肝癌细胞与内皮细胞的粘附力学特性研究

采用细胞同步技术和微管吸吮技术,从细胞周期的角度研究不同细胞周期肝癌细胞(hepatocellularcarcinoma cells,HCC)与脐静脉内皮细胞(HUVEC)的粘附力学特性。结果表明,未同步化肝癌细胞各周期时相的细胞百分比为:G_0/G_1期,53.51％;G_2/M期,11.01％;S期,35.48％。采用胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法可分别获得G_1期和S期的肝癌细胞,其平均同步率分别为69.02％和96.50％。G_1期肝癌细胞与人脐静脉内皮细胞的粘附力比S期相应值明显降低(P<0.01),与未同步化肝癌细胞组比较也得到同样结果,而S期与未同步化肝癌细胞组的粘附力值无明显差别。肝癌细胞与脐静脉内皮细胞的粘附力随着粘附时间的变化而变化,在30～60min内迅速增长,60min之后维持在较稳定的水平,即300×10~(10)N左右。提示:在肝癌细胞与内皮细胞的粘附过程中,S期细胞可能起的作用更大;肝癌细胞和内皮细胞上粘附分子表达呈现时间效应,从而体现出粘附和去粘附的行为特征。     

沉默EVL表达抑制肝癌细胞SMMC-7721的增殖和迁移能力

Ena/VASP 样蛋白（Ena/VASP like protein,EVL）是Ena/VASP家族成员之一,它参与肌动蛋白细胞骨架重组,以及细胞迁移、收缩环形成和细胞间附着.EVL在肝癌SMMC-7721细胞中高表达. 抑制EVL蛋白表达后,SMMC-7721细胞的增殖与迁移能力降低.为研究EVL在肝癌细胞的功能,构建了靶向shRNA干扰表达载体,稳定转染肝癌SMMC-7721细胞. MTT实验和细胞集落形成实验显示,与转染对照比较,沉默EVL蛋白表达可明显抑制SMMC-7721肝癌细胞的增殖、集落形成能力. Transwell实验证明,沉默EVL表达导致SMMC-7721细胞迁移能力降低. 进而,流式细胞术揭示,沉默EVL表达的SMMC-7721细胞G0/G1期细胞比例增多.研究结果提示,EVL蛋白可促进肝癌细胞的增殖与迁移;该结果可解释EVL在肝癌细胞中高表达的意义.     

TGFα对肝癌细胞SMMC-7721增殖和ERK蛋白的影响

目的观察TGFα诱导肝癌细胞增殖和对信号传导因子ERK蛋白表达的影响.方法应用MTT比色法观察不同浓度TGFα对肝癌细胞SMMC-7721的增殖作用.用流式细胞术检测TGFα对肝癌细胞凋亡和细胞周期的影响.用免疫组化方法检测TGFα对ERK蛋白表达影响.结果 1μg/L TGFα作用24h SMMC-7721细胞增殖率为3%(P>0.05);作用48h后增殖率达16%(P<0.05).5μg/L TGFα作用24h增殖率达18%(P<0.05);作用48h增殖率达24%(P<0.01),增殖效应呈时间、剂量依赖性.5μg/L TGFα作用肝癌细胞48h能抑制肝癌细胞凋亡,使细胞滞留于G2M期,增加PI.5μg/L TGFα能促进ERK蛋白在细胞核中的表达.结论 TGFα能促进肝癌细胞增殖,增加ERK蛋白在细胞核中的表达.     

紫杉醇对人肝癌SMMC7721细胞NDRG1表达及增值的影响

旨在探索紫杉醇对人肝癌SMMC7721细胞NDRG1表达的影响,及紫杉醇对肝癌SMMC7721细胞系增殖的抑制作用。分别提取紫杉醇处理前后SMMC7721细胞的RNA,进行逆转录-聚合酶链反应(RT-PCR),判断紫杉醇处理前后肝癌细胞中NDRG1表达的情况;采用蛋白印迹技术(Western blotting)分析紫杉醇处理前后肝癌细胞中NDRG1蛋白表达的情况;应用不同浓度紫杉醇处理肝癌细胞,以MTT法检测处理前后肝癌细胞的抑制率,流式细胞术(FCM)观察细胞周期变化的况。结果表明紫杉醇处理后的肝癌SMMC7721细胞中NDRG1表达下降,紫杉醇浓度越高,NDRG1表达水平越低,具有浓度依赖性。以MTT法观察紫杉醇对肝癌细胞的抑制作用,试验结果表明不同浓度的紫杉醇处理肝癌SMMC7721细胞后,癌细胞被明显抑制;以流式细胞术观察紫杉醇作用后肝癌SMMC7721细胞周期的变化,结果显示G2-M期细胞比例升高的程度随浓度增高而升高,细胞越来越多地被阻滞在G2-M期,不能继续分裂增殖。分化相关基因NDRG1的表达可能是肝癌的发病机制之一,紫杉醇可抑制肝癌SMMC 7721细胞中NDRG1的表达;同时紫杉醇能使肝癌SMMC7721细胞的生长阻滞在G2-M期,从而显著抑制SMMC7721细胞的增殖,并且具有剂量、时间依赖效应。     

重楼皂苷Ⅰ对肝癌细胞的增殖及凋亡的影响

探讨重楼皂苷Ⅰ(paris saponinⅠ,PSⅠ)在体外对人肝癌SMMC-7721细胞株增殖和凋亡的影响及相关机制.MTT法检测PSⅠ对肝癌SMM-C7721细胞株的增殖抑制作用,Hoechst 33528染色法观察细胞核的形态学变化,流式细胞术Propidium iodide(PI)染色检测细胞周期及凋亡的情况,免疫印迹的方法检测Fas、Bcl-2、Bax、细胞周期素D1(cell cycle regulatory factor D1,Cyclin D1)和细胞周期素E(cell cycle regulatory factor E,Cy-clin E)的表达情况.PSⅠ能时间和浓度依赖性的抑制肝癌SMMC-7721细胞的增殖,与对照组比较,差异有统计学意义(P<0.05).干预后的SMMC-7721细胞染色质浓缩,核碎裂,凋亡小体形成,呈典型的凋亡变化.细胞周期阻滞于G1期,并且有凋亡峰形成,呈浓度依赖性.PSⅠ能浓度依赖性地上调Fas和Bax的表达,下调Bcl-2、Cyclin D1和Cyclin E蛋白的表达水平.PSⅠ可能是通过阻滞肿瘤细胞的生长及诱导细胞凋亡等机制,从而抑制肝癌细胞的增殖.     

不同细胞周期大鼠肝实质细胞癌细胞粘弹特性研究

以胸腺嘧啶核苷和秋水仙碱顺序阻断法及胸腺嘧啶核苷双阻断法分别获得同步化Ｇ１期和Ｓ期细胞,从细胞周期角度出发,采用微管吸吮技术对大鼠肝实质细胞癌细胞的粘弹特性进行了测定并以标准线性固体模型对实验数据进行了拟合,结果表明：该细胞具有高弹性和低粘性的总体特征;Ｇ１期细胞与Ｓ期细胞相比具有高Ｋ１值和低μ值的特点,从而显示Ｇ１期细胞比Ｓ期细胞具有更大的强度和更快的被动变形能力。这些结果不仅反映了同步化细胞存在的细胞骨架状态的周期性差异,也提示Ｇ１期细胞可能比Ｓ期细胞更适于在血流中存活和转移。     

斑褐孔菌石油醚提取物体外抗肿瘤活性研究

探讨斑褐孔菌石油醚提取物(PEFP)体外抗肿瘤活性及其机制。采用磺酰罗丹明染色法(SRB法)考察PEFP对人肝癌细胞SMMC-7721、人胃癌细胞SGC-7901、人喉癌上皮细胞Hep-2增殖的影响。倒置相差显微镜、透射电镜观察PEFP作用SMMC-7721后的细胞形态及超微结构的变化;流式细胞术检测PEFP作用SMMC-7721后细胞周期的改变情况。实验结果显示PEFP对上述三种人恶性肿瘤细胞均表现出明显的抑制效果,且呈剂量效应关系,其半数抑制浓度(IC50)分别为52.72、69.18、58.88μg/mL。取1/2 IC50浓度的PEFP作用于SMMC-7721 48 h后,细胞形态结构及细胞周期发生改变,G0/G1期细胞增多,S期细胞明显减少。PEFP可能通过阻滞SMMC-7721细胞周期于G0/G1期来抑制肿瘤细胞的增殖,具有较明显体外抗肿瘤活性。     

HMBA诱导人肝癌SMMC-7721细胞分化的观察

本文研究HMBA对人肝癌SMMC-7721细胞的诱导分化作用.细胞生长曲线测定和细胞分裂指数观察显示HMBA可明显抑制细胞增殖,细胞生长抑制率达64.14%,分裂指数抑制率为53.88%.光镜和透射电镜观察可见HMBA能诱导人肝癌SMMG-7721细胞形态和超微结构发生恢复性改变.生化检测或免疫细胞化学方法观察显示,HM-BA处理后细胞γ.谷氨酸转肽酶(γ-GT)活性和甲胎蛋白(AFP)、增殖细胞核抗原(PCNA)表达均降低,而酪氨酸.酮戊二酸转氨酶(TAT)活性增强.流式细胞仪分析表明HMBA引起细胞发生G0/G1期阻滞.以上结果表明HMBA能有效抑制人肝癌细胞恶性增殖活性,逆转肝癌细胞恶性形态与超微结构特征,改变肝癌细胞相关酶活性和抗原表达,引发G0/G1期阻滞,从而对肝癌细胞具有明显的诱导分化作用.     

Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth.

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.     

Synchronization of the cell cycle using Lovastatin

Synchronization by Lovastatin arrests many cell types reversibly in the G1 phase of the cell cycle. Here we show that Lovastatin (10 µM) mediates cell cycle arrest in human breast cancer cells, MCF-7 and MDA-MB-231, where 85% of cells accumulate in the G1 phase of the cell cycle. Addition of mevalonate (at 100X the Lovastatin concentration) releases the cells from the G1 arrest and allows for synchronous entry into late G1, S and G2/M phases of the cell cycle. The expressions of different cyclins as a marker for different phases of the cell cycle are detected by western blot analysis and indicative of synchronous transition into each of cell cycle phases following the initial G1 arrest. Due to its level of synchrony and high yield of synchronous populations of cells, Lovastatin method of cell synchronization can be used for examining gene expression patterns in a variety of different cell lines.     

Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells

Sialoglycans on the cell surface of human colon cancer (HCC) cells have been implicated in cellular adhesion and metastasis. To clarify the role of N-acetylneuraminic acid (NeuAc) linked alpha2,3 to galactose (Gal) on the surface of HCC cells, we studied the intercellular adhesion of HCC cell lines expressing increasing NeuAcalpha2,3Gal-R. Our model system consisted of the HCC SW48 cell line, which inherently possesses low levels of cell surface alpha2,3 and alpha2,6 sialoglycans. To generate SW48 clonal variants with elevated cell surface NeuAcalpha2,3Gal-R linkages, we transfected the expression vector, pcDNA3, containing either rat liver cDNA encoding Galbeta1,3(4)GlcNAc alpha2,3 sialyltransferase (ST3Gal III) or human placental cDNA encoding Galbeta1,3GalNAc/Galbeta1,4GlcNAc alpha2,3 sialyltransferase (ST3Gal IV) into SW48 cells. Selection of neomycin-resistant clones (600 microgram G418/ml) having a higher percentage of cells expressing NeuAcalpha2,3Gal-R (up to 85% positive Maackia amurenis agglutinin staining compared with 30% for wild type cells) was performed. These ST3Gal III and ST3Gal IV clonal variants demonstrated increased adherence to IL-1beta-activated human umbilical vein endothelial cells (HUVEC) (up to 90% adherent cells compared with 63% for wild type cells). Interestingly, ST3Gal III and ST3Gal IV clonal variants also bound non-activated HUVEC up to 4-fold more effectively than wild type cells. Cell surface NeuAcalpha2,3Gal-R expression within the various SW48 clonal variants correlated directly with increased adhesion to HUVEC (r=0.84). Using HCC HT-29 cells, which express high levels of surface NeuAcalpha2,3Gal-R, addition of synthetic sialyl, sulfo or GalNAc Lewis X structures were found to specifically inhibit intercellular adhesion. At 1.0mM, NeuAcalpha2,3Galbeta1,3(Fucalpha1, 4)GlcNAc-OH and Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(SE-6Galbeta1++ +, 3)GalNAcalpha1-O-methyl inhibited HT-29 cell adhesion to IL-1beta-stimulated HUVEC by 100% and 68%, respectively. GalNAcbeta1, 4(Fucalpha1,3)GlcNAcbeta1-O-methyl and GalNAcbeta1,4(Fucalpha1, 3)GlcNAcbeta1,6Manalpha1,6Manbeta1-0-C30H61, however, did not possess inhibitory activity. In conclusion, these studies demonstrated that cell surface NeuAcalpha2,3Gal-R expression is involved in HCC cellular adhesion to HUVEC. These specific carbohydrate-mediated intercellular adhesive events may play an important role in tumor angiogenesis, metastasis and growth control.     

Diarsenic and tetraarsenic oxide inhibit cell cycle progression and bFGF- and VEGF-induced proliferation of human endothelial cells

Arsenic trioxide (As2O3, diarsenic oxide) has recently been reported to induce apoptosis and inhibit the proliferation of various human cancer cells derived from solid tumors as well as hematopoietic malignancies. In this study, the in vitro effects of As2O3 and tetraasrsenic oxide (As4O6) on cell cycle regulation and basic fibroblast growth factor (bFGF)- or vascular endothelial growth factor (VEGF)-stimulated cell proliferation of human umbilical vein endothelial cells (HUVEC) were investigated. Significant dose-dependent inhibition of cell proliferation was observed when HUVEC were treated with either arsenical compound for 48 h, and flow cytometric analysis revealed that these two arsenical compounds induced cell cycle arrest at the G1 and G2/M phases--the increases in cell population at the G1 and G2/M phase were dominantly observed in As2O3- and As4O6-treated cells, respectively. In both arsenical compounds-treated cells, the protein levels of cyclin A and CDC25C were significantly reduced in a dose-dependent manner, concomitant to the reduced activities of CDK2- and CDC2-associated kinase. In G1-synchronized HUVEC, the arsenical compounds prevented the cell cycle progression from G1 to S phase, which was stimulated by bFGF or VEGF, through the inhibition of growth factor-dependent signaling. These results suggest that arsenical compounds inhibit the proliferation of HUVEC via G1 and G2/M phase arrest of the cell cycle. In addition, these inhibitory effects on bFGF- or VEGF-stimulated cell proliferation suggest antiangiogenic potential of these arsenical compounds.     

Early replicating DNA involved in the G2 to M transition in Allium cepa L meristematic cells

Summary— The involvement of genome portions replicating at different times of the S phase in the control of cell cycle events was analysed in Allium cepa L meristematic cells. 5-Azacytidine (5-azaC) was incorporated into discrete replicating DNA portions in synchronous cells. Cells treated with 5-azaC during the late S phase went through normal cell cycles while cells treated with 5-azaC during the early S phase were able to go through the immediately succeeding mitosis, as well as to begin the subsequent S phase. However, they were unable to enter a second mitosis. Thus, sequences replicating in the early S phase appear to code for a mitotic inhibitor which represses the emergence of a positive mitotic signal. This inhibition took place in the first half of the interphase (ie during G1) and resulted in a cell cycle blockage in G2.     

Arginine vasopressin inhibition of cyclin D1 gene expression blocks the cell cycle and cell proliferation in the mouse Y1 adrenocortical tumor cell line

Arginine vasopressin (AVP) is a nonapeptide long known as an endocrine and paracrine regulator of important systemic functions, namely, vasoconstriction, gluconeogenesis, corticosteroidogenesis, and excretion of water and urea. Here we report, for the first time, that AVP specifically inhibits expression of the cyclin D1 gene, leading to cell cycle blockage and halting cell proliferation. In G0/G1-arrested mouse Y1 adrenocortical tumor cells, maintained in serum-free medium (SFM), AVP mimics FGF2, promoting rapid ERK1/2 activation (5 min) followed by c-Fos protein induction (2 h). PKC inhibitor Go6983 and PI3K inhibitors wortmannin and LY294002 all inhibit ERK1/2 activation by AVP, but not by FGF2. Thus, AVP and FGF2 concur to activate ERK1/2 by different regulatory pathways. However, AVP is not a mitogenic factor for Y1 cells. On the contrary, AVP strongly antagonizes FGF2 late induction (2-5 h) of the cyclin D1 gene, down-regulating both cyclin D1 mRNA and protein. AVP inhibition of cyclin D1 expression is sufficient to block G1 phase progression and cell entry into the S phase, monitored by BrdU nuclear labeling. In addition, AVP completely inhibits proliferation of Y1 cells in 10% fetal calf serum (10% FCS) medium. On the other hand, ectopic expression of the cyclin D1 protein renders Y1 cells resistant to AVP for both entry into the S phase in SFM and continuous proliferation in 10% FCS medium. In conclusion, inhibition of cyclin D1 expression by AVP is an efficient mechanism of cell cycle blockage and consequent proliferation inhibition in Y1 adrenocortical cells.     

Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes

Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive.     

IL-4 inhibits cell cycle progression of human umbilical vein endothelial cells by affecting p53, p21(Waf1), cyclin D1, and cyclin E expression

IL-4 is emerging as a candidate cytokine for the treatment of inflammatory and autoimmune diseases. We have reported that IL-4 has anti-angiogenic activity and inhibits the growth of human umbilical vein endothelial cells (HUVEC) in response to vascular endothelial growth factor (VEGF) or fibroblast growth factor-2 (FGF-2). Cell cycle analysis of this effect revealed that IL-4 arrests the growth of FGF-2-stimulated HUVEC in G0 + G1 phases. The absence of subdiploid cells showed that it did not induce apoptosis. Growth arrest was dose-dependent, but the percentage of G0 + G1 phase cells never exceeded 85%. An immunoblot analysis demonstrated that expression of p53 and p21(Waf1) was increased and that of cyclin D1 and cyclin E decreased by IL-4. These results show that IL-4 inhibits endothelial cell growth by altering the expression of cell cycle regulatory molecules.     

Glioblastoma cells block radiation-induced programmed cell death of endothelial cells

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co-culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP-U87) exhibit resistance to radiation-mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor-related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin alphav, integrin beta1, MAPK(p42), Rad51, DNA-PK(CS), and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP-U87 cells compared with HUVEC grown in mono-culture. Treatment with anti-VEGF antibody decreases the expression of integrin alphav, integrin beta1, DNA-PK(CS) and ATM with a corresponding increase in ionizing radiation (IR)-induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up-regulation by tumor cells of endothelial cells genes associated with survival.