Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption

Aquaporin-1 (AQP1) is a water channel protein expressed widely in vascular endothelia, where it increases cell membrane water permeability. The role of AQP1 in endothelial cell function is unknown. Here we show remarkably impaired tumour growth in AQP1-null mice after subcutaneous or intracranial tumour cell implantation, with reduced tumour vascularity and extensive necrosis. A new mechanism for the impaired angiogenesis was established from cell culture studies. Although adhesion and proliferation were similar in primary cultures of aortic endothelia from wild-type and from AQP1-null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfection of non-endothelial cells with AQP1 or with a structurally different water-selective transporter (AQP4) accelerated cell migration and wound healing in vitro. Motile AQP1-expressing cells had prominent membrane ruffles at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. Our findings support a fundamental role of water channels in cell migration, which is central to diverse biological phenomena including angiogenesis, wound healing, tumour spread and organ regeneration.     

MiR-205 inhibits the invasion and migration of esophageal squamous cell carcinoma by modulating SMAD1 expression

Esophageal squamous cell carcinoma （ESCC） is one of the most lethal cancers worldwide. In this study, we aimed to investigate the underlying mechanisms of metastasis inhibition by miR-205 in ESCC. In microRNA （miRNA） array and quantitative RT-PCR analyses, we found that the expression level of miR-205 was significantly lower in patients with lymph node metastasis compared with that in patients without lymph node metastasis. After transfection of miR-205 mimics or inhibitors into ESCC cell lines, a significant negative correlation was observed between the expression level of miR-205 and Smad 1. In luciferase reporter assays, we revealed that miR- 205 inhibited the expression of SMAD1 by targeting the 3＇ untranslated region （3＇-UTR） of SMAD1 mRNA in ESCC cells. Furthermore, our results showed that miR-205 sup- pressed the invasion and migration of ESCC cells, whereas Smadl increased their invasion and migration. Taken together, our study demonstrates that miR-205 functions as a suppressor of tumor metastasis by regulating SMAD1 expression through targeting the 3＇-UTR of SMAD1 mRNAin ESCC. Therefore, miR-205 may be a potential therapeutic target for miRNA-based therapy of ESCC.     

Importance of NPA motifs in the expression and function of water channel aquaporin-1

The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and effi-cient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 de-letion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water per-meation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.     

自噬对肝癌SMMC-7721细胞侵袭迁移能力的影响

为探讨自噬对照射过程中肝癌SMMC-7721细胞侵袭和迁移能力的影响及其潜在的作用机制,将肝癌SMMC-7721细胞分为6组,分别为阴性对照(NC)组、8 Gy X-线照射(IR)组、自噬激活剂雷帕霉素(Rapa)组、自噬抑制剂三甲基腺嘌呤(3-MA)组、照射+雷帕霉素(IR+Rapa)组和照射+三甲基腺嘌呤(IR+3-MA)组,利用划痕实验和Transwell实验检测自噬对肝癌SMMC-7721细胞迁移和侵袭的影响;用CCK8实验检测照射、雷帕霉素和3-MA对肝癌细胞增殖的影响;用蛋白免疫印迹(Western blot)检测N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和LC3B蛋白表达情况。研究结果显示,照射可增强肝癌SMMC-7721细胞侵袭迁移的能力(P<0.05),同时,照射可抑制细胞的增殖能力(P<0.05);雷帕霉素激活自噬可增强细胞的侵袭迁移能力(P<0.05),3-MA抑制自噬可抑制细胞侵袭迁移和细胞的增殖能力(P<0.05)。照射可上调N-cadherin、Vimentin表达水平(P<0.05),激活或抑制自噬可分别增加或抑制N-cadherin、Vimentin蛋白表达水平(P<0.05)。表明X-线照射可激活自噬,促进细胞上皮-间充质转化(Epithelial-Mesenchymal Transition,EMT)的发生。激活自噬可以提高肝癌细胞上皮间质转化进而促进肝癌细胞侵袭迁移;反之,抑制自噬可阻碍肝癌细胞的侵袭迁移。     

钾通道阻断剂对胶质瘤细胞迁移和侵袭的影响

为了检测电压门控钾通道阻断剂4-氨基吡啶(4-AP)、四乙胺(TEA)和ATP敏感钾通道阻断剂格列苯脲(Glibenclamide,Gli)对胶质瘤细胞迁移和侵袭的影响,选用人胶质瘤细胞系U87和U251,其中钾通道阻断剂4-AP、TEA及Gli处理作为实验组,未处理的作为对照组.采用划痕实验和Transwell小室法检测钾通道阻断剂对U87和U251细胞迁移和侵袭能力的影响; Western blot检测药物处理后细胞高迁移率蛋白B1(high mobility group protein B1,HMGB1)表达水平.结果表明:5 mmol·L^-1的4-AP、40 mmol·L^-1的TEA及400 μmol·L^-1的Gli可以显著抑制胶质瘤细胞的迁移、侵袭,并降低HMGB1表达水平.电压门控钾通道和ATP敏感钾通道对胶质瘤细胞迁移和侵袭具有重要调控作用,3种钾通道阻断剂对胶质瘤细胞迁移和侵袭有不同程度的抑制作用,可能通过调控HMGB1相关通路实现.     

The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions

Aquaporins, found in virtually all living organisms, are membrane-intrinsic proteins that form water-permeable complexes. The mammalian aquaporin AQP1 has also shown CO2 permeability when expressed heterologously in Xenopus oocytes, although whether this is a biochemical curiosity or of physiological significance is a matter of debate. Here we report that, in the same expression system, a CO2 permeability comparable to that of the human AQP1 is observed for the tobacco plasma membrane aquaporin NtAQP1. NtAQP1 facilitates CO2 membrane transport in the homologous plant system at the cellular level, and has a significant function in photosynthesis and in stomatal opening. NtAQP1 overexpression heightens membrane permeability for CO2 and water, and increases leaf growth. The results indicate that NtAQP1-related CO2 permeability is of physiological importance under conditions where the CO2 gradient across a membrane is small, as is the case between the atmosphere and the inside of a plant cell.     

结直肠癌细胞过表达tricellulin促进人脐静脉内皮细胞侵袭转移

三细胞间紧密连接蛋白tricellulin在结直肠癌组织中高表达,且与结直肠癌的不良预后相关。血管生成是肿瘤侵袭转移的重要因素之一,血管内皮细胞在血管新生过程中起决定作用。研究tricellulin与内皮细胞的关系对探索肿瘤侵袭转移机制具有重要意义。本研究通过western blot观察结直肠癌细胞株与正常结肠上皮细胞株中tricellulin的差异表达,通过基因工程技术调控结直肠癌细胞HCT116中tricellulin的表达,transwell小室检测过表达tricellulin前后结直肠癌细胞侵袭能力变化及其上清液对人脐静脉内皮细胞（HUVEC）侵袭能力的影响,ELISA实验检测过表达tricellulin前后MMP2、MMP7的表达变化。结果显示：过表达tricellulin可增强HCT116细胞的侵袭能力,并且其上清液可增强HUVEC细胞的侵袭能力;过表达tricellulin可增加MMP2、MMP7的表达。说明人结直肠癌细胞过表达tricellulin可促进结直肠癌细胞侵袭迁移,并通过影响MMP2、MMP7的表达调控HUVEC细胞的侵袭转移。     

Overexpression of PITPNM3 promotes hepatocellular carcinoma cell metastasis

A previous study indicated that C–C chemokine(C–C motif)ligand 18(CCL18)is capable of inducing tumor cell invasion and metastasis by interacting with receptor membrane-associated phosphatidylinositol transfer protein 3(PITPNM3)in breast cancer cells.The present study aims to investigate the correlation between the PITPNM3 expression and metastasis in hepatocellular carcinoma(HCC).Real-time quantitative polymerase chain reaction and Western blot were performed to detect the expression pattern of PITPNM3 in patient samples and HCC cell lines.Wound-healing and transwell chamber assays were performed to assess the migration and invasiveness of HCC cells,and the activation of the signaling protein downstream of PITPNM3 was also detected by Western blot and immunofluorescence.The results revealed that PITPNM3 was upregulated in HCC tissue compared to matched normal liver tissue.Silencing the expression of PITPNM3 by specific siRNAs markedly attenuated the invasive and metastatic abilities of HCC cells,whereas the upregulation of PITPNM3 significantly increased HCC cell mobility.Furthermore,inhibiting the expression of PITPNM3 suppressed the activation of Pyk2,FAK,and Src,while overexpression of PITPNM3enhanced the phosphorylation of FAK and Src in HCC cells.Besides,suppression of Pyk2 can also impair the clustering of integrin.These results imply that PITPNM3 is a vital determinant of HCC migration and invasion.     

Effects of Genistein and Daidzein on the Proliferation, Invasion, Migration and Adhesion of Melanoma Cells

IntroductionGenistein and daidzein (Fig.1 ) ,two majorisoflavonoids in soybeans,were reported to playimportant roles in cancer prevention[1 ,2 ] .Previousstudies demonstrated that genistein and daidzeininhibited the growth of leukemia,breast,colonand prostate cancers[38] .Our earlier studies alsodemonstrated that daidzein enhanced the immunefunction in mice[9,1 0 ] . These functions madegenistein and daidzein promising candidates forcancer prevention. However,it is usually themetastatic disse…     

CXCR7对HeLa细胞增殖、粘附和侵袭能力的影响

基质细胞衍生因子-1(SDF-1)在肿瘤侵袭、转移过程中起着重要的调控作用.此前认为SDF-1是通过其唯一受体CXCR4来起作用.近年来发现SDF-1还有另一作用受体——CXCR7,SDF-1/CXCR7在部分肿瘤侵袭转移过程中起重要作用,但其在宫颈癌HeLa细胞中的作用目前尚未明确.通过Western blotting检测HeLa细胞中CXCR4和CXCR7的表达,阻断CXCR4或CXCR7后,通过MTT法评价细胞增殖能力,细胞粘附实验评价细胞粘附能力,Transwell实验评价细胞侵袭能力.结果表明,CXCR4和CXCR7在HeLa细胞中表达.阻断CXCR4或CXCR7后,SDF-1诱导的HeLa细胞增殖、侵袭和与内皮细胞的粘附能力均被阻断.结果提示CXCR7在SDF-1诱导HeLa细胞增殖、侵袭和与内皮细胞的粘附过程中起着重要作用,将有望成为治疗宫颈癌转移的新靶点.     

Cell adhesion receptors and cancer

Cell-to-cell and cell-to-extracellular matrix (ECM) interactions in the functions of cell adhesion and signal transduction are important in global control of cell phenotypes and cell behavior and are crucial for maintenance of homeostasis and structural/functional stabilization of tissues and organs. Cell adhesion receptors are recognized as the molecular basis of cell adhesion. Cadherin and Integrin are widely expressed adhesion receptors in most tissues. They are transmembrane glycoproteins which, through their cytoplasmic domain, bind to many proteins at the inner surface of cell membrane to form molecule-linkage complexes and then connect with the cytoskeleton. Through cell adhesion receptors a network functioning as cell adhesion and signal transduction is organized between tissue cells and cell-ECM. In this regard cell adhesion receptors play an important role in regulation of morphogenesis, cell-cell recognition, cell migration, cell sorting and the determination of cell's fate in development. They mediate cell functions and their fault expression is intimately correlated with development of disorders like cancer. Several isoforms of Integrin were found to have tumor suppressor effect. Some components in the molecule-linkage of focal contact are actin-binding proteins as well as substrates of kinase in the Integrin initiated signal pathway to play a role as signal transducer. Some of these molecules exhibited tumor suppressor effect too. Decreased expression of E-Cadherin has been demonstrated in many epithelium originated carcinomas. Cadherin associated membrane adhesion plaque molecule β-Catenin is also involved in the oncogene Wnt signal pathway. Both E-Cadherin and β-Catenin were proved respectively with tumor suppressor effect against invasiveness and metastasis. That Cadherin is important for the posttranslationally functional expression of Connexin has been supported by evidence from developmental biology and cancer cell differentiation studies to suggest that some sort of interrelation feedback control exists between the two signal pathways.     

低氧条件下巨噬细胞分泌CCL22促进三阴乳腺癌转移

三阴乳腺癌(Triple Negative Breast Cancer, TNBC)是乳腺癌中恶性程度最高的一种亚型，表现为很高的转移潜能。巨噬细胞，即肿瘤相关巨噬细胞(Tumor-Associated Macrophages, TAM)，在促进TNBC转移中起了重要作用。乳腺癌作为一种实体肿瘤，往往处于缺氧环境中。低氧环境能够促进癌细胞的转移，然而低氧环境中巨噬细胞在促进肿瘤转移中的作用仍然不清楚。在该研究中，THP1细胞被诱导成TAM，经过缺氧培养后，通过Transwell实验检测其促进三阴乳腺癌细胞BT-549和MDA-MB-231的细胞迁移能力；通过尾静脉注射，将MDA-MB-231细胞移植于祼鼠体内，CT扫描，分析了TAM促进TNBC细胞的器官转移能力；通过ELISA实验检测低氧对TAM分泌的肿瘤转移相关因子的影响，通过GDSC在线软件分析了CCL22受体CCR4和其他CCR在乳腺癌组织与正常组织中表达的差异。结果表明低氧条件下巨噬细胞通过分泌CCL22的表达来促进三阴乳腺癌细胞迁移:经过缺氧培养后的TAM显著增强了TNBC细胞迁移能力，以及促进癌细胞在体内向肺转移；低氧诱导TAM分泌CCL22；CCL22受体CCR4在乳腺癌组织中的表达显著高于在正常组织中的。     

木棉皮抗消化道肿瘤活性部位筛选及抑制肿瘤转移机制

为筛选木棉皮醇提物抗消化道肿瘤活性部位,探究其抑制敏感肿瘤细胞增殖、转移作用机制,通过采用CCK-8(cell counting kit-8)法考查木棉皮醇提物不同极性萃取部位对人肝癌HepG2细胞、人胃癌SGC7901细胞、人结肠癌SW480细胞和人胰腺癌PANC-1细胞这4种肿瘤细胞的抑制作用,筛选出木棉皮醇提物抗肿瘤的活性部位和敏感细胞。采用细胞划痕实验、Transwell实验和细胞黏附实验,研究木棉皮醇提物抗肿瘤活性部位对敏感肿瘤细胞迁移、侵袭和黏附能力的影响。定量聚合酶链反应法(quantitative polymerase chain reaction, qPCR)检测基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)基因的mRNA转录水平。结果表明,木棉皮醇提物不同极性萃取部位中石油醚部位对人胃癌SGC7901细胞和人肝癌HepG2细胞的抑制率最大,且对人胃癌SGC7901细胞最为敏感。随着木棉皮醇提物石油醚部位浓度增加,在24、48、72 h时间段人胃癌SGC7901细胞的迁移面积相对于空白组有所减小(P<0.05)。与空白组相比,木棉皮醇提物石油...     

FEZF1在结直肠癌组织和癌旁正常组织中的表达及临床意义

本文旨在探究FEZF1蛋白在结直肠癌组织和癌旁组织中的表达和其临床意义.通过免疫组化实验和Western Blot实验以及细胞实验,发现FEZF1在结直肠癌组织中低表达,并且其表达与肿瘤淋巴结转移(tumor node metastasis,TNM)分期具有显著相关性,在FEZF1低表达时,RKO细胞增殖和迁移能力提高,FEZF1高表达时RKO细胞增殖能力减弱.本研究证明了FEZF1是结直肠癌中的一个低表达蛋白,其表达与结直肠组织癌变的发生、发展相关,并且FEZF1会抑制结直肠癌细胞的增殖和迁移.     

海参皂苷Echinoside A通过MMP-9信号通路抑制肿瘤转移

从富含海参皂苷的革皮氏海参中分离纯化出Echinoside A（EA）,并研究了其抗肿瘤细胞转移活性及其作用机制。通过MTT法检测了EA对肿瘤细胞（HepG2）和人脐静脉内皮细胞生长的影响;采用细胞黏附实验、划痕愈合实验和Transwell小室侵袭模型研究了EA对肿瘤细胞黏附、迁移和侵袭能力的影响;采用体外小管形成实验...     

Rapid gating and anion permeability of an intracellular aquaporin

Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting alpha-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.     

重组绿豆胰蛋白酶抑制剂片段对肠癌细胞SW480迁移的影响

探讨重组胰蛋白酶抑制剂活性片段(LysGP33)的抗肠癌效应.大肠杆菌原核表达LysGP33活性片段,GST亲和层析柱对表达蛋白进行纯化.MTT比色和细胞划痕方法检测LysGP33活性片段对SW480细胞增殖和迁移的影响.结果表明:10 μmol/L LysGP33活性片段对SW480细胞的增殖活性无明显影响,但能够有效地抑制SW480细胞的迁移,并呈现时间依赖效应.绿豆胰蛋白酶抑制剂抑制了肠癌细胞的迁移,在抗肿瘤浸润和转移中具有一定的应用前景.     

免疫缺陷动物在肿瘤细胞侵袭研究中的应用

近10余年来利用免疫缺陷动物从不同角度进行了人类瘤细胞侵袭性的观察。共进行了以下几个方面的工作:(1)侵袭模型的建立:共建立了6个体内侵袭模型(包括背侧皮下,肌肉内,腹腔内,肾包膜下,睾丸包膜下和爪垫皮下等移植侵袭模型),不同模型具有不同用途。这6种侵袭模型均可作侵袭力的比较研究。(2)侵袭和转移之间关系的观察;利用爪垫皮下和睾丸包膜下移植侵袭模型,以不同时间截肢(切除局部肿瘤),待动物存活到最终时间和不同时间处死动物,观察局部肿瘤侵袭程度和转移出现时间的关系。初步证明局部肿瘤侵袭发展到Ⅲ级侵袭以上时方出现转移,侵袭Ⅲ级是危险期,这个结果对临床肿瘤病人手术时机的选择有重要参考价值。(3)对瘤细胞侵袭程度的定量分析做了初步观察,为今后深入研究侵袭定量打下了基础。(4)瘤细胞侵袭性和器官微环境关系的观察,初步证明了肿瘤细胞侵袭性和转移一样,也具有器官特异性。(5)同时也证明了瘤细胞侵袭的表达与移植方式的关系,找出了对瘤细胞侵袭最敏感的部位和器官,如肾包膜下和腹腔内为敏感的部位;背侧部皮下为不敏感的部位。因此鉴定一个肿瘤是否具有侵袭性,其侵袭程度如何,就不能仅用一个部位和器官。本文总结了近10余年的初步结果,提出了一些待深入研究的问题,这为今后深入研究人类肿瘤细胞侵袭机制和实验治疗,建立了良好的基础和条件。     

实时无标记肿瘤细胞迁移筛选技术体系的建立

肿瘤转移是导致肿瘤患者死亡的最主要原因。细胞迁移在多步骤、多阶段的肿瘤转移过程中发挥着重要的作用。尽管有许多迁移相关分子的研究,但细胞迁移的分子机制仍不清楚。研究将xCELLigence细胞分析系统和RNA干扰技术结合,初步建立了实时无标记的肿瘤细胞迁移筛选技术体系。该技术体系的建立将为大规模的肿瘤细胞迁移筛选工作奠定基础,还将有助于发现肿瘤细胞迁移信号通路中新的调控分子,揭示肿瘤转移的分子机制。