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At least 775 records · Page 43Linked to original sources
PubMed2006

Reduced angiogenic responses in adult Endoglin heterozygous mice.

OBJECTIVE: To determine if angiogenesis is altered in adult Endoglin heterozygous (Eng(+/-)) mice, the animal model for the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). METHODS: Primary cultures of endothelial cells were generated from Eng(+/-) and Eng(+/+) mice and analyzed for proliferation, migration, and ability to form capillary-like tubes. Endothelial cells derived from umbilical veins of newborns (HUVEC) with an HHT1 genotype were also tested for capillary formation. Two in vivo models of angiogenesis were tested in the Eng(+/-) and Eng(+/+) mice: Matrigel implant-dependent angiogenesis and reperfusion following hindlimb ischemia. RESULTS: The Eng(+/-) endothelial cells displayed significantly reduced proliferation and migration, increased collagen production, and decreased NO synthase expression and vascular endothelial growth factor (VEGF) secretion. They also showed impaired capillary tube formation in vitro, as did the HHT1 HUVEC. These endothelial cell-specific abnormalities were associated with impaired Matrigel-dependent capillary tube formation in vivo and delayed reperfusion following hindlimb ischemia. CONCLUSIONS: Although vascular development is normal in Eng(+/-) mice, angiogenic abnormalities were observed in the adult mice and their isolated endothelial cells. These results suggest that a normal level of endoglin is required for full angiogenic activity.

Animals↗
PubMed2006

Adipokine resistin promotes in vitro angiogenesis of human endothelial cells.

OBJECTIVE: Resistin may be associated with obesity and cardiovascular diseases. However, it is unknown whether resistin directly contributes to angiogenesis. In the present study, we evaluated the effects of resistin on angiogenic potential, including endothelial cell proliferation, migration, and capillary-like tube formation. METHODS: Human coronary artery endothelial cells (HCAECs) were treated with resistin. Cell proliferation was evaluated by [3H]thymidine incorporation and MTS assays. Cell migration was assessed by a modified Boyden chamber assay. Capillary-like tube formation was studied with a Matrigel model. Several gene expression levels were determined by real-time PCR. Activation of mitogen-activated protein kinases (MAPKs) was determined by Bio-Plex luminex analyzer. Basic fibroblast growth factor (bFGF) was used as a control. Human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVEC-L) were also included. RESULTS: Resistin induced both endothelial proliferation and migration in a dose- and time-dependent manner with the maximal effect at 40 ng/ml. Both resistin-induced cell proliferation and migration could be effectively blocked by a resistin-neutralization antibody. In addition, resistin promoted capillary-like tube formation of HCAECs on Matrigel. Resistin also significantly upregulated the mRNA expression of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) and matrix metalloproteinases (MMP-1 and MMP-2) at both mRNA and protein levels. Furthermore, transient phosphorylation of ERK1/2 and p38 was observed after the addition of resistin to HCAECs. The resistin-induced cell proliferation and migration were both completely blocked by specific ERK1/2 and p38 inhibitors. CONCLUSIONS: Resistin induces human endothelial cell proliferation and migration, promotes capillary-like tube formation, upregulates the expression of VEGFRs and MMPs, and activates ERK1/2 and p38 pathways. Thus, resistin may play an important role in angiogenesis-associated vascular disorders.

Antibodies, Monoclonal↗
PubMed2006

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture.

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.

Breast↗
PubMed2006

Establishment and characterization of a spontaneously immortalized porcine mammary epithelial cell line.

We have established a spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) from the mammary gland of a lactating sow and maintained it long-term in culture by continuous subculturing. SI-PMEC cells were maintained for more than 8 months (70 passages) in DMEM/F12 medium supplemented with 10% fetal calf serum, insulin, and hydrocortisone without obvious signs of senescence. When grown at low density on a plastic substratum, SI-PMEC cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobblestone morphology of epithelial cells. The subcultured SI-PMEC cells appeared to proliferate without changes in morphology or growth pattern, with an estimated population doubling time of 20-22 h. With increasing density, SI-PMEC cells organized into lumen-like structures with elongated cells at the margins. SI-PMEC cells from stocks frozen at Passage 30 were subcultured up to 20 times without changes in cell viability, proliferation rate, or morphology. Furthermore, SI-PMEC cells remained immunopositive to an antibody against cytokeratin AE3 and immunonegative to an antibody against a human fibroblast antigen. The SI-PMEC cells could form functional structures resembling ducts, lateral-buds, and alveoli in a Matrigel matrix-dependent manner in vitro. When grown on the Matrigel and stimulated by prolactin, the cells differentiated and formed mammary gland structures and strongly expressed transcripts encoding the milk proteins alpha-lactalbumin, beta-casein, and beta-lactoglobulin. Our results indicate that the SI-PMEC cell line can be subpassaged many times and still form functional differentiated secretory structures. To our knowledge, this is the first report of an immortalized mammary epithelial cell line from pig.

Animals↗
PubMed2006

Nucleotide exchange factor ECT2 regulates epithelial cell polarity.

Cell polarity regulates diverse biological events such as localization of embryonic determinants and establishment of tissue and organ architecture. Epithelial cell polarity is regulated by the polarity complex Par6/Par3/atypical protein kinase C (aPKC). We previously found that the nucleotide exchange factor ECT2 associates with this polarity complex and regulates aPKC activity, but the role of ECT2 in cell polarity is still unclear. Here we show that expression of a dominant negative (ECT2-N2) or constitutively active (ECT2-DeltaN5) form of ECT2 inhibits normal cyst formation of MDCK cells in 3-dimensional collagen gels. Central lumens were not observed in cysts formed by cells expressing either ECT2-DeltaN5 or ECT2-N2. Apical localization of ZO-1 and basolateral localization of beta-catenin were no longer observed in these cells. Interestingly, cells expressing ECT2-N2 did form normal cysts when cultured in the basement membrane matrix Matrigel instead of collagen gels. Addition of a major Matrigel component, laminin, partially rescued the normal cyst formation inhibited by ECT2-N2 in 3-dimensional collagen gels. Thus, signaling through laminin might override the defects of signaling through collagen and ECT2. Whereas ECT2-N2 inhibited the lumen formation of MDCK cysts, caspase-3, which is reportedly involved in lumen formation through apoptosis, was activated at various locations of cells in the cysts. It is likely that perturbation of ECT2 signaling inhibits the establishment of epithelial cell polarity leading to the inhibition of selected elimination of cells at the center of cysts. Thus, ECT2 appears to play a critical role in epithelial cell polarity.

Animals↗
PubMed2004

Smad2 suppresses the growth of Mv1Lu cells subcutaneously inoculated in mice.

Smad2 and Smad3 are intracellular signal transduction proteins of importance in transforming growth factor-beta (TGFbeta)-mediated inhibition of epithelial cell proliferation. Inactivating mutations in the Smad2 and Smad3 genes have been found in various human malignancies. Here, we show that expression of Smad2 leads to the inhibition of growth of Mv1Lu cells inoculated with Matrigel subcutaneously (s.c.) in severe combined immunodeficient (SCID) mice. In histological appearance, the Matrigel plugs with Smad2-transfected cells showed strongly reduced cell density, proliferation and angiogenesis compared with the small tumour nodules of similar size formed by the vector- or Smad3-transfected cells. The histological appearance of vector- and Smad3-transfected cells inoculated in mice was identical. Overexpression of Smad2 and Smad3 in Mv1Lu cells led to the inhibition of cell growth in three-dimensional cultures when compared with vector-transfected cells. Overexpression of Smad2 and Smad3 also decreased the hyperphosphorylation of pRb in Smad-transfected cells. Thus, increased expression of Smad2 leads to inhibition of Mv1Lu cell proliferation and a reduction in the growth of the Smad2-expressing cells inoculated in mice.

Animals↗
PubMed2004

Antitumor activities of a novel indolin-2-ketone compound, Z24: more potent inhibition on bFGF-induced angiogenesis and bcl-2 over-expressing cancer cells.

The present study was designed to select the effective dosage range of Z24 [3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one], a novel synthetic indolin-2-ketone small-molecule compound, against tumorigenesis and angiogenesis in vitro and in vivo and to investigate the primary action mechanism of Z24 on the angiogenesis by comparing with SU5416 [3-[(2,4-dimethylpyrrol-5-yl)methyllidenyl]-indolin-2-one] in the selective effects on vascular endothelial growth factor (VEGF)/basic fibroblast growth factor (bFGF) signaling and Bcl-2-related cell vitality because Z24 is a potential inhibitor of the Bcl-2 that inhibits growth of multiple tumor types in vivo in our previous study. Per os Z24 inhibited dose-dependently the mouse S180 xenograft tumor growth and angiogenesis in mouse subcutaneous (s.c.) Matrigel plugs in vivo. The maximum growth inhibitory rate was 56.1% by 80 mg/kg/day on S180 mouse sarcoma cells; however, the maximum inhibitory potency on angiogenesis in C57BL/6 mouse subcutaneous Matrigel plug model was 50 mg/kg/day. Z24 inhibited angiogenesis in chicken chorioallantoic membrane (CAM) and invasion and inhibited tube formation of endothelial cells in a dose-dependent manner. Compared with SU5416, the IC50 (50% inhibition concentration) of Z24 on the proliferation of ECV-304 carcinoma cells induced by VEGF or bFGF was 24.4 and 17.99 microM, respectively, which is higher or lower, respectively, than that of SU5416 (14.2 microM for VEGF and 22.7 microM for bFGF). Furthermore, the IC50 of Z24 on the proliferation of Bcl-2 over-expressing HeLa cells and non-Bcl-2-expressing (wild-type) HeLa cells are 11.9 and 24.8 microM, respectively. SU5416 did not exert such a selective inhibiting effect on Bcl-2 over-expressing HeLa cells. These results suggest that Z24 per os has dose-dependent antitumor and antiangiogenesis pharmacological activity. The higher selectivity of Z24 on Bcl-2 protein and on bFGF other than VEGF signaling path may contribute to its efficiency against tumor and tumor-associated angiogenesis.

Animals↗
PubMed2004

Pseudolarix acid B inhibits angiogenesis by antagonizing the vascular endothelial growth factor-mediated anti-apoptotic effect.

Angiogenesis is controlled by a number of growth factors, including vascular endothelial growth factor (VEGF). In this study, pseudolarix acid B, isolated from the traditional Chinese medicinal plant Pseudolarix kaempferi and originally identified as an early pregnancy-terminating agent, was evaluated for its potential as an angiogenesis inhibitor, using in vitro and in vivo models. After exposure to pseudolarix acid B 0.625-5 microM for 72 h, the proliferation of human umbilical vein endothelial cells was significantly inhibited. Pseudolarix acid B 0.313-2.5 microM for 24 h potently blocked the VEGF-induced tube formation of human umbilical vein endothelial cells in a dose-dependent manner. Matrigel plug assays disclosed that pseudolarix acid B reduced angiogenesis induced by VEGF in vivo. In addition, pseudolarix acid B antagonized VEGF-mediated anti-apoptotic effects on serum-deprived human umbilical vein endothelial cells and increased apoptosis of endothelial cells induced by VEGF in Matrigel plug assays. Moreover, pseudolarix acid B significantly inhibited VEGF-induced tyrosine phosphorylation of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1), in correlation with a marked decrease in the phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings collectively suggest that pseudolarix acid B possesses anti-angiogenic activity. One of the main anti-angiogenesis mechanisms of pseudolarix acid B may involve antagonism of the VEGF-mediated anti-apoptosis effect via inhibition of KDR/flk-1, ERK1/2, and Akt phosphorylation in endothelial cells.

Angiogenesis Inhibitors↗
PubMed2005

Antiangiogenic activity of beta-eudesmol in vitro and in vivo.

Abnormal angiogenesis is implicated in various diseases including cancer and diabetic retinopathy. In this study, we examined the effect of beta-eudesmol, a sesquiterpenoid alcohol isolated from Atractylodes lancea rhizome, on angiogenesis in vitro and in vivo. Proliferation of porcine brain microvascular endothelial cells and human umbilical vein endothelial cells (HUVEC) was inhibited by beta-eudesmol (50-100 microM). It also inhibited the HUVEC migration stimulated by basic fibroblast growth factor (bFGF) and the tube formation by HUVEC in Matrigel. beta-eudesmol (100 microM) blocked the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 induced by bFGF or vascular endothelial growth factor. Furthermore, beta-eudesmol significantly inhibited angiogenesis in subcutaneously implanted Matrigel plugs in mice and in adjuvant-induced granuloma in mice. These results indicate that beta-eudesmol inhibits angiogenesis, at least in part, through the blockade of the ERK signaling pathway. We considered that beta-eudesmol may aid the development of drugs to treat angiogenic diseases.

Angiogenesis Inhibitors↗
PubMed2005

Expression analysis and potential functional role of the CXCR4 chemokine receptor in bladder cancer.

OBJECTIVE: Recent analysis gave evidence for the fact that the chemokine receptor CXCR4 is of functional significance in the multistep procedure of metastasis directing tumor cells to their metastatic target organs. In our study we investigated the expression of the CXCR4 receptor on bladder cancer cells and the functional activation of the CXCR4 receptor in bladder cancer cell lines regarding signal transduction pathways, which are involved in metastasis. METHODS: Receptor transcript expression was analysed and quantified by reverse transcription PCR with RNA extracted from different samples of native human bladder cancer tissue, normal urothelium and the bladder cancer cell lines J82 and T24. Measurement of intracellular [Ca(++)](i) and analysis of intracellular stress fiber formation, chemotaxis, Matrigel invasion and proliferation were performed in the bladder cancer cell lines J82 and T24 upon stimulation with the specific agonist SDF-1. RESULTS: Specific CXCR4 receptor transcripts were detected in normal urothelium, the bladder cancer cell lines J82 and T24 and in all bladder carcinoma specimens. We did not observe a quantitative correlation of transcript expression and tumor stage. While SDF-1 did not evoke increases in [Ca(++)](i) in bladder cancer cell lines, we observed a distinct rise in intracellular stress fiber formation, chemotactic activity, invasion through Matrigel coated membranes and cell growth upon stimulation with SDF-1, which was blocked in the presence of a specific CXCR4 receptor antibody. CONCLUSION: Our results support that the chemokine receptor CXCR4 is an interesting candidate for the future investigation of metastasis of bladder cancer in vivo.

Chemokine CXCL12↗
PubMed2006

Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy.

OBJECTIVE: Cytokine-induced killer (CIK) cells are ex vivo expanded cells enriched in CD3(+)CD56(+) natural killer T (NKT) cells with major histocompatibility-unrestricted cytotoxicity against several tumoral targets, except B-lineage acute lymphoblastic leukemia (B-ALL). We redirected CIK cells cytotoxicity toward B-ALL with a chimeric receptor specific for the CD19 antigen and then explored if modified-CIK cells maintain the same chemotactic properties of freshly isolated NKT cells, whose trafficking machinery reflects their preferential localization into the sites of B-ALL infiltration. MATERIAL AND METHODS: CIK cells were expanded ex vivo for 21 days and analyzed for expression of adhesion molecules and chemokine receptors regulating adhesion and homing toward leukemia-infiltrated tissues. CIK cells were then transduced with the anti-CD19-zeta-internal ribosomal entry site-green fluorescent protein retroviral vector and characterized for their cytotoxicity against B-ALL cells in a (51)Cr-release assay and for their trafficking properties, including chemotactic activity, adhesion and transendothelial migration, and metalloproteases-dependent invasion of Matrigel. RESULTS: Similarly to freshly isolated NKT cells, CD49d and CD11a were highly expressed on CIK cells. Moreover, CIK cells expressed CXCR4, CCR6, and CCR7 (mean expression 72%, 60%, and 32%, respectively), presenting chemotactic activity toward their respective ligands. Anti-CD19 chimeric receptor-modified CIK cells became cytotoxic against B-ALL cells (mean lysis, 60%) and showed, after exposure to a CXCL12 gradient, high capacity to adhere and transmigrate through endothelial cells and to invade Matrigel. CONCLUSION: The potential capacity to localize into leukemia-infiltrated tissues of anti-CD19 chimeric receptor-redirected CIK cells, together with their ability to efficiently kill B-ALL cells, suggests that modified-CIK cells represent a valuable tool for leukemia immunotherapy.

Antigens, CD↗
PubMed2006

Inhibition of tumor invasion and metastasis by aqueous extract of the radix of Platycodon grandiflorum.

Platycodon grandiflorum is a traditional oriental herbal medicine that is known for its immunostimulatory and anti-tumor effects. This study examined the anti-metastatic activities of an aqueous extract from the root of P. grandiflorum (Changkil: CK) using in vitro and in vivo metastasis assays. CK inhibited the invasion of B16-F10 melanoma cells through a reconstituted basement membrane (Matrigel)-coated filter, and strongly inhibited the adhesion of B16-F10 melanoma cell to extracellular matrices such as Matrigel, fibronectin and laminin substrates. CK also inhibited an experimentally induced lung cancer and prolonged the survival time in vivo. In addition, CK augmented NK cell activity. These results show that CK can reduce the extent of a lung metastasis of B16-F10 melanoma cells by inhibiting the adhesion of tumor cells to the basement membrane possibly and activating NK cells.

Animals↗
PubMed2005

Involvement of matrix metalloproteinase-9 in platelet-activating factor-induced angiogenesis.

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF-kappaB. In this study, we investigated the role of the matrix metalloproteinase (MMP)-9, in PAF-induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP-9 activity in ECV304 cells, in a NF-kappaB-dependent manner. PAF increased MMP-9 promoter activity in ECV304, which was inhibited by WEB2107, and NF-kappaB inhibitors. Transfected NF-kappaB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP-9, resulting in an increase not only of MMP-9 luciferase activity, but also of mRNA expression in MMP-9. MMP-9 or NF-kappaB inhibitors significantly inhibited PAF-induced angiogenesis, in a dose-dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP-9 or NF-kappaB inhibitors inhibited PAF-induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF-kappaB-dependent MMP-9 plays a key role in PAF-induced angiogenesis.

Animals↗
PubMed2006

Enhanced survival in perineural invasion of pancreatic cancer: an in vitro approach.

Pancreatic cancer (PanCa) is characterized by perineural invasion (PNI), early lymph node and liver metastasis, and poor prognosis. PNI is one of the important causes of local recurrence. Little is known about the mechanism of PNI in PanCa. We presented a novel model system that may shed light on the mystery of PNI in PanCa. In this study, mouse dorsal root ganglia (DRGs) and human PanCa cell line (MIA PaCa-2) were cocultured in Matrigel matrix (BD Biosciences, San Jose, CA) to build this PNI model. MIA PaCa-2 cell line alone (control 1) or DRG alone (control 2) was cultured with Matrigel matrix as controls. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were observed under inverted microscopy and then were photographed and quantitated with the Optimas imaging system (Optimas Corp., Bothell, MA). At day 14, both the experimental and control 2 samples were harvested and subjected to total RNA isolation and fixed in paraffin-embedded blocks. Slides cut from paraffin blocks were studied with Ki-67 immunostaining and TUNEL assay. Gene profiling was performed using complementary DNA microarray. Overexpressed target genes were verified by quantitative reverse transcriptase polymerase chain reaction. The results showed that reciprocity was observed between neurites and MIA PaCa colonies with 24 hours of coculture. Neurite outgrowth was stimulated in the presence of pancreatic carcinoma cells, which showed 2-fold more area than did control 2. After 72 hours, MIA PaCa colonies cocultured with DRG exhibited 58% more colony area than did control 1. The Ki-67 index of the DRG/MIA PaCa cells (mean, 5.02%) was significantly higher than that in control 1 (mean, 1.18%) (P < .05); in contrast, the apoptotic index in the DRG/MIA PaCa cells was significantly lower (mean, 0.45%) than that in the control 1 (mean, 1.85%) (P < .001). Prosurvival genes MALT1 and TRAF were increased 2-fold in DRG/MIA PaCa compared with controls. We demonstrated that neural-epithelial interaction is a mutually beneficial process for the growth of nerves and PanCa cells. It is possible that oncogenes and growth factors might act synergistically in promoting proliferation and/or inhibiting apoptosis, a survival strategy crucial to the development of PNI in PanCa.

Animals↗
PubMed2006

Quantitative characterization of binding of small molecules to extracellular matrix.

Extracellular matrix (ECM) is a major tissue component that, besides its cell support function, is implicated in cell-cell signaling, wound repair, cell adhesion, and other cell and tissue functions. For small molecules acting in tissues, including chemicals, signaling peptides, effectors, inhibitors, and other man-made and physiological compounds, non-specific binding to ECM is a critical phenomenon affecting their disposition. We describe here a method for a quantitative characterization of the ECM binding, using a solidified ECM layer incubated with medium containing studied small molecules. Working conditions of Matrigel, a commercial basement membrane preparation, were optimized in terms of the protein concentration, surface area, gel layer thickness, solidification time, and mixing speed. The release of proteins from the solidified layer into the buffer was monitored and taken into account. Two major proteins, laminin and collagen IV, dissolve at different rates. The Matrigel stability data, obtained under varying incubation conditions and gentle mixing, can also be useful in other ECM-related research. The experimental binding data, averaged over all binding sites, were analyzed assuming a fast linear binding. The binding constants were determined for 10 small organic molecules for both dissolved proteins and the solidified layer. The binding constants tend to increase with lipophilicity of the compounds, as characterized by the 1-octanol/water partition coefficients.

Animals↗
PubMed2005

Growth and differentiation of colony-forming human hepatocytes in vitro.

BACKGROUND/AIMS: Parenchymal hepatocytes (PHs) of rat contain colony-forming parenchymal hepatocytes (CF-PHs) as a small fraction. We aimed to demonstrate the presence of CF-PHs in humans and characterize them with respect to growth and differentiation potential. METHODS: Human PHs were co-cultured with Swiss 3T3 cells in the medium containing human serum, EGF, nicontinamide, and ascorbic acid 2-phosphate. To examine differentiation potential hepatocytes were cultured on gels of Matrigel Matrix. RESULTS: Few PHs formed colonies, the colony-forming efficiency being as low as 0.01-0.09%. The CF-PHs could be subcultured up to 7 passages. They showed a liver epithelial cell-like morphology, and immunocytochemically positive for albumin (ALB), cytokeratin (CK) 7, 8, 18, and 19 in a pre- and early phase-confluence, whereas they showed a typical differentiated hepatocyte-like morphology, and positive for alpha(1)-antitrypsin, but negative for CK7 and 19 in condensed regions at confluence. The CF-PHs at late confluence expressed mRNAs of ALB, HNF4, and isoforms of cytochrome P450 at low levels. However, when cultured on Matrigel, these cells expressed them at high levels comparable to those of original PHs. CONCLUSIONS: We concluded that the human liver contains highly replicative hepatic progenitor-like cells as a minute population that retain a normal differentiation potential.

Adolescent↗
PubMed2004

A novel method for isolation of endothelial cells and macrophages from murine tumors based on Ac-LDL uptake and CD16 expression.

Analysis of specific properties of tumor endothelium should be useful for development of novel antiangiogenic strategies. However, the isolation of pure endothelial cells from tumor tissues is still a fundamental problem. In this study, we have attempted to develop a reliable method for the isolation of endothelial cells from murine tumors. We found that the labeling with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated-low density lipoprotein (Dil-Ac-LDL), commonly used for this purpose, can result in the contamination of isolated endothelium by macrophages due to the overlapping staining patterns of these two distinct cell types. Therefore, we chose the CD16, which is expressed on macrophages but not endothelial cells, to better distinguish them when labeled with Dil-Ac-LDL. By using this method, we obtained pure populations of endothelial cells and macrophages from murine colorectal cancer tissues, showing characteristic morphological and functional properties of the either cell type. The endothelial cells were long spindle-shaped, spread on gelatin, formed tube-like structures on Matrigel and expressed MECA-32 but not CD68. In contrast, the macrophages were round-shaped, partially spread on gelatin, formed unorganized aggregates on Matrigel and expressed CD68 but not MECA-32. The additional analysis of normal and tumor tissues revealed a positive correlation between the relative numbers of tumor endothelial cells and macrophages, calculated as % total cells, as well as the respective relative number and tumor weight. The present method is hoped to be useful for the evaluation of tumor angiogenesis and antitumor immunity.

Animals↗
PubMed2006

Differential effects of photofrin, 5-aminolevulinic acid and calphostin C on glioma cells.

The invasive nature of malignant gliomas makes treatment by surgery alone extremely difficult. However, the preferential accumulation of photosensitisers in neoplastic tissues suggests photodynamic therapy (PDT) may be useful as an adjuvant therapy following tumour resection. In this study, the potential use of three different photosensitisers, namely Photofrin, 5-aminolevulinic acid (5-ALA) and calphostin C in the treatment of glioma was investigated. The uptake, cytotoxicity on U87 and GBM6840 glioma cell lines were determined by flow cytometry and MTT assay respectively. Their effect on glioma cell invasiveness was evaluated by (1) measuring the levels of matrix degradation enzymes matrix metalloproteinase (MMP)-2 and -9 using gelatin zymography, and (2) Matrigel invasion assay. The results showed that uptake of calphostin C reached saturation within 2 h, while Photofrin and 5-ALA induced protoporphyrin IX (PpIX) levels elevated steadily up to 24 h. Photocytotoxic effect on the two glioma cell lines was similar with LD50 at optimal uptake: 1 microg/mL Photofrin at 1.5 J/cm(2); 1 mM 5-ALA at 2 J/cm(2) and 100 nM calphostin C at 2 J/cm(2). The inhibition in cell proliferation after Photofrin treatment was similar for both cell lines, which correlated to more cells being arrested in the G0/G1 phase of the cell cycle (P<0.01). By contrast, U87 was more sensitive to calphostin C whereas GBM6840 was more susceptible to 5-ALA treatment. The ability of both cell lines to migrate through the Matrigel artificial basement membrane was significantly reduced after PDT (P<0.001). This might be due to a decreased production in MMP-2 and MMP-9, together with the reduction of adhesion molecule expression. Photofrin was most superior in inhibiting cell invasion and calphostin C was least effective in reducing adhesion molecule expression. Taken together, PDT could be useful in the treatment of gliomas but the choice of photosensitisers must be taken into consideration.

Aminolevulinic Acid↗