Lysosomal and cytoskeletal events in epithelial salivary tumours as assessed by imunohistochemistry for CD63 and HSP27.

Little attention has been paid to immunohistochemically assessed, lysosomal activities in salivary neoplasia. In an attempt to remedy this, the present investigation applied immunohistochemistry for CD63 antigen (a lysosomal membranous protein) and HSP27 (a molecular chaperone with roles in intracellular homeostasis) to archival paraffin-embedded surgical specimens of 101 benign and malignant, epithelial salivary tumours. Diffuse cytoplasmic CD63 immunoreactivity was seen in serous cells in acinic cell carcinoma, and mucous cells in mucoepidermoid carcinoma, pleomorphic adenoma (PA) and Warthin tumour. Apical rims or bands of CD63 immunoreactivity were also seen in simple cells lining tubular structures in PA, acinic cell carcinoma, mucoepidermoid carcinoma and polymorphous (low-grade) adenocarcinoma; and, occasionally, oncocytic luminal cells in Warthin tumour. HSP27 immunoreactivity was usually seen in non-luminal cells of PA, basal cells of oncocytic tumours, epidermoid cells of mucoepidermoid carcinoma and PA, and cells outlining aggregates or pseudolumina of adenoid cystic carcinoma. Expression of CD63 is preferentially associated with differentiated or simple luminal cell phenotypes in epithelial salivary tumours and possibly reflects autophagy of secretory granules or absorption of luminal material. Expression of HSP27 is preferentially associated with non-luminal cells and possibly reflects remodelling of the cytoskeleton.

Neuroprotective Effects of Asparagus officinalis Stem Extract in Transgenic Mice Overexpressing Amyloid Precursor Protein.

To mimic Alzheimer's disease, transgenic mice overexpressing the amyloid precursor protein (APP) were used in this study. We hypothesize that the neuroprotective effects of ETAS®50, a standardized extract of Asparagus officinalis stem produced by Amino Up Co., Ltd. (Sapporo, Japan), are linked to the inhibition of the apoptosis cascade through an enhancement of the stress-response proteins: heat shock proteins (HSPs). APP-overexpressing mice (double-transgenic APP and PS1 mouse strains with a 129s6 background), ages 6-8 weeks old, and weighing 20-24 grams were successfully bred in our laboratory. The animals were divided into 5 groups. APP-overexpressing mice and wild-type (WT) mice were pretreated with ETAS®50 powder (50% elemental ETAS and 50% destrin) at 200 mg/kg and 1000 mg/kg body weight. Saline, the vehicle for ETAS®50, was administered in APP-overexpressing mice and WT mice. ETAS®50 and saline were administered by gavage daily for 1 month. Cognitive assessments, using the Morris Water Maze, demonstrated that memory was recovered following ETAS®50 treatment as compared to nontreated APP mice. At euthanization, the brain was removed and HSPs, amyloid ß, tau proteins, and caspase-3 were evaluated through immunofluorescence staining with the appropriate antibodies. Our data indicate that APP mice have cognitive impairment along with elevated amyloid ß, tau proteins, and caspase-3. ETAS®50 restored cognitive function in these transgenic mice, increased both HSP70 and HSP27, and attenuated pathogenic level of amyloid ß, tau proteins, and caspsase-3 leading to neuroprotection. Our results were confirmed with a significant increase in HSP70 gene expression in the hippocampus.

Apoptosis, autophagy and atherosclerosis: Relationships and the role of Hsp27.

Atherosclerosis is a multifactorial chronic inflammatory disease of the arterial wall, and an important pathological basis of coronary heart disease. Endothelial cells, vascular smooth muscle cells, and macrophages play important roles in the development of atherosclerosis. Of note, apoptosis and autophagy, two types of programmed cell death, influence the development and progression of atherosclerosis via the modulation of such cells. The small heat shock protein Hsp27 is a multifunctional protein induced by various stress factors and has a protective effect on cells. A large number of studies have demonstrated that Hsp27 plays an important role in regulating apoptosis. Recently, some studies have suggested that Hsp27 also participates in the autophagic process. Moreover, Hsp27 is closely related to the occurrence and development of atherosclerosis. Here, we summarize the molecular mechanisms of apoptosis and autophagy and discuss their effects on endothelial cells, vascular smooth muscle cells, and macrophages in the context of atherosclerotic procession. We further explore the involvement of Hsp27 in apoptosis, autophagy, and atherosclerosis. We speculate that Hsp27 may exert its anti-atherosclerotic role via the regulation of apoptosis and autophagy; this may provide the basis for the development of new approaches for the prevention and treatment of atherosclerosis.

Potential effects of long-term abuse of anabolic androgen steroids on human skeletal muscle.

BACKGROUND: We have previously evaluated muscle functions and morphology in power athletes of long term (5 to15 years) abuse of anabolic androgen steroids (AAS; Doped) and in clean power athletes (Clean), and observed significant improvements in both muscle morphology and muscle functions in Doped. To our knowledge, the effects of long term AAS abuse on human muscle protein profile have never been studied. METHODS: The study examined further the muscle biopsies using a two-dimensional difference gel electrophoresis (2D DIGE) for proteomic screening and protein expression. Cellular localization/distribution of specific proteins identified by proteomic analysis was examined using immunohistochemistry (IHC). RESULTS: Different protein profiles were observed between Doped and Clean, and a valid orthogonal projection of latent structure discriminant analysis model was built (N.=16, x=5, R2=0.88/Q2=0.84, P=0.0005), which separated Doped from Clean. Liquid chromatography followed by tandem spectrometry identified 14 protein spots (representing nine different proteins) of significant difference in relative quantity (P<0.05), of which nine spots were down-regulated in Doped compared with Clean. IHC revealed no significant alteration in cellular localization in phosphoglucomutase-1 and heat shock protein beta-1, but indeed in two reference proteins desmin and F-actin in Doped. CONCLUSIONS: Long term abuse of AAS in combination with training is potentially associated with alterations in skeletal muscle protein profile and protein expression, and structural proteins rather than non-structural proteins are preferentially affected in cellular localization/distribution.

Profiling of heat shock proteins 27 and 70 in adenoids of children.

PURPOSE: Heat shock protein (HSP)27 and 70 are molecular chaperones that may have immunomodulatory functions. We determined if and at what levels each are expressed in the adenoids of pediatric subjects. We also examined tissue distributions, associated clinical characteristics, and antibacterial effects. METHODS: Western blot, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry were applied to adenoidal tissues and lavage fluids obtained from children (N = 40) undergoing adenotonsillectomy. RESULTS: Via western blot and ELISA, both HSP27 and 70 were regularly detected in adenoidal tissue and in lavage fluid samples. HSP27 was highly expressed in epithelium, whereas HSP70 showed strong subepithelial positivity and bore a significant relation to adenoidal size. Assayed levels of HSP27 and 70 correlated inversely, and their addition to culture media independently increased bacterial numbers (Staphylococcus aureus). Upon the precipitation of each from adenoidal lavage fluids, bacterial counts declined. CONCLUSIONS: HSP27 and 70 are readily expressed in the adenoids of children and may be implicated in immunologic responses.

Catechin Weakens Diabetic Retinopathy by Inhibiting the Expression of NF-κB Signaling Pathway-Mediated Inflammatory Factors.

OBJECTIVE: The aim of the present study was to explore the role of catechin on the expression of heat shock protein 27 (HSP27), nuclear factor-kappa beta (NF-κB) and inflammatory factors IL-1ß, IL-6 and TNF-α in streptozotocin (STZ)-induced diabetic retinopathy (DR) rats. METHODS: A total of 150 rats were selected and randomly assigned to five groups: control group, STZ group, and three groups with different concentrations of catechin (low, middle and high concentrations). After STZ induction, DR rats were treated with different concentrations of catechin (50 mg/kg/day, low catechin group; 100 mg/kg/ day, middle catechin group; 200 mg/kg/day, high catechin group) by intravitreal injection for eight weeks. Hematoxylin and eosin (H&E) staining was used to observe the pathological changes in retinal tissues. The mRNA and protein levels of HSP27 in the retina were determined by RT-PCR and western blotting. Furthermore, the expression of NF-κB p65 and p-NF-κB p65 were determined by western blotting, while the expression of IL-1ß, IL-6, and TNF-α were determined by ELISA. RESULTS: HSP27 levels increased in STZ-induced DR rats, and became further upregulated after catechin treatment. Furthermore, IL-1ß, IL-6, and TNF-α levels were upregulated in the retinas of STZ-induced DR rats, but these changes were partially inhibited after treatment with catechin. Moreover, the application of catechin inhibited the activation of NF-κB, which was upregulated in STZ-induced DR. A negative correlation was observed between the concentrations of catechin and the expression of inflammatory factors. CONCLUSION: Catechin can weaken DR induced by STZ by increasing HSP27 levels and decreasing the production of associated inflammatory factors. This could help to treat DR in clinic.

Cytoprotective effect of eupatilin against indomethacin-induced damage in feline esophageal epithelial cells: relevance of HSP27 and HSP70.

Indomethacin is a non-steroidal anti-inflammatory drug with clearly known side effects on the gastrointestinal tract. The purpose of the present study was to investigate whether eupatilin inhibit cell injury induced by indomethacin in cultured feline esophageal epithelial cells (EECs). EECs were used to investigate the ability of eupatilin to induce the expression of heat shock proteins (HSP27 and HSP70) and analyze its cytoprotective effect against indomethacin-induced damage. The treatment of EECs with indomethacin for 8 h decreased cell viability. Western blot analysis showed that the levels of HSPs gradually decreased in cells treated with indomethacin, while eupatilin treatment increased the levels of HSPs. When treated with both indomethacin and eupatilin, the levels of HSPs increased rapidly, and were maintained at 130-140%. In addition, treatment with the specific inhibitors of PTK, PKC, PLC, p38 MAPK, JNKs, and PI3K attenuated the eupatilin-induced expression of HSPs. Pretreatment of EECs with the inhibitors of protein synthesis, actinomycin D or cycloheximide, attenuated the cytoprotective effect of eupatilin on indomethacin-induced cell damage. Reactive oxygen species production was upregulated by indomethacin, but downregulated by eupatilin. Taken together, it was suggested that HSPs were partly responsible for the eupatilin-mediated cytoprotective activity against the indomethacin-induced damage in EECs.

Phosphorylated Hsp27 is mutually exclusive with ATRX loss and the IDH1R132H mutation and may predict better prognosis among glioblastomas without the IDH1 mutation and ATRX loss.

AIM: To identify biomarkers for accurate classification of glioma. PATIENTS AND METHODS: We evaluated the heat shock protein 27 (Hsp27), phosphorylated Hsp27 (p-Hsp27), ATRX and IDH1R132Hproteins using immunohistochemistry in 421 glioma tissues. The χ2 test was used to assess the relationship between molecular alterations and clinico-pathological parameters. Kaplan-Meier survival curves were constructed, and differences were detected by the log-rank test. RESULTS: We found that Hsp27 and p-Hsp27 were mainly expressed in aggressive astrocytic gliomas. However, neither Hsp27 nor p-Hsp27 expression was related to survival time for any grade of glioma. Interestingly, p-Hsp27 was mutually exclusive with ATRX loss (ATRX-) and the IDH1R132H mutation, except for one case of anaplastic astrocytoma. We classified glioblastomas (GBMs) into three subtypes: ATRX-/IDH1R132H, high p-Hsp27 expression (p-Hsp27+) and none of these three markers. ATRX-/IDH1R132Hshowed the longest median survival (19.6 months). The prognostic difference between p-Hsp27+ and none of these three markers was significant (15.0 vs 13.1 months, P=0.045). Moreover, p-Hsp27+ predicted better sensitivity for standard therapy among GBMs without the IDH1 mutation and ATRX loss (26.3 vs 15.5 months, P=0.008). CONCLUSION: p-Hsp27 is a novel biomarker of glioma and might have important clinical value for further classification of patients with wild-type IDH1 and normal ATRX expression, for evaluating prognosis and for guidance for adjuvant therapy.

Effect of liposome­mediated HSP27 transfection on collagen synthesis in alveolar type II epithelial cells.

To investigate the effect of liposome Lipofectamine® 2000­mediated HSP27 plasmid transfection in A549 human alveolar type II epithelial cell line on collagen synthesis during transforming growth factor­ß1 (TGF­ß1)­induced type II epithelial cell transition to myofibroblasts. Cells were transfected with varying ratios of the Lipofectamine® 2000­mediated heat shock protein 27 (HSP27) plasmid and the transfection efficiency was determined using flow cytometry. The maximum transfection efficacy was confirmed by laser confocal microscopy. HSP gene expression and the most efficient HSP27 plasmid were determined using reverse transcription­quantitative polymerase chain reaction. Western blot analysis was used to examine HSP27 and collagen expression levels. With a transfection efficiency of 83%, the 8 µg:20 µl ratio of liposome: Plasmid had the highest transfection levels. Among the four different interference sequences in the HSP27 plasmid, the D sequence had the highest interference effect with 70% silencing of the HSP27 gene. The expression of type I and III collagen in TGF­ß1­induced transition of A549 human alveolar type II epithelial cell line to myofibroblasts was significantly downregulated by the successful transfection with HSP27­interfering plasmid. The expression of type I and III collagen in the TGF­ß1­induced transition of A549 cells to myofibroblasts was significantly downregulated by transfection of A549 cells with HSP27 plasmid D­interfering sequence and optimal ratio of Lipofectamine® 2000 and HSP27 plasmid.

Analysis of HspB1 (Hsp27) Oligomerization and Phosphorylation Patterns and Its Interaction with Specific Client Polypeptides.

Human HspB1 (also denoted as Hsp27) belongs to the family of small (or stress) proteins (sHsps). The family, which contains ten members including αA,B-crystallin polypeptides, is characterized by a conserved C-terminal α-crystallin domain and molecular weights ranging from 20 to 40 kDa. Here, procedures are described for analyzing the dynamic oligomerization and phosphorylation patterns of HspB1 in cells exposed to different environments. Changes in the structural organization of HspB1 can reprogram its interaction with specific partner/client polypeptides. Methods are presented to analyze these interactions using tissue culture cells genetically modified to express different levels of this protein. In addition, the laboratory approaches presented here could be used to test the nine other human sHsp members as well as sHsps from other species.

Avaliação do papel de HSPB1 na modulação da autofagia induzida por PRL em células-beta / Unveiling the role of HSPB1 in PRL-induced autophagy modulation in beta-cells

O diabetes mellitus tipo 1 é uma doença metabólica, caracterizada pela desregulação glicêmica, que ocorre devido a um ataque autoimune. A insulinoterapia é o tratamento clássico para o DM1. Contudo, alguns pacientes que apresentam essa doença não respondem de forma eficiente a este tratamento e apresentam episódios frequentes de hipoglicemia severa e despercebida (pacientes hiperlábeis). Essas complicações comprometem de forma significativa a qualidade de vida dessas pessoas. O transplante de ilhotas é uma importante alternativa para o tratamento de pacientes hiperlábeis com DM1. No entanto, essa terapia apresenta restrições como a necessidade de mais de um doador por transplante e significativa morte das ilhotas devido ao estresse provocado pelo procedimento de isolamento, além da morte promovida pelo sistema imune do paciente nos primeiros momentos pós-transplante. A autofagia é um mecanismo de reciclagem de componentes citoplasmáticos que é fundamental para a homeostase celular. Em condições de estresse, este mecanismo é ativado acima do seu nível basal, promovendo a degradação de agregados proteicos e organelas defeituosas, evitando assim, danos celulares que comprometam a viabilidade da célula. Trabalhos realizados por nosso grupo têm mostrado a citoproteção que PRL promove em células-beta, reduzindo a apoptose induzida por citocinas pró-inflamatórias. Também demonstramos o papel essencial de HSPB1 na inibição de apoptose induzida por PRL após o tratamento com citocinas. Além disso, resultados recentes de nosso laboratório mostraram um aumento nos níveis de autofagia em células-beta após sua exposição a citocinas, bem como uma restauração a níveis normais na presença de PRL. Visando um melhor entendimento do papel da PRL na modulação da autofagia em células-beta, o objetivo desse projeto foi estudar se HSPB1 também é essencial no mecanismo de regulação da autofagia induzido por PRL.Para tal, fizemos experimentos em modelos de células-beta MIN6, MIN6 silenciadas para HSPB1 (MIN6-shHSPB1) e MIN6 com sequencia short hairpin aleatória (MIN6- SsC), medindo a morte celular através de ensaios de viabilidade, e ensaios de western blot para avaliar os níveis de marcadores de autofagia e fluxo autofágico (degradação de autofagossomos), tratando as células com citocinas, prolactina e indutores ou inibidores de autofagia. Os resultados mostraram que a modulação da autofagia ocasionada pela prolactina em células-beta se dá, em parte, através de HSPB1. O tratamento com prolactina foi capaz de inibir a morte celular induzida por citocinas, mesmo na presença de cloroquina, um bloqueador de autofagia, o que nos levou a concluir que a autofagia não é uma via envolvida na citoproteção de células beta induzida por PRL. Os resultados gerados nesse estudo contribuíram para uma melhor compreensão dos eventos moleculares induzidos por PRL em células-beta, e poderão permitir a inferência de novas abordagens que melhorem a citoproteção, cultura e transplante dessas células em pacientes com diabetes tipo 1

Type 1 diabetes mellitus is a metabolic disease characterized by glycemic dysregulation, which occurs due to an autoimmune destruction of beta-cells. Insulin therapy is the gold standard treatment for DM1. However, some DM1 patients do not respond efficiently to this treatment and suffer frequent episodes of severe hypoglycemia unawareness. Since this complication jeopardizes the quality of life of these people, Islet transplantation is a therapeutic alternative indicated to treat these patients. However, besides the lack of enough organ donors, the loss of beta cells during both the isolation as well as the infusion of islets into the recipient induce a great estresse and thus a significant cell death is one of the drawbacks of this procedure. Autophagy is a mechanism of recycling cytoplasmic components and is essential for cellular homeostasis. Under estresse conditions, this mechanism is activated above basal levels, promoting the degradation of protein aggregates and defective organelles, thus avoiding cell damage that could compromise cell viability. Studies carried out by our group have shown not only that PRL promotes cytoprotection in beta-cells, reducing pro-inflammatory cytokines-induced apoptosis, but also that HSPB1 plays an essential role in this inhibition of apoptosis mediated by PRL after treatment with cytokines. Moreover, recent results from our laboratory showed an increase in autophagy levels in beta-cells after exposure to cytokines, as well as a restauration to normal levels in the presence of PRL. In order to better understand the role of PRL in the modulation of autophagy in these cells, the aim of this project is to study whether HSPB1 is also essential in the mechanism of autophagy regulation induced by PRL. Using MIN6 beta cell models where HSPB1 was silenced (MIN6-shHSPB1) or not (MIN6-SsC), we studied cell death by viability assays. Moreover, western blot assays were performed in order to assess levels of autophagy and autophagic flux markers in the cells.Our results showed that HSPB1 in one of the mediators of PRL-induced modulation of autophagy. Nevertheless, since hormonal treatment was still able to inhibit cytokinesinduced cell death even in the presence of chloroquin, an autophagy blocker, we conclude that autophagy is not a signaling pathway involved in PRl-induced beta-cell cytoprotection. Altogether, the results shown in this study may help to increase the knowledge of the molecular events induced by PRL in beta-cells, and may allow to infer new approaches to improve cytoprotection, culture and transplantation of these cells into type 1 diabetic patients

Functional role of inflammation in the surgical injury induced vascular remodeling of male albino rats.

Our present study investigates the cellular and molecular inflammatory events in male albino rat arterial injury. Male albino rats were subjected to longitudinal incision and carotid artery clamping for the duration of 45 days. Heat shock protein (HSP) 27, HSP70, HSP47 and Nuclear Factor kappa B (NF-κB) expressions were determined by qPCR and Western blot method. The morphology of vessel wall alteration was studied by the light microscopy. The expression of NF-κB was found to be increased after ten days of carotid artery injury. The qPCR and Western blot analysis showed elevation in HSP47, HSP27, and HSP70 expression, ten days following the surgical injury. The neointima-formation and the media layer discontinuity were evidenced by light microscopy. The dendritic-like cells were in close contact with the lymphocytes. Our study reports that the surgical injury induces an inflammatory response through the increased NF-κB and HSPs expression.

The small heat shock protein Hsp27: Present understanding and future prospects.

Heat shock proteins are important for maintaining protein homeostasis and cell survival. Among different classes of highly conserved Hsps, low molecular weight Hsps (sHsps) have significant place, particularly Hsp27, whose role has been demonstrated in wide range of biological processes, including development, immunity, diseases and therapy. In this review, the structure and functions of Hsp27 and related genes, their role in different cellular processes as well as in stress tolerance, is highlighted.

Upregulation and phosphorylation of HspB1/Hsp25 and HspB5/αB-crystallin after transient middle cerebral artery occlusion in rats.

Ischemic stroke leads to cellular dysfunction, cell death, and devastating clinical outcomes. The cells of the brain react to such a cellular stress by a stress response with an upregulation of heat shock proteins resulting in activation of endogenous neuroprotective capacities. Several members of the family of small heat shock proteins (HspBs) have been shown to be neuroprotective. However, yet no systematic study examined all HspBs during cerebral ischemia. Here, we performed a comprehensive comparative study comprising all HspBs in an animal model of stroke, i.e., 1 h transient middle cerebral artery occlusion followed by 23 h of reperfusion. On the mRNA level out of the 11 HspBs investigated, HspB1/Hsp25, HspB3, HspB4/αA-crystallin, HspB5/αB-crystallin, HspB7/cvHsp, and HspB8/Hsp22 were significantly upregulated in the peri-infarct region of the cerebral cortex of infarcted hemispheres. HspB1 and HspB5 reached the highest mRNA levels and were also upregulated at the protein level, suggesting that these HspBs might be functionally most relevant. Interestingly, in the infarcted cortex, both HspB1 and HspB5 were mainly allocated to neurons and to a lesser extent to glial cells. Additionally, both proteins were found to be phosphorylated in response to ischemia. Our data suggest that among all HspBs, HspB1 and HspB5 might be most important in the neuronal stress response to ischemia/reperfusion injury in the brain and might be involved in neuroprotection.

A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma.

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis.

Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins.

Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component.

HSPB1 Inhibits the Endothelial-to-Mesenchymal Transition to Suppress Pulmonary Fibrosis and Lung Tumorigenesis.

The endothelial-to-mesenchymal transition (EndMT) contributes to cancer, fibrosis, and other pathologic processes. However, the underlying mechanisms are poorly understood. Endothelial HSP1 (HSPB1) protects against cellular stress and has been implicated in cancer progression and pulmonary fibrosis. In this study, we investigated the role of HSPB1 in mediating the EndMT during the development of pulmonary fibrosis and lung cancer. HSPB1 silencing in human pulmonary endothelial cells accelerated emergence of the fibrotic phenotype after treatment with TGFß or other cytokines linked to pulmonary fibrosis, suggesting that HSPB1 maintains endothelial cell identity. In mice, endothelial-specific overexpression of HSPB1 was sufficient to inhibit pulmonary fibrosis by blocking the EndMT. Conversely, HSPB1 depletion in a mouse model of lung tumorigenesis induced the EndMT. In clinical specimens of non-small cell lung cancer, HSPB1 expression was absent from tumor endothelial cells undergoing the EndMT. Our results showed that HSPB1 regulated the EndMT in lung fibrosis and cancer, suggesting that HSPB1-targeted therapeutic strategies may be applicable for treating an array of fibrotic diseases.

Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

Reduced expression of HSP27 following HAD-B treatment is associated with Her2 downregulation in NIH:OVCAR-3 human ovarian cancer cells.

The Korean traditional medicine, HangAmDan (HAD), was developed in 1996 for use as an antitumor agent, and has since been modified to HAD­B (an altered form of HAD), in order to potentiate its therapeutic effects. In the present study, the effect of HAD­B on the proliferation and invasion of NIH:OVCAR­3 and SKOV­3 human ovarian cancer cell lines was investigated. In addition, the expression of major signal transduction molecules and changes in the proteome in these cells were measured. HAD­B treatment effectively induced a reduction in the levels of cell proliferation in serum­free conditioned media. However, unaltered levels of PARP and caspase­3 indicated that HAD­B does not reduce proliferation by inducing apoptotic cell death. Fluorescence­activated cell sorting analysis revealed no significant change in apoptosis following HAD-B treatment. Invasion assay results indicated a reduced rate of invasion following HAD­B treatment. HAD­B also influenced the expression of major signal transduction molecules; the phosphorylation of mTOR and AKT was reduced, while that of ERK was increased. Alterations in the proteomes of the two cell lines were investigated following HAD­B treatment. Among the 9 proteins with differential expression, heat­shock protein ß­1 (HSP27) was downregulated in NIH:OVCAR­3 cells treated with HAD­B. The reduced expression of HSP27 was associated with human epidermal growth factor receptor 2 (Her2) downregulation in these cells. In conclusion, the results of the current proteome assessment suggest that HAD­B has the potential to suppress the proliferation and invasion of human ovarian cancer cells. HAD­B treatment of NIH:OVCAR­3 cells suppressed HSP27 expression and was also associated with Her2 downregulation.