Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Matrigel”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed2003

Experimental study of anti-tumor effects of polysaccharides from Angelica sinensis.

AIM: To investigate the in vivo anti-tumor effects of total polysaccharide (AP-0) isolated from Angelica sinensis (Oliv.) Diels (Danggui) on mice and the in vitro inhibitory effects of AP-0 and the sub-constituents (AP-1, AP-2 and AP-3) separated from AP-0 on invasion and metastasis of human hepatocellular carcinoma. METHODS: Three kinds of murine tumor models in vivo, sarcoma 180 (S180), leukemia L1210 and Ehrlich ascitic cancer (EAC) were employed to investigate the anti-tumor effects of AP-0. For each kind of tumor model, three experimental groups were respectively given AP-0 at doses of 30, 100 and 300 mg/kg by ip once a day for 10 days. Positive control groups were respectively given Cy at a dose of 30 mg/kg for S180 and leukemia L1210, and 5-FU at a dose of 20 mg/kg for EAC. On d 11, mice bearing S180 were sacrificed and the masses of tumors, spleens and thymus weighed. The average living days of mice bearing EAC and of mice bearing L1210 were observed, and the rates of life prolongation of each treatment were calculated, respectively. The inhibitory effects of APs on hepatoma invasion and metastasis in vitro were investigated by employing human hepatocellular carcinoma cell line (HHCC) with the Matrigel invasion chamber, adhesion to extracellular matrix and chemotatic migration tests, respectively. RESULTS: AP-0 had no obviously inhibitory effect on the growth of S180, but it could significantly decrease the thymus weights of the mice bearing S180. AP-0 could significantly reduce the production of ascitic liquids and prolong the life of mice bearing EAC. AP-0 could also increase the survival time of mice bearing L1210. AP-0 and AP-2 had significantly inhibitory effects on the invasion of HHCC into the Matrigel reconstituted basement membrane with the inhibitory rates of 56.4 % and 68.3 %, respectively. AP-0, AP-1, AP-2 and AP-3 could influence the adhesion of HHCC to extracellular matrix proteins (Matrigel and fibronectin) at different degrees, among them only AP-3 had significant blocking effect on the adhesion of HHCC to fibronectin with an inhibitory rate of 30.3 %. AP-0, AP-1 and AP-3 could partially inhibit the chemotactic migration abilities of HHCC. CONCLUSION: The experimental findings suggest that the total polysaccharide of Angelica sinensis (Oliv.) Diels (Chinese Danggui) possesses anti-tumor effects on experimental tumor models in vivo and inhibitory effects on invasion and metastasis of hepatocellular carcinoma cells in vitro.

Angelica sinensis↗
PubMed1998

Early events in angiogenesis: cloning an alpha-prolyl 4-hydroxylase-like gene.

To gain further insight into angiogenesis we sought to clone genes which are actively expressed during this complex process. Using the Matrigel-induced in vitro model we were able to show that although several cell-types form reticular arrays of cells on the gels (align), only endothelial cells were able to go on and form the capillary-like structures reminiscent of patent vessels. Although this alignment process did not require gene activation we show that tube formation was ultimately dependent upon gene expression occuring during the first few hours that cells are seeded onto Matrigel. We generated a cDNA library enriched for the expression of those genes and have sequenced an alpha-prolyl 4-hydroxylase-like clone (angio 0.9). This clone shares 66% overall homology to the carboxy-terminal 106 amino-acids of the published human sequence. In the region corresponding to the co-factor binding domains, His 1 and His 2, angio 0.9 has >90% homology to the published sequence. Using an RNAse protection assay we show that the level of expression of the message of this clone is five fold elevated in endothelial cells which have aligned on Matrigel. The dependence of collagen, and collagen hydroxylation in angiogenesis is well documented. Thus, our results are demonstrable proof that the principle of this approach has the potential to generate novel discoveries.

Amino Acid Sequence↗
PubMed2001

Evidence of IL-18 as a novel angiogenic mediator.

Angiogenesis, or new blood vessel growth, is a key process in the development of synovial inflammation in rheumatoid arthritis (RA). Integral to this pathologic proliferation are proinflammatory cytokines. We hypothesized a role for IL-18 as an angiogenic mediator in RA. We examined the effect of human IL-18 on human microvascular endothelial cell (HMVEC) migration. IL-18 induced HMVEC migration at 1 nM (p < 0.05). RA synovial fluids potently induced endothelial cell migration, but IL-18 immunodepletion resulted in a 68 +/- 5% decrease in HMVEC migration (p < 0.05). IL-18 appears to act on HMVECs via alpha(v)beta(3) integrin. To test whether IL-18 induced endothelial cell tube formation in vitro, we quantitated the degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM, resulted in a 77% or 87% increase in tube formation compared with control (p < 0.05). To determine whether IL-18 may be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice, and IL-18 at 1 and 10 nM induced angiogenesis (p < 0.05). The angiogenesis observed appears to be independent of the contribution of local TNF-alpha, as evidenced by adding neutralizing anti-TNF-alpha Ab to the Matrigel plugs. In an alternative in vivo model, sponges embedded with IL-18 or control were implanted into mice. IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control (p < 0.05). These findings support a novel function for IL-18 as an angiogenic factor in RA and may elucidate a potential therapeutic target for angiogenesis-directed diseases.

Angiogenesis Inducing Agents↗
PubMed2004

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A.

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

Angiogenesis Inducing Agents↗
PubMed1999

Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane.

The mechanism(s) underlying the release of stem/progenitor cells from bone marrow into the circulation is poorly understood. We hypothesized that matrix metalloproteinases (MMPs), especially gelatinases, which are believed to participate in the proteolysis of basement membranes and in the migration of leukocytes, may facilitate this process. First, we investigated whether CD34(+) stem/progenitor cells express gelatinases A (MMP-2) and/or B (MMP-9) and whether growth factors and cytokines (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], stem cell factor [SCF], macrophage colony-stimulating factor [M-CSF], interleukin-3 [IL-3], IL-6, IL-8, and tumor necrosis factor-alpha [TNF-alpha]) are able to modulate their expression. Next, we examined the transmigration of these stem/progenitor cells through reconstituted basement membrane (Matrigel) and its modulation by growth factors and cytokines. CD34(+) cells were obtained from steady-state bone marrow and peripheral blood (from leukapheresis products collected either in steady-state hematopoiesis or after mobilization with G-CSF plus chemotherapy or G-CSF alone). We found that peripheral blood CD34(+) cells, regardless of whether they were mobilized or not, strongly expressed both gelatinases (MMP-2 and MMP-9) in contrast to steady-state bone marrow CD34(+) cells, which did not. However, all the growth factors and cytokines tested could induce MMP-2 and MMP-9 secretion by the latter cells. Moreover, the stimulatory effects of G-CSF and SCF on both MMP-2 and MMP-9 secretion were found to be significantly higher in CD34(+) cells isolated from bone marrow than in those from peripheral blood. In addition TNF-alpha, GM-CSF, and IL-6 increased the secretion of a partially active form of MMP-2. Basal transmigration of bone marrow CD34(+) cells through Matrigel was lower than that of peripheral blood CD34(+) cells (P <.0001), but growth factors and cytokines increased it by 50% to 150%. Positive correlations were established between expression of gelatinases and CD34(+) cell migration (r >.9). The stimulatory effect of G-CSF was significantly greater on the migration of CD34(+) cells from bone marrow than on those from peripheral blood (P =.004). Moreover, CD34(+) cell migration was reduced to approximately 50% by antibodies to MMP-2 and MMP-9, tissue inhibitors of metalloproteinases (rhTIMP-1 and -2), and o-phenanthroline. TNF-alpha-induced gelatinase secretion and migration of CD34(+) cells and of clonogenic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], and colony-forming unit-megakaryocyte [CFU-MK]) were dose-dependent. Therefore, this study demonstrated that CD34(+) cells that are circulating in peripheral blood express both MMP-2 and MMP-9 and transmigrate through Matrigel. In contrast, CD34(+) cells from steady-state bone marrow acquire similar properties after exposure to growth factors and cytokines, which upregulate expression of gelatinases and transmigration of these cells when they enter the bloodstream. Hence, we suggest that growth factors and cytokines induce release of stem/progenitor cells from bone marrow into peripheral blood during mobilization, as well as during steady-state hematopoiesis, by signaling through gelatinase pathways.

Antigens, CD34↗
PubMed1999

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.

Apoptosis↗
PubMed1999

Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes.

The effect of medium formulation, composition of extracellular matrix overlay, and culture dish material on liver microsomal cytochrome P-450 (CYP) 2B induction by phenobarbital (PB) was investigated in primary cultures of rat hepatocytes. When hepatocytes were maintained on Permanox dishes with an overlay of either collagen (type I) or Matrigel, Williams' E medium was superior to other medium formulations in terms of the magnitude of induction of CYP2B on a per milligram microsomal protein basis. Modified Chee's medium (MCM) and hepatocyte culture medium were intermediate in their capacity to sustain induction of CYP2B by PB, and Dulbecco's modified Eagle's medium was slightly less effective. The overall induction of CYP2B activity by PB was, on average, 50% lower in hepatocytes cultured on polystyrene dishes (LUX). Little or no difference was observed between hepatocytes overlaid with collagen and those overlaid with Matrigel. MCM was superior to Williams' E medium in terms of the yield of microsomal protein and the ultrastructural features of the hepatocyte monolayers. CYP2B induction by PB was optimal after 3 days of treatment in either medium. CYP1A, CYP3A, and CYP4A activities could be induced in vitro by prototypical inducing agents in hepatocytes cultured on Permanox dishes with MCM and a Matrigel overlay to comparable levels observed in vivo. The results of these studies show that medium formulation and culture vessel material, but not the type of extracellular matrix overlay, have significant effects on the induction of CYP enzymes in cultured rat hepatocytes maintained in a sandwich configuration.

Animals↗
PubMed1999

Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors.

Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.

Animals↗
PubMed2000

Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases.

Eicosanoids modulate the interaction of tumor cells with various host components in cancer metastasis. Their synthesis involves the release of arachidonic acid (AA) from cellular phospholipids by phospholipase A2 (PLA2), followed by metabolism by cyclooxygenases (COXs) and lipooxygenases (LOXs). This study aimed to identify the pathway(s) of AA metabolism that are required for the invasion of prostate tumor cells. DU-145 and PC-3 human prostate cancer cell lines were used to test the effect of inhibitors of PLA2, COX, or LOX on the invasion of prostate tumor cells through Matrigel in vitro using the Boyden chamber assay and fibroblast-conditioned medium as the chemoattractant. We used nontoxic doses that did not inhibit simple cell motility and did not decrease clonogenic survival. All of the inhibitors caused a significant reduction in AA release from treated cells compared with control cells, which indicated that the treatments were biochemically active. Invasion through Matrigel was inhibited by the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB), the general COX inhibitor ibuprofen (IB), and the highly selective COX-2 inhibitor NS398. Inhibition of cell invasiveness by 4-BPB (1.0 microM), IB (10.0 microM), and NS398 (10.0 microM) was reversed by the addition of prostaglandin E2 (PGE2). PGE2 alone, however, did not stimulate invasiveness, which suggests that its production is necessary for rendering the cells invasive-permissive but not sufficient for inducing invasiveness. In contrast, we found no significant inhibition of invasion of prostate tumor cells treated with esculetin (1.0 microM) or nordihydroguiaretic acid (1.0 microM), which are specific inhibitors of LOX. We also tested the effect of 4-BPB, IB, NS398, and esculetin on the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), as key enzymes in the proteolysis of Matrigel during invasion, using gelatin zymograms and Western blots. Cells that received 4-BPB, IB, or NS398, but not esculetin showed a significant reduction in the levels of proMMP-2, MMP-9, and proMMP-9 in the culture medium. DU-145 cells did not secrete TIMP-1, and the drugs did not alter the secretion of TIMP-2. This work highlights the role played by COX in disturbing the balance between MMPs and TIMPs in prostate cancer cells, and it points to the potential use of COX inibitors, especially COX-2 selective inhibitors, in the prevention and therapy of prostate cancer invasion.

3T3 Cells↗
PubMed2001

An intracellular form of cathepsin B contributes to invasiveness in cancer.

Cathepsin B is a lysosomal cysteine proteinase whose expression and trafficking are frequently altered in cancer, and plasma membrane and secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of cathepsin B in several tumor cell lines and measured their capacity to invade through a reconstituted extracellular (Matrigel) matrix. Transient expression of human cathepsin B in a poorly metastatic B16F1 murine melanoma variant produced a 3-5-fold increase in cathepsin B activity and a comparable increase in invasiveness. Stable antisense cathepsin B-expressing clones of the highly metastatic human melanoma A375M and prostate carcinoma PC3M cell lines produced 40-50% less cathepsin B than control cells and were proportionately less invasive. In contrast, manipulating cathepsin B levels had no effect on cell migration across an uncoated membrane. The anionic cathepsin B inhibitor (L-3-trans-propylcarbamoyloxirane-2-carbony)-L-isoleucyl-L-proline (CA-074), at a concentration of 1 microM, caused a nearly quantitative inhibition of extracellular cathepsin B but had no effect on Matrigel invasion. In contrast, the equally potent but less selective inhibitor, trans-epoxysuccinyl-L-leucylamino(4-guanidino)butane (E-64) inhibited invasion by 75%. Surprisingly, at a concentration of 10 microM, CA-074 slowly permeated the cells, causing an 80-95% inhibition of intracellular cathepsin B after 12 h, the duration of the invasion assay. The membrane-permeant cathepsin B inhibitor, CA-074 methyl ester, and the higher concentration of CA-074 that inhibited intracellular cathepsin B both significantly reduced Matrigel invasion. Collectively, these results identify an intracellular role for cathepsin B in matrix degradation. They also indicate that caution should be exercised in assuming that CA-074 is unable to enter cells when it is used to inhibit biological processes of long duration.

3T3 Cells↗
PubMed1997

[The influence of basement membrane matrix on the attachment of human gingival epithelial cell to titanium: a scanning electron microscopic study].

In order to investigate the factors which could enhance the integrity of the biological seal between implant and epithelial cells, basement membrane matrigel was coated on the surface of smooth pure titanium. SEM observations showed that the spreading of primary cultured human gingival epithelial cells on Matrigel coated titanium surface was better than that of control groups on the third and the sixth day. This result suggests that the attachment of human gingival epithelial cells to titanium can be enhanced by the basement membrane matrigel.

Biocompatible Materials↗
PubMed2002

The chemopreventive agent oltipraz possesses potent antiangiogenic activity in vitro, ex vivo, and in vivo and inhibits tumor xenograft growth.

Angiogenesis plays a pivotal role in tumor growth and represents a key target for chemopreventive intervention. On the basis of the structural features and lack of target organ specificity of the synthetic dithiolethione oltipraz, inhibition of angiogenesis was assessed as a potential mechanism for its broad-based chemopreventive activity. The effects of oltipraz on the development and maturation of a vascular network was determined in vitro using two-dimensional capillary tube formation assays with human umbilical vein endothelial cells plated on Matrigel and ex vivo using primary rat aortic ring explant cultures in three-dimensional collagen gels, respectively. The antiangiogenic and antitumor efficacy of oltipraz administration in vivo in nude mice was evaluated by determining its effects on neovascularization in s.c. Matrigel implants seeded with vascular endothelial growth factor and basic fibroblast growth factor-stimulated porcine aortic endothelial cells and on tumor growth and angiogenesis in SVR murine angiosarcoma xenografts implanted s.c. A dose-dependent reduction (0.4-100 microM) in microvessel formation was observed in both human and rodent bioassays after oltipraz exposure, with inhibition approaching 100% in the rat aortic ring assay at the highest concentration (P < 0.01). Similarly, oltipraz (40 microM) inhibited complete capillary tube formation by human umbilical vein endothelial cells by 62% (P < 0.05) relative to control cultures. p.o. administration of oltipraz (250 mg/kg/day for 6 days) to nude mice implanted with porcine aortic endothelial cell-Matrigel plugs resulted in a 42% reduction in neovascularization (P < 0.05) relative to vehicle-treated control mice. Administration of the same dose of oltipraz to athymic mice bearing established s.c. SVR angiosarcoma xenografts for 10 days resulted in a significant inhibition of tumor growth as early as day 4 of dosing (P < 0.005), with a maximum inhibition of tumor growth (81%, P < 0.001) relative vehicle-treated mice by day 10. The observed efficacy of oltipraz in this model is comparable with that of SU 5416 and TNP-470, known antiangiogenic agents currently under clinical development. Plasma levels of oltipraz at the termination of in vivo efficacy studies were 66.4 +/- 7 microM as determined by reversed phase high-performance liquid chromatography, a concentration range associated with significant antiangiogenic activity of oltipraz in vitro and ex vivo. These data suggest that the chemopreventive agent oltipraz may be effective in the treatment of advanced stage cancers and metastases, in part, because of its antiangiogenic activity in vivo.

Angiogenesis Inhibitors↗
PubMed2002

Modulation of radiation response and tumor-induced angiogenesis after epidermal growth factor receptor inhibition by ZD1839 (Iressa).

ZD1839 ("Iressa") is an orally-active, selective epidermal growth factor receptor-tyrosinekinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with radiation in human squamous cell carcinomas (SCCs) of the head and neck. ZD1839 produced a dose-dependent inhibition of cellular proliferation in human SCCs grown in culture. Flow cytometry analysis of cell cycle progression confirmed the accumulation of cells in G(1) phase after exposure to ZD1839. Clonogenic analysis demonstrated that treatment of SCCs with ZD1839 reduced cell survival after radiation exposure. Flow cytometric analysis further demonstrated that treatment of SCCs with ZD1839 amplified radiation-induced apoptosis. Tumor xenograft studies confirmed that oral administration of ZD1839, or focal radiation, resulted in partial and transient tumor regression in both SCC-1 and SCC-6 xenografts. In contrast, profound tumor regression and regrowth delay was observed in mice treated with the combination of ZD1839 and radiation. To examine antiangiogenic effects, we studied the impact of ZD1839 on human umbilical vascular endothelial cells (HUVECs). In the presence of reconstituted Matrigel matrix, HUVECs established a capillary-like network structure (tube formation). Treatment with ZD1839 reduced the cell-to-cell interaction of HUVECs, resulting in disruption of tube formation. The effect of ZD1839 was further examined using an in vivo tumor xenograft model of angiogenesis (Matrigel plug) in athymic mice. Systemic treatment with ZD1839 significantly inhibited tumor-induced neovascularization across the Matrigel plug. Taken together, these results suggest that the antitumor activity of ZD1839 in combination with radiation appears to derive from not only proliferative growth inhibition (with associated cell cycle arrest and enhancement of radiation-induced apoptosis) but also from inhibition of tumor angiogenesis.

Animals↗
PubMed2002

Histidine-proline-rich glycoprotein has potent antiangiogenic activity mediated through the histidine-proline-rich domain.

Histidine-proline-rich glycoprotein (HPRG) is an abundant multidomain plasma protein evolutionarily related to high-molecular-weight kininogen. The cleaved form of high-molecular-weight kininogen has recently been demonstrated to exhibit antiangiogenic activities in vitro (J. C. Zhang et al., FASEB J., 14: 2589-2600, 2000), mediated primarily through domain 5. HPRG contains a histidine-proline-rich (H/P) domain with sequence and functional similarities to HKa-D5. We hypothesized that HPRG may also have antiangiogenic properties, localized within its H/P domain. The H/P domain is highly conserved among species, and because rabbit H/P domain is more resistant to internal proteolytic cleavage than the human domain, the rabbit HPRG (rbHPRG) was primarily used to assess the antiangiogenic activity of HPRG. Rabbit HPRG inhibited human umbilical vein endothelial cell (HUVEC) tube formation stimulated by fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor on a Matrigel surface as well as cell proliferation of FGF-2 stimulated HUVECs. The antiangiogenic activity of rbHPRG was localized to the H/P domain by use of proteolytic fragments of rbHPRG and was further confirmed and characterized in two in vivo models of angiogenesis: the chorioallantoic membrane of the chick assay and the mouse Matrigel plug assay. Caspase-3 activation was observed in HUVECs stimulated with FGF-2 in the presence of rbHPRG, suggesting that apoptosis of activated endothelial cells may be one of the mechanisms underlying its antiangiogenic activity. Finally, the H/P domain of rbHPRG reduced tumor cell number when tumor cells were co-inoculated in the Matrigel plug assay. In conclusion, the H/P domain within HPRG induces the apoptosis of activated endothelial cells leading to potent antiangiogenic effects.

Angiogenesis Inhibitors↗
PubMed2002

Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): association with impaired repositioning of the microtubule organizing center.

A number of cancer chemotherapeutic drugs designed to have cytotoxic actions on tumor cells have recently been shown to also have antiangiogenic activities. Endothelial cell migration and proliferation are key components of tumor angiogenesis, and agents that target the microtubule cytoskeleton can interfere with these processes. In this study, the effect on endothelial cell functions of the microtubule-stabilizing drugs Taxotere and Taxol were evaluated in three in vitro assays: a chemokinetic migration assay, an angiogenesis factor-mediated chemotactic migration assay, and a three-dimensional Matrigel tubule formation assay, using rat fat pad endothelial cells (RFPECs) and/or human umbilical vein endothelial cells (HUVECs). Taxotere was active in all three assays at concentrations that were not cytotoxic and did not inhibit endothelial cell proliferation. In the RFPEC chemokinetic migration and in vitro tubule formation assays, the IC50 values were approximately 10(-9) M for both Taxotere and Taxol. HUVEC migration, however, was more sensitive to Taxotere, with an observed IC50 of 10(-12) M in a chemokinetic assay. In a Boyden chamber assay, HUVEC chemotaxis stimulated by either of two angiogenic factors, thymidine phosphorylase or vascular endothelial growth factor, was inhibited by Taxotere with an IC50 of 10(-11) M and was ablated at 10(-9) M. Taxotere was also up to 1000-fold more potent than Taxol in inhibiting either chemokinetic or chemotactic migration. When the microtubule cytoskeleton was visualized using immunofluorescence staining of alpha-tubulin, there were no gross morphological changes observed in HUVECs or RFPECs treated with Taxotere at concentrations that inhibited endothelial cell migration but not proliferation. The effects of Taxotere on migration were associated with a reduction in the reorientation of the cell's centrosome, at concentrations that did not affect gross microtubule morphology or proliferation. Reorientation of the centrosome, which acts as the microtubule organizing center, in the intended direction of movement is a critical early step in the stabilization of directed cell migration. These data indicate that endothelial cell migration correlates more closely with changes in microtubule plasticity than with microtubule gross structure. The antiangiogenic activity of Taxotere in vivo was assessed in a Matrigel plug assay. In this assay, the angiogenic response to fibroblast growth factor 2 was inhibited in vivo by Taxotere with an ID50 of 5.4 mg/kg when injected twice weekly over a 14-day period, and angiogenesis was completely blocked in mice that received 10 mg/kg Taxotere. The in vivo data further suggested that Taxotere had selectivity for endothelial cell migration and/or microvessel formation because infiltration of inflammatory cells into the Matrigel plug was much less sensitive to inhibition by Taxotere. In conclusion, Taxotere is a potent and potentially specific inhibitor of endothelial cell migration in vitro and angiogenesis in vitro and in vivo.

Animals↗
PubMedDate not supplied

Antitumor and antimetastatic effects on liver of triterpenoid fractions of Ganoderma lucidum: mechanism of action and isolation of an active substance.

The triterpenoid fraction (100 and 200 mg/kg) of the fruit bodies of Ganoderma lucidum inhibited primary solid-tumor growth in the spleen, liver metastasis and secondary metastatic tumor growth in the liver in intrasplenic Lewis lung carcinoma (LLC)-implanted mice. In addition, the triterpenoid fraction (800 micrograms/mL) inhibited angiogenesis induced by Matrigel (a soluble basement membrane extract of the Engelbreth-Holm-Swam (EHS) tumor) supplemented with vascular endothelial growth factor (VEGF) and heparin in an in vivo model. This suggested that the antitumor and antimetastatic activities of the triterpenoid fraction of G. lucidum might be due to the inhibition of tumor-induced angiogenesis. Next, we attempted to isolate the active substance(s) using the in vivo assay system of Matrigel-induced angiogenesis. The acidic fraction of the triterpenoid fraction inhibited the Matrigel-induced angiogenesis. Compound I was isolated from the acidic fraction as an active substance that inhibited the Martigel-induced angiogenesis. Compound I was identified as ganoderic acid F based on the data of IR, 1H- and 13C-NMR and MS analyses.

Animals↗
PubMedDate not supplied

Anti-metastatic activities of all-trans retinoic acid, indole-3-carbinol and (+)-catechin in Dunning rat invasive prostate adenocarcinoma cells.

Dunning rat invasive prostate adenocarcinoma cells were employed to investigate the anti-metastatic potential and probable mechanisms of action of all-trans retinoic acid (ATRA), indole-3-carbinol (13C) and (+)-catechin (CAT). The invasive parameters studied include: matrigel membrane invasion; zymography and Northern analysis for matrix metalloproteinases (MMPs) activity and gene expression; and Western analysis for the membrane-associated proteins alpha-, beta- and gamma-catenins. ATRA significantly and dose-dependently inhibited matrigel membrane invasion of the cells by 53%, inhibited MMP-2 activity by 71%, MMP-9(80%), alpha-(59%) and beta-(65%) catenin expression at 10 microM (p < 0.01). gamma-Catenin expression was completely inhibited by ATRA even at 2 microM. Catechin at 25 microM decreased matrigel membrane invasion by 24% and also inhibited gamma-catenin protein levels by 58% (p < 0.01). Loss of E-cadherin was implicated in the exacerbation of the anti-metastatic effects of ATRA and CAT by the use of E-cadherin-positive, non-invasive cells. In conclusion, ATRA and CAT show anti-metastatic potential in the invasive rat prostate adenocarcinoma model and gamma-catenin appears to play a mechanistic role.

Adenocarcinoma↗
PubMed2003

Isolation and characterization of murine retinal endothelial cells.

PURPOSE: To isolate and characterize primary retinal endothelial cells (REC) from wild type and transgenic mice to facilitate the study of their properties in vitro. METHODS: REC were isolated from wild type or transgenic-immortomouse by collagenase digestion of retina and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (anti-PECAM-1). The bound cells were plated on fibronectin-coated wells and expanded. The REC were characterized for expression and localization of endothelial cell markers by fluorescence-activated cell sorting (FACS) analysis and indirect immunofluorescence staining. The ability of these cells to form capillary like networks was assessed on Matrigel while the migration properties were examined in wound closure assays. RESULTS: Isolation of REC from mouse has been very difficult and has not been previously reported. Here, we describe a method for isolation of retinal endothelial cells from wild type and thrombospondin-1 deficient (TSP1-/-) immortomice. Our results indicate that nearly 100% of selected cells express the endothelial cell marker PECAM-1 and vascular endothelial-cadherin (VE-cadherin). The cells were successfully passaged and maintained in culture for several months without a significant loss in expression of endothelial cell markers. The wild type REC, like most primary endothelial cells, formed capillary-like networks on Matrigel. The ability of the REC from TSP1-/- mice to form capillary-like networks on Matrigel was severely compromised. This may be attributed, at least in part, to the enhanced migratory and less differentiated phenotype of these cells. CONCLUSIONS: The retinal endothelial cells can be readily obtained from wild type and transgenic mice, which facilitate the comparison and identification of the physiologic role of specific genes in endothelial cell function.

Animals↗