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1.
Br J Haematol ; 190(3): 418-429, 2020 08.
Article in English | MEDLINE | ID: mdl-32130729

ABSTRACT

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is driven by complex genetic and epigenetic alterations from an aberrant clone of hematopoietic stem/progenitor cells (HSPCs). Ubiquitin-specific protease 7 (USP7) has been demonstrated to have an important oncogenic role in the development of several cancer types, but its role in MDS is unknown. Here, we demonstrate that USP7 expression is elevated in MDS cell lines and patient samples. The USP7-selective small-molecule inhibitors P5091 and P22077 inhibited cell proliferation and induced megakaryocytic differentiation in both cell lines and primary cells. Furthermore, pharmacological inhibition of USP7 markedly suppressed the growth of MDS cell lines in xenograft mouse models. To explore the mechanisms underlying the observed phenotypic changes, we employed RNA-seq to compare the differences in genes after USP7 inhibitor treatment and found that gelsolin (GSN) expression was increased significantly after USP7 inhibitor treatment. Furthermore, knockdown of GSN attenuated the proliferation inhibition, apoptosis induction and megakaryocyte differentiation induced by USP7 inhibitors in MDS cells. Collectively, our findings identify previously unknown roles of USP7 and suggest that the USP7/GSN axis may be a potential therapeutic target in MDS.


Subject(s)
Gelsolin/physiology , Megakaryocytes/drug effects , Myelodysplastic Syndromes/pathology , Protease Inhibitors/pharmacology , Thiophenes/pharmacology , Thrombopoiesis/drug effects , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line/transplantation , Enzyme Induction/drug effects , Gelsolin/biosynthesis , Gelsolin/genetics , Heterografts , Humans , Megakaryocytes/pathology , Mice , Mice, Inbred NOD , Neoplasms, Experimental/etiology , Risk , Transcriptome/drug effects , Ubiquitin-Specific Peptidase 7/physiology , Up-Regulation/drug effects
2.
Protein Expr Purif ; 135: 33-36, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28465181

ABSTRACT

A mutation (D187N/Y) in human plasma gelsolin (GSN) leads to the generation of an 8 kDa GSN fragment (8 kDa-GSN), and consequently causes the familial amyloidosis of Finnish type. Because of its faster kinetics of amyloid formation under physiologically relevant conditions, 8 kDa-GSN is used to explore gelsolin amyloidosis and screen small molecules that can disaggregate amyloids. However, the synthetic 8 kDa-GSN is expensive, and substantial quantities of 8 kDa-GSN are needed for the screen. Here we report a study to obtain recombinant 8 kDa-GSN with high yield from Escherichia coli. Firstly, 8 kDa-GSN in fusion with Mxe GyrA intein was purified by Ni-affinity chromatography. Then 8 kDa-GSN was released by intein-mediated protein cleavage, and separated from intein by ion-exchange chromatography. The yield of 8 kDa-GSN was only 1.5 mg/L from bacterial culture in the previous report, while it was improved to 4.25 mg/L in our study. Finally, the amyloidogenic property of 8 kDa-GSN was validated by circular dichroism spectrometry and dynamic light scattering.


Subject(s)
Amyloidogenic Proteins/biosynthesis , DNA Gyrase/genetics , Gelsolin/biosynthesis , Inteins/genetics , Mycobacterium xenopi/chemistry , Recombinant Fusion Proteins/biosynthesis , Amyloidogenic Proteins/genetics , Amyloidogenic Proteins/isolation & purification , Chromatography, Ion Exchange , Cloning, Molecular , DNA Gyrase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gelsolin/genetics , Gelsolin/isolation & purification , Gene Expression , Humans , Molecular Weight , Mutation , Mycobacterium xenopi/enzymology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification
3.
Pathol Res Pract ; 211(7): 505-12, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25908108

ABSTRACT

OBJECTIVES: To examine the prognostic significance of Gelsolin, NF-κB, and p53 in clear cell renal cell carcinoma (CRCC), which has an unpredictable behavior and tendency for recurrence and metastasis. MATERIALS AND METHODS: Immunohistochemistry was performed on 100 consecutive cases of CRCC using antibodies against Gelsolin, NF-κB, and p53. Tumors were grouped by nuclear grade (NG) as low NG (NG1, 2) or high NG (NG3, 4), and by pathological stage as localized (pT1, 2) or locally invasive (pT3, 4). Clinical stage was grouped as early stage (stage I, II) or late stage (stage III, IV). Evaluation was based on cytoplasmic (NF-κB(Cyt)) and nuclear (NF-κB(Nuc)) expression for NF-κB, nuclear expression for p53, membranous and cytoplasmic expression for Gelsolin. RESULTS: Gelsolin expression correlated with high NG (p = 0.001), metastasis (p = 0.003), late stage (p = 0.008), and cancer death (p = 0.001). NF-κB(Cyt) expression correlated with high NG (p = 0.002), perirenal invasion (p = 0.010), local invasion (p = 0.020), and late stage (p= 0.003). NF-κB(Nuc) expression failed to predict the prognosis of CRCC. p53 expression correlated with high NG (p = 0.045), lymphovascular invasion (p = 0.05), metastasis (p = 0.001), late stage (p = 0.028), and cancer death (p = 0.034). However, only Gelsolin was found to correlate with disease-specific survival, (p = 0.006), and neither NF-κB nor p53 showed such relation. CONCLUSION: Expressions of Gelsolin, NF-κB(Cyt), and p53 associated with aggressive behavior of CRCC, while Gelsolin expression specifically indicated poor disease-specific survival. The results of the present study served to determine biomarkers for predicting high-risk patients with CRCC, expected to show aggressive phenotype.


Subject(s)
Carcinoma, Renal Cell/pathology , Gelsolin/biosynthesis , Kidney Neoplasms/pathology , NF-kappa B/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Adult , Aged , Biomarkers, Tumor/analysis , Carcinoma, Renal Cell/mortality , Disease-Free Survival , Female , Gelsolin/analysis , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Kidney Neoplasms/mortality , Male , Middle Aged , NF-kappa B/analysis , Proportional Hazards Models , Tumor Suppressor Protein p53/analysis
4.
Sci Rep ; 5: 7901, 2015 Jan 20.
Article in English | MEDLINE | ID: mdl-25600697

ABSTRACT

Recent studies show that targeting gene promoter or 3' terminal regions of mRNA with siRNA induces target gene transcription. However, the ability of exon-targeting siRNA to affect transcription has yet to be demonstrated. We designed and synthesized siRNA against various exons in the gelsolin gene (GSN) to knockdown GSN transcript in KYSE150 and KYSE450 cells. Surprisingly, we found that siGSN-2, targeting the GSN twelfth exon, induced GSN gene transcription detected by real time RT-PCR. An siGSN-2 co-precipitation assay was performed and H3 histone, previously shown to correlate with gene transcription, was detected in the siGSN-2 pull-down pellet. However, H3 histone was not detected in an siGSN-1-precipitated pellet, which resulted in GSN knockdown. In addition, siGSN-2 decreased stress fibers, lamellipodia and filopodia, demonstrating that siGSN-2 induced GSN transcription activation and exerted biological function. In conclusion, our finds reveal siRNA, which is derived from target gene exon, can form the complex with H3 histone to be involved in the regulation of gene expression.


Subject(s)
Esophageal Neoplasms/genetics , Gelsolin/genetics , RNA, Small Interfering/genetics , Transcriptional Activation/genetics , Apoptosis/genetics , Cell Line, Tumor , Esophageal Neoplasms/pathology , Exons/genetics , Gelsolin/biosynthesis , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Histones/genetics , Humans
5.
J Endod ; 41(4): 493-500, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25583547

ABSTRACT

INTRODUCTION: Adseverin is an actin-severing and actin-capping protein that is primarily expressed in secretory cells, where it regulates the filamentous actin cytoskeleton during cell differentiation and exocytosis. However, little is known regarding its regulatory role in dental pulp cells (DPCs). This study examined the expression and function of adseverin in the proliferation, migration, and odontoblastic differentiation of DPCs. METHODS: DPCs were assayed for morphologic changes, proliferation, migration, alkaline phosphatase activity, and dentin sialoprotein and dentin matrix protein-1 protein levels in vitro after knockdown of adseverin by using small interfering RNA. Tooth germs isolated from Sprague-Dawley rats were processed for immunohistochemistry analysis of adseverin. RESULTS: Adseverin expression was increased in a time-dependent fashion in the early stage of odontoblastic differentiation. When adseverin expression was suppressed in DPCs, their cellular morphology was altered, and their proliferation, migration, and odontoblastic differentiation were substantially decreased in vitro. Secretory odontoblasts in the tooth germ at day 5 post partum expressed a stronger adseverin signal compared with those at days 1 and 3 post partum. CONCLUSIONS: Adseverin may play a crucial role in the proliferation, migration, and odontoblastic differentiation of DPCs via filamentous actin cytoskeleton regulation. However, further investigations are required to clarify the underlying mechanisms.


Subject(s)
Dental Pulp/cytology , Gelsolin/physiology , Odontoblasts/cytology , Cell Differentiation , Cell Movement , Cell Proliferation , Cells, Cultured , Gelsolin/biosynthesis , Humans
6.
Int J Oncol ; 44(1): 161-6, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24212916

ABSTRACT

SCIN is a calcium regulated actin severing and capping protein. Its homologue in zebrafish is found to be related with cell death. In the present study, we found that SCIN is highly expressed in human prostate cancer specimens. However, the functions of SCIN in human prostate carcinoma cells are largely unknown. To address the function of SCIN in prostate carcinoma cells, we used lentivirus-mediated RNAi to knock down SCIN expression in PC3 cells, a prostate carcinoma cell line. We found that in vitro silencing of SCIN could inhibit the proliferation and colony formation ability of PC3 cells. Furthermore, cell cycle analysis showed that reduced SCIN expression lead to G0/G1 cell cycle arrest through the regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin-dependent kinase inhibitor 2A (CDKN2A, p16Ink4A) and cyclin A2. These results suggest that SCIN plays an important role in the proliferation of prostate cancer cells and lentivirus-mediated inhibition of SCIN expression may be a potential therapeutic method for the treatment of prostate cancer.


Subject(s)
Cell Proliferation , G1 Phase Cell Cycle Checkpoints/genetics , Gelsolin/biosynthesis , Prostatic Neoplasms/genetics , Apoptosis/genetics , Gelsolin/antagonists & inhibitors , Gelsolin/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Lentivirus , Male , Prostatic Neoplasms/pathology
7.
PLoS One ; 8(12): e81099, 2013.
Article in English | MEDLINE | ID: mdl-24312525

ABSTRACT

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation.


Subject(s)
Ceratitis capitata/metabolism , Diet , Gelsolin/biosynthesis , Gene Expression Regulation, Developmental/physiology , Insect Proteins/biosynthesis , Proteome/biosynthesis , Animals , Ceratitis capitata/embryology , Ceratitis capitata/genetics , Gelsolin/genetics , Humans , Insect Proteins/genetics , Larva/metabolism , Proteome/genetics , Proteomics/methods
8.
Biomed Pharmacother ; 67(7): 557-60, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23809371

ABSTRACT

We have previously demonstrated that Ech1 is involved in the lymphatic metastasis of tumors in vitro. Here, we gain an insight into the role that Ech1 is playing in Hca-F cell. The expression of Annexin A7, Gelsolin and Clic1 genes, which were also relevant to tumor lymphatic metastasis, had been inhibited due to downregulation Ech1 gene by Western blot analysis. And downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo. Our work indicates although the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that.


Subject(s)
Carbon-Carbon Double Bond Isomerases/metabolism , Liver Neoplasms/metabolism , Lymphatic Metastasis/prevention & control , Animals , Annexin A7/biosynthesis , Cell Line, Tumor , Chloride Channels/biosynthesis , Down-Regulation , Gelsolin/biosynthesis , Gene Expression Regulation, Neoplastic , Liver Neoplasms/pathology , Lymphatic Metastasis/pathology , Mice
9.
PLoS One ; 8(6): e65809, 2013.
Article in English | MEDLINE | ID: mdl-23799049

ABSTRACT

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.


Subject(s)
Heart Ventricles/metabolism , MicroRNAs/metabolism , Transcriptome , Animals , Base Sequence , Gelsolin/biosynthesis , Gelsolin/genetics , Genes, Reporter , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , MAP Kinase Signaling System , Male , MicroRNAs/genetics , Molecular Sequence Annotation , Molecular Sequence Data , RNA Interference , RNA Isoforms/genetics , RNA Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Sequence Analysis, RNA
10.
Cytoskeleton (Hoboken) ; 70(7): 385-93, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23595955

ABSTRACT

Integrins are functionally regulated by "inside-out" signaling, in that stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs, but the mechanisms that regulate inside out signaling are not completely understood. Previously, we identified gelsolin in a proteomics screen to identify proteins involved in inside-out control of integrins using the lymphocytic leukemia cell line L1210. Furthermore, we showed that gelsolin was involved in affinity regulation of ß1 -integrins in the leukemic cell line U937. Here, we examined how gelsolin regulates ß1 -integrin affinity in the leukemia cell line L1210. We show that gelsolin is mainly expressed at the cell membrane and is present near ß1 -integrins. The role for actin polymerization in integrin affinity regulation was examined using the actin modulating agent jasplakinolide, which decreased ß1 -integrin affinity. Similarly, knock-down of gelsolin in L1210 cells also decreased ß1 -integrin affinity and cell adhesion to collagen. These data suggest that increased actin polymerization through gelsolin regulates ß1 -integrin affinity and cell adhesion.


Subject(s)
Cell Adhesion/physiology , Gelsolin/biosynthesis , Integrin beta1/metabolism , Actins/metabolism , Animals , Cell Membrane/metabolism , Gelsolin/antagonists & inhibitors , Gelsolin/deficiency , Gelsolin/metabolism , Gene Knockdown Techniques , Humans , Integrin beta1/biosynthesis , Leukemia L1210 , Mice , Signal Transduction
11.
Neuroscience ; 228: 60-72, 2013 Jan 03.
Article in English | MEDLINE | ID: mdl-23079629

ABSTRACT

Gelsolin is an actin filament-severing and capping protein, affecting cellular motility, adhesiveness and apoptosis. Whether it is expressed in the brain of burned mice has not yet been characterized. Mice were subjected to a 15% total body surface area scald injury. Neuropathology was examined by hematoxylin and eosin staining. Cerebral gelsolin mRNA, distribution and cleavage were demonstrated by quantitative polymerase chain reaction (QPCR), immunohistochemistry and Western blot, respectively. Cysteinyl aspartate-specific protease (caspase)-3-positive cells and activity were also measured. Burn injury could induce pathological alterations in the brain including leukocyte infiltration, necrosis, microabscess and gliosis. Compared with sham-injured mice, gelsolin mRNA was up-regulated at 8h post-burn (pb) in a transient manner in the cortex and striatum of burned mice, while it remained at higher levels in the hippocampus up to 72 hpb. Of interest, gelsolin was further cleaved into 42 and 48 kDa (kilo Dalton) fragments as illustrated in the hippocampus at 24 hpb, and was widely expressed in the brain by activated monocyte/macrophages, astrocytes and damaged neurons. In the meantime, caspase-3-positive cells were noted in the striatum of burned mice and its activity peaked at 24 hpb. To clarify inflammation-induced gelsolin expression and cleavage in the brain, rat pheochromocytoma cells were exposed to lipopolysaccharide to show increased gelsolin expression and caspase-3-dependent cleavage. The results suggest that burn-induced cerebral gelsolin expression would be involved in the activation of both the monocytes and astroglial cells, thereby playing a crucial role in the subsequent inflammation-induced neural apoptosis following burn injury.


Subject(s)
Brain/metabolism , Brain/pathology , Burns/metabolism , Gelsolin/biosynthesis , Gene Expression Regulation , Animals , Astrocytes/metabolism , Burns/pathology , Inflammation Mediators/physiology , Male , Mice , Mice, Inbred BALB C , Neurons/metabolism , Neurons/pathology , PC12 Cells , Random Allocation , Rats
12.
Can J Gastroenterol ; 26(1): 41-7, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22288069

ABSTRACT

OBJECTIVE: To identify and validate potential biomarkers of colorectal adenocarcinoma using a proteomic approach. METHODS: Multidimensional liquid chromatography/mass spectrometry was used to analyze biological samples labelled with isobaric mass tags for relative and absolute quantitation to identify differentially expressed proteins in human colorectal adenocarcinoma and paired normal mucosa for the discovery of cancerous biomarkers. Cancerous and noncancerous samples were compared using online and offline separation. Protein identification was performed using mass spectrometry. The downregulation of gelsolin protein in colorectal adenocarcinoma samples was confirmed by Western blot analysis and validated using immunohistochemistry. RESULTS: A total of 802 nonredundant proteins were identified in colorectal adenocarcinoma samples, 82 of which fell outside the expression range of 0.8 to 1.2, and were considered to be potential cancer-specific proteins. Immunohistochemistry revealed a complete absence of gelsolin expression in 86.89% of samples and a reduction of expression in 13.11% of samples, yielding a sensitivity of 86.89% and a specificity of 100% for distinguishing colorectal adenocarcinoma from normal tissue. CONCLUSIONS: These findings suggest that decreased expression of gelsolin is a potential biomarker of colorectal adenocarcinoma.


Subject(s)
Adenocarcinoma/metabolism , Biomarkers, Tumor/biosynthesis , Chromatography, Liquid/methods , Colorectal Neoplasms/metabolism , Gelsolin/biosynthesis , Tandem Mass Spectrometry/methods , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Aged , Asian People/genetics , Blotting, Western , China , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Down-Regulation/physiology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Indicators and Reagents , Male , Middle Aged , Proteomics/methods , Rectal Neoplasms/genetics , Rectal Neoplasms/metabolism , Rectal Neoplasms/pathology
13.
Int J Immunopathol Pharmacol ; 25(4): 911-22, 2012.
Article in English | MEDLINE | ID: mdl-23298482

ABSTRACT

Sepsis triggered by endotoxinemia may impair cardiac function. A decline in tolerance to septic shock occurs with aging. This study addressed the hypothesis that aging negatively impairs expression of gelsolin, and axerts the regulatory effects on the water channel protein aquaporin-1 (AQP-1) and endotoxin-inducible nitric oxide synthase (iNOS). We explored whether the age-related gene changes are associated with the cardiac dysfunction induced by endotoxic stress exposure. Male mice at young (3-month) and old (12-month) ages received intraperitoneal injections of saline or lipopolysaccharide (LPS, 30mg/Kg). Cardiac performance and morphology were analyzed by echocardiography at baseline and 2 and 24 h after injection. At the end of treatment, the animals were sacrificed, and cardiac tissues were collected for assessing expression of gelsolin, AQP-1, iNOS, and transcription-3 (STAT3). LPS administration led to a decreased contractility while increasing cardiac dimensions in both young and old mice. LPS also markedly induced expression of gelsolin in both animal groups. However, compared to young mice, old mice showed compromised induction of gelsolin and cardiac performance in response to endotoxin. Meanwhile, the LPS-exposed old animals exhibited higher levels of AQP-1, iNOS, and phosphorylated STAT3. Gelsolin-null mice had increased expression of glycosylated AQP-1 and STAT3 phosphorylation as well as cardiac dysfunction. Thus, endotoxin administration induces expression of gelsolin, AQP-1 and pro-inflammatory genes, such as iNOS. Our data suggest that changed expression of gelsolin, AQP-1 and iNOS may contribute to dysfunction of hearts in aged subjects with septic endotoxinemia.


Subject(s)
Aging/metabolism , Aquaporin 1/biosynthesis , Gelsolin/biosynthesis , Heart Failure/metabolism , Lipopolysaccharides/toxicity , Myocardium/metabolism , Actins/biosynthesis , Animals , Cells, Cultured , Humans , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase/biosynthesis , Phosphorylation , STAT3 Transcription Factor/metabolism
14.
Gynecol Oncol ; 120(1): 135-44, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21035170

ABSTRACT

OBJECTIVES: Cervical carcinoma is the second most common cause of death from gynecological cancers worldwide. Knowledge of the molecular mechanisms underlying the tumorigenesis of cervical cancer cell, except human papilloma virus infection, is limited. METHODS: A microarray was used to study the differential expression of genes in cancerous tissues to identify new molecular markers for diagnosis and prognosis. Their differential expression was confirmed with Western blotting and immunohistochemical analyses. The clinical correlations and prognostic significance of the aberrantly expressed proteins were evaluated to identify novel biomarkers of cervical cancer. RESULTS: The expression of gelsolin was significantly upregulated in 78% of patients with cervical cancer, and gelsolin was selected for further study. Gelsolin expression was stronger in cervical tumor tissues than in the surrounding noncancerous tissues (P<0.001). Gelsolin expression in the plasma of cervical cancer patients was increased 2.2-fold compared with that of healthy control subjects (P<0.001). The levels of plasma gelsolin in the early and late stages were significantly different (P=0.006). According to immunohistochemical analysis, increased gelsolin expression was associated with histological type and FIGO stage II. The 5-year overall survival and recurrence-free survival rates for the low-expression group (cut-off=115) were significantly higher than those of the high-expression group. Cancer cells with reduced gelsolin expression exhibited reduced migration and proliferation. CONCLUSIONS: These results provide strong evidence that gelsolin plays an important role in cellular proliferation and migration in cervical cancer and suggest that gelsolin is a promising marker for cervical cancer screening and prognosis.


Subject(s)
Biomarkers, Tumor/biosynthesis , Gelsolin/biosynthesis , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Adult , Aged , Biomarkers, Tumor/genetics , Cadherins/metabolism , Case-Control Studies , Cell Growth Processes/genetics , Cell Movement/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Epithelial-Mesenchymal Transition , Female , Fibronectins/metabolism , Gelsolin/blood , Gelsolin/genetics , Gene Expression Profiling , Gene Knockdown Techniques , HeLa Cells , Humans , Matrix Metalloproteinase 2/metabolism , Middle Aged , Neoplasm Staging , Prognosis , Retrospective Studies , Survival Rate , Up-Regulation , Uterine Cervical Neoplasms/genetics , Vimentin/metabolism
15.
Neurochem Res ; 35(7): 1075-82, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20339915

ABSTRACT

Gelsolin plays an important role in the regulation of amyloid beta-protein fibrillogenesis. We report here that calcium ionophore A23187 induces the expression of cytoplasmic gelsolin (c-gelsolin), and that protein kinase C (PKC) is involved in the up-regulation of c-gelsolin. In the presence of calcium, both SH-SY5Y and HEK-293 cells upon treatment with A23187 showed an increase in c-gelsolin expression in a concentration-dependent manner. Calcium-mediated up-regulation of c-gelsolin was inhibited by cycloheximide (a general inhibitor of protein synthesis). When cells were pretreated with staurosporine (an inhibitor of a variety of protein kinases including PKC), the up-regulation of c-gelsolin induced by A23187 was inhibited. Calphostin C, an inhibitor of PKC, blocked the up-regulation of c-gelsolin induced by A23187, while inhibitors of mitogen-activated protein kinases had no effect on c-gelsolin expression. In addition, phorbol-12-myristate-13-acetate, an activator of PKC, up-regulated c-gelsolin expression. These results suggest that calcium mediates up-regulation of c-gelsolin in a PKC-dependent manner.


Subject(s)
Calcium/metabolism , Cytoplasm/metabolism , Gelsolin/biosynthesis , Calcimycin/pharmacology , Cell Line , Cell Line, Tumor , Humans , Ionophores/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/physiology , Staurosporine/pharmacology , Up-Regulation
16.
Alzheimer Dis Assoc Disord ; 23(4): 319-22, 2009.
Article in English | MEDLINE | ID: mdl-19561443

ABSTRACT

Neuronal dysfunctions in several neurodegenerative diseases such as Down syndrome (DS) have been linked to oxidative stress. In this study, we observed that lipid peroxidation, a marker of oxidative stress, is significantly increased in the frontal cortex of brains of individuals with DS as compared with control subjects. We report here that gelsolin levels are increased in the frontal cortex of individuals with DS as compared with controls during early developmental ages (5 to 13 y). Interestingly, the levels of gelsolin in the frontal cortex were increased as a function of age in both DS and control subjects. Because cytoplasmic gelsolin has 5 free thiol groups (cysteine), and its levels are increased in response to oxidative stress, we propose that gelsolin may serve as an antioxidant protein.


Subject(s)
Aging/metabolism , Down Syndrome/metabolism , Frontal Lobe/growth & development , Frontal Lobe/metabolism , Gelsolin/metabolism , Adolescent , Aging/pathology , Aging/physiology , Antioxidants/metabolism , Antioxidants/physiology , Biomarkers/metabolism , Child , Child, Preschool , Down Syndrome/pathology , Female , Frontal Lobe/physiopathology , Gelsolin/biosynthesis , Humans , Infant , Male , Oxidative Stress/physiology , Up-Regulation/physiology
17.
J Proteome Res ; 7(8): 3572-82, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18549265

ABSTRACT

Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.


Subject(s)
Lipoproteins, LDL/metabolism , Macrophages/metabolism , Proteome/metabolism , Actins/biosynthesis , CapZ Actin Capping Protein/biosynthesis , Carbocyanines , Catalase/biosynthesis , Cells, Cultured , Cytoskeletal Proteins/biosynthesis , Electrophoresis, Gel, Two-Dimensional , Fluorescent Dyes , Gelsolin/biosynthesis , Heat-Shock Proteins/biosynthesis , Humans , Intracellular Space/metabolism , Lipoproteins, LDL/pharmacology , Microfilament Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Pyruvate Kinase/biosynthesis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
18.
BMC Cancer ; 8: 39, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-18237446

ABSTRACT

BACKGROUND: Gelsolin-like actin-capping protein (CapG) is a ubiquitous gelsolin-family actin-modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis, and motility. CapG has generated great interest due to its oncogenic function in the control of cell migration or invasion in a variety of cancer cells. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous-cell carcinoma (OSCC) cells and detected significantly high expression levels of CapG in OSCC-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of CapG in OSCC, we evaluated the status of CapG protein and mRNA expression in human oral premalignant lesions (OPLs) and primary OSCCs and correlated the results with clinicopathologic variables. METHODS: Matched normal and tumour tissue sections of 79 human primary OSCCs and 28 OPLs were analyzed for CapG expression by immunohistochemistry (IHC). Correlations between CapG-immunohistochemical staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to estimate CapG expression at the mRNA level. RESULTS: In IHC, substantial up-regulation of CapG protein was observed in primary OSCCs (52%) and OPLs (64%), whereas corresponding normal tissues showed consistently weak or absent immunoreactivity of CapG. qRT-PCR data were consistent with the protein expression status. Moreover, CapG expression was correlated with the TNM stage grading of OSCCs. CONCLUSION: Our finding of frequent dysregulated expression of CapG in premalignant and malignant lesions together with an association with an advanced clinical disease stage suggests that CapG could contribute to cancer development and progression and that CapG may have potential as a biomarker and a therapeutic target for OSCC.


Subject(s)
Carcinoma, Squamous Cell/metabolism , Gene Expression Regulation, Neoplastic/physiology , Microfilament Proteins/biosynthesis , Mouth Neoplasms/metabolism , Nuclear Proteins/biosynthesis , Precancerous Conditions/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Carrier Proteins/analysis , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cell Line, Tumor , Gelsolin/analysis , Gelsolin/biosynthesis , Gelsolin/genetics , Humans , Immunohistochemistry , Microfilament Proteins/analysis , Microfilament Proteins/genetics , Mouth/metabolism , Mouth/pathology , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Precancerous Conditions/genetics , Precancerous Conditions/pathology
19.
Cancer Lett ; 255(1): 57-70, 2007 Sep 18.
Article in English | MEDLINE | ID: mdl-17493746

ABSTRACT

Gelsolin and CapG are both actin binding proteins that modulate a variety of physiological processes by interacting differently with the actin cytoskeleton. Several studies suggest that overexpression of these proteins promotes invasion in vitro. In this study we explored the contribution of these proteins in human cancer cell invasion and motility. We show that down regulation of CapG or gelsolin in several types of cancer cells, including MDA-MB 231 and PC-3 cells, significantly reduces the invasive and motile properties of cells, as well as cell aggregation. These results point to a role for CapG and gelsolin as tumor activator.


Subject(s)
Gelsolin/biosynthesis , Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Actins/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Collagen/chemistry , Collagen Type I/metabolism , Drug Combinations , Humans , In Vitro Techniques , Laminin/chemistry , Microfilament Proteins/metabolism , Neoplasm Invasiveness , Proteoglycans/chemistry , RNA Interference , Signal Transduction , Wound Healing
20.
J Neurochem ; 98(4): 1019-31, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16895577

ABSTRACT

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.


Subject(s)
Enzyme Inhibitors/pharmacology , Gelsolin/deficiency , Glucose/physiology , Histones/metabolism , Hydroxamic Acids/pharmacology , Hypoxia/pathology , Neurons/physiology , Neuroprotective Agents , Acetylation/drug effects , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Animals , Cell Death/drug effects , Chromatin/metabolism , Dealkylation , Female , Gelsolin/biosynthesis , Gelsolin/genetics , Glucose/deficiency , Immunoassay , Immunoblotting , Immunohistochemistry , Microscopy, Confocal , Pregnancy , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/physiology
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