Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay

Garrido, Teresa; Riese, H H; Aracil, Miguel; Pérez-Aranda, A.

doi:10.1038/bjc.1995.149

Cited by 33 publications

(23 citation statements)

References 20 publications

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“…Previous studies have shown endothelial rearrangement into capillary-like structures when treated with condition media from tumor cell lines. 45,46 In our present study, 2 in vitro models of angiogenesis (co-culture and Matrigel assay) using rAAV-TFPI-2 inhibited capillary-like structure formation. Collectively, these data suggest the novel role of TFPI-2 in the anti-angiogenic therapy of gliomas.…”

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confidence: 99%

Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Yanamandra

Kondraganti

Gondi

et al. 2005

Intl Journal of Cancer

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Recombinant adeno-associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long-term transgene expression and the ability to transduce both dividing and nondividing cells. We have shown previously the anti-cancer role of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8-kb fragment of human TFPI-2 was cloned into the adeno-associated viral vectors (rAAA-TFPI-2), rAAV-TFPI-2 infection of SNB19 cells significantly increased TFPI-2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV-TFPI-2 significantly reduced migration and invasion in a dosedependent manner. Tumor spheroids infected with rAAV-TFPI-2 and co-cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and AAV-CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co-cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary-like structure formation in rAAV-TFPI-2-treated cells as compared to parental and mock-transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV-TFPI-2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV-TFPI-2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV-TFPI-2-mediated gene therapy offers a novel tool for the treatment of brain tumors. ' 2005 Wiley-Liss, Inc.

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confidence: 99%

Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Yanamandra

Kondraganti

Gondi

et al. 2005

Intl Journal of Cancer

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“…Thus, tumor cells appear to control, not only the proliferation and invasion steps, but also the differentiation of endothelial cells during tumor-induced angiogenesis. Other investigators have examined endothelial cell differentiation into capillary-like structures in response to the conditioned media of different tumors, and observed that bovine aortic endothelial cells formed capillarylike structures on Matrigel in the presence of B16F0 and B16F10 murine melanoma cells (Garrido et al, 1995). Conditioned media of several fibrosarcoma cell lines are also mitogenic for bovine capillary endothelial cells, and the resulting solid fibrosarcoma formed from cells injected into transgenic mice are highly vascularized (Kandel et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…The average number of capillaries and small arterioles per field was calculated microscopically in the tissue surrounding the tumors, within *200 mm from the infiltrating edges of the neoplasms. As a further control we used sections derived from murine B16F10 melanoma tumors which are highly vascularized (Garrido et al, 1995). Quantization of the data showed a marked reduction in the number of blood vessels in the decorin-expressing In these experiments, *4610 4 HUVECs were seeded onto Matrigel-coated wells; after a 1-h incubation at 378C, inserts with a 3 mm-pore size, containing various numbers of tumor cells as indicated were placed in the wells.…”

Section: Effects Of Decorin On the Growth And Vascularization Of Tumomentioning

confidence: 99%

Decorin suppresses tumor cell-mediated angiogenesis

et al. 2002

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The progressive growth of most neoplasms is dependent upon the establishment of new blood vessels, a process regulated by tumor-secreted factors and matrix proteins. We examined the in vitro and in vivo angiogenic ability of conditioned media obtained from fibrosarcoma, carcinoma, and osteosarcoma cells and their decorintransfected counterparts. Human endothelial cells were investigated in vitro by evaluating three essential steps of angiogenesis: migration, attachment, and differentiation. On the whole, wild-type tumor cell-secretions enhanced endothelial cell attachment, migration, and differentiation, whereas their decorin-expressing forms inhibited these processes. Similarly, decorin-containing media suppressed endothelial cell sprouting in an ex vivo aortic ring assay. Since angiogenesis is an important component of tumor expansion, the growth rate of these cells as tumor xenografts was examined by implantation in nude mice. In vivo, the decorin-expressing tumor xenografts grew at markedly lower rates and showed a significant suppression of neovascularization. Immunohistochemical, Northern and Western blot analyses indicated that the decorin-expressing cells produced vascular endothelial growth factor (VEGF) at markedly reduced rates vis-a´-vis their wild-type counterparts. Specificity of this process was confirmed by experiments where addition of recombinant decorin to the wild-type tumor cells caused 80 -95% suppression of VEGF mRNA and protein. These results provide a novel mechanism of action for decorin, and indicate that decorin could adversely affect in vivo tumor growth by suppressing the endogenous tumor cell production of a powerful angiogenic stimulus.

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“…β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ERK kinase inhibitor PD98059 was purchased from CalBiochem (La Jolla, CA, USA).Cell culture MDA-MB 231 cells (poorly differentiated adenocarcinoma, grade III), a human breast cancer cell line, appearing phenotypically as spindle shaped cells exhibiting highly invasive phenotype [10,11] and differentiation markers such as E-cadherin and keratin, β-catenin, and vimentin [12] , were grown in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere under 5% CO 2 . Cells were collected and passaged using a 0.05% trypsin-0.02% ethylenediamine tetra-acetic acid (EDTA) solution, and cell counts were performed in triplicate using a hemocytometer.…”

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confidence: 99%

Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells

et al. 2011

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Aim: Protein L-isoaspartyl O-methyltransferase (PIMT) regulates cell adhesion in various cancer cell lines through activation of integrin αv and the PI3K pathway. The epithelial mesenchymal transition (EMT) enables epithelial cells to acquire the characteristics of mesenchymal cells, and to allow them to migrate for metastasis. Here, we examined the relationship between PIMT and EMT with attached or detached MDA-MB 231 cells. Methods: Human breast cancer cell line MDA-MB-231 cells were maintained in a suspension on poly-HEMA in the presence or absence of PIMT siRNA or ERK inhibitor PD98059. The mRNAs and proteins were analyzed using RT-PCR and immunoblotting, respectively. Results: During cellular incubation under detached conditions, PIMT, integrin αv and EMT proteins, such as Snail, Slug and matrix metalloproteinase 2 (MMP-2), were significantly increased in correlation with the phosphorylation of ERK1/2. The ERK inhibitor PD98059 (25 µmol/L) strongly suppressed the expression of the proteins and PIMT. Interestingly, PIMT siRNA blocked the phosphorylation of ERK and the expression of the EMT proteins. Additionally, PIMT and ERK phosphorylation were both co-activated by treatment with TGF-β (10 ng/mL) and TNF-α (10 ng/mL). Conclusion: A tight cross-regulation exists between ERK and PIMT in regards to their activation and expression during the EMT. Because the role of PIMT in the EMT and metastatic processes remains unclear, in this study, we explored the involvement of PIMT in the regulation of the detachment and attachment of the anoikis-resistant cell line MDA-MB-231, an aggressive breast cancer cell line with a highly invasive, migrative, and metastatic characters [10] , by culturing the cells in poly-HEMA (2-hydroxyethylmethacrylate)-coated dishes and introducing siRNA specific for PIMT. Materials and methods MaterialsLiCl and poly-HEMA were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin solution (10 000 unit/mL and 10 mg/mL, respectively), fetal bovine serum (FBS) and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Gibco BRL (Gaithersburg, MD, USA). MDA-MB 231 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Moloney murine leukemia virus (M-MLV) reverse transcriptase, polymerase chain reaction (PCR) premix, and Sapphire Super Taq were purchased from Rexgene Biotech Co, Ltd (Ochang, Korea). All primers used for PCR were purchased from Bioneer (Taejeon, Korea). A mixture of Stealth TM /siRNA duplex oligoribonucleotides against PIMT and Lipofectamine TM RNAiMAX was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-PIMT antiserum was produced against recombinant PIMT proteins of porcine brain as previously described (Koh and Hong, unpublished data). Antibodies against MAPK, MMP-2, MMP-9, N-cadherin, integrin αv, phospho-GSK3 (Tyr 279/216 ), phospho-ERK1/2 (Thr 202 /Tyr 204 ), phospho-MEK1 (Ser 218/222 )/MEK2 (Ser 222/226 ), phospho-Akt1/PkBα (Ser 473 ) and phospho-p90RSK (Ser 380 ) were...

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Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay

Cited by 33 publications

References 20 publications

Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Decorin suppresses tumor cell-mediated angiogenesis

Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells

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