Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor

GPR4 is a member of the proton-sensing G protein-coupled receptor family. Within tumor microenvironments, the interstitial acidic pH may activate GPR4 to regulate the behavior of tumor cells. Mouse B16F10 melanoma cells and TRAMP-C1 prostate cancer cells, genetically engineered to overexpress GPR4 or the control vector, were subject to a series of cell migration, invasion and metastasis assays. Upon GPR4 overexpression and activation in an acidic pH, the migration of B16F10 and TRAMP-C1 cells was substantially inhibited in comparison to the vector control. Similar results were observed in the Matrigel invasion and transendothelial invasion assays. At the molecular level, stimulation of GPR4 by acidosis induced the activation of RhoA and the formation of actin stress fibers. In addition, treating B16F10 cells with the known Rho activator CN01 (calpeptin) strongly inhibited cell migration, recapitulating the acidosis/GPR4-induced motility inhibition phenotype. To examine the biological effects in vivo, B16F10 melanoma cells were intravenously injected into syngeneic C57BL/6 mice and pulmonary metastasis was inhibited by approximately 80% in GPR4-overexpressing B16F10 cells in comparison to the vector control. Upon treatment with the Rho activator CN01, the phenotype of the B16F10 vector cells paralleled that of the GPR4-overexpressing cells in cell migration and metastasis assays. These findings suggest that GPR4 activation by an acidic pH inhibits tumor cell migration and invasion, and the Rho GTPase is at least partly responsible for this phenotype.     

Cytoprotective role of astaxanthin against glycated protein/iron chelate‐induced toxicity in human umbilical vein endothelial cells

Astaxanthin (ASX), a red carotenoid pigment with no pro‐vitamin A activity, is a biological antioxidant that occurs naturally in a wide variety of plants, algae and seafoods. This study investigated whether ASX could inhibit glycated protein/iron chelate‐induced toxicity in human umbilical‐vein endothelial cells (HUVEC) by interfering with ROS generation in these cells. Glycated fetal bovine serum (GFBS) was prepared by incubating fetal bovine serum (FBS) with high‐concentration glucose. Stimulation of cultured HUVECs with 50 mm 1 mL of GFBS significantly enhanced lipid peroxidation and decreased antioxidant enzyme activities and levels of phase II enzymes. However, preincubation of the cultures with ASX resulted in a marked decrease in the level of lipid peroxide (LPO) and an increase in the levels of antioxidant enzymes in an ASX concentration‐dependent manner. These results demonstrate that ASX could inhibit LPO formation and enhance the antioxidant enzyme status in GFBS/iron chelate‐exposed endothelial cells by suppressing ROS generation, thereby limiting the effects of the AGE–RAGE interaction. The results indicate that ASX could have a beneficial role against glycated protein/iron chelate‐induced toxicity by preventing lipid and protein oxidation and increasing the activity of antioxidant enzymes. Copyright © 2009 John Wiley & Sons, Ltd.     

内皮细胞生长因子的分离纯化及生物活性的研究

介绍了内皮细胞生长因子(ECGF)一种分离纯化的方法,经硫酸铵分级盐析和肝素亲和层析可得到高纯度的ECGF。经SDS—PAGE电泳分析其分子量为17000,等电点为4.8。肝素与ECGF的生物活性密切相关,并可使已失活的(ECGF)恢复70%～80%的生物活性。     

Proteomic characterization of human early pro-angiogenic cells

Early pro-angiogenic cells (EPCs) have been shown to be involved in neovascularization, angiogenesis and re-endothelialization and cathepsin L inhibition blunted their pro-angiogenic effect. In the present study, we have analysed and mapped the proteome and secretome of human EPCs, utilizing a combination of difference in-gel electrophoresis (DIGE) and shotgun proteomics. A population of 206 protein spots were analysed, with 171 being identified in the cellular proteome of EPCs. 82 proteins were identified in their conditioned medium, including the alternative macrophage markers C-C motif chemokine 18 (CCL18) and the hemoglobin scavenger receptor CD163 as well as platelet factor 4 (CXCL4) and platelet basic protein (CXCL7) with “platelet alpha granule” being returned as the top category according to the Gene Ontology Annotation. Apart from cathepsin L, the cathepsin L inhibitor also attenuated the release of a wide range of other cathepsins and lysosomal proteins such as legumain, but stimulated the secretion of members of the S100 protein family. The data presented here are the most comprehensive characterization of protein expression and secretion in human EPCs to date and highlight the potential importance of cysteine proteases in the processing of platelet factors for their pro-angiogenic potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

LTBP-2 confers pleiotropic suppression and promotes dormancy in a growth factor permissive microenvironment in nasopharyngeal carcinoma

This study identified LTBP-2 as a pleiotropic tumor suppressor in nasopharyngeal carcinoma, which safeguards against critical malignant behaviors of tumor cells. LTBP-2 expression was significantly decreased or lost in up to 100% of NPC cell lines (7/7) and 80% of biopsies (24/30). Promoter hypermethylation was found to be involved in LTBP-2 silencing. Using a tetracycline-regulated inducible expression system, we unveiled functional roles of LTBP-2 in suppressing colony formation, anchorage-independent growth, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. Three-dimensional culture studies suggested the involvement of LTBP-2 in maintenance of tumor cell dormancy in a growth factor favorable microenvironment.     

3-去氮腺苷对脐静脉内皮细胞超氧化物歧化酶表达及甲基化的影响

<正>既往研究显示,高蛋氨酸摄入和B族维生素缺乏等膳食因素、以及先天性蛋氨酸代谢酶缺失等均可导致血浆中总同型半胱氨酸     

替沃扎尼抑制甲状腺癌细胞和血管内皮细胞的增殖

目的 研究新型酪氨酸激酶抑制剂替沃扎尼对甲状腺癌细胞株SW579和血管内皮细胞株HUVEC增殖的影响及作用机制。方法 SW579和HUVEC暴露于不同浓度（1、2、4、8和16 μM）的替沃扎尼至72 h,无药物处理的细胞作为对照。采用CCK-8细胞活度测定、图像分析技术及分裂细胞荧光免疫染色观察评价替沃扎尼对细胞增殖的影响,应用流式细胞仪进行细胞周期时相分析。结果细胞活度测定显示,替沃扎尼对两种细胞的生长增殖均有明显的抑制作用（P<0.05）,并呈浓度依赖性及时间依赖性,两种细胞半数抑制浓度（IC50）分别约为4 μM和8 μM。图像分析显示,在IC50作用下,替沃扎尼使两种细胞的细胞密度降低,分裂指数下降（P<0.05）。细胞周期时相分析表明,替沃扎尼可使SW579和HUVEC发生细胞周期休止,但分别休止于G1期和G2/M期（P<0.05）。结论 替沃扎尼对甲状腺癌细胞和血管内皮细胞的增殖均有显著的抑制效应,其作用是籍细胞周期休止作用而实现,但在两种细胞所诱发的细胞周期休止时相不同。替沃扎尼作为一种新型酪氨酸激酶抑制剂可通过靶向癌细胞和血管内皮细胞来治疗甲状腺癌。     

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells

Background:  Secretion of Weibel–Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. Objective and method:  In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. Results:  We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the α and β isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. Conclusions:  These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.     

Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10)

Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0-20 microM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not the CDK2-p27, CDK4-p21, and CDK4-p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.     

芎芍胶囊对人脐静脉内皮细胞分泌血管新生因子的影响

目的探讨芎芍胶囊对人脐静脉内皮细胞（HUVEC）分泌血管新生因子影响的机制。方法采用血清药理学和体外干预培养HUVEC的方法,通过MTF法和酶联免疫吸附分析法观察芎芍胶囊低、中、高剂量对HUVEC的体外增殖和分泌Ang、Ang-2、VEGF、bFGF、EGF和TGF-β1的影响。结果芎芍胶囊各剂量组均有促HUVEC增殖的作用。低剂量组有促进HUVEC分泌Ang、Ang-2、EGF和VEGF的作用,而对bFGF、TGF-β1分泌的影响无明显差异;中剂量组有促进HUVEC分泌Ang、Ang-2、EGF和bFGF的作用,抑制TGF-β1的分泌,对VEGF的分泌无明显影响;高剂量组有促进HUVEC分泌Ang、EGF和bFGF的作用,并抑制VEGF与TGF-β1的分泌,而对Ang-2的影响无明显差异。结论芎芍胶囊能促进HUVEC的增殖,增加HUVEC分泌促血管新生因子和降低抑血管生成因子表达,且与剂量有一定的关系。