PAF Produced by Human Breast Cancer Cells Promotes Migration and Proliferation of Tumor Cells and Neo-Angiogenesis
2000; Elsevier BV; Volume: 157; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64808-0
ISSN1525-2191
AutoresBenedetta Bussolati, Luigi Biancone, Paola Cassoni, Simona Russo, Marek Rola‐Pleszczynski, Giuseppe Montrucchio, Giovanni Camussi,
Tópico(s)Cell Adhesion Molecules Research
ResumoPlatelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response. Considerable experimental evidence indicates that the formation of new blood vessels penetrating into solid tumors is required for their growth and metastatic dissemination.1Folkman J Angiogenesis in cancer, vascular, rheumatoid and other diseases.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7290) Google Scholar, 2Weidner N Semple JP Welch WR Folkman J Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma.N Engl J Med. 1991; 324: 3-8Crossref Scopus (5444) Google Scholar In human breast cancer, it was extensively demonstrated that the tumor vascularization is strictly related to its growth and invasion, providing the basis for the use of the intratumor microvessel density as an independent prognostic marker.3Gasparini G Harris AL Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool.J Clin Oncol. 1995; 13: 765-782Crossref PubMed Scopus (456) Google Scholar Several mediators responsible for tumor angiogenesis have been identified in human breast cancer.4Hanahan D Folkman J Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6177) Google Scholar Studies on tumor angiogenesis have primarily focused on the role of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), interleukin-8, transforming growth factor-β, basic fibroblast growth factor (bFGF), and tissue factor.5Relf M LeJeune S Scott PAF Fox S Smith K Leek R Moghaddan A Withouse R Bicknell R Harris AL Expression of the angiogenic factors vascular endothelial cell growth factor, acid and basic fibroblast growth factor, tumor growth factor-b, platelet derived endothelial cell growth factor, placental growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis.Cancer Res. 1997; 57: 963-969PubMed Google Scholar These mediators are likely to be produced from the tumor cells themselves and/or by inflammatory infiltrating cells, such as mast cells and macrophages.6Leek RD Harris AL Lewis CE Cytokine networks in solid human tumors: regulation of angiogenesis.J Leukocyte Biol. 1994; 56: 423-435Crossref PubMed Scopus (203) Google Scholar, 7Sunderskotter C Steinbrink K Goebeler M Bhardway C Sorg C Macrophages and angiogenesis.J Leukocyte Biol. 1994; 55: 410-422PubMed Google Scholar Moreover, the simultaneous presence of growth factors and their receptors within breast cancer suggests that autocrine and/or paracrine loops are involved in the stimulation of cell proliferation and angiogenesis in this tumor.8De Jong JS van Diest PJ van der Valk P Baak JP Expression of growth factors, growth inhibiting factors, and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops.J Pathol. 1998; 184: 44-52Crossref PubMed Scopus (165) Google Scholar We recently demonstrated that platelet-activating factor (PAF), a phospholipid mediator of inflammation,9Prescott SM Zimmerman GA McIntyre GA Platelet-activating factor.J Biol Chem. 1990; 268: 17381-17384Google Scholar that may also trigger angiogenesis,10Camussi G Montrucchio G Lupia E DeMartino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar is present in breast cancer tissues and correlates with the tumor microvessel density.11Montrucchio G Sapino A Bussolati B Ghisolfi G Rizea-Savu S Silvestro L Lupia E Camussi G Potential angiogenic role of platelet-activating factor in human breast cancer.Am J Pathol. 1998; 153: 1589-1596Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar PAF acts through a specific receptor belonging to the family of seven membrane-spanning domain receptors.12Honda Z Nakamura M Miki I Minami M Watanabe T Seyama Y Okado H Toh H Ito K Myamoto T Shimizu T Cloning by functional expression of platelet activating factor receptor from guinea pig lung.Nature. 1991; 349: 342-346Crossref PubMed Scopus (574) Google Scholar The receptor interacts with a G protein that activates phosphatidyl-inositol-specific phospholipase C.13Liu B Nakashima S Takano T Shimizu T Nozawa Y Implication of protein kinase C alpha in PAF-stimulated phospholipase D activation in Chinese hamster ovary (CHO) cells expressing PAF receptor.Biochem Biophys Res Commun. 1995; 214: 418-423Crossref PubMed Scopus (16) Google Scholar The development of potent PAF-receptor (PAF-R) antagonists allows investigation on the role of this mediator in several pathophysiological conditions.14Barnes PJ Page CP Henson P Platelet Activating Factor and Human Disease. Blackwell Scientific Publications, London1989Google Scholar A role for PAF in tumor development has been recently suggested by the spontaneous development of skin tumors in transgenic mice overexpressing PAF-R.15Ishii S Nagase T Tashiro F Ikuta K Sato S Waga I Kume K Miyazaki J Shimizu T Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor.EMBO J. 1997; 16: 133-142Crossref PubMed Scopus (131) Google Scholar Moreover, in a murine model of melanoma, the blockade of PAF-R has been shown to reduce the melanoma cell dissemination into the lung.16Im SY Ko HM Kim JW Lee HK Ha TY Lee HB Oh SJ Bai S Chung KC Lee YB Chun SB Augmentation of tumor metastases by platelet-activating factor.Cancer Res. 1996; 56: 2662-2665PubMed Google Scholar In vitro, PAF induced an autocrine proliferative loop in the endometrial cancer cell line HEC-1A,17Maggi M Bonaccorsi L Finetti G Carloni V Muratori M Laffi G Forti G Serio M Baldi E Platelet-activating factor mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A.Cancer Res. 1994; 54: 4777-4784PubMed Google Scholar the migration of Kaposi's cells,18Biancone L, Cantaluppi V, Boccellino M, Bussolati B, Del Sorbo L, Conaldi PG, Albini A, Toniolo A, Camussi G. Motility induced by human immunodeficiency virus-1 tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis. Am J Pathol, 155:1731--1739Google Scholar and calcium influx in HT29 cells19Lohmeyer M McNaughton L Hunt SP Workman P Stimulation of intracellular free calcium increases by platelet-activating factor in HT29 colon carcinoma cells. Spectrofluorimetric and preliminary spatio-temporal analysis using confocal laser scanning fluorescence imaging microscopy.Biochem Pharmacol. 1994; 47: 975-985Crossref PubMed Scopus (8) Google Scholar and N1E-115 cells.20Diserbo M Cand F Ziade M Verdetti J Stimulation of platelet-activating factor (PAF) receptors increases inositol phosphate production and cytosolic free Ca2+ concentration in N1E-115 neuroblastoma cells.Cell Calcium. 1995; 17: 442-452Crossref PubMed Scopus (14) Google Scholar The aim of the present study was to investigate the biological significance of inducible PAF synthesis in the invasiveness of breast cancer cells and in the neo-angiogenesis. In vitro, we evaluated the production of PAF by two estrogen-dependent breast cancer cell lines (MCF7 and T-47D) and by an estrogen-independent invasive breast cancer cell line (MDA-MB231) in response to proinflammatory, angiogenic, and hormonal stimuli. Moreover, we studied the expression of PAF-R by these cell lines and the effect of PAF on tumor cell motility and growth. In vivo, we evaluated, using two structurally unrelated PAF-R antagonist WEB 2170 and CV 3988, the potential role of tumor-synthesized PAF on the angiogenesis occurring in MDA-MB231 cells implanted subcutaneously into SCID mice. Synthetic C16 PAF (1-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) and carbamyl PAF (1-hexadecyl-2-methylcarbamyl-sn-glyceryl-3-phosphorylcholine) were obtained from Bachem Feinchemikalien (Bubendorf, Switzerland). Stock solutions in chloroform were stored at −20°C until use. The chloroform was evaporated, and saline containing 0.25% bovine serum albumin (BSA) fraction V, low endotoxin, was added immediately before use. CV 3988 was from Takeda Chemical Industries (Kyoto, Japan).21Terashita Z Tsushima S Yoshioka S Namoto H Inada Y Nishikawa K CV 3988: a specific antagonist of platelet-activating factor (Paf-acether).Life Science. 1983; 32: 1975-1982Crossref PubMed Scopus (343) Google Scholar WEB 2170 was obtained from Boehringer (Ingelheim, Germany).22Heuer HO Casals-Stenzel J Muacevic G Weber KH Pharmacologic activity of bepafant (WEB 2170), a new and selective hetrazepinoic antagonist of platelet-activating factor.J Pharmacol Exp Ther. 1990; 255: 962-968PubMed Google Scholar Silica gel 60F254 thin-layer chromatography plates were obtained from Merck (Darmstadt, Germany). μPorasil high-pressure liquid chromatography columns were provided from Millipore chromatographic Division (Waters, Milford, MA). RPMI 1640 medium and bovine calf serum were from Life Technologies, Inc. (Grand Island, NY). Polymyxin B, phospholipase A2, phospholipase A1, BSA fraction V (tested for not >1 ng endotoxin per mg), fibronectin, bovine type-I collagen, thrombin, A23187, human recombinant TNF, hepatocyte growth factor (HGF) and bFGF, and FITC-conjugated anti-mouse and anti-rabbit IgG were all purchased from Sigma Chemical Company (St Louis, MO), as well as 17-β-estradiol, progesterone, and oxytocin. The anti-PAF-R monoclonal antibody (mAb) was kindly provided by Marek Rola-Pleszczynski (Sherbrooke, QC, Canada.). The irrelevant isotypic control was from Cederlane (Hornby, Ontario, Canada). The anti-PAF-R polyclonal antibody (pAb) was obtained from Alexis (San Diego, CA). Matrigel basement membrane matrix was obtained from Becton Dickinson Labware (Bedford, MA). Recombinant human VEGF165 was obtained from R&D Systems (Abington, UK). To investigate the production of PAF from breast cancer cells, two hormone-dependent (MCF-7 and T-47D) and a hormone-independent (MDA-MB231) breast adenocarcinoma cell lines (American Type Culture Collection, Rockville, MD) were used. Cells were equilibrated for 15 minutes in Tris-buffered Tyrode containing 0.25% delipidized BSA (fraction V), as previously described23Montrucchio G Lupia E Battaglia E Passerini G Bussolino F Emanuelli G Camussi G Tumor necrosis factor induced angiogenesis depends on in situ platelet-activating factor biosynthesis.J Exp Med. 1994; 180: 377-382Crossref PubMed Scopus (139) Google Scholar and incubated at 37°C for the indicated time with the agonists. Selected experiments were conducted in the presence of 5 μg/ml Polymixin B for 30 minutes at 37°C to exclude lipopolysaccharide contamination. The supernatants and the cell pellets were extracted according to a modification of the Bligh and Dyer procedure,24Bligh EG Dyer WJ A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44552) Google Scholar with formic acid added to lower the pH of the aqueous phase to 3.0. Each individual experiment was performed in triplicate. PAF was quantified, after extraction and purification by thin-layer chromatography and high-pressure liquid chromatography, by aggregation of washed rabbit platelets, as previously reported.10Camussi G Montrucchio G Lupia E DeMartino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar, 23Montrucchio G Lupia E Battaglia E Passerini G Bussolino F Emanuelli G Camussi G Tumor necrosis factor induced angiogenesis depends on in situ platelet-activating factor biosynthesis.J Exp Med. 1994; 180: 377-382Crossref PubMed Scopus (139) Google Scholar The biologically active material extracted from cells and supernatants in different experiments was characterized by comparison with synthetic PAF according to the following criteria:25McManus LM Woodard DS Deavers SI Pinckard SI PAF molecular heterogeneity: pathobiological implications.Lab Invest. 1993; 69: 639-648PubMed Google Scholar 1) induction of platelet aggregation by a pathway independent from both ADP and arachidonic acid/thromboxane A2-mediated pathways; 2) specificity of platelet aggregation as inferred from the inhibitory effect of 5 μmol/L WEB 2170 or CV 3988, two different PAF-R antagonists; 3) thin-layer chromatography and high-pressure liquid chromatography behavior and physicochemical characteristics, such as inactivation by strong bases and by phospholipase A2 treatment, but resistance to phospholipase A1, acids, weak bases, and 5 minutes heating in boiling water. The ovarian cancer cells CHO (ATCC) were transfected with a vector containing the neomycin resistance gene only (CHO neo) or with a pRC/RSV vector containing human PAF-R cDNA (CHO PAF-R).26Ye RD Prossits ER Zua A Cochrane CG Characterization of a human cDNA that encodes a functional receptor for platelet-activating factor.Biochem Biophys Res Commun. 1991; 180: 105-111Crossref PubMed Scopus (180) Google Scholar Transfectants were generated by electroporation at 1,000 μF in 4-mm electroporation cuvettes. Clones were selected for neomycin resistance, isolated for limiting dilution, and screened for expression of PAF-R by using reverse transcriptase-polymerase chain reaction. PAF-R-specific mRNA and PAF-AH mRNA were detected in total RNA extracted from cells by guanidinium thiocyanate phenol-chloroform and precipitated with isopropanol. One μg of RNA was treated with 6 U of RNase-free DNase for 1 hour at 37°C and then for 5 minutes at 94°C. complementary DNA was obtained by using random hexamer primers (Perkin-Elmer Cetus, Norwalk, CT). Reverse transcription was performed at 42°C for 60 minutes; in addition to 1 μg of RNA, the reaction mixture (20 μl) contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 1.0 mmol/L dNTPs, 20 U ribonuclease inhibitor, and 50 U of Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus). For PAF-R mRNA detection, cDNA was subjected to 35 cycles of amplification by the polymerase chain reaction in an automated DNA thermal cycler (Perkin-Elmer Cetus. by using the PAF-R mRNA-specific primer pairs18Biancone L, Cantaluppi V, Boccellino M, Bussolati B, Del Sorbo L, Conaldi PG, Albini A, Toniolo A, Camussi G. Motility induced by human immunodeficiency virus-1 tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis. Am J Pathol, 155:1731--1739Google Scholar forward: 5′ CAC GGG CTC GAG ACC AAC ACA GTG CCC GAC AGT GCT 3′; and reverse: 5′ CGC GGG ATC CCG GGT GAC CTG ATG TGC ATC ATT AAT 3′. The polymerase chain reaction mixture (50 μl) contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 20 pmol of (+) and (−) primers and 2 U thermostable DNA polymerase (Perkin-Elmer Cetus). Times and temperatures for denaturation, annealing, and extension were 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, respectively. Amplification product was analyzed in 2% agarose gels containing 0.5 mg/ml of ethidium bromide. As control, B16 cells (ATCC) untransfected or transfected with the human PAF-R-specific cDNA (kindly provided by Dr. R. D. Ye, La Jolla, CA)18Biancone L, Cantaluppi V, Boccellino M, Bussolati B, Del Sorbo L, Conaldi PG, Albini A, Toniolo A, Camussi G. Motility induced by human immunodeficiency virus-1 tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis. Am J Pathol, 155:1731--1739Google Scholar were used. To detect PAF-AH mRNA, cDNA was subjected to 35 cycles of amplification by the polymerase chain reaction by using PAF-AH mRNA-specific primer pairs: forward: 5′ TTTTCACTGGCAAGACACATCTTCTTTTGACTTC 3′; and reverse: 5′ CGTCAAAGTTCTGGTGCCTGAGCCCTTGATTGTA 3′. Times and temperatures for denaturation, annealing, and extension were 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, respectively. Amplification product was analyzed in 2% agarose gels containing 0.5 mg/ml of ethidium bromide. The presence of the PAF-R on the membrane of breast tumor cells or of a nontumor mammary gland cell line MCF-10A (Michigan Cancer Foundation, Detroit, MI) was evaluated by cytofluorimetric analysis using the murine anti-PAF-R mAb. Cells (2 × 106) were fixed in 1% paraformaldehyde at 4°C for 20 minutes. After washing in phosphate-buffered saline (PBS. containing 2% heat-inactivated human serum and incubation for another 15 minutes with whole heat-inactivated human serum to block remaining nonspecific sites, cells were incubated for 30 minutes with the anti-PAF-R mAb (IgG2a, 1:500 dilution) or with the irrelevant isotypic control, in PBS containing 1% BSA. The intracellular staining for the PAF-R was evaluated in permeabilized cells. Briefly, cells were incubated with the anti-PAFR mAb at 4°C for 45 minutes in permeabilizing solution (PBS containing 0,1% saponin, 1% BSA, and 0.1% Na azide). After appropriate washings, cells were stained by the addition of fluorescein-conjugated goat-anti-mouse IgG antibodies (Sigma). All incubations were performed at 4°C. To selectively stain the intracellular pool of PAF-R, cells were first incubated with the rabbit anti-PAF-R pAb (1:250 dilution) for 30 minutes at 4°C, to saturate the membrane pool of receptors. After fixation, cells were incubated in permeabilizing solution with the monoclonal anti-PAFR mAb and then with the fluorescein-labeled anti-mouse IgG. The stained cells were analyzed on a flow cytometer (Becton-Dickinson). As a positive control, umbilical vein endothelial cells, prepared as previously described,10Camussi G Montrucchio G Lupia E DeMartino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar were used. The acquisition was done with 10,000 events per sample. The Kolmogorov-Smirnov statistic analysis was performed in each individual experiment. Cell motility in response to PAF was investigated both as chemotaxis in Boyden's chambers and as random cell movement of adherent cells. Chemotaxis across a polycarbonate filter (8-μm pore size) was performed as previously described.10Camussi G Montrucchio G Lupia E DeMartino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar RPMI medium containing 0.25% BSA and the stimulus or the vehicle alone was placed in the lower compartment of the chamber. Cells (2 × 105), suspended in the same medium, were then seeded in the upper compartment of the Boyden's chamber. In selected experiments, cells were preincubated for 30 minutes at 37°C with the PAF-R antagonists 3 μmol/L WEB 2170 or 3 μmol/L CV3988. After 5 hours incubation at 37°C, the upper surface of the filter was scraped with a rubber policeman. The filters were then fixed and stained with Diff-Quick (Harleco, Gibbstown, NJ) and 10 fields at ×200 magnification were counted. Cell migration of quiesced adherent MDA-MB231 cells or of CHO cells, transfected or not for PAF-R (105 cells/well in RPMI plus 0.25% BSA) was studied throughout a 4-hour period under a Nikon Diaphot (Tokyo, Japan) inverted microscope with a ×10 phase-contrast objective, as previously described.18Biancone L, Cantaluppi V, Boccellino M, Bussolati B, Del Sorbo L, Conaldi PG, Albini A, Toniolo A, Camussi G. Motility induced by human immunodeficiency virus-1 tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis. Am J Pathol, 155:1731--1739Google Scholar Cells were kept in an attached, hermetically sealed Plexiglas Nikon NP-2 incubator at 37°C. Cell migration was recorded using a Panasonic, CCTV (Matsushita Communication, Neumũnster, Germany) video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging srl, Venice, Italy) and an IBM-compatible system equipped with a video card (Targa 2000, Truevision, Santa Clara, CA). Image analysis was performed by digital saving of images at 30 minutes of interval. Migration tracks were generated by marking the position of nucleus of individual cells on each image. The net migratory speed (velocity straight line) was calculated by the MicroImage software based on the straight line distance between the starting and ending points divided by the time of observation. Migration of at least 30 cells was analyzed for each experimental condition. Values are given as mean ± SD. Cell division did not start to any significant degree during the experiments. In selected experiments, MDA-MB231 cells were seeded on plates previously coated with 10 μg/ml of bovine fibronectin, type I collagen, or reconstituted basement membrane (Matrigel), overnight at 37°C. PAF-R-positive breast cancer cells and PAF-R-negative COS cells were seeded at 8,000 to 10,000 cells/well into 24-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 10. fetal calf serum. Stimulation was initiated by addition of different concentrations of carbamyl-PAF or of the PAF-R antagonists WEB 2170 and CV 3988. In the 96-hour experiments, media containing the tested substances was replaced after 48 hours. After 48 or 96 hours of incubation, cells were washed with PBS before addition of 1 ml Hepes (1.19 g/L), MgCl2 (0.153 g/L) solution plus ZapoglobinR (Coulter Electronics Ltd., Luton Beds, UK). After 10 minutes of incubation at 37°C, cell suspensions were added to 9 ml of NaCl solution with 0.05% formalin in optically clear pots and stored at 4°C until counted. Cell number was determined by triplicate readings per each well of triplicate samples using a Coulter Counter (Coulter Electronics Ltd.). Three experiments were performed in triplicate. Statistical analysis was performed by one-way analysis of variance followed by Bonferroni correction. For the in vivo studies, MDA-MB231 cells were implanted subcutaneously into SCID mice (Charles River, Wilmington MA) within growth factor-depleted Matrigel, as previously described.27Mullen P Ritchie A Langdon SP Miller WR Effect of Matrigel on tumorigenicity of human breast and ovarian carcinoma cell lines.Int J Cancer. 1996; 67: 816-820Crossref PubMed Scopus (43) Google Scholar The use of Matrigel is necessary for the initial establishment of tumors deriving from this cell line.27Mullen P Ritchie A Langdon SP Miller WR Effect of Matrigel on tumorigenicity of human breast and ovarian carcinoma cell lines.Int J Cancer. 1996; 67: 816-820Crossref PubMed Scopus (43) Google Scholar MDA-MB231 cells were harvested using trypsin-ethylenediaminetetraacetic acid, washed with PBS, counted in a microcytometer chamber, and resuspended in DMEM (4 × 106 in 250 μl DMEM). Cells were chilled on ice, added to 250 μl of Matrigel at 4°C, and injected subcutaneously into the left back of SCID mice via a 26-gauge needle using a 1-ml syringe. For PAF-R inhibition studies, WEB2170 and CV 3988, two structurally different PAF-R antagonists, were added to the Matrigel (final concentration, 250 ng/ml) and to drinking water (3 mg/kg/day), as previously described.28Summers JB Albert DAH Platelet activating factor antagonists.Adv Pharmacol. 1995; 32: 67-168Crossref PubMed Scopus (57) Google Scholar In selected experiments, VEGF (20 ng/ml) was also added to Matrigel. At day 7, mice (controls, n = 12; WEB 2170, n = 10; CV 3988, n = 5; VEGF, n = 5; and VEGF+WEB 2170, n = 5) were sacrificed and tumor plugs were recovered and processed for histology. Typically, the overlying skin was removed, and gels were cut out by retaining the peritoneal lining for support, fixed in 10% buffered formalin, and embedded in paraffin. Sections (3 μm) were cut and stained with hematoxylin and eosin or with a Masson trichromic reaction and examined under a light microscope system. Morphometric analysis was performed to count vessels that were expressed as number/mm2. Vessel structures were counted only if showing a patented lumen with red globuli and/or leukocytes. The mean size of vessels was planimetrically assessed using the computing integral area calculation of the Lucia digital system (Nikon UK Limited Instrument Division, Kingston, UK). Endothelial cells in the neoformed vessels were stained with fluorescein-labeled Griffonia simplicifolia lectin (Sigma)29Sahagun G Moore SA Fabry Z Schelper RL Hart MN Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled Griffonia simplicifolia agglutinin.Am J Pathol. 1989; 134: 1227-1232PubMed Google Scholar and von Willebrand factor pAb (Sigma) by fluorescence. The direct angiogenic effect of PAF released from MDA-MB231 cells was evaluated in the murine Matrigel angiogenesis assay.28Summers JB Albert DAH Platelet activating factor antagonists.Adv Pharmacol. 1995; 32: 67-168Crossref PubMed Scopus (57) Google Scholar PAF, extracted from the supernatant of 1 × 106 unstimulated cells cultured for 8 hours in DMEM containing 0.25% BSA, was resuspended in 50 μl saline with 0.25% BSA, added to growth factor depleted Matrigel and injected into mice. Briefly, Matrigel (0.5 ml) was subcutaneously injected into the abdominal tissue of female C57 mice along the peritoneal midline. After 6 days, mice were sacrificed and tumor plugs were recovered and processed for histological analysis of neoangiogenesis, as described above. In a previous study, we showed that MCF-7 breast cancer cells release bioactive PAF in basal conditions.11Montrucchio G Sapino A Bussolati B Ghisolfi G Rizea-Savu S Silvestro L Lupia E Camussi G Potential angiogenic role of platelet-activating factor in human breast cancer.Am J Pathol. 1998; 153: 1589-1596Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar In the present study we compare basal and stimulated PAF synthesis in two estrogen-dependent cell lines (MCF-7 and T47-D) and one estrogen-independent cell line (MDA-MD231). Unstimulated cells produced a small amount of PAF, detectable both as associated to the cell fraction and as released in the supernatant (Figure 1). No significant difference in the amount of PAF produced in basal conditions was observed among these cell lines. In contrast MCF-10A, a nontumor breast cell line did not synthesized PAF (data not shown). Stimulation for 1 hour with calcium ionophore A23187, used as nonspecific inducer of PAF synthesis, increased PAF levels in all of the cell lines (Figure 1). In MCF7 and T-47D cells PAF was detected mainly as cell associated, whereas in MDA-MB231 cells it was almost completely released into the supernatan
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