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1.
There is a current interest in dietary compounds, such as green tea polyphenols, that can favor protection against a variety
of brain disorders, including Alzheimer’s disease, ischemia, and stroke. The objective of the present study was to investigate
the effects of ( _)-epicatechin-3-gallate (ECG), one of three three major green tea antioxidants, on C6 lineage cells. Here, we evaluated cell
morphology and integrity and specific astrocyte activities; glutamate uptake and secretion of S100B in the presence of 0.1,
1 and 10 μM ECG. During 6 h of incubation, cell morphology was altered only at 10 μM ECG; however, after 24 h of treatment,
cells become stellate in the presence of all concentrations of ECG. Loss of cell integrity was observed after 24 h with 10 μM
ECG and represented only 6% of cells, in contrast with 2% observed at basal conditions. ECG (1–10 μM) induced a decrease (about
36%) in glutamate uptake after 1 h of incubation. After 6 h, an opposite effect occurred and ECG induced a sustained increase
in glutamate uptake of about 70% from 0.1 μM. In addition, a significant increase in S100B was observed at 1 μM ECG (36%)
and 10 μM ECG (69%) after 1 h, in contrast to 6 h of treatment, where all doses of ECG induced a significant increase (about
60%) in S100B secretion. These data demonstrate that ECG induces a significant improvement in glutamate uptake and S100B secretion
in C6 cells, indicating that ECG could contribute to the neuroprotective role of astroglial cells. 相似文献
2.
S100B is an astrocyte calcium-binding protein that plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, has a trophic or apoptotic effect on neurons, depending on its concentration. Hyperglycemic rats show changes in glial parameters, including S100B expression. Here, we investigated cell density, morphological and biochemical alterations in primary cortical astrocytes from rats and C6 glioma cells cultured in high-glucose medium. Astrocytes and C6 glioma cells have a reduced content of S100B and glial fibrillary acidic protein when cultured in a high-glucose environment, as well as a reduced content of glutathione and cell proliferation rate. Although these cells have been used indistinctly to study S100B secretion, we observed a contrasting profile of S100B secretion in a high-glucose medium: a decrease in primary astrocytes and an increase in C6 glioma cells. Based on the in vitro neurotrophic effects of the S100B protein, our data suggest that chronic elevated glucose levels affect astrocyte activity, reducing extracellular secretion of S100B and that this, in turn, could affect neuronal activity and survival. Such astrocyte alterations could contribute to cognitive deficit and other impairments observed in diabetic patients. 相似文献
3.
The phenotypic effects of selectively decreasing the levels of S100 beta in cultured glial cells were analyzed. Two separate antisense approaches were utilized for inhibition of S100 beta production: analysis of clonal isolates of rat C6 glioma cells containing an S100 beta antisense gene under the control of a dexamethasone-inducible promoter, and analysis of C6 cells treated with S100 beta antisense oligodeoxynucleotides. Both antisense methods resulted in a decrease in S100 beta levels in the cell, as measured by RIA. The inhibition of S100 beta production correlated with three alterations in cellular phenotype: (a) a flattened cell morphology; (b) a more organized microfilament network; and (c) a decrease in cell growth rate. The studies describe here provide direct evidence for an involvement of S100 beta in glial cell structure and function, and suggest potential in vivo roles for S100 beta in regulation of glial cell morphology, cytoskeletal organization, and cell proliferation. 相似文献
4.
Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABAA receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABAA communication between astrocytes and neurons. The effects of two putative agonists of GABAA, β-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABAA receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response. 相似文献
5.
S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin β1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin β1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin–dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and β tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin β1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin β1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways. 相似文献
6.
为研究hS100A6对人骨肉瘤细胞系143B的体内体外作用,利用整合有S100A6基因和SiS100A6基因的腺病毒(AdS100A6和AdSiS100A6)作用于143B细胞,MTT法和台盼蓝染色法检测其对细胞的增殖作用,Hoechst染色法检测其对凋亡的作用,Transwell实验检测其对迁移的作用,同时以143B裸鼠皮下移植瘤为动物模型,检测S100A6对143B的体内作用,结果显示AdS100A6干预后出现细胞增殖活性的减弱、凋亡率增高、迁移率降低,在体内试验中瘤体体积明显较对照组减小,而AdsiS100A6干预后出现时间依赖性地细胞增殖活性的增强、凋亡率降低、迁移率增高,体内试验中瘤体体积较对照组明显增大.提示hS100A6能抑制人骨肉瘤细胞株143B细胞的增殖和迁移,并促进细胞凋亡,同时能抑制人骨肉瘤细胞株143B的裸鼠皮下移植瘤的生长. 相似文献
7.
Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells. 相似文献
8.
探讨体外共培养环境中小鼠胚胎干细胞对小鼠黑色素瘤B16细胞的影响。建立C57BL/6小鼠胚胎干细胞系,通过小鼠胚胎干细胞与肿瘤细胞体外共培养模型观察小鼠胚胎干细胞对肿瘤细胞的形态及生长行为的影响,MTT法与transwell小室法分别检测共培养后肿瘤细胞粘附性、迁移性及侵袭性的变化。共培养中小鼠胚胎干细胞能够侵入并推开小鼠黑色素瘤细胞形成自己的生长空间,与对照组比较共培养后肿瘤细胞的粘附性、迁移性及侵袭性均显著降低(P<0.05,P<0.01)。结果表明体外共培养体系中小鼠胚胎干细胞能够侵袭肿瘤细胞,并降低细胞粘附、迁移及侵袭相关恶性生物学行为。 相似文献
9.
Several molecules have been shown to be involved in glial-neuronal communication, including S100B, an astrocyte-derived neurotrophic cytokine. Extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by an unknown mechanism. Here, we investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. DCG-IV, an agonist of group II metabotropic glutamate receptors, caused a smaller decrease in S100B secretion when compared to 1 mM glutamate. d-aspartate partially reverted the glutamate effect on S100B release and two other inhibitors, PDC and DIDS, reverted it completely. These findings suggest that S100B secretion is inversely coupled to glutamate uptake. Decrease in S100B secretion may be considered as direct excitotoxic damage, but a beneficial mechanism effect cannot be ruled out, because S100B elevation could cause an additional cell death.The authors Francine Tramontina and Marina C. Leite are equally contributed to this work. 相似文献
10.
S100A6 (calcyclin) is a calcium binding protein with two EF‐hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non‐transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by β‐galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function. J. Cell. Biochem. 109: 576–584, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
11.
We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human β-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammtory cytokine secretion (IL-1β and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent. 相似文献
12.
In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5°C). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha- ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5°C, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G 1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5°C that is phenotypically indistinguishable from p53-mediated G 1 arrest at the permissive temperature (32°C). S100B-REF cells proceeding from G 1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis. 相似文献
13.
Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide, comprised of three amino acids, that has shown antitumor effects on human hepatocarcinoma BEL-7402 in vitro and in vivo. In this study, we evaluated the inhibition of YSL on invasion and adhesion of the mouse B16-F10 melanoma cell line by injecting B16-F10 cells into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model. YSL inhibited B16-F10 cell metastasis to lung, reducing the number and area of metastasis lesions. When we treated B16-F10 cells with YSL (0.01, 0.1, 1, 10, or 100 microg/mL) in vitro, we found that YSL inhibited the proliferation of B16-F10 cells with a 28.11% rate of inhibition. YSL significantly decreased the adhesiveness of B16-F10 cells to Matrigel with a 29.15% inhibition rate; YSL also significantly inhibited the invasion of B16-F10 cells, producing an inhibition of 35.31%. By analyses with Western blot and real-time RT-PCR, we found that YSL markedly inhibited the expression of ICAM-1 in B16-F10 cells. These data suggest that YSL inhibits the growth, invasion, and adhesion of B16-F10 cells. 相似文献
14.
S100 proteins are EF-hand calcium-binding proteins with various intracellular functions including cell proliferation, differentiation, migration, and apoptosis. Some S100 proteins are also secreted and exert extracellular paracrine and autocrine functions. Experimental results suggest that the receptor for advanced glycation end products (RAGE) plays important roles in mediating S100 protein-induced cellular signaling. Here we compared the interaction of two S100 proteins, S100B and S100A6, with RAGE by in vitro assay and in culture of human SH-SY5Y neuroblastoma cells. Our in vitro binding data showed that S100B and S100A6, although structurally very similar, interact with different RAGE extracellular domains. Our cell assay data demonstrated that S100B and S100A6 differentially modulate cell survival. At micromolar concentration, S100B increased cellular proliferation, whereas at the same concentration, S100A6 triggered apoptosis. Although both S100 proteins induced the formation of reactive oxygen species, S100B recruited phosphatidylinositol 3-kinase/AKT and NF-kappaB, whereas S100A6 activated JNK. More importantly, we showed that S100B and S100A6 modulate cell survival in a RAGE-dependent manner; S100B specifically interacted with the RAGE V and C(1) domains and S100A6 specifically interacted with the C(1) and C(2) RAGE domains. Altogether these results highlight the complexity of S100/RAGE cellular signaling. 相似文献
15.
The differential adhesion of cultured mammalian clonal cell lines to components of the extracellular matrix was examined by kinetic adhesion and long-term growth assays. Uniform artificial matrices were prepared by air drying collagen Type I solution (C) onto a microtiter well and then air drying a solution containing a single glycosaminoglycan (GAG): hyaluronic acid (HA), chondroitin sulfate-4 (CHS-4), or chondroitin sulfate-6 (CHS-6). The adhesion of [3H]thymidine-prelabeled cells suspended in fibronectin (FN) depleted medium was measured at 2 and 6 hr. Neuroblastoma (N18, Lan 1) and melanoma (B16, G361, S91) cell lines exhibited a significantly greater percentage of cells adhering to one or more C-GAG matrices compared with C matrices. Maximal adhesion at 2 hr was to C-HA. In contrast at 2 hr, two glial, two epithelial, and one fibroblastic cell line showed unchanged or significantly decreased binding to C-GAG compared with C matrices. Further experiments using a neuroblastoma (N18) and a glioma (C6) cell line indicated that the adhesion patterns were not altered either by the method of dissociation from the tissue culture dish, preincubation with exogenous GAG, or the addition of exogenous fibronectin. Assays of N18 and C6 adhesion to matrices made from a non-GAG polyanionic compound, polygalacturonic acid (PGA), did not yield the same adhesion patterns as C-HA matrices. Long-term growth studies of a neuroblastoma (N18) melanoma (S91), and glioma (C6) cell line on nonuniform matrices deliberately prepared with GAG-rich and GAG-poor regions complemented the observations from the kinetic adhesion assays. N18 and S91 cells did not grow on areas which did not contain GAG by toluidine blue staining. However, the C6 cells did not grow on areas which did strongly stain for GAG. A quantitative analysis of the long term growth of N18 and C6 cells substantiated these observations. All these data indicate that the cellular phenotype may be correlated with matrix adhesion. Neuroblastomas and melanomas have a greater affinity for GAG-containing matrices while glial, epithelial, and fibroblastic cells appear to have a greater or equal affinity for collagen matrices. 相似文献
16.
To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100 beta polypeptide. The level of S100 beta in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about two-fold. The subcellular distribution of S100 beta and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100- and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100 beta is the predominant S100 polypeptide in C6 cells and suggest that changes in S100 beta and S100 beta-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells. 相似文献
17.
Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis. 相似文献
18.
Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment. 相似文献
20.
Initial adhesion of B16 melanoma variants to non-activated endothelial cells is mediated through specific interaction between GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer) expressed on melanoma cells and lactosylceramide (LacCer, Gal beta 1----4Glc beta 1----Cer) expressed on endothelial cells. This adhesion is predominant over integrin- or lectin-mediated adhesion in a dynamic flow experimental system employing a parallel plate laminar flow chamber (Lawrence, M. B., Smith, C. W., Eskin, S. G., and McIntire, L. V. (1990) Blood 75, 227-237). In this system, a tumor cell suspension flows over a glass plate coated with glycosphingolipid, lectin, or fibronectin, and adhesion is recorded on videotape. These conditions were designed to mimic the microvascular environment in which tumor metastatic deposition takes place. In contrast, lectin- and fibronectin-based mechanisms are predominant in previously used static adhesion systems. Under static conditions, the relative degree of adhesion of the four B16 variants to endothelial cells or to LacCer-coated plates was the same as their relative degree of GM3 expression (i.e. BL6 approximately F10 greater than F1 greater than WA4), and adhesion was inhibited in the presence of methyl-beta-lactoside, or liposomes containing LacCer or GM3. Adhesion was also inhibited by pretreatment of B16 cells with anti-GM3 antibody DH2 or sialidase and by pretreatment of endothelial cells with anti-LacCer antibody T5A7. Under dynamic flow conditions, WA4 cells did not adhere to mouse endothelial cells at high shear stress (greater than 2.5 dynes/cm2) but did adhere at lower shear stress. In contrast, BL6 and F10 cells adhered strongly at both low and high shear stress. BL6 cell adhesion to endothelial cells at both low and high shear stress was inhibited in the presence of antibody DH2, ethyl-beta-lactoside, or lactose, as well as by pretreatment of BL6 cells with sialidase. Thus, some clear differences, as well as similarities, in cell adhesion under static versus dynamic conditions are demonstrated. These findings suggest that melanoma cell adhesion to endothelial cells, based on GM3/LacCer interaction, initiates metastatic deposition, which may trigger a series of "cascade" reactions leading to activation of endothelial cells and expression of Ig family or selectin receptors, thereby promoting adhesion and migration of tumor cells. 相似文献
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