expression. Our results showed that HSP90α overexpression might be associated with disease progression and poorer survival in patients with GC. Therefore, HSP90α could be used as possible biomarker for the prognosis of GC.

Proteomic Profile of Sorafenib Resistance in Hepatocellular Carcinoma; GRP78 Expression Is Associated With Inferior Response to Sorafenib.

The outcome of patients with advanced hepatocellular carcinoma (HCC) remains poor and therapeutic options, including sorafenib, the first anti-cancer drug proved to prolong survival in patients with advanced HCC, are limited. However, no clinically useful predictive biomarker for sorafenib has been reported. We exploited two-dimensional gel electrophoresis coupled with mass spectrometry to find de-regulated proteins by using conditioning of a sorafenib-resistant HCC cell line, Huh7. Tumor samples from 60 patients with HCC treated with sorafenib were analyzed and correlated with survival outcome. Comparative proteomics indicated three proteins including, 78 kDa glucose related protein (GRP78), 14-3-3ε, and heat shock protein 90β (HSP90β). The three proteins were over-expressed in sorafenib-resistant Huh7 cells. In HCC tumor samples from patients treated with sorafenib, 73% of tumor samples had a high expression of GRP78, 18% had high 14-3-3ε expression and 85% had high HSP90β expression. Among these, GRP78 was associated with the shortest progression-free survival of HCC patients treated with sorafenib. GRP78 can be a predictive biomarker in HCC patients treated with sorafenib. Strategies designed to inhibit the GRP78-related pathway may overcome sorafenib resistance.

Jujuboside A promotes Aβ clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway.

Rationale: It has been reported that peroxisome proliferator activated receptor γ (PPARγ) level decreases significantly in the brains of Alzheimer's disease (AD) patients and mice models, while the mechanism is unclear. This study aims to unravel the mechanism that amyloid β (Aβ) decreases PPARγ and attempted to discover lead compound that preserves PPARγ.Methods: In APP/PS1 transgenic mice and Aβ treated microglia, the interaction between HSP90 and PPARγ were analyzed by western blot. Using a PPRE (PPARγ responsive element) containing reporter cell line, compounds that activate PPARγ activity were identified. After genetic ablation or pharmacological inhibition of potential target pathways, the target of jujuboside A (JuA) was discovered through Axl/HSP90β. After oral administration or intrathecal injection, the anti-AD activity of JuA was evaluated by Morris water maze (MWM) test and object recognition test. Soluble Aβ42 levels and plaque numbers after JuA treatment were detected by thioflavin S staining, and the activation of microglia was assayed by immunofluorescence staining against Iba-1.Results: We found that Aβ stress decreased heat shock protein 90 β (HSP90β), subsequently reduced the abundance of PPARγ, and down-regulated Aβ clearance-related genes in BV2 cells and primary microglia. We identified that JuA stimulated the expression of HSP90β, strengthened the interaction between HSP90β and PPARγ, preserved PPARγ levels, and thus effectively promoted the clearance of Aβ42. We demonstrated that JuA increased HSP90β expression through Axl/ERK pathway. JuA significantly ameliorated cognitive deficiency in APP/PS1 transgenic mice, meanwhile, JuA significantly reduced the soluble Aβ42 levels and plaque numbers in the brain. Notably, the therapeutic effects of JuA were dampened by R428, an Axl inhibitor.Conclusions: This study suggests that the up-regulation of HSP90β by JuA through Axl is a potential therapeutic strategy to facilitate Aβ42 clearance and ameliorate cognitive deficiency in AD.

Hsp90\u03b2 promoted endothelial cell-dependent tumor angiogenesis in hepatocellular carcinoma

BackgroundVascular endothelial growth factor receptors (VEGFRs) are the major receptors involved in endothelial cell-dependent tumor angiogenesis. There are studies account for the effects of Hsp90 on angiogenesis, but the role and mechanism of Hsp90β isoforms and NVP-BEP800, a specific inhibitor of Hsp90β, in tumor angiogenesis is rarely mentioned.MethodsImmunohistochemistry and statistical analysis was used to evaluate the correlation between Hsp90β expression, CD31 endothelial cell-dependent vessel density, and VEGFRs expression in tissue samples of 96 HCCs. Kaplan-Meier survival analysis and COX proportional hazards analysis the relation of Hsp90β and prognosis. HUVEC cells were transfected with Hsp90β or treated with NVP-BEP800, and then cell proliferation, migration, invasion and tube formation were investigated. The VEGFR1 and VEGFR2 expression was determined by Western blot and immunofluorescence. The VEGFR1 and VEGFR2 promoter activities were detected by dual luciferase report system. In vivo, the angiogenesis promotion of Hsp90β and anti-angiogenesis efficacy of NVP-BEP800 was tested in HCC xenograft models. Histological analysis was performed on tumor samples to evaluate Hsp90β, VEGFRs expression and MVD.ResultsThis study investigated the correlation between Hsp90β expression and CD31+ endothelial cell-dependent vessel density. Hsp90β promoted VEGFRs expression by increasing their promoter activities. The proliferation, migration, invasion, and tube formation activities of human endothelial cells significantly increased when Hsp90β was overexpressed. NVP-BEP800 down-regulated VEGFRs expression to significantly reduce tubular differentiation, as well as endothelial cell proliferation, migration, and invasion. Furthermore, NVP-BEP800 decreased VEGFR1 and VEGFR2 promoter activities. In vivo, Hsp90β promoted VEGFRs and CD31 expression in human hepatocellular carcinoma tumor xenografts and was associated with increased tumor microvessel density. After 18 days of treatment with 30 mg/kg/day NVP-BEP800, VEGFRs and CD31 expression significantly decreased.ConclusionHsp90β induced endothelial cell-dependent tumor angiogenesis by activating VEGFRs transcription. NVP-BEP800 has potential as a therapeutic strategy for inhibiting tumor angiogenesis by decreasing endothelial cell progression and metastasis. It can help develop a therapeutic strategy for tumor treatment through the inhibition of endothelial cell progression and metastasis.

The cloning of a heat shock protein 90β gene and expression analysis in Botia reevesae after ammonia-N exposure and Aeromonas hydrophila challenge

The objective of this study was to clone the full-length cDNA of Heat shock protein 90 (HSP90) in Botia reevesae and to determine the effects of pathogenic bacterial challenge after acute sublethal ammonia exposure on HSP90β expression. The HSP90β cDNA of B. reevesae contained 2358bp, including a 1947bp open reading frame, a 36bp 5′-untranslated region (UTR), a 375bp 3′-UTR. Sequence comparisons indicated that the predicted amino acid sequence of B. reevesae HSP90 showed a high degree of similarity with other known HSP90β genes, and contained five HSP superfamily signatures, thus suggesting that B. reevesae HSP90β is a cytosolic member of the HSP90 family. Quantitative real-time PCR analysis revealed that HSP90 transcripts could be detected in all of the tissues tested, and was strongly expressed in liver, gill and kidney tissues (p<0.05) of B. reevesae after sublethal ammonia-N exposure and Aeromonas hydrophila challenge. Additionally, following sublethal ammonia-N exposure and A. hydrophila challenge, expression of HSP90β mRNA transcripts was increased in gill and kidney tissues by 6–24h (p<0.05), and was more reduced in the liver than the levels observed in response to ammonia-N exposure or A. hydrophila challenge alone. This result indicated that after ammonia-N stress, B. reevesae could trigger elevated HSP90β expression in specific tissues to respond to pathogenic bacteria.

High-level expression of Hsp90β is associated with poor survival in resectable non-small-cell lung cancer patients.

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). 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. Hsp90β expression might be a useful marker of poor OS, although further large prospective studies are warranted to validate our findings.

Profiling Hsp90 differential expression and the molecular effects of the Hsp90 inhibitor IPI-504 in high-grade glioma models.

Retaspimycin hydrochloride (IPI-504), an Hsp90 (heat shock protein 90) inhibitor, has shown activity in multiple preclinical cancer models, such as lung, breast and ovarian cancers. However, its biological effects in gliomas and normal brain derived cellular populations remain unknown. In this study, we profiled the expression pattern of Hsp90α/β mRNA in stable glioma cell lines, multiple glioma-derived primary cultures and human neural stem/progenitor cells. The effects of IPI-504 on cell proliferation, apoptosis, motility and expression of Hsp90 client proteins were evaluated in glioma cell lines. In vivo activity of IPI-504 was investigated in subcutaneous glioma xenografts. Our results showed Hsp90α and Hsp90β expression levels to be patient-specific, higher in high-grade glioma-derived primary cells than in low-grade glioma-derived primary cells, and strongly correlated with CD133 expression and differentiation status of cells. Hsp90 inhibition by IPI-504 induced apoptosis, blocked migration and invasion, and significantly decreased epidermal growth factor receptor levels, mitogen-activated protein kinase and/or Akt activities, and secretion of vascular endothelial growth factor in glioma cell lines. In vivo study showed that IPI-504 could mildly attenuate tumor growth in immunocompromised mice. These findings suggest that targeting Hsp90 by IPI-504 has the potential to become an active therapeutic strategy in gliomas in a selective group of patients, but further research into combination therapies is still needed.

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence-related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12-13° C) and warm (18-19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold-treated fish. Analysis of single genes with real-time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90β and hsp30 as well as interferon-inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90β expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm-treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once-in-the-lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations.

Heat shock protein 90β inhibits apoptosis of intestinal epithelial cells induced by hypoxia through stabilizing phosphorylated Akt

Intestinal epithelial cell (IEC) apoptosis induced by hypoxia compromise intestinal epithelium barrier function. Both Akt and Hsp90 have cytoprotective function. However, the specific role of Akt and Hsp90β in IEC apoptosis induced by hypoxia has not been explored. We confirmed that hypoxia-induced apoptosis was reduced by Hsp90β overexpression but enhanced by decreasing Hsp90β expression. Hsp90β overexpression enhanced BAD phosphorylation and thus reduced mitochondrial release of cytochrome C. Reducing Hsp90β expression had opposite effects. The protective effect of Hsp90β against apoptosis was negated by LY294002, an Akt inhibitor. Further study showed that Akt phosphorylation was enhanced by Hsp90β, which was not due to the activation of upstream PI3K and PDK1 but because of stabilization of pAkt via direct interaction between Hsp90β and pAkt. These results demonstrate that Hsp90β may play a significant role in protecting IECs from hypoxia-induced apoptosis via stabilizing pAkt to phosphorylate BAD and reduce cytochrome C release. [BMB Reports 2013;46(1): 47-52]

Simultaneous Inhibition of Hsp90 and Akt Disable Immune Functions of Alloreactive T Cells in a Synergistic Manner

Abstract 4697 Introduction:Despite advances in prevention and post transplant immuno-suppressive strategies Graft versus Host Disease (GvHD) remains a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Recently we have shown, that heat shock protein 90 (Hsp90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) selectively depletes alloreactive T cells in vitro. Here, we demonstrate that DMAG and isoform-specific Akt1 and 2 inhibitor Akti-1/2 interact in a synergistic manner to suppress essential immune functions of alloreactive human T cells in vitro. Methods:Human CD3+ T cells were activated by coculture for 5 days with allogeneic dendritic cells (DC) in the presence of DMAG and Akti-1/2 alone and in combination. In another setting the inhibitors were added for the last 24 hours of T cell culture. Subsequently, the effect of Hsp90, Akt and Akt/Hsp90 inhibition on proliferation, apoptosis and cell cycle of alloreactive T cells was determined. ResultsHere, we demonstrate that human CD3+ T cells activated in a physiological manner by coculture with allogeneic DCs have an increased expression of Akt2 isoform compared to unstimulated human T cells. Inhibition of Akt activity by isoform-specific Akt1 and 2 inhibitor Akti-1/2 decreased proliferation of alloreactive T cells in a dose-dependent manner. This effect can be attributed to modest apoptosis induction and induction of cell cycle arrest in G1-phase. Cell cycle arrest is accompanied by accumulation of cell cycle regulator proteins p21WAF/CIP1 and p27KIP1. Simultaneous inhibition of Akt and Hsp90 induced a synergistic effect on proliferation suppression of alloreactive T cells. Akti-1/2 (1 μM) caused for example 26% proliferation decrease that was reinforced to 65% and 69% when combined with 0.5 μM and 2 μM DMAG, respectively. Similar results were obtained, when inhibitors were added at the beginning of the T cell culture. To exclude that the observed effect relies on inhibitor-induced DC dysfunction, we selectively target Hsp90 and Akt2 function in the T cell population by Hsp90β- and Akt2-specific siRNA. T cells transfected with Hsp90β- or Akt2-specific siRNA showed only moderate down-regulation of Hsp90β and Akt2 expression as determined by western blot. However, siRNA-mediated knockdown resulted in a significant decrease of proliferation in alloreactive T cells compared to negative control siRNA transfected T cells. Furthermore, T cells transfected with Hsp90β- and Akt2-specific siRNA in combination demonstrated no distinct down-regulation of Akt2 or Hsp90β expression compared to T cells transfected with Akt2- or Hsp90β-specific siRNA alone, but showed significant reduction of proliferation in comparison to T cells transfected with only one specific siRNA.To identify the underlying mechanism of increased proliferation suppression of agent combination, we investigated by western blot analysis expression of Akt substrate proteins and Hsp90 client proteins which are known to regulate proliferation of T cells. Increased effect on proliferation suppression of agent combination compared to single agent administration is accompanied by accumulation of negative cell cycle regulator p21WAF/CIP1, whereas abundance of positive cell cycle regulator PLK1 and cyclin B1 decrease. We also show that simultaneous inhibition of Hsp90 and Akt causes a slight increase of PARP cleavage suggesting an enhancement in caspase-mediated apoptosis induction in alloreactive T cells. In addition, we determined phosphorylation state of Akt substrate proteins in CD3/CD28-activated T cells by western blot analysis and observed a considerable decrease of GSK3α phosphorylation induced by combination of Akti-1/2 and DMAG compared to single agent use.In contrast, we provide data that inhibitor combination maintains viral immunity by showing that combination treatment have no effect on number of CMV-specific CD8+ T cells. Conclusion:We suggest that combinational action of Hsp90 and Akt inhibition can be defined as a new immunosuppressive strategy suited for the treatment of GVHD and other deregulated and unwanted T cell-mediated immune responses. Disclosures:Topp:Micromet: Consultancy, Honoraria.

Impact of butyrate on PKM2 and HSP90β expression in human colon tissues of different transformation stages: a comparison of gene and protein data

Due to protection of oncogenic proteins from degradation and enhancement of glycolytic phosphometabolites for synthetic processes, respectively, heat shock protein 90 (HSP90) and pyruvate kinase type M2 (PKM2) are important proteins for tumor growth. The present study was undertaken to investigate the susceptibility of both proteins and their encoding genes to the chemopreventive agent butyrate in human colon cells. Matched tissue of different transformation stages derived from 20 individual colon cancer patients was used for the experiments. The results of quantitative real-time PCR revealed a moderate increase of HSP90β and PKM2 mRNA in colon tumors (P<0.01) compared to normal tissues without relation to clinical parameters. The expression pattern could be confirmed for PKM2 protein by Western blot but not for HSP90β. During culturing with butyrate, the amount of PKM2 transcripts decreased in all three tissue types with the strongest effects observed in tumors (median fold decrease 45%, P<0.05). The protein data have not reflected this influence supposing a more gradual degradation rate due to a longer half-life of PKM2. In contrast, the mRNA expression of HSP90β in normal tissue was found 1.38-fold increased by butyrate (P<0.05), but not the corresponding protein level. HSP90β expression in adenomas and tumors remained generally insensitive. Only in malignant tissue, however, a significant correlation was found between the individual effects observed on gene and protein expression level. In conclusion, the present study identified PKM2 as a potential direct target of butyrate in neoplastic colon tissue, whereas HSP90β is none of it.