Resveratrol increases glutamate uptake and glutamine synthetase activity in C6 glioma cells

Resveratrol, a phytoalexin found mainly in grapes, is a promising natural product with anti-cancer and cardio-protective activities. Here, we investigated, in C6 glioma cells, the effect of resveratrol on some specific parameters of astrocyte activity (glutamate uptake, glutamine synthetase and secretion of S100B, a neurotrophic cytokine) commonly associated with the protective role of these cells. Cell proliferation was significantly decreased by 8% and 26%, following 24h of treatment with 100 and 250 microM resveratrol. Extracellular S100B increased after 48 h of resveratrol exposure. Short-term resveratrol exposure (from 1 to 100 microM) induced a linear increase in glutamate uptake (up to 50% at 100 microM resveratrol) and in glutamine synthetase activity. Changes in these glial activities can contribute to the protective role of astrocytes in brain injury conditions, reinforcing the putative use of this compound in the therapeutic arsenal against neurodegenerative diseases and ischemic disorders.     

S100/RAGE在实验性自身免疫性重症肌无力中对T细胞的作用及其机制研究

目的:探讨RAGE及其配体S100B在实验性自身免疫性重症肌无力(EAMG)中对T细胞的作用及其相关机制。方法:选取体重160-180g的Lewis大鼠,并将其随机分为EAMG模型组和CFA对照组。EAMG模型组大鼠通过尾根部注入200μL含有R-ACh R97-116肽段的免疫乳剂,并于初始免疫后的第30天尾根部追加免疫一次;CFA对照组大鼠为免疫乳剂中不含有R-ACh R97-116肽段。采用流式细胞术检测和比较两组大鼠CD4+T细胞上RAGE的表达情况;ELISA方法检测淋巴细胞培养上清中IFN-γ、IL-4、IL-17、TGF-β、IL-6和S100B的表达水平;采用S100B体外干预淋巴细胞,进一步检测S100B对EAMG大鼠T细胞增殖、亚型分布、细胞因子分泌的影响。结果:在疾病发生的晚期时相(初次免疫后第45天),EAMG组淋巴细胞CD4+T细胞上RAGE的表达明显高于CFA组(P0.001),血清中RAGE的配体S100B的表达也高于CFA组(P0.001);体外加入S100B干预能促进T淋巴细胞的增殖,与未干预组相比较差异显著(P0.05);S100B刺激后,Th1细胞和Th17细胞的百分比进一步增高,Th2细胞和Treg细胞百分比下降(PTh10.05,PTh170.05,PTh20.05,PTreg0.05),IFN-γ和IL-17的表达上调(PIFN-γ0.05,PIL-170.05),而IL-4和TGF-β表达下降(PTGF-β0.01,PIL-40.05),Th17细胞的调控因子IL-6表达升高(PIL-60.05)。结论:RAGE及其配体S100B参与EAMG的发病过程,在晚期时相中表现出明显的致病性;RAGE与S100B相互作用可以上调T细胞的致病性作用,加重四种辅助性T细胞之间的网络失衡。     

S100 protein translocation in response to extracellular S100 is mediated by receptor for advanced glycation endproducts in human endothelial cells

The extracellular functions of S100 proteins have attracted more attention in recent years. S100 proteins are a group of calcium-binding proteins which exhibit cell- and tissue-specific expression, and different expression levels of members from this family have been observed in various pathological conditions. The reported extracellular functions of S100 proteins include the ability to enhance neurite outgrowth, involvement in inflammation, and motility of tumour cells. In our previous study, we reported translocation of S100A13 in response to the elevated intracellular calcium levels induced by angiotensin II. In order to investigate potential effects of extracellular S100A13, recombinant S100A13 was used here to stimulate human endothelial cells. Addition of extracellular S100A13 to the cells resulted in both endogenous protein translocation and protein uptake from the extracellular space. To test specificity of this effect, addition of various other S100 proteins was also performed. Interestingly, translocation of specific S100 proteins was only observed when the cells were stimulated with the same extracellular S100 protein. Since the receptor for advanced glycation end products (RAGE) is a putative cell surface receptor for S100 proteins and is involved in various signal transduction pathways, we next investigated the interaction between the receptor and extracellular S100 proteins. We show here that NF-kappaB which is a downstream regulator in RAGE-mediated transduction pathways can be activated by addition of extracellular S100 proteins, and translocation of S100 proteins was inhibited by soluble RAGE. These experiments suggest a common cell surface receptor for S100 proteins on endothelial cells even though intracellular translocation induced by extracellular S100 proteins is specific.     

Expression analysis of S100 proteins and RAGE in human tumors using tissue microarrays

Microarray technology provides important information for diagnostic, prognostic, and even therapeutic applications. Several S100 proteins have been proposed to play important roles in tumor progression and are recognized as potential tumor markers. To substantiate these limited earlier findings, we screened hundreds of tumor specimens from patients of eight different tumor types using tissue microarrays. The results validated the expression of S100A4, S100A6, and S100B in specific tumor types. A significant S100A2 expression was observed in lymphoma biopsies, which implies a possible link between this S100 protein and lymphoma development. In contrast, S100A5 and S100A12 were not significantly expressed in any of the tumor tissues tested. Interestingly, expression of RAGE (receptor for advanced glycation end products) was found in breast and lung tumor tissues where abundant S100A4 and S100A6 expression was also observed. This suggests a possible role of RAGE-mediated signal transduction in the development of these particular cancers.     

hS100A6对人骨肉瘤细胞株143B的体内体外作用

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.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

Assignment and secondary structure of calcium-bound human S100B

The NMR assignments of backbone ¹H, ¹³C,and ¹⁵N resonances for calcium-bound human S100B werecompleted via heteronuclear multidimensional NMR spectroscopic techniques.NOE correlations, amide exchange, ³J_HN_Hcoupling constants, and CSI analysis were used to identify the secondarystructure for Ca-S100B. The protein is comprised of four helices (helix I,Glu²-;Arg²⁰; helix II,Glu³¹-;Asn³⁸; helix III,Gln⁵⁰-;Thr⁵⁹; helix IV,Phe⁷⁰-;Phe⁸⁷), three loops (loop I,Glu²¹-;His²⁵; loop II,Glu³⁹-;Glu⁴⁹; loop III,Leu⁶⁰-;Gly⁶⁶), and two -strands(strand I, Lys²⁶>-;Lys²⁸; strand II,Glu⁶⁷-;Asp⁶⁹) which form a shortantiparallel -sheet. Helix IV is extended by approximately one turnwhen compared to the secondary structures of apo-rat [Drohat et al. (1996)Biochemistry, 35, 11577-;11588] and bovine S100B [Kilby et al. (1996)Structure, 4, 1041-;1052]. In addition, several residues outside thecalcium-binding loops in S100B undergo significant backbone chemical shiftchanges upon binding calcium which are not observed in the related proteincalbindin D_9k. Together these observations support previoussite-directed mutagenesis, absorption spectroscopy, and cysteine chemicalreactivity experiments, suggesting that the C-terminus in Ca-S100B isimportant for interactions with other proteins.     

Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.