Copolymères solubles inhibiteurs de l'angiogenèse in vitro

Water-soluble statistic copolymers composed of sodium styrene sulfonate (NaSS) and methacrylic acid (MA) were synthesized. They inhibited adherence and proliferation of human breast cancer cells MCF7. One polymer, chemical composition 30%mol MA and 70%mol NaSS (MA–NaSS 30–70), shown a growth inhibition rate of 42% at day 6 at 100 μg/mL. Effects on human endothelial cells were then tested. The activity of four polymers MA–NaSS 30–70, 50–50, 60–40 and poly(NaSS), at various concentrations on human umbilical vein endothelial cells (HUVECs) was investigated. Results showed an inhibition of 80% on HUVECs growth. The effect of copolymers on cell morphology was investigated. When HUVECs were cultivated in the presence of MA–NaSS 30–70 and poly(NaSS) at 10 μg/mL, cells remained rounded-shaped and did not spread. Moreover, cell area was systematically smaller for cells cultivated in the presence of copolymers versus control. The cytoskeleton of HUVECs was sensitively affected. Actin stress fibers were neither elongated nor well differentiated and the number of focal contacts was dramatically decreased in comparison with control. The effect on angiogenesis was studied. After 24 hs of contact with MA–NaSS 30–70 at 10 μg/mL, HUVECs did not form tubules. In contrast, tubules were numerous and elongated on control samples. The expression of integrins mediating HUVECs adhesion or angiogenesis was studied. DNA electrophoresis of adherent and non-adherent cells were performed after 72 hours of incubation with MA–NaSS 30–70. For adherent cells β₁, β₃, and α_V were less expressed α₅ was more expressed and α₂ expression was unchanged when compared to the controls. For non adherent cells β₁and α₅ were more expressed than in controls and α₂, α_V and β₃ were almost the same as control. Our copolymers are able to in vitro inhibit cancer cells growth and angiogenesis. Their activity is based on the inhibition of integrins mediated cell adherence to the extracellular matrix.     

C/EBPα inhibits proliferation of breast cancer cells via a novel pathway of miR-134/CREB

C/EBPα plays an important role in the modulation of cell proliferation, differentiation or apoptosis in various tissues. Most recently, reduced expression of C/EBPα and growth inhibitory effect was found in primary mammary carcinomas. However, the underlying mechanism is still not fully aware. Here, we firstly identified miR-134 as a target of C/EBPα in MCF7 breast cancer cell lines. C/EBPα overexpression promoted miR-134 expression, causing suppression of apoptosis- protective genes CREB and Bcl-2, and resulted in the proliferation inhibition of MCF7 cells. Moreover, anti-miR-134 rescued the proliferation inhibition of MCF7 cells and the suppression of anti-apoptotic genes CREB and Bcl-2 caused by C/EBPα overexpression. Collectively, C/EBPα inhibited cell growth in breast cancer cells via a novel pathway miR-134/CREB.     

Retinal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from synthetic biodegradable polymers

Novel synthetic biodegradable polymer substrates with specific chemical micropatterns were fabricated from poly(DL-lactic-coglycolic acid) (PLGA) and diblock copolymers of poly(ethylene glycol) and poly(DL-lactic acid) (PEG/PLA). Thin films of PLGA and PEG/PLA supported and inhibited, respectively, retinal pigment epithelial (RPE) cell proliferation, with a corresponding cell density of 352,900 and 850 cells/cm2 after 7 days (from an initial seeding density of 15,000 cells/cm2). A microcontact printing technique was used to define arrays of circular (diameter of 50 microm) PLGA domains surrounded and separated by regions (width of 50 microm) of PEG/PLA. Reversed patterns composed of PEG/PLA circular domains surrounded by PLGA regions were also fabricated. Both micropatterned surfaces were shown to affect initial RPE cell attachment, limit cell spreading, and promote the characteristic cuboidal cell morphology during the 8-h period of the experiments. In contrast, RPE cells on plain PLGA (control films) were elongated and appeared fibroblast-like. The reversed patterns had continuous PLGA regions that allowed cell-cell interactions and thus higher cell adhesion. These results demonstrate the feasibility of fabricating micropatterned synthetic biodegradable polymer surfaces to control RPE cell morphology.     

Interactions between self-assembled polyelectrolyte shells and tumor cells

Layer-by-layer self-assembled polyelectrolyte shells are a new class of micro/nanocapsules with unique physicochemical properties for potential applications in drug/gene delivery. The objective of this study was to investigate the interactions of polyelectrolyte shells ( approximately 1 mum in diameter) with MCF-7 breast cancer cells and identify key parameters that affect such interactions. Tailoring of surface properties of polyelectrolyte shells was achieved by choosing different outermost layer materials, including cationic polymers, anionic polymers, and lipid bilayers. Different surface compositions led to a wide range of electrostatic potentials from -46 to +47 mV in phophate-buffered saline buffer. Confocal microscopy studies showed that the polyelectrolyte shells were internalized into the cell cytoplasm, but not into the nuclei. Correlation of cell uptake with shell surface compositions was complicated by the adsorption of serum proteins on the surface of polyelectrolyte shells, particularly polycation-coated shells. To prevent protein adsorption, poly(ethylene glycol) (PEG) grafted poly(ethyleneimine) (PEI) copolymers (1:1, 1:5, 1:10 graft ratios) were synthesized and introduced on the shell surface. Shells coated with PEI-PEG copolymers effectively reduced protein adsorption whereas PEI-PEG copolymers with lower graft ratios achieved higher cell uptake efficiency after 24 h of incubation with MCF-7 cells.     

The uptake of N-(2-hydroxypropyl)-methacrylamide based homo, random and block copolymers by human multi-drug resistant breast adenocarcinoma cells

A series of well-defined, fluorescently labelled homopolymers, random and block copolymers based on N-(2-hydroxypropyl)-methacrylamide were prepared by reversible addition–fragmentation chain transfer polymerization (RAFT polymerization). The polydispersity indexes for all polymers were in the range of 1.2–1.3 and the number average of the molar mass (M_n) for each polymer was set to be in the range of 15–30 kDa. The cellular uptake of these polymers was investigated in the human multi-drug resistant breast adenocarcinoma cell line MCF7/ADR. The uptake greatly depended on the polymer molecular mass and structure. Specifically, smaller polymers (approx. 15 kDa) were taken up by the cells at much lower concentrations than larger polymers (approx. 30 kDa). Furthermore, for polymers of the same molar mass, the random copolymers were more easily internalized in cells than block copolymers or homopolymers. This is attributed to the fact that random copolymers form micelle-like aggregates by intra- and interchain interactions, which are smaller and less stable than the block copolymer structures in which the hydrophobic domain is buried and thus prevented from unspecific interaction with the cell membrane. Our findings underline the need for highly defined polymeric carriers and excipients for future applications in the field of nanomedicine.     

Effect of the chemical composition of Ricinus communis polyurethane on rat bone marrow cell attachment,proliferation, and differentiation

Alterations in the chemical composition of a polymer may be undertaken to improve its biological properties. The aim of this study was to investigate the in vitro biocompatibility of Ricinus communis polyurethane (RCP) with three different chemical compositions: RCPp (pure RCP), RCP+CaCO(3), and RCP+Ca(3)(PO(4))(2). Rat bone marrow cells were cultivated under conditions that allowed osteoblastic differentiation and were evaluated for cell attachment, cell proliferation, cell morphology, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. For the evaluation of attachment, cells were cultured for 4 h. After 3 days, cell morphology was evaluated. Cell proliferation was evaluated after 7 and 14 days. Total protein content and ALP activity were evaluated after 14 days. For bonelike nodule formation, cells were cultured for 21 days. Data were compared with an analysis of variance and Duncan's multiple range test when appropriate. Cell attachment and ALP activity were not affected by RCP chemical composition. Proliferation, total protein content, and bonelike nodule formation were all affected by RCP chemical composition. These results suggest that initial cell events are not affected by RCP chemical composition, whereas RCPs blended with calcium carbonate or, better yet, calcium phosphate, by favoring events that promote matrix mineralization, are more biocompatible materials.     

Interaction of human skin fibroblasts with moderate wettable polyacrylonitrile--copolymer membranes

The development of a bioartificial skin is a step toward the treatment of patients with deep burns or nonhealing skin ulcers. One possible approach is based on growing dermal cells on membranes to obtain appropriate living cellular stroma (sheets) to cover the wound. New membrane-forming copolymers were synthesized, based on acrylonitrile (AN) copolymerization with hydrophilic N-vinylpyrrolidone (NVP) monomer, in different percentage ratios, such as 5, 20, and 30% w/w, and with two other relatively high polar comonomers--namely, sodium 2-methyl-2-propene-1-sulfonic acid (NaMAS) and aminoethylmethacrylate (AeMA). All these copolymers were characterized for their bulk composition and number average molecular weight, and used to prepare ultrafiltration membranes. Water contact angles and water uptake were estimated to characterize the wettability and scanning force microscopy to visualize the morphology of the resulting polymer surface. Cytotoxicity was estimated according to the international standard regulations, and the materials were found to be nontoxic. The interaction of the membranes with human skin fibroblasts was investigated considering that these cells are among the first to colonize membranes upon implantation or with prolonged external contact. The overall cell morphology, formation of focal adhesion contacts, and cell proliferation were estimated to characterize the cell material interactions. It was found that the pure polyacrylonitrile homopolymer (PAN) membrane provides excellent conditions for seeding with fibroblasts, comparable only to a copolymer containing AeMA. In contrast, the presence of NaMAS with acidic ionic groups decreased both the attachment and proliferation of fibroblasts. Low content of NVP in the copolymer, up to about 5%, still enabled good attachment and spreading of cells, as well as subsequent proliferation of fibroblasts, but higher ratios of 20 and 30% resulted in a significant decrease of these cellular activities.     

布托啡诺调控PBX3基因抑制乳腺癌细胞系MCF7增殖、迁移和侵袭

目的探讨布托啡诺(butorphanol)对乳腺癌细胞增殖、迁移和侵袭的影响以及相关的分子机制。方法用MTT法检测不同浓度布托啡诺对人乳腺癌细胞系MCF7的抑制作用;用Transwell迁移及侵袭实验检测不同浓度布托啡诺对人乳腺癌MCF7细胞迁移及侵袭的影响;RT-qCR与Western blot法分别检测乳腺癌细胞系、正常乳腺上皮细胞以及布托啡诺对MCF7细胞中PBX3 mRNA及蛋白表达的影响;观察转染si-PBX3或si-con后,MCF7细胞增殖、迁移及侵袭能力的变化;PBX3过表达验证布托啡诺对乳腺癌增殖、迁移及侵袭的作用机制。Western blot检测cyclin D1和MMP-2蛋白表达。结果PBX3在乳腺癌细胞系中的表达上调,沉默PBX3表达可明显抑制MCF7细胞增殖、迁移及侵袭,同时抑制cyclin D1和MMP-2的表达;不同浓度的布托啡诺干预能显著抑制MCF7细胞增殖、迁移及侵袭且具有浓度依赖性,还可抑制PBX3、cyclin D1和MMP-2的表达;过表达PBX3可逆转布托啡诺对乳腺癌细胞增殖、迁移及侵袭的抑制作用。结论布托啡诺可通过抑制PBX3降低乳腺癌细胞增殖、迁移及侵袭能力。     

Vitronectin is significant in the adhesion of lens epithelial cells to PMMA polymers

A major complication of intraocular lens surgery is diminished visual acuity caused by the regrowth of lens epithelial cells (secondary cataract). Polymethylmethacrylate (PMMA) is a commonly used intraocular lens material. This study addresses the mechanisms underlying the initial adhesion of lens epithelial cells to PMMA and a functionalized PMMA-based terpolymer known to inhibit cell proliferation. Rabbit lens epithelial cells were cultured on the test polymer surfaces in medium containing serum depleted of either fibronectin or vitronectin (or both) to identify the role of these proteins in the initial process of cell adhesion. Adherent cells were quantitated after 60 min, and the actin cytoskeleton and focal contact formation were compared in each serum treatment on both polymers. Vitronectin was significantly more effective for initial cell attachment to both polymers than fibronectin. Normal cell spreading on PMMA required vitronectin and was independent of fibronectin, whereas cell spreading on the terpolymer was abnormal and required the presence of fibronectin and vitronectin together. Together, these results help to explain the inhibition of cell proliferation previously shown on the functionalized PMMA. This work contributes to the design of a polymer for use in intraocular lenses that inhibits proliferation of the target cells.     

Interrelationship of micromechanics and morphology of fibroblasts adhered on different polymeric surfaces

The membrane stiffness (ε) of rat lung fibroblasts (RLFs) adhered on different polymeric surfaces was probed by atomic force microscopy. The corresponding cell morphology was also analyzed to probe its interrelationship with ε. Two tyrosine-derived polymer families, poly(DTR glutarate)s and poly(DTE-co-PEG₁₀₀₀ carbonate)s with systematic variations in the chemical composition and physical properties, notably surface hydrophilicity, were used. The cell membrane of adhered RLFs was indented by a probe tip. ε was obtained by best-fitting the relationship of applied tip forces and the indentation depth with the Hertz model. Excluding tissue culture polystyrene, non-PEG-containing polymers are generally hydrophobic and the changes in chemical composition do not elicit significant changes in ε. In contrast, polymers containing as little as 2 mol.% PEG display a major increase in surface hydrophilicity and invoke a substantial decrease in ε. Additionally, RLFs show a high degree of spreading and fibroblastic appearance on non-PEG-containing polymers, but much less spreading and axial morphology when PEG is present. A mechanism is proposed to explain how a cell maintains its structural integrity on different polymeric surfaces: the degree of cell spreading is higher on non-PEG-containing surfaces than on PEG-containing ones, resulting in more extended cytoskeletal filaments and hence a stiffer cell membrane. Our studies shed light on the use of cellular micromechanics, and in particular membrane stiffness, to characterize cell response as a function of the chemical composition of the underlying substrata.     

Microtubular architecture of biodegradable polymer scaffolds

It is a relatively new approach to generate tissues with mammalian cells and scaffolds (temporary synthetic extracellular matrices). Many tissues, such as nerve, muscle, tendon, ligament, blood vessel, bone, and teeth, have tubular or fibrous bundle architectures and anisotropic properties. In this work, we have designed and fabricated highly porous scaffolds from biodegradable polymers with a novel phase-separation technique to generate controllable parallel array of microtubular architecture. Porosity as high as 97% has been achieved. The porosity, diameter of the microtubules, the tubular morphology, and their orientation are controlled by the polymer concentration, solvent system, and temperature gradient. The mechanical properties of these scaffolds are anisotropic. Osteoprogenitor cells are seeded in these three-dimensional scaffolds and cultured in vitro. The cell distribution and the neo-tissue organization are guided by the microtubular architecture. The fabrication technique can be applied to a variety of polymers, therefore the degradation rate and cell--matrix interactions can be controlled by the chemical composition of the polymers and the incorporation of bioactive moieties. These microtubular scaffolds may be used to engineer a variety of tissues with anisotropic architecture and properties.     

Endothelial cell intracellular Ca concentration is increased upon breast tumor cell contact and mediates tumor cell transendothelial migration

Tumor cell extravasation is a determinant step in the process of hematogenous metastasis. The signal transduction pathways involved in the interactions between tumor cells and the vascular endothelium during transendothelial migration are still undefined. In the present study, we have investigated the influence of human breast adenocarcinoma cells (MCF7) on human umbilical vein endothelial cell (HUVEC) intracellular Ca²⁺ concentration ([Ca²⁺]i). We show that the contact between MCF7 cells and a confluent HUVEC monolayer induces an immediate and transient increase in HUVEC [Ca²⁺]i. This [Ca²⁺]i rise could not be elicited by tumor cell-conditioned medium, isolated tumor cell membranes, inert beads or normal breast epithelial cells, demonstrating the involvement of specific recognition mechanisms between MCF7 cells and HUVEC. Depletion of HUVEC intracellular Ca²⁺ stores by the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin as well as the selective depletion of inositol 1,4,5-tri phosphate (IP3)-sensitive Ca²⁺ stores by prior activation of HUVEC using histamine resulted in a complete inhibition of tumor cell-induced [Ca²⁺]_i elevation. Similar results were obtained when HUVEC monolayers were treated with the tyrosine kinase inhibitor herbimycin A, suggesting a role for tyrosine kinase-associated cell surface receptors in tumor cell-endothelial cell interactions. The depletion of HUVEC intracellular Ca²⁺ stores by thapsigargin was also shown to delay MCF7-induced endothelial cell disjunction, to prevent their spreading on the subendothelial extracellular matrix and transendothelial migration in vitro. These results suggest that transient changes in endothelial [Ca²⁺]_i may govern multiple steps of tumor cell extravasation. © Rapid Science 1998     

沉默GRP94 基因对乳腺癌MCF7 细胞增殖和凋亡的影响

目的： 研究沉默葡萄糖调节蛋白GRP94 (Glucose regulated protein,GRP94)对乳腺癌MCF7细胞增殖、凋亡的影响及潜在机制。方法：设计并化学合成靶向沉默GRP94基因的小干扰RNA,通过脂质体转染入MCF7细胞中,采用qRT-PCR和Western blot分别检测GRP94、cyclinD1、Bax和Bcl-2 mRNA和蛋白的表达水平;通过流式细胞术检测细胞凋亡比例变化,Hoechst 33258染色检测凋亡细胞核变化、CCK8实验检测细胞增殖能力的变化。结果： GRP94 siRNA组GRP94基因的表达水平被有效抑制;与对照组相比,GRP94-siRNA转染组的细胞凋亡比例明显增加;凋亡细胞核形态发生变化;增殖能力明显下降;mRNA及蛋白水平cyclinD1、Bcl-2表达明显下调,Bax表达增加。结论：沉默GRP94基因可明显抑制乳腺癌MCF7细胞增殖能力,促进细胞凋亡的发生,且其可能通过下调cyclinD1、Bcl-2和上调Bax表达参与其中。     

Adhesion and proliferation of bovine aortic endothelial cells on monoamine- and diamine-containing polystyrene derivatives.

Adhesion and proliferation of bovine aortic endothelial cells on polystyrene derivatives having monoamine or diamine side chain was investigated focusing on the chemical structure of amino groups. Copolymers, SE8.5, is composed of polystyrene with 8.5 mol% of monoamine side chains, and SED8, which is with 8 mol% of diamine side chains, were estimated to contain almost the same amount of protonated amino groups in bulk composition at physiological pH (pH 7.4). There observed significant difference in cellular spreading of attached endothelial cells between these two types of copolymer surfaces. Spreading-% of attached cells on SED8 surfaces was approximately 1.6 times greater than that on SE8.5 6 h after seeding. This difference in cellular spreading influenced to subsequent cell growth. Cellular growth on each polymer surface was featured by parameter k, which corresponds to the 'rate constant' of cellular proliferation. While the k-value for SE8.5 decreased with decreasing seeding density as well as the case for polystyrene, SED8 maintained a high k-value even at low seeding density as 2 x 10(3) cells/cm2. These results suggest that cells may recognize the difference in the chemical structure of amine side chains of SE and SED copolymers.     

Experimental and computational analysis of cellular interactions with nylon-3-bearing substrates

The ability to design biomaterials that interact with biological environments in a predictable manner necessitates an improved understanding of how surface chemistry influences events such as protein adsorption and cell adhesion. In this work, we examined mechanisms governing the interactions between 3T3 fibroblasts and nylon-3 polymers, which have a protein-like polyamide backbone and are highly amenable to tuning of chemical and physical properties. Protein adsorption and cell adhesion to a library of nylon-3 polymers were characterized and analyzed by partial least squares regression. This analysis revealed that specific chemical features of the nylon-3 polymers correlated with the extent of protein adsorption, which, in turn, correlated with cell adhesion in a serum-containing environment. In contrast, in a serum-free environment, cell adhesion could be predicted solely from chemical properties. Enzymatic treatments of 3T3 cells before plating indicated that proteins bound to the cell surface mediated cell-nylon-3 polymer interactions under serum-free conditions, with additional analysis suggesting that cell-associated fibronectin played a dominant role in adhesion in the absence of serum. The mechanistic insight gained from these studies can be used to inform the design of new polymer structures in addition to providing a basis for continued development of nylon-3 copolymers for tissue engineering applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2750-2759, 2012.     

Oxytocin inhibits proliferation of human breast cancer cell lines

In this study we show that treatment of MDA-MB231 hormone-independent human breast cancer cells with oxytocin (OT) or with the OT analogue F314 induces significant growth inhibition together with a change in cell phenotype. In MCF7 and T47D human breast cancer cells, OT inhibits oestrogen-induced cell growth. In these same cells, OT administration significantly enhances the inhibitory effect of tamoxifen on cell proliferation. MDA-MB231, MCF7 and T47D cells all express mRNA specific for the OT receptor. These data suggest that it may be possible to inhibit breast cancer growth using OT and OT analogues.     

Die Fraktionierung von Styrol-Acrylnitril-Copolymeren nach dem Acrylnitrilgehalt in zweiphasigen Lösungsmittelgemischen

Liquid phase pairs were found in which polystyrene (PS) and styrene-acrylonitrile (SAN)-copolymers with an AN-content up to 13 wt.-% can be partitioned. They could be used for the fractionation of the copolymers according to chemical composition. The phase pairs are composed of 4 solvents, cyclohexane and nitromethane to form the phases and two solvents for the polymers. The partition ratio was measured as a function of the composition of the phase pairs, the molecular weight of PS, and of the AN-content of the copolymers. The phase composition at which a polymer is equally distributed into both phases depends on the chemical composition of the polymer but not on its molecular weight. With other phase compositions the partition equilibrium is shifted to one phase or the other the more the degree of polymerisation increases. Based on the partition equilibrium, fractionation methods are proposed to separate the SAN-copolymers according to their chemical composition if their molecular weight is high enough (>about 10⁵). The conditions which determine fractionation more by molecular weight or more by chemical composition are discussed.     

Attachment and spreading of human gingival fibroblasts on potentially bioactive glasses in vitro

The attachment and spreading of cultured fibroblasts on potentially bioactive glasses (bioglasses) of ten different compositions were studied. Human gingival fibroblasts were allowed to attach and spread on bio-glasses for 1-72 h. Unreactive silica glass and cell culture polystyrene served as controls. The attachment and spreading of cells were examined by 3H-thymidine labeling of cells, planimetric analysis, cytological staining, immunocytochemistry, and scanning electron microscopy. The cell attachment to bioglasses and silica glass and the cell spreading on bioglasses were slower and cell morphology more elongated compared to control plastic. In spite of great differences in bioglass compositions no great differences in cell behavior on these surfaces were detected. Thus the initial events in the tissue-implant interface might be independent on the bioglass composition, and furthermore the differences in the organization of the tissue-implant interface in vivo might depend on the nature of the surrounding tissues and subsequent changes of the implant surface and the extracellular environment.     

金雀黄素抑制人乳腺癌细胞系体外增殖作用机理的实验研究

目的 研究金雀黄素抑制人乳腺癌细胞株体外增殖作用的机理。方法 选用2种不同的人乳腺癌细胞株MCF-7和MDA-MB-23l体外培养，MTT法检测细胞增殖作用，Giemsa染色观察细胞形态学改变，流式细胞仪检测细胞周期及凋亡率，同时进行了原位细胞凋亡的检测。结果 金雀黄素对MCF-7细胞和MDA-MB-231细胞体外生长具有明显的抑制作用。Giemsa染色显示，金雀黄素处理的MCF-7细胞呈明显的凋亡形态改变，细胞固缩、发泡，染色质凝集呈块状，并沿核周分布。流式细胞仪检测在G1峰前可见凋亡峰，且随用药时间的延长，凋亡比率递增。而金雀黄素处理的MDA-MB-231细胞未见明显的凋亡形态改变和凋亡峰的出现，细胞周期明显地阻滞于G2～M期。结论 金雀黄素通过诱导细胞凋亡或阻滞细胞于G2-M期，从而抑制乳腺癌细胞的体外增殖，为其减缓人乳腺癌细胞的体内生长提供理论依据。