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Heat shock protein 27 (HSP27) is a multifunctional protein and belongs to the small HSP family. It has been shown that HSP27 is involved in viral replication as a cellular chaperone, but the function of HSP27 during porcine reproductive and respiratory syndrome virus (PRRSV) infections remains unexplored. Here, we found that PRRSV replication can induce HSP27 expression and phosphorylation in vitro. HSP27 overexpression promoted PRRSV replication, whereas its knockdown reduced PRRSV proliferation. Additionally, suppressing HSP27 phosphorylation reduced PRRSV replication and the level of viral double-stranded RNA (dsRNA), a marker of the viral replication and transcription complexes (RTCs). Furthermore, HSP27 can interact with multiple viral nonstructural proteins (nsps), including nsp1α, nsp1ß, nsp5, nsp9, nsp11 and nsp12. Suppressing the phosphorylation of HSP27 almost completely disrupted its interaction with nsp1ß and nsp12. Altogether, our study revealed that HSP27 plays an important role in PRRSV replication.

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Expression of heat shock protein (HSP) correlates with the oncogenic status of malignant cells and plays an important role in tumorigenesis. HSP27 is constitutively expressed at specific stages of cancer development, and several clinical trials have reported correlations between HSP27 expression and tumor progression, metastasis, and chemoresistance in various types of cancer cells. These findings indicate that HSP27 is a major drug target, particularly in chemo-resistant cancers. As part of our ongoing efforts to improve the previously identified J2, a HSP27 cross-linker, we, in this study, report the identification of NK16 as a novel inducer of abnormal HSP27 dimers that did not affect the expression of HSP90 in an NCI-H460 lung cancer cell model. When NCI-H460 cells were treated with NK16 in combination with the anticancer drug cisplatin or paclitaxel, cleavage of PARP and caspase-3 was increased compared to administration of cisplatin or paclitaxel alone. Similar results were obtained in an NCI-H460-xenografted mouse model, in which tumor growth was suppressed more by co-administration of NK16 and paclitaxel than by paclitaxel alone. We propose NK16 as a meaningful strategy to improve the anticancer efficacy of cisplatin and paclitaxel.

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PURPOSE: Although epidermal growth factor receptor (EGFR)-activating mutations in non-small cell lung cancer (NSCLC) usually show sensitivity to first-generation EGFR-tyrosine kinase inhibitors (TKIs), most patients relapse because of drug resistance. Heat shock protein 27 (HSP27) has been reported to be involved in the resistance of EGFR-TKIs, although the underlying mechanism is unclear. Here, we explore the mechanisms of HSP27-mediated EGFR TKI resistance and propose novel therapeutic strategies. METHODS: To determine the mechanism of HSP27 associated gefitinib resistance, differences were assessed using gefitinib-sensitive and -resistant NSCLC cell lines. In vivo xenograft experiments were conducted to elucidate the combinatorial effects of J2, a small molecule HSP27 inhibitor, and gefitinib. Analyses of human NSCLC tissues and PDX tissues were also used for comparison of HSP27 and phosphorylated AKT expression. RESULTS: Large-scale cohort analysis of NSCLC cases revealed that HSP27 expression correlated well with the incidence of EGFR mutations and affected patient survival. Increased pAKT and HSP27 was observed in gefitinib-resistant cells compared with gefitinib-sensitive cells. Moreover, increased phosphorylation of HSP27 by gefitinib augmented its protein stability and potentiated its binding activity with pAKT, which resulted in increased gefitinib resistance. However, in gefitinib-sensitive cells, stronger binding activity between EGFR and HSP27 was observed. Moreover, these phenomena occurred regardless of EGFR mutation including secondary mutations, such as T790M. AKT knockdown switched HSP27-pAKT binding to HSP27-EGFR, which promoted gefitinib sensitivity in gefitinib-resistant cells. Functional inhibition of HSP27 yielded sensitization to gefitinib in gefitinib-resistant cells by inhibiting the interaction between HSP27 and pAKT. CONCLUSIONS: Our results indicate that combination of EGFR-TKIs with HSP27 inhibitors may represent a good strategy to overcome resistance to EGFR-TKIs, especially in cancers exhibiting AKT pathway activation.

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Initially discovered to be induced by heat shock, heat shock protein 27 (HSP27, also called HSPB1), a member of the small HSP family, can help cells better withstand or avoid heat shock damage. After years of studies, HSP27 was gradually found to be extensively engaged in various physiological or pathophysiological activities. Herein, revisiting the previously published data concerning HSP27, we conducted a critical review of the literature regarding its role in squamous cell carcinoma (SCC) from the perspective of clinicopathological and prognostic significance, excluding studies conducted on adenocarcinoma, which is very different from SCC, to understand the enigmatic role of HSP27 in the tumorigenesis of SCC, including normal mucosa, dysplasia, intraepithelial neoplasm, carcinoma in situ and invasive SCC.

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Small heat shock protein 27 is a critically important chaperone, that plays a key role in several essential and varied physiological processes. These include thermotolerance, apoptosis, cytoskeletal dynamics, cell differentiation, protein folding, among others. Despite its relatively small size and intrinsically disordered termini, it forms large and polydisperse oligomers that are in equilibrium with dimers. This equilibrium is driven by transient interactions between the N-terminal region, the α-crystallin domain, and the C-terminal region. The continuous redistribution of binding partners results in a conformationally dynamic protein that allows it to adapt to different functions where substrate capture is required. However, the intrinsic disorder of the amino and carboxy terminal regions and subsequent conformational variability has made structural investigations challenging. Because heat shock protein 27 is critical for so many key cellular functions, it is not surprising that it also has been linked to human disease. Charcot-Marie-Tooth and distal hereditary motor neuropathy are examples of neurodegenerative disorders that arise from single point mutations in heat shock protein 27. The development of possible treatments, however, depends on our understanding of its normal function at the molecular level so we might be able to understand how mutations manifest as disease. This review will summarize recent reports describing investigations into the structurally elusive regions of Hsp27. Recent insights begin to provide the required context to explain the relationship between a mutation and the resulting loss or gain of function that leads to Charcot-Marie Tooth disease and distal hereditary motor neuropathy.

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BACKGROUND: There is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC). METHODS: We conducted whole-transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression. RESULTS: In this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27-JNK/P38 mitogen-activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27-JNK/P38 MAPK signaling and promoted tumor progression. CONCLUSIONS: Our results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27-JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

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Heat shock protein 27 (HSP27) is one of the small molecular chaperones and is involved in many cell mechanisms. Besides the known protective and helpful functions of intracellular HSP27, very little is known about the mode of action of extracellular HSP27. In a previous study, we showed that intravitreal injection of HSP27 led to neuronal damage in the retina and optic nerve after 21 days. However, it was not clear which degenerative signaling pathways were induced by the injection. For this reason, the pathological mechanisms of intravitreal HSP27 injection after 14 days were investigated. Histological and RT-qPCR analyses revealed an increase in endogenous HSP27 in the retina and an activation of components of the intrinsic and extrinsic apoptosis pathway. In addition, an increase in nucleus factor-kappa-light-chain-enhancer of activated B cells (NFκB), as well as of microglia/macrophages and T-cells could be observed. In the optic nerve, however, only an increased apoptosis rate was detectable. Therefore, the activation of caspases and the induction of an incipient immune response seem to be the main triggers for retinal degeneration in this intravitreal HSP27 model.

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AIMS: This study aimed to examine the anti-fibrotic role of Nuclear Factor-Erythroid derived 2 (NF-E2) in human renal tubule (HK-11) cells and in type 1 and type 2 diabetic (T1D, T2D) mouse kidneys. MAIN METHODS: Anti-fibrotic effects of NF-E2 were examined in transforming growth factor-ß (TGF-ß) treated HK-11 cells by over-expressing/silencing NF-E2 expression and determining its effects on profibrotic signaling. NF-E2 proteasomal degradation was confirmed by proteasome inhibition in HK-11 cells and diabetic mice. Clinical relevance of changes in NF-E2 expression to fibrotic changes in the kidney were assessed in T1D and T2D mouse kidneys. KEY FINDINGS: NF-E2 expression was significantly decreased in TGF-ß treated HK-11 cells and in kidneys of diabetic mice with concurrent increase in expression of fibrotic proteins. TGF-ß treatment of HK-11 cells did not inhibit NF-E2 mRNA expression, suggesting that the post-translational changes may contribute to NF-E2 protein degradation. The down-regulation of NF-E2 expression was attributed to its proteasomal degradation, as TGF-ß- and diabetes-induced NF-E2 down regulation was prevented by proteasome inhibitor treatment. In HK-11 cells TGF-ß treatment decreased E-cadherin expression and induced pSer82Hsp27/NF-E2 association, likely to promote NF-E2 degradation, as Hsp27 can target proteins to the proteasome. A critical role for NF-E2 in regulation of renal fibrosis was demonstrated as over-expression of NF-E2 or silencing NF-E2 expression, decreased or increased profibrotic proteins in TGF-ß-treated HK-11 cells, respectively. SIGNIFICANCE: NF-E2, a novel anti-fibrotic protein, is down-regulated in diabetic kidneys. Preserving/inducing NF-E2 expression in diabetic kidneys may provide a therapeutic potential to combat DN.

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Bladder cancer (BC) is one of the most common cancers worldwide, with a high rate of recurrence and poor outcomes. High-mobility group nucleosome-binding domain 5 (HMGN5) is overexpressed in many cancers and could cause carcinogenesis in BC. By protein-protein-interaction (PPI) analysis, we found that heat shock protein 27 (Hsp27), also a crucial functional factor in BC carcinogenesis, is significantly related to HMGN5. Hsp27 is required for IL-6-mediated EMT via STAT3/Twist signaling in prostate cancer. Here, we hypothesize that HMGN5 may interact with Hsp27 to affect IL-6-induced EMT and invasion in BC via STAT3 signaling. In the present study, we found that HMGN5 and Hsp27 are highly expressed in BC tissues and positively correlated with each other. HMGN5 interacts with Hsp27 in vitro, to modulate the cell invasion and EMT in BC. Moreover, HMGN5 could modulate IL-6-Hsp27-induced EMT and invasion in BC cells by regulating STAT3 phosphorylation and STAT3 targeting of the Twist promoter. HMGN5 interacts with Hsp27 to promote tumor growth in a human BC xenograft model in nude mice. In summary, HMGN5 interacts with Hsp27 to promote IL-6-induced EMT, therefore promoting invasion in BC and contributing to the progression of BC.

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Gastric cancer is one of the most common types of cancer worldwide. Nevertheless, effective therapeutic strategies have not yet been discovered. Several studies have shown that tanshinone IIA (TIIA), which is extracted from the traditional herbal medicine plant Danshen (Salvia miltiorrhiza), has potential activity against many kinds of cancer. Our previous research demonstrated that TIIA can induce cell death in gastric cancer. However, the exact signaling pathway response is still unclear. Post-translational modification (PTM) plays a significant role in a wide range of physiological processes in cancer, via regulation of both signal transduction cascades and many cellular pathways. Here, we integrated multilayer omics-transcriptomics and dynamic phosphoproteomics-to elucidate the regulatory networks triggered by TIIA in gastric cancer. We identified the phosphorylation of heat shock protein 27 (HSP27) at serine 82 in response to TIIA, which caused reactive oxygen species (ROS) production and unfolded protein response (UPR). Moreover, the accumulation of cellular stress increased the expression of heat shock factor 1 (HSF1). In addition, the downstream targets of HSF1, which were involved in heat shock stress and apoptosis, were also activated in TIIA-treated cells. In conclusion, this study performs a multiomic approach to clarify a comprehensive TIIA-responsive network leading to cell death in gastric cancer.

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Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats (n = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.

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Heat Shock Protein 27 (HSP27) is a small molecular chaperone that reduces the development of atherosclerosis by lowering plasma cholesterol levels as well as inflammation. Human studies show an inverse correlation between atherosclerotic burden and HSP27 expression, and are supported by murine models in which augmenting HSP27 levels curbs experimental atherogenesis. Natural HSP27 auto-antibodies (AAb) are found in human plasma, however their role in modulating the athero-protective effects of HSP27 is unknown. The purpose of this study is to characterize the biophysical interaction between human recombinant HSP27 and AAb. A validated polyclonal anti-HSP27 IgG antibody (PAb) was used to mimic natural AAb. Homology modeling and secondary structure prediction tools facilitated the design of HSP27 truncation and phosphorylation mutants. Secondary structural changes were identified using Circular Dichroism (CD) and Dynamic Light Scattering (DLS). Similar to prior structural investigations of HSP27, there was a predominance of α-helical content in the N-terminal truncation and dephosphorylation ("AA") mutants. The α-crystallin domain (ACD) predominantly consists of ß-strands, with the addition of the N-terminal increasing helical content and the C-terminal maintaining ß structure. With increasing ratios of PAb to HSP27 ß structure abundance and particle size increased, with a similar trend observed with the N-terminus, C-terminus and ACD peptides but an opposite trend with the phosphorylation peptides. Taken together, these studies provide insights into the interaction of HSP27 and its AAb that ultimately may aid in optimizing the design of HSP27 peptidomimetics with anti-atherogenic potential.

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BACKGROUND: The precise role of heat shock protein 27 (HSP27), as a type of small molecular protein in HSPs, in pancreatic ductal adenocarcinoma (PDAC) remains to be elucidated. The aim of the present study was to investigate the expression and function of HSP27 in PDAC cells. METHODS: We first detected the expression of HSP27 in PDAC tissues. Combining with the clinical pathology characteristics of PDAC patients, the relationship between them was analyzed. Then, we knocked down HSP27 using short interfering RNA (siRNA) and observed its biological functions using scratch assay and matrigel invasion and migration assays in ASPC-1 and PANC-1 cells. In mechanism, we verified the ß-catenin/MMP-3 pathway associated proteins in ASPC-1 and PANC-1 cells. RESULTS: We found that HSP27 was highly expression in PDAC tissues, and was positively correlated with tumor differentiation, TNM staging and poor prognosis of PDAC patients. In vitro, we down-regulated the expression of HSP27 in ASPC-1 and PANC-1 cells and found that the invasion and migration ability of PDAC cells were significantly depressed, meanwhile, the activation of the ß-catenin/MMP-3 pathway was inhibited. CONCLUSIONS: HSP27 may be used as a tumor biomarker for diagnosis of PDAC, and HSP27 can promote the invasion and migration of PDAC by activating the ß-catenin/MMP3 Pathway. Therefore, inhibition of HSP27 has therapeutic potential for the treatment of PDAC.

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OBJECTIVE: To clarify the mechanism of heat shock protein 27 (HSP27) as a diagnostic biomarker in coronary heart disease (CHD) and atherosclerosis (AS). METHOD: Expressions of HSP27 in patients with CHD and healthy controls were determined by enzyme-linked immunosorbent assay and the expressions of HSP27 in aortas of patients with CHD and healthy controls were measured by immunohistochemistry. Receiver operating characteristic curve was applied to assess the diagnostic performance of HSP27 in CHD. ApoE-/- mice were included and accordingly grouped. The expressions of HSP27 in AS plaque were measured by quantitative real-time polymerase chain reaction, immunohistochemistry, and Western blot analysis. AS plaque was observed using hematoxylin and eosin staining. DHE was used to detect reactive oxygen species (ROS) levels in aortas. The expressions of mitochondrial apoptosis-related proteins were measured by Western blot analysis. Cell apoptosis was determined by TUNEL staining. RESULTS: HSP27 was highly expressed in patients with CHD than in healthy controls ( P < 0.01). In comparison to the normal group, the model group had increased the relative positive area of HSP27 and higher expressions of HSP27, Bax, caspase-3, and apoptosis index (AI) but decreased Bcl-2 expression in AS plaque, as well as larger plaque areas and elevated ROS levels in the aorta (all P < 0.05). The HSP27-small interfering RNA group had increased expressions of Bax, caspase-3, and AI but decreased Bcl-2 and HSP27 expressions in AS plaque, as well as larger plaque areas, the relative positive area of HSP27 and higher ROS levels in aorta when compared with those in the model group (all P < 0.05). CONCLUSION: HSP27 exerts its protective role by suppressing ROS and AS progression by inhibiting mitochondria apoptosis pathway in CHD.

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BACKGROUND: In response to various stimuli, heat shock protein 27 (Hsp27) functions as an anti-apoptotic or/and anti-inflammatory factor which confers a survival advantage to cells. This study was aimed to explore whether Hsp27 also has a cytoprotective role in human renal tubular epithelial cells, and to evaluate its potential in treating septic acute kidney injury (septic AKI). METHODS: HK-2 cells were subjected to different concentrations (0-10 µg/mL) of lipopolysaccharide (LPS) for various times (0-24 h) to establish a septic AKI model in vitro. Before LPS administration, HK-2 cells were transfected either with vectors or siRNA against Hsp27, and the changes in cell viability and apoptotic cells rate were assessed using CCK-8 and flow cytometry. The expression changes in apoptosis-related proteins, proinflammatory cytokines and chemokine, as well as main factors in NF-κB and JNK pathways were mainly determined by Western blotting. Besides, the relationship between Hsp27 and Bcl-2 was detected by co-immunoprecipitation. RESULTS: LPS remarkably damaged HK-2 cells by reduction of cell viability, induction of apoptosis, and stimulation of proinflammatory cytokines and chemokine release. Hsp27 overexpression significantly impaired LPS-induced damage in HK-2 cells. Hsp27 overexpression couldn't alter the mRNA level of Bcl-2, but could interact with Bcl-2 at an endogenous level. Both NF-κB and JNK pathways were activated by LPS, while were blocked in Hsp27-overexpressing cells. CONCLUSION: Hsp27 overexpression conferred a survival advantage to LPS-injured HK-2 cells by controlling cell viability, apoptosis and inflammation, possibly via interaction with Bcl-2 and modulation of NF-κB and JNK pathways.

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Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 µM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

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CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures.

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The aim of the present study was to investigate whether the inhibition of HSP27 phosphorylation, which affects certain cellular functions, modulates sensitivity to 5-fluorouracil (5-FU) in colorectal cancer cells. Exposure to 5-FU in HCT116 and HCT15 cells expressing high levels of HSP27 with a low 5-FU sensitivity caused a minimal change in HSP27 expression, but induced the upregulation of HSP27 phosphorylation, particularly at Ser78. By contrast, exposure to 5-FU in HT29 cells expressing a low level of HSP27 with a high 5-FU sensitivity marginally increased HSP27 expression, with minimal phosphorylation. Treatment with a selective inhibitor, p38 mitogen-activated protein kinase (MAPK; SB203580), caused the dose-dependent suppression of HSP27 phosphorylation, which was upregulated by 5-FU, reducing the half maximal inhibitory concentration values of 5-FU in the HCT116 and HCT15 cells. However, treatment with SB203580 exhibited no significant effect on cell growth or survival. In conclusion, this study indicated that the inhibition of HSP27 phosphorylation by a selective inhibitor of p38 MAPK promotes 5-FU sensitivity without causing cytotoxicity in colorectal cancer cells.

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To identify the molecular target of diallyl trisulfide (DATS) in human leukemic cell line U937, we examined modification of thiol group(s) of cellular proteins by the redox 2D PAGE. A unique protein spot appeared by DATS treatment was identified to be heat shock protein 27 (HSP27). Hsp27 is suggested to be one of the molecular target of DATS in U937.

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One missing puzzle piece to study heat shock protein 27 (HSP27) in P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) was the amount of HSP27 and the extent of its phosphorylation in the biological context. Liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based targeted proteomics allows researchers to monitor associated proteins and their modification simultaneously and quantitatively. In this study, a targeted proteomics assay was first developed and validated for the quantification of HSP27 and its phosphorylated forms. Using this assay, the level of HSP27 was determined in non-tumoral cells MCF-10A, parental drug-sensitive cancer cells MCF-7/WT and drug-resistant cancer cells MCF-7/ADR. A decrease of HSP27 expression was observed in P-gp overexpressed MCF-7/ADR cells. A quantitative time-course analysis of both HSP27 and P-gp in doxorubicin (DOX)-treated MCF-7/WT cells also implied that HSP27 may participate in the P-gp modulation. Furthermore, stoichiometry of site-specific HSP27 phosphorylation indicated that DOX treatment rapidly induced the HSP27 phosphorylation at Ser82. Moreover, conventional analytical methods were also performed for a comparison. BIOLOGICAL SIGNIFICANCE: LC/MS/MS-based targeted proteomics turns out to be a promising quantification approach for the study of proteins in the preclinical and clinical environment. Unfortunately, rare studies applied this technology to detect multiple associated proteins or protein modification in one experiment. This study demonstrated the potential of LC/MS/MS-based targeted proteomics to understand the cell events in a more accurate context of biological system. By the quantitative time-course analysis of HSP27 and its phosphorylated forms at sites of Ser15 and Ser82, the possible role of HSP27 in P-gp mediated MDR was suggested. Further development of targeted proteomics in future may provide more insight into signal transduction pathways upon perturbation of a protein network or changes to a panel of proposed biomarkers in a given disease state.

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