5-AIQ对大肠癌细胞系HT-29细胞PARP活性抑制的生物学作用

背景与目的:聚(腺苷二磷酸核糖)聚合酶[poly(adenosine 5'-diphosphate ribose)polymerase,PARP]抑制剂5-氨基异喹啉酮(5-aminoisoquinolinone,5-AIQ)在炎症中具重要作用,但在肿瘤中的作用尚不清楚.本研究初步探讨5-AIQ对大肠癌PARP活性抑制的生物学作用.方法:链霉生物素-过氧化物酶法(Streptavidin-Peroxidase,SP)免疫组化法和免疫荧光双标法用于检测人大肠癌组织聚(腺苷二磷酸核糖)[poly(adenosine 5'-diphosphate ribose),PAR],及其与P-选择素(P-selectin)、细胞间粘附分子(intercellular adhesion molecule-1,ICAM-1)的共表达,大肠癌切缘肠粘膜为对照.通过粘附实验观察5-AIQ对结肠癌HT-29细胞系与人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)粘附的影响;同时采用SP法观察5-AIQ对HT-29细胞PAR、P-selectin和ICAM-1表达的影响.结果:45例大肠癌组织中,PAR阳性率(77.8%,35/45)明显高于对照肠粘膜(10.0%,1/10)(P＜0.05);其中伴转移者PAR阳性率(86.7%,26/30)高于不伴转移者(60.0%,9/15).PAR阳性与P-selectin和ICAM-1表达相关.结肠癌HT-29细胞与HUVEC粘附实验显示,5-AIQ浓度为100、300、500 μmol/L时,细胞粘附率分别为56.79%、46.31%和39.77%,明显低于对照组(未加5-AIQ者)(粘附率为100%).经5-AIQ处理的HT-29细胞PAR、P-selectin和ICAM-1表达HSCOR得分分别为1.41±0.12、1.57±0.13和1.23±0.13,明显低于未经5-AIQ处理的HT-29细胞(HSCOR得分分别为2.61±0.10、2.73±0.16和2.30±0.12)(P＜0.05).结论:大肠癌组织内PARP活性增强.PARP抑制剂5-AIQ可抑制大肠癌HT-29细胞与HUVEC的粘附,并可抑制大肠癌HT-29细胞PAR、P-selectin和ICAM-1表达.     

Inhibition of cellular adhesion in human umbilical vein endothelial cells by NF-kappaB inhibitor DHMEQ under flow

We previously designed and synthesized DHMEQ as an inhibitor of NF-kappaB. In the present study, we looked into the effect of DHMEQ on the cell adhesion in human umbilical vein endothelial cells (HUVEC) under flow. We used freshly prepared HUVEC and human mononuclear cells throughout the experiment. DHMEQ inhibited TNF-alpha-, IL-1beta-, and LPS-induced NF-kappaB activation in HUVEC. It also inhibited TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. DHMEQ also inhibited TNF-alpha-induced mononuclear cell-HUVEC adhesion. The effect of DHMEQ was more prominent when the cells were under shear stress. DHMEQ inhibited the adhesion between HUVEC and HT-29 colon cancer cells more clearly under the flow condition than under the static condition of the culture medium. These results suggest that DHMEQ, being a unique inhibitor of NF-kappaB, may be effective in suppressing atherosclerosis and metastasis by inhibiting the expression of adhesion molecules.     

Sialyl Lewis-X抗原在大肠癌肝转移中的作用研究及临床意义

目的探讨SialylLewis-X(SLeX)抗原在大肠癌肝转移中的作用及临床意义。方法采用大肠癌Lovo、HT29细胞与人脐静脉血管内皮及内皮细胞的粘附试验,分别用扫描电镜及透射电镜观察Lovo、HT29细胞与脐静脉血管内皮及内皮细胞的粘附性及SLeX单抗封闭大肠癌细胞后,这种粘附性的改变;采用实验性裸鼠大肠癌肝转移模型分别观察SLeX单抗封闭Lovo、HT29细胞前后对实验性裸鼠大肠癌肝转移的影响。结果高表达SLeX抗原的Lovo细胞与脐静脉血管内皮的粘附性较低表达SLeX抗原的HT29细胞强,Lovo细胞与脐静脉血管内皮细胞的连接方式与HT29细胞明显不同;高表达SLeX抗原的Lovo细胞引起裸鼠肝转移率高于低表达SLeX抗原的HT29细胞。结论大肠癌细胞表面SLeX抗原在大肠癌细胞与脐静脉血管内皮细胞的粘附及实验性裸鼠肝转移中起重要作用,SLeX单抗能有效地抑制肿瘤细胞与脐静脉血管内皮细胞的粘附,并能降低实验性裸鼠肝转移的形成。     

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40°C for 6-12h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1 &#103 , TNF- &#102 , IFN- &#110 , IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1 &#103 , MIG). This is in contrast to the stimulatory effects of TNF- &#102 or 43°C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the &#102 4 &#103 7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.     

Influence of tumor-derived interleukin 1 on melanoma-endothelial cell interactions in vitro.

Human melanoma cell lines that express high constitutive levels of the metastasis-associated marker intercellular adhesion molecule 1 (ICAM-1) were found to secrete interleukin 1 (IL-1) in vitro. Experiments with neutralizing antibodies showed that this cytokine was responsible for their expression of ICAM-1 but not that of two other progression/metastasis markers, Muc-18 and Gp IIb/IIIa. The IL-1 present in melanoma-conditioned medium induced the expression of vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 on human umbilical vein endothelial cells (ECs) in culture and increased the rate at which melanoma cells and ECs adhered to each other. IL-1-producing melanoma lines adhered significantly more rapidly to ECs than did non-IL-1-producing lines, and this enhancement was reduced by prior incubation of the melanoma cells with neutralizing anti-IL-1 antibodies. Similarly, endothelial cells treated with conditioned medium from IL-1-producing melanoma lines adhered significantly more rapidly to melanoma cells than did ECs treated with medium from non-IL-1-producing melanoma lines, and this enhancement was abolished by addition of anti-IL-1 antibodies to EC cultures in conditioned medium. Blocking antibodies to endothelial vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 failed to inhibit melanoma-EC adhesion, but an antibody to tumor cell GpIIb/IIIa did block adhesion by up to 44%. The ability to secrete IL-1 could increase the metastatic potential of melanoma cells by stimulating tumor cell-EC adhesion.     

蛋白激酶C调控大肠癌HT-29细胞粘附、侵袭能力的研究

目的探讨蛋白激酶C(ProteinKinaseC,PKC)对大肠癌HT-29细胞体外粘附、侵袭能力的调控作用。方法采用体外细胞培养的方法,通过PKC激动剂佛波酯PMA和抑制剂Staurosporine(SP)对大肠癌HT-29细胞粘附人脐带静脉内皮细胞(HUVECs)能力、侵袭血管小块的形态学和侵袭人羊膜能力影响的研究,探讨PKC对HT-29细胞体外粘附、侵袭能力的调控作用。结果采用3H-TdR标记的同位素液闪测定发现,PMA不同浓度处理HT-29细胞可使其粘附HUVECs的能力增强,在一定浓度范围内,随着PMA浓度的增加,粘附作用也逐渐增强,SP可拮抗HT-29细胞粘附HUVECs的作用。侵袭血管小块的扫描电镜观察发现,PMA处理细胞后,细胞侵袭血管的能力增强,细胞伸出伪足,向血管内侵袭,而SP可拮抗PMA作用,细胞侵袭性下降。PMA可显著增强HT-29细胞侵袭人羊膜的能力,100nmol/LPMA诱导4h,可使HT-29细胞在体外获得最大的侵袭羊膜的能力,而SP则可拮抗PMA的这种诱导作用。结论PKC对HT-29细胞体外粘附、侵袭能力存在调控作用,PKC的激活使HT-29细胞粘附性、侵袭性增强,而PKC的抑制使HT-29细胞粘附性、侵袭性下降。     

Cisplatin up-regulates ICAM-1 expression in endothelial cell via a NF-kappaB dependent pathway

The anticancer drug cis -diammindichloroplatin (cisplatin) can cause severe side-effects, but to date, the mechanisms of action of these dangerous side-effects have not been completely elucidated. Since cellular adhesion molecules (CAMs), by mediating the recruitment of circulating leukocytes to the blood vessel wall and their subsequent migration into the subendothelial spaces, play a crucial role in several pathophysiologic processes, we sought potential proof for CAMs in the pathophysiology of cisplatin-induced vascular damage. In vitro , human umbilical vein endothelial cells (HUVECs) were subjected to various concentrations of cisplatin, considerable up-regulation of intercellular adhesion molecule-1 (ICAM-1) but not P-selectin, E-selectin, and vascular cell adhesion molecule 1 at both messenger mRNA and protein expression levels were observed. Electrophoretic mobility shift assays and Western blotting analysis revealed that cisplatin up-regulates ICAM-1 expression in HUVECs via an NF-kappaB-dependent pathway. Further intravital microscopy study demonstrated that significantly higher ( P  < 0.01) numbers of rolling and sticking leukocytes on the wall of postcapillary venules in male Golden Syrian hamster's cheek pouch bearing a human cervical carcinoma were observed, while inhibition of ICAM-1 by using specific anti-ICAM-1 antibody can attenuate cisplatin-stimulated leukocyte/endothelium interactions. These data suggest that ICAM-1 involves in the pathophsiologic process of cisplatin-induced vascular toxicity and may be exploited for therapeutic advantage. ( Cancer Sci 2008; 99: 391–397)     

In vitro Study of the Antagonistic Effect of Low-dose Liquiritigenin on Gemcitabine-induced Capillary Leak Syndrome in Pancreatic Adenocarcinoma via Inhibiting ROSMediated Signalling Pathways

Background: To investigate in-vitro antagonistic effect of low-dose liquiritigenin on gemcitabine-inducedcapillary leak syndrome (CLS) in pancreatic adenocarcinoma via inhibiting reactive oxygen species (ROS)-mediated signalling pathways. Materials and Methods: Human pancreatic adenocarcinoma Panc-1 cells andhuman umbilical vein endothelial cells (HUVECs) were pre-treated using low-dose liquiritigenin for 24 h, thenadded into gemcitabine and incubated for 48 h. Cell viability, apoptosis rate and ROS levels of Panc-1 cellsand HUVECs were respectively detected through methylthiazolyldiphenyl-tetrazoliumbromide (MTT) andflow cytometry. For HUVECs, transendothelial electrical resistance (TEER) and transcellular and paracellularleak were measured using transwell assays, then poly (ADP-ribose) polymerase 1 (PARP-1) and metal matrixproteinase-9 (MMP9) activity were assayed via kits, mRNA expressions of p53 and Rac-1 were determinedthrough quantitative polymerase chain reaction (qPCR); The expressions of intercellular adhesion molecule1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and PARP-1 were measured via western blotting.Results: Low-dose liquiritigenin exerted no effect on gemcitabine-induced changes of cell viability, apoptosis rateand ROS levels in Panc-1 cells, but for HUVECs, liquiritigenin (3 μM) could remarkably elevate gemcitabineinduceddecrease of cell viability, transepithelial electrical resistance (TEER), pro-MMP9 level and expressionof ICAM-1 and VCAM-1 (p<0.01). Meanwhile, it could also significantly decrease gemcitabine-induced increaseof transcellular and paracellular leak, ROS level, PARP-1 activity, Act-MMP9 level, mRNA expressions of p53and Rac-1, expression of PARP-1 and apoptosis rate (p<0.01). Conclusions: Low-dose liquiritigenin exertsan antagonistic effect on gemcitabine-induced leak across HUVECs via inhibiting ROS-mediated signallingpathways, but without affecting gemcitabine-induced Panc-1 cell apoptosis. Therefore, low-dose liquiritigeninmight be beneficial to prevent the occurrence of gemcitabine-induced CLS in pancreatic adenocarcinoma.     

烟曲霉素抑制小鼠结直肠癌生长及转移的体内外研究

背景与目的：血管新生与肿瘤生长密切相关,烟曲霉素（Fumagillin）可特异性抑制血管内皮细胞的增殖,但其对结直肠癌的治疗作用尚不十分清楚。本研究旨在探讨Fumagillin对结直肠癌细胞系生长的抑制作用及其作用机制。方法：将WiDr或HT-29细胞以5&#215;105/L的量分别接种于20只SCID小鼠背部皮下,接种4周后每2日腹腔注射Fumagillin（0.1mg/kg）或Cyclo（1mg/kg）（Arg-Gly-Asp-D-Phe-Val）持续4周,然后处死动物并测量原发瘤质量及原发瘤内微血管密度。在体外培养脐带静脉内皮细胞（HUVECs）,向培养基内加入Fumagillin（0.01mg/kg）或Cyclo（0.1mg/kg）,观察其对HUVECs增殖及微管形成的抑制作用。以基因芯片技术筛选Fumagillin处理后HUVECs的基因表达变化,并以定量PCR和免疫印迹技术对基因芯片的结果做进一步验证。结果：Fumagillin处理组小鼠的结直肠癌原发瘤质量及肿瘤内CD105阳性的微血管数量显著小于对照组小鼠（P〈0.05）,体外实验显示,Fumagillin处理后HUVECs的增殖和微管形成受到明显抑制,基因芯片检测显示Fumagillin处理后HUVECs有71个基因表达上调和143个基因表达下调,表达变化的基因多与细胞黏附、移动、增殖和基因转录有关。定量PCR和免疫印迹技术发现Fumagillin抑制HUVECs表达cyclinE2,白细胞活化黏附因子（activated leukocyte cell adhesion molecule,ALCAM）和细胞间黏附分子-1（intercellular adhesion molecule-1,ICAM-1）。结论：Fumagillin通过抑制新生血管的形成来抑制结直肠癌的生长。     

The preparation and characterization of anti-VEGFR2 conjugated, paclitaxel-loaded PLLA or PLGA microspheres for the systemic targeting of human prostate tumors

Purpose The objective of this study was to manufacture paclitaxel (PTX) loaded polymeric microspheres, that were surface conjugated with antibodies to vascular endothelial growth factor receptor 2 (anti-VEGFR2), for systemic targeting to angiogenic sites in prostate tumors. Methods Microspheres were manufactured in the 1–3 μm size range from poly (l-lactic acid) (PLLA) or poly (lactide-co-glycolide) (PLGA) by a modified solvent evaporation method using Polytron homogenization followed by high speed dispersion in poly vinyl alcohol. Antibodies were conjugated to the surface of these microspheres using cyanogen bromide activation of the polymer surface. Cell Binding was determined using human umbilical vein endothelial cells (HUVECs) in vitro. Efficacy determinations were made using human prostate tumors (PC-3) grown subcutaneously in mice. Results Antibodies were effectively bound to the surface of PLLA and PLGA micropsheres. Anti-VEGFR2 conjugated PLLA microspheres bound strongly to HUVEC’s. Pilot efficacy studies in mice showed variability but demonstrated a significant inhibition of tumor growth following the systemic administration of a single dose of PTX-loaded anti-VEGFR2 conjugated PLLA microspheres as compared to non-antibody-conjugated PTX-loaded microspheres. Conclusion Anti-VEGFR2 conjugated PLLA microspheres containing PTX may offer an effective way of administering a controlled release formulation of the drug to target prostate tumors.     

Hyperthermia decreases cytokine-mediated adhesion molecule expression on human umbilical vein endothelial cells

Although hyperthermia has been used as an effective cancer treatment modality, its effects on metastasis of tumour cells are not clear. Since adhesion molecules play a key role in metastasis, we evaluated how the expression of adhesion molecules is influenced by hyperthermia. Human umbilical vein endothelial cells were incubated in vitro for 1 h. at 39, 42, 43 and 44°C with and without addition of tumour-necrosis factor (TNF) or interferon-gamma (IFN-γ) and the expression of endothelial cell leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class-II molecule was measured. Expression of MHC class-II molecules and expression of unstimulated constituent ICAM-1's was not reduced by heat treatment. In contrast, expression of cytokine-induced ELAM-1's and ICAM-1's was significantly lower after heat treatment. The adhesion to HUVEC in vitro of HL-60 leukemia cells, which express sialyl-Lewis-x antigen as a ligand to ELAM-1, was diminished after incubation at 42°C and totally lost after treatment at 44°C. This suggests that any decrease in metastasis formation after heat treatment, which is occasionally observed, could be due to a reduced action of TNF or related cytokines on adhesion molecule induction and subsequent membrane expression by the endothelial cell. A possible underlying mechanism involved is a heat-induced alteration or blockage of the biosynthetic pathways required for synthesis of ELAM-1 and ICAM-1 proteins.     

Influence of dose-rate on inflammatory damage and adhesion molecule expression after abdominal radiation in the rat

PURPOSE: The goal of this study was to assess the effects of two clinically relevant radiation dose-rates on endothelial adhesion molecule expression, inflammatory response, and microvascular dysfunction. METHODS AND MATERIALS: Rats were irradiated with 10 Gy at low (0.9 Gy/min) or high (3 Gy/min) dose-rates. Control animals received sham irradiation. Leukocyte rolling, adhesion, emigration, and microvascular permeability were assessed in mesenteric venules by intravital microscopy 6 hours after irradiation. P-selectin and intercellular adhesion molecule-1 (ICAM-1) expression were measured using radiolabeled monoclonal antibodies. RESULTS: Low dose-rate (LDR) abdominal irradiation increased leukocyte adhesion compared with sham-irradiated animals, whereas high dose-rate (HDR) irradiation resulted in enhanced leukocyte rolling, adhesion, and emigration, compared with the LDR or with sham-irradiated rats. Both dose-rates increased microvascular permeability, although this effect was significantly greater after radiation with the high (8-fold) than the low (5-fold) dose-rate. HDR radiation induced significantly larger increments in P-selectin expression in splanchnic organs than LDR, whereas in most organs ICAM-1 expression was only upregulated by the HDR. Blockade of ICAM-1, but not P-selectin, abrogated leukocyte adhesion at both dose-rates. CONCLUSIONS: The magnitude of upregulation of endothelial adhesion molecules, leukocyte recruitment, and endothelial barrier dysfunction elicited by radiation therapy is dependent on the dose-rate at which the radiation is delivered.     

Cyclooxygenase-2 activity altered the cell-surface carbohydrate antigens on colon cancer cells and enhanced liver metastasis

Cyclooxygenase-2 (COX-2) was recently reported (M. Tsujii and R. N. DuBois, Cell, 83: 493-501, 1995) to affect the metastatic potential of cells. Previous studies (M. Fukuda, Cancer Res., 56: 2237-2244, 1996) indicated that sialyl Lewis antigen expression is correlated with hematogenous metastasis of colon cancer. In the present study, we investigated the interaction between COX-2 activity, expression of sialyl Lewis antigens, in vitro cancer cell adhesion to endothelial cells, and in vivo metastatic potential. Effects of COX-2 activity and prostaglandin E(2) on cell adhesion, expression of sialyl Lewis antigens, and glycosyltransferase genes were determined in Caco-2-m (COX-2 low level), Caco-2-COX-2 (programmed to overexpress COX-2), and HT-29 (COX-2 high level) cells. Metastatic spread of these cells to the liver was also investigated. Caco-2-COX-2 cells had increased SPan-1 levels and increased adherence to endothelial cells via SPan-1 compared with Caco-2-m cells. HT-29 cells expressed sialyl Lewis a and adhered to endothelial cells via sialyl Lewis a. Treatment with a COX-2 inhibitor, celecoxib, decreased SPan-1 and sialyl Lewis a expression and adherence to endothelial cells. beta 3Gal-T5 and ST3Gal III and IV expression was inhibited by celecoxib and was enhanced by prostaglandin E(2) treatment. Caco-2-COX-2 and HT-29 cells metastasized to the liver, whereas Caco-2-m cells did not. Pretreatment with celecoxib reduced the metastatic potential as well as anti-sialyl Lewis antibodies. Our results indicate a direct link between COX-2 and enhanced adhesion of carcinoma cells to endothelial cells, and enhanced liver metastatic potential via accelerated production of sialyl Lewis antigens. COX-2 inhibitors may suppress metastasis.     

Tumor-Endothelium Cross Talk Blocks Recruitment of Neutrophils to Endothelial Cells: A Novel Mechanism of Endothelial Cell Anergy

Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs) and/or peripheral blood lymphocytes (PBLs). Human umbilical vein endothelial cells (HUVECs) and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours with and without HUVEC conditioning, PMNs or PBLs were added to the HUVEC monolayer. Adhesion to conditioned HUVEC versus adhesion to nonconditioned HUVEC was compared. Effects on endothelial CD44v4, CD44v5, CD44v7, intercellular adhesion molecule 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule 1 (VCAM-1) adhesion receptor expression were analyzed by flow cytometry, intracellular signaling proteins of the mitogen-activated protein kinase pathway and protein kinase C (PKC) subtypes quantified by Western blot analysis. Endothelial conditioning led to a distinct reduction in PMN but not in PBL adhesion to HUVEC. CD44 was significantly reduced, whereas ICAM-1, E-selectin, and VCAM-1 were not altered during HUVEC conditioning. Antibody blockade against CD44v4, CD44v5, and CD44v7 inhibited PMN but not PBL binding. The observed effects were caused by direct tumor cell-HUVEC contact because addition of isolated tumor cell membrane fragments but not of soluble cell culture supernatant to HUVEC induced the CD44 receptor loss. PKCα activity was strongly enhanced in conditioned HUVEC. Blocking PKC prevented the reduction in PMN binding, indicating that this protein is involved in PMN adhesion regulation. A novel tumor escape strategy is presented here. Cell contact-dependent adhesion of tumor cells to the vascular wall promotes down-regulation of endothelial CD44 receptor expression, impairing an effective neutrophil attack.     

Different adhesion properties of highly and poorly metastatic HT-29 colon carcinoma cells with extracellular matrix components: role of integrin expression and cytoskeletal components.

Integrin-mediated tumour cell adhesion to extracellular matrix (ECM) components is an important step in the development of metastatic lesions. Thus, integrin expression and integrin-mediated adhesion of colon carcinoma cells to various ECM components was examined. Poorly (HT-29P) and highly (HT-29LMM) liver-metastatic colon carcinoma cells were used to study the rates of adhesion to collagen I (C I), collagen IV (C IV), laminin (LN), fibronectin (FN), or vitronectin (VN) in a static adhesion assay (10-120 min). Cells were untreated or treated with oligopeptides (RGD, GRGDS, YIGSR, RGES), anti-integrin antibodies, or colchicine, nocodazole, cycloheximide, acrylamide or cytochalasin D (to disrupt cytoskeletal structures). Both cell lines expressed similar patterns of integrin expression (alpha2, alpha3, ,alpha6, alphav, beta1, beta4, and beta5) by immunocytochemistry and immunoprecipitation. HT-29LMM cells showed significantly higher rates of adhesion to LN (P < 0.001) and FN (P < 0.001), but significantly poorer rates of adhesion to C I (P < 0.05) and C IV (P < 0.001) than HT-29P cells, respectively, adhesion to VN was insignificant. RGD and GRGDS inhibited HT-29LMM cell adhesion to FN only. Pretreatment with anti-beta, or anti-alpha2 integrin subunits suppressed adhesion to C I and C IV, and adhesion to LN was inhibited with anti-beta1 or anti-alpha6 integrin. Anti-beta1 or anti-alphav blocked adhesion to FN. Pretreatment of cells with cytochalasin D, cycloheximide or acrylamide inhibited adhesive interactions of both cell lines to the ECM components. In contrast, colchicine and nocodazole had no effect. The results demonstrate that adhesion of HT-29 cells to ECM is mediated, in part, by different integrins, depending on the substrate. Poorly and highly metastatic HT-29 cells possessed different patterns of adhesion to the various ECM substrates, but these differences were not due to different expression of integrin subunits. The results also suggested that the initial adhesion of poorly or highly metastatic HT-29 cells to ECM components requires, in part, the presence of native action and intermediate filaments, but not of microtubules. Thus the adhesion of tumour cells to ECM components may be dependent on signal transduction and assembly of microfilaments.     

Requirement of both mucins and proteoglycans in cell-cell dissociation and invasiveness of colon carcinoma HT-29 cells

Human colon carcinomas are characterized by an aberrant expression of mucins, which in some case leads to an abundant presence of mucus such as in mucinous and signet ring cell carcinomas. Cellular cloning of the human colon carcinoma cell line HT-29 (HT-29 STD), which is mainly composed of undifferentiated cells, yielded a highly mucin-secreting variant (HT-29 5M21). The latter cloned cells cultured on plastic display a polarized organization with an apical secretion of MUC5AC mucin (Lesuffleur et al., Int J Cancer 1998;76:383-92.). Our aim was to study these 2 cell-types as for the invasive and adhesive properties with regard to the function of E-cadherin. HT-29 STD cells were noninvasive in collagen type I, whereas HT-29 5M21 cells were invasive, and the latter behavior was connected to a loss of function of E-cadherin. Likewise, HT-29 5M21 cells were characterized by a cell-cell adhesion independent of E-cadherin, in contrast to the E-cadherin dependent cell-cell adhesion of HT-29 STD cells. Immunofluorescence of HT-29 5M21 cells cultured on collagen type I showed the disappearance of the polarized organization, with a redistribution of apical mucins to the entire cell surface. Treatment of HT-29 5M21 cells by 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn) or by beta-D-xyloside revealed that both mucins and proteoglycans were involved in the loss of E-cadherin function. The use of specific antibodies allowed to show that MUC5AC, MUC1 and heparan sulfate proteoglycans cooperated in the formation of a biological inhibitory complex towards the function of E-cadherin in this invasive HT-29 clone.     

端粒酶逆转录酶在脐静脉内皮细胞耐受曲古抑菌素A诱导凋亡中的作用

目的观察曲古抑菌素A（TSA）对脐静脉内皮细胞及官颈癌细胞凋亡、端粒酶逆转录酶（hTERT）表达的影响,并探讨hTERT在脐静脉内皮细胞耐受TSA中的作用。方法磺酰罗丹明B （RSB）法检测药物动力学特征;流式细胞仪检测周期改变和凋亡;RT-PCR检测hTERT和p21^Waf1基因表达变化;免疫荧光结合流式细胞术检测hTERT蛋白表达变化;转染hTERT质粒后,PCR-TRAP- ELISA法检测转染细胞端粒酶活性;AnnexinV／PI检测转染细胞在TSA作用下的早期凋亡。结果在大剂量TSA作用脐静脉内皮细胞后,增殖抑制、周期阻滞,但凋亡并不显著;HeLa细胞在相同剂量的TSA作用下凋亡明显。脐静脉内皮细胞经TSA诱导后,hTERT表达上调,p21^Waf1则无明显变化;而HeLa细胞p21^Waf1表达上升,hTERT表达下降。转染显性负突变hTERT的脐静脉内皮细胞,其端粒酶活性显著低于对照组。TSA作用转染不同质粒的脐静脉内皮细胞的凋亡率与对照组差异有统计学意义。结论脐静脉内皮细胞可以耐受大剂量TSA诱导的凋亡,hTERT表达上调可能是脐静脉内皮细胞耐受TSA诱导凋亡的重要机制之一。     

Influence of proinflammatory cytokines on the adhesion of human colon carcinoma cells to lung microvascular endothelium

In this experimental study, the influence of surgery-induced proinflammatory cytokines on tumor recurrence in the lung was investigated. A reproducible human in vitro assay was developed to study the adhesion of HT29 colon carcinoma cells to monolayers of microvascular endothelial cells of the lung (HMVECs-L) or human umbilical venous endothelial cells (HUVECs). Preincubation of HMVECs-L with maximally active concentrations of IL-1beta and TNF-alpha, but not with IL-6, resulted in at least 250% adhesion compared to control adhesion (p 相似文献