Potential Angiogenic Role of Platelet-Activating Factor in Human Breast Cancer
1998; Elsevier BV; Volume: 153; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65747-1
ISSN1525-2191
AutoresGiuseppe Montrucchio, Anna Sapino, Benedetta Bussolati, G Ghisolfi, Simona Rizea-Savu, Luigi Silvestro, Enrico Lupia, Giovanni Camussi,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoThis study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34- and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesisin vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivoneoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested. This study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34- and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesisin vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivoneoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested. 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The aim of the present study was to evaluate whether the production of PAF within human breast cancer correlates with the extent of neovascularization of the tumor and whether tumor-extracted PAF induces neoangiogenesis in an in vivomurine model. Specimens from 18 patients with primary invasive breast carcinoma (11 ductal, 5 lobular, and 2 mixed ductal/lobular carcinomas; age range, 45 to 85 years; Table 1) and from 20 controls (7 normal breast tissues and 13 benign breast lesions such as fibroadenomas, fibrocystic changes, and sclerosing adenosis; age range, 42 to 75 years) were obtained after surgery. Adjacent cross sections of specimens were processed for PAF extraction and histological analysis.Table 1Clinical and Immunohistochemical Characteristics of Breast CancersPatientsTypeTNGER (%)PR (%)K67 (%)Cat.S (%)Cat.N (%)p53 (%)cErb (%)PAF (pg/mg) 1DIC1c01959014550501 2DIC1c1295704515605071 3LIC20300438090801.5 4DIC20380603550000.9 5DIC+ LIC4126005053010027 6DIC+ LIC1c029050250606557 7DIC30290903335603800 8DIC1c010048040609013.3 9DIC1c0175801900000.210LIC1c0190301000000.411DIC201507012000013712DIC20195602000004013DIC1c117090600000.114DIC203006020209000.215DIC1c01001920802801616LIC213006010000017DIC1c1190159000753418LIC1c03909031005800.4DIC, ductal infiltrating carcinoma; LIC, lobular infiltrating carcinoma; ER, estrogen receptor; PR, progesteron receptor; Cat., cathepsin. PAF extracted from breast cancers was expressed as pg/mg dry tissue. Open table in a new tab DIC, ductal infiltrating carcinoma; LIC, lobular infiltrating carcinoma; ER, estrogen receptor; PR, progesteron receptor; Cat., cathepsin. PAF extracted from breast cancers was expressed as pg/mg dry tissue. Samples were fixed in buffered formalin and paraffin embedded. Sections were stained with hematoxylin and eosin (H&E) for histological diagnosis. Representative sections of the tumors including the infiltrating edges were selected and processed for immunohistochemistry. For the purpose of heat-induced antigen retrieval, sections were pretreated in a pressure cooker in a 10 nmol/L citrate buffer (pH 6.0) solution. A standard avidin-biotin-peroxidase complex procedure was applied. The assessment of vascularization was performed using anti-CD31 monoclonal antibody (MAb; diluted 1:30; Dako, Glostrup, Denmark) and anti-CD34 MAb (diluted 1:50; Novocastra Laboratories, New Castle upon Tyne, UK). After staining, sections were scanned at low power for hot spots of angiogenesis. Microvessels were evaluated on three ×200 fields. Any endothelial cell or cluster of endothelial cells positive for CD31 or CD34 was counted. Cathepsin D expression was evaluated using MAb M1G8 (diluted 1:1; CIS BIO International, Gif Sur Yvette, France). The percentage of the overall staining contributed solely by carcinoma cells and the percentage contributed only by stromal staining were scored. Hormonal receptor expression was evaluated using estrogen receptor MAb ER-1D5 (diluted 1:50; Dako) and progesterone receptor MAb PGR (diluted 1:50; Abbott Diagnostic, Wiesbaden-Delkenheim, Germany). p53 protein expression was studied using the MAb DO-7 (diluted 1:100; Biogenex, San Ramon, CA), and c-erbB-2 oncoprotein was demonstrated with a MAb HER-2/neu (diluted 1:100; Pabish, Pero, Italy). Only cell membrane immunoreactivity was regarded as specific for c-erbB-2 overexpression. Proliferation rate was assessed using a Ki67-related MAb (MIB1; diluted 1:10; Immunotech, Marseille, France). The T-47D and MCF7 breast adenocarcinoma cell lines were obtained from American Type Culture Collection (Manassas, VA) and nontumor mammary gland MCF10A cell line was obtained from the Michigan Cancer Foundation (Detroit, MI). Cells were cultured in RPMI containing 10% fetal calf serum (Sigma Chemical Co, St. Louis, MO). Cells grown at confluence in 35-mm Petri dishes were washed twice and cultured overnight in RPMI containing 0.25% bovine serum albumin (BSA fraction V; Sigma). PAF was extracted from the cell supernatant as previously described.48Camussi G Aglietta M Malavasi F Tetta C Piacibello W Sanavio F Bussolino F The release of platelet activating factor from human endothelial cells in culture.J Immunol. 1983; 131: 2397-2403PubMed Google Scholar Lipids were extracted from breast specimen homogenates or from culture medium by chloroform/methanol/water acidified to pH 3.0 to 3.5 with formic acid as previously described.47Lupia E Montrucchio G Battaglia E Modena V Camussi G Role of tumor necrosis factor-a and platelet activating factor in neoangiogenesis induced by sinovial fluids of patients with rheumatoid arthritis.Eur J Immunol. 1996; 26: 1690-1694Crossref PubMed Scopus (54) Google Scholar PAF was quantified by bioassay on washed rabbit platelets after purification from the lipid extract by thin layer chromatography (TLC; silica gel 60, F254, Merck; solvent system: chloroform/methanol/water, 65:35:6, v:v) and high-pressure liquid chromatography (HPLC; μPorasil Millipore Chromatographic Division, Waters, Bedford, MA; mobile phase: chloroform/methanol/water, 60:55:5, v:v; flow rate, 1 ml/minute) as previously described.48Camussi G Aglietta M Malavasi F Tetta C Piacibello W Sanavio F Bussolino F The release of platelet activating factor from human endothelial cells in culture.J Immunol. 1983; 131: 2397-2403PubMed Google Scholar, 49Bussolino F Arese M Silvestro L Soldi R Benfenati E Sanavio F Aglietta M Bosia A Camussi G Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-α or interleukin-1α.Eur J Immunol. 1994; 24: 3131-3139Crossref PubMed Scopus (29) Google Scholar The recovery of radioactive standards (Du Pont-NEN, Brussels, Belgium), submitted to the same procedures of extraction and TLC and HPLC purification of biological samples was, respectively, 96 to 98% for 1-O-[3H]-alkyl-PAF C16:O, 96 to 98% for 1-O-[3H]-alkyl-PAF C18:O, and 79 to 82% for [14C]-acyl-PAF C16:O. The specificity of platelet aggregation was inferred from the inhibitory effect of 3 μmol/L WEB 2170, a PAF receptor antagonist (Boehringer, Ingelheim, Germany).50Heuer HO Casals-Stenzel J Muacevic G Weber KH Pharmacologic activity of bepafant (WEB2170), a new and selective tetrazepinoic antagonist of platelet-activating factor.J Pharmacol Exp Ther. 1990; 225: 962-968Google Scholar PAF bioactivity was not inhibited by phospholipase A1 (Sigma), thus suggesting that it is related to alkyl-PAF rather than to acyl-PAF, which is known to be more than 1000 times less active than alkyl species of PAF.37McManus LM Woodard DS Deavers SI Pinckard SI PAF molecular heterogeneity: pathobiological implications.Lab Invest. 1993; 69: 639-648PubMed Google Scholar The bioactive material was further characterized as PAF on the basis of TLC and HPLC behavior and of physicochemical characteristics,45Pitton C Lanson M Besson P Fetissoff F Lansac J Benveniste J Bougnoux P Presence of PAF-acether in human breast carcinoma: relation to axillary lymph node metastasis.J Natl Cancer Inst. 1989; 81: 1298-1302Crossref PubMed Scopus (28) Google Scholar, 49Bussolino F Arese M Silvestro L Soldi R Benfenati E Sanavio F Aglietta M Bosia A Camussi G Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-α or interleukin-1α.Eur J Immunol. 1994; 24: 3131-3139Crossref PubMed Scopus (29) Google Scholar such as inactivation by strong bases and phospholipase A2 (Sigma) but resistance to phospholipase A1 and acidic treatment. After TLC and HPLC purification, PAF-bioactive material was also analyzed by a recently developed technique based on HPLC (normal phase silica column μPorasil, 250 × 4.6 mm internal diameter (Millipore Waters), eluted under isocratic conditions at 1.0 ml/minute, with a mobile phase composed of chloroform/methanol/water, 60:55:5, v:v) tandem mass spectrometry (HPLC-MS/MS).51Rizea Savu S Silvestro L Sorgel F Montrucchio G Lupia E Camussi G Determination of 1-O-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, platelet-activating factor and related phospholipids in biological samples by high-performance liquid chromatography by tandem mass spectrometry.J Chromatogr. 1996; 628: 35-42Crossref Scopus (11) Google Scholar To reduce the mobile phase flow rate to a level compatible with the MS system, post-column splitting was performed by connecting a silica capillary of adequate length to the splitting port of the MS interface. The samples were injected dissolved in the mobile phase using an injection volume of 250 ml and a 200-ml sample loop. Mass spectrometric analysis was performed on a Perkin-Elmer-Sciex (Thornhill, Canada) API III-Plus triple quadrupole mass spectrometer, equipped with an atmospheric pressure articulated ion spray source. High-purity nitrogen served both as the nebulizer gas (operative pressure was 0.5 MPa) and curtain gas (flow rate was 0.8 L/minute). Argon was used as the target gas for the MS/MS experiments, at a collision gas thickness of 3 × 1015 atoms/cm2. The ion spray needle voltage was set at 5 kV, the orifice voltage at 50 V, and the MS collision energy at 25 V, which were previously shown optimal conditions for these analyses.51Rizea Savu S Silvestro L Sorgel F Montrucchio G Lupia E Camussi G Determination of 1-O-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, platelet-activating factor and related phospholipids in biological samples by high-performance liquid chromatography by tandem mass spectrometry.J Chromatogr. 1996; 628: 35-42Crossref Scopus (11) Google Scholar The quantitative analysis was performed in multiple reaction monitoring (MRM). Parent ion spectra, positive mode, were obtained from daughter ions with mass-to-charge ratio (m/z) 184 corresponding to phosphocholine fragment; the scanning range was m/z 100 to 600. In the MRM analysis, acquired in positive mode, the study of different PAF molecular species was done using the following reactions (parent ions→daughter ions): 552→184 (C18-alkylPAF), 524→184 (C16-alkylPAF), 524→184 (C18-lysophosphatidylcholine (LPC)), 496→184 (C16-LPC, lyso-PAF), 538→184 (C16-acylPAF), 566→184 (C18-acylPAF), and 594→210 (CV3988, used as internal standard). CV3988 was kindly provided by Takeda (Osaka, Japan). All other standards were purchased from Sigma. Female C57 mice at 6 to 8 weeks of age were used. Angiogenesis was assayed as growth of blood vessels from subcutaneous tissue into a solid gel of basement membrane (Matrigel, Becton Dickinson Labware, Bedford, MA) containing heparin (64 U/ml; Sigma) and the test sample.52Passaniti A Taylor RM Pili R Guo Y Long PV Haney JA Pauly RR Grant DS Martin GR A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor.Lab Invest. 1992; 67: 519-528PubMed Google Scholar Matrigel (8 mg/ml), in liquid form at 4°C, was mixed with the experimental substances and injected (0.5 ml) into the abdominal subcutaneous tissue of mice, along the peritoneal midline. The Matrigel used was extracted according to the procedure described by Taub et al,53Taub M Wang Y Szczesny TM Kleinman HK Epidermal growth factor or transforming growth factor alpha is required for kidney tubulogenesis in Matrigel cultures in serum-free medium.Proc Natl Acad Sci USA. 1990; 87: 4002-4006Crossref PubMed Scopus (213) Google Scholar which has been previously shown to efficiently deplete Matrigel of basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor I, and platelet-derived growth factor.54Vukicevic S Kleinman HK Luyten FP Roberts AB Roche NS Reddi AH Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.Exp Cell Res. 1992; 202: 1-8Crossref PubMed Scopus (553) Google Scholar, 55Santos OFP Nigam SK HGF-induced tubulogenesis and branching of epithelial cells is modulated by extracellular matrix and TGF-β.Dev Biol. 1993; 160: 293-302Crossref PubMed Scopus (189) Google Scholar The angiogenic effect of 25 μl of TLC-purified lipid extract from breast tumor, control breast, or culture media from breast cell lines was evaluated in the absence or in the presence of WEB 2170. WEB 2170 was included in the Matrigel plug (final concentration, 250 ng/ml)
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