10.1245/s10434-006-9225-3
Annals of Surgical Oncology 14:862-868 (2007)
© 2007 Society of Surgical Oncology
Cancer Cells Cause Vascular Endothelial Cell (vEC) Retraction via 12(S)HETE Secretion; The Possible Role of Cancer Cell Derived Microparticle
Keiji Uchide, MD,
Masato Sakon, MD,
Hideo Ariyoshi, MD,
Syouji Nakamori, MD,
Masaru Tokunaga, MD and
Morito Monden, MD
Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suitacity, Osaka, Japan
Correspondence: Address correspondence and reprint requests to: Keiji Uchide, MD; E-mail: k-uchide{at}jb3.so-net.ne.jp
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ABSTRACT
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Background: Cancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7.
Methods and Results: In order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM.
Conclusion: These findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.
Key Words: Endothelial cell retraction • 12(S)HETE • Cancer cell • Cancer metastasis • Microparticles
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INTRODUCTION
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Cancer metastasis requires several steps such as invasion through basement membrane, passage through extracellular matrix, intravasation, interaction with vascular endothelial cells (vEC), extravasation, and growth in the new site.1,2 Among these steps, tumor invasion into the subendothelial layer has been considered one of the critical steps for the development of tumor metastasis. Since the integrity of vEC works as a barrier against cancer cell migration, cancer cell mediated retraction of vEC plays a key role in this process of cancer cell extravasation.3
Cancer cells are considered to induce vEC retraction through two different mechanisms; one is the direct stimulation of vEC by cell to cell contact via cell surface ligand-receptor interaction.4,5 This mode of cancer cell-vEC interaction has been intensively studied especially in the field of cell surface adhesive ligands like E-selectin.6 The other possible mechanism is the indirect stimulation of vECs by soluble factors secreted from cancer cells.7 In general, soluble factors like glycoprotein or lipids are known to cause vEC retraction.8,9 However there have been no reports investigating cancer cell derived bioactive molecules causing vEC retraction. In this report, we investigated on cancer cell secreted lipids involved in vEC retraction.
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MATERIALS AND METHODS
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Materials
Thin layer chromatography plate, LK6DF Silica Gel 60 a° , was purchased from Whatman International Ltd (England). Monoclonal antibody against 12(S) hydroxyeicosatetraenoic acid (HETE) was purchased from Assay Designs Inc. (USA). Authentic 12(S)HETE and 12(R)HETE was purchased from Cayman Chemical (USA). Transwell chamber, Costar 3413, was purchased from Corning Incorporated (USA). Matrigel was purchased from Becton Dickinson Labware, Bredford, (MA, USA). Trypsin, acetylsalicylic acid (ASA), FITC-dextran and Nordihydroguaiaretic acid (NDGA) were purchased from Sigma Chemical Co. (USA). Other reagents were of the highest analytical grade available.
Cell Culture
Calf pulmonary arterial endothelial cells (CPAE) were obtained from Japan Cell Resources Bank (Osaka, Japan). CPAE were prepared and passaged as described previously.10 For retraction experiments, cells were grown on the transwell chamber, Costar 3413, coated with Matrigel in medium (D-MEN) with 10% fetal bovine serum (FBS), 5 µg/ml of recombinant human basic fibroblast growth factor (b-FGF, Pepro Tech EC, London, UK), penicillin (100 units/ml), and streptomycin (100 µg/ml). Confluent cells were rinsed twice with modified HEPES Tyrode’s buffer (MHTB:129.0 mM NaCl, 10.0 mM HEPES, 8.9 mM NaHCO3, 5,6 mM dextrose, 0.8 mM MgCl2, 0.8 mM KH2PO4, pH 7.4).
Preparation of Culture Medium (CM) derived from MCF-7
MCF-7 cell line was obtained from the Japanese Cancer Research Bank (Tokyo, Japan). CM was prepared using MCF-7 cells, as described previously.10 Briefly, cells were grown on culture dishes and incubated at 37°C in DMEM/F12 supplemented with 10% FBS, penicillin, and streptomycin. After incubation for 3 to 4 days to form a confluent monolayer, cells were washed three times with PBS in order to remove serum complements, and incubated with 20 ml serum free DMEM/F12. After incubation for 48 hr, CM was collected and centrifuged at 12000 g for 10 min. The collected supernatant was dialyzed against RPMI 1640 medium for 24 hr at 4°C and passed through a 0.45 µm filter to remove cells and cell debris. The filtered supernatant was concentrated by ultrafiltration membrane PM10 (Amicon, Danvers, MA, USA). The concentrate was dialyzed against PBS for 24 hr at 4°C and filtered through a 0.45 µm filter again. After protein concentration was adjusted to 1mg/ml by adding PBS, it was subjected to vEC retraction assays.
Vascular Endothelial Cell (vEC) Retraction Assay
The extent of vEC retraction was measured as the amount of fluorescein-isothiocyanate-labeled dextran (FITC-Dx: average molecular weight, 70kDa) (Sigma) that passed across an endothelial cell monolayer. CPAE cells were cultured to form a monolayer for 3 days on the polycarbonate membranes with a 0.4 µm pore size coated with the reconstituted basement membrane MATRIGELTM (Becton Dickinson Labware, Bredford, MA, USA) of each upper chamber of the transwell chamber (Costar, Cambridge, MA, USA) (Fig. 1
). After the removal of culture medium, 200 µl of the medium containing 1mg/ml of FITC-Dx with and without crude and treated CM was added into the each upper transwell chamber. The lower transwell chamber was filled with 1 ml of the same medium without FITC-Dx and these transwells were cultured at 37°C for a given period. Following culture, the amount of FITC-DX in the lower chamber was quantified by Microplate reader M-T max (Wako, Osaka, JAPAN) under excision and emission wave lengths of 490 nm and 550 nm, respectively. The concentration of FITC-Dx in the lower chamber was calculated from the relative fluorescence intensity against 1mg/ml FITC dextran in culture medium. The endothelial cell retraction activity was represented by the mean concentration of FITC-Dx in the lower chamber. Each assay was performed in triplicate. As shown in Fig. 2, our transwell vEC retraction assay clearly showed a good time- and dose-dependency, suggesting that our system is suitable for measuring vEC retracting activity in CM. We employed incubation time of 24 hours for later experiments.
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FIG. 1. CPAE-cell retraction assay. A schematic represents the transwell chamber assay for measuring the endothelial cell retraction activity. CM derived from tumor cells was added into the upper chamber to induce CPAE-cell retraction. When endothelial cells are retracted exposing inter endothelial spaces, the amount of FITC-Dx diffused from the upper to the lower chamber is increased. The activity of CPAE-cell retraction is expressed as percent activity of untreated CM, designating the difference in the amount of FITC-Dx between the absence and the presence of untreated CM as 100%.
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FIG. 2. Effects of MCF-7 CM on the CPAE cell retraction activity. The extent of retraction was determined by measuring the amount of FITC-Dx, which passed through a CPAE monolayer on Matrigel-coated polycarbonate. Membrane only (square). CPAE-cell monolayer on a Matrigel-coated polycarbonate membrane (circle). Addition of CM (2mg/ml) derived from MCF-7 cells on CPAE cell monolayer (triangle). Addition of CM (1 mg/ml) derived from MCF-7 cells on CPAE cell monolayer (star). Values are mean ± SD from triplicate experiments.
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Treatment of CM with Tripsin
CM was incubated with 0.1% tripsin at 37°C for 1hr. Then, the amount of sample was adjusted to be equivalent to that of the untreated, control experiment.
Lipid Extraction and Separation
Lipids were extracted from each aqueous sample according to the method of Bligh and Dyer’s,11 and samples were kept at –20°C until use. Briefly, each sample was added chloroform (250 µl) and shaken for 30 seconds, were added distilled water (300 µl), and shaken for another 30 seconds, and then, were centrifuged at 20g for 5 minutes at 4°C. After complete separation to chloroform layer, fluff layer, and alcoholic layer, chloroform layer was removed by aspiration. The chloroform layers were evaporated under nitrogen stream to form crystals. Thin layer chromatography using TLC plate, LK6DF was carried out using the solvent system (chloroform/methanol/ acetic acid/water = 90/8/1/0.8;v/v/v/v) at 4°C according to the method of Okuma.12 After the visualization of separated lipids by iodine vapor, Rf value was calculated. In some experiments, separated lipid was recovered by scratching this part of the silica gel and lipid was re-extracted by Bligh and Dyer’s Methods.
Flow Cytometric Analysis of 12(S)HETE
One ml of MCF-7 CM with or without addition of monoclonal antibody against 12(S)HETE were ultracentrifuged at 105000g for 2.5 hours at 4°C. The resultant pellets were incubated with 1 µg of FITC-labeled mAb against 12(S)HETE or control IgG for 30 min at 4°C. Then, samples were analyzed on a fluorescence-activated cell sorter (FACScan, Beckton Dickinson) according to the methods described by Yamamoto et al.13 Data were processed using Cell QuestTM software (BD).
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RESULTS
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1. Effects of proteolytic digestion, lipid extraction, and heat treatment on CPAE-cell retraction by crude MCF-7 cell CM.
Addition of MCF-7 cell CM (0 to 2 mg/ml) enhanced CPAE-cell retraction in a dose or time related manner as shown in Fig. 1
. About 75% of the cell retraction activity was obtained with 2 mg/ml of CM. In order to determine what component of CM causes CPAE-cell retraction, crude MCF-7 cell CM was subjected to trypsin digestion, lipid extraction, and heat treatment (Fig. 3). The CPAE cell retraction activity was decreased up to 38 ± 6% and 46 ± 11% by 0.1% trypsin digestion and lipid extraction, respectively. On the other hand, heat treatment at 95°C for 5 min completely abolished CPAE-cell retraction activity.
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FIG. 3. Effects of trypsin digestion, lipid extraction, and heat treatment on CPAE-cell retraction activity of MCF-7 CM. Crude MCF-7 CM was treated with 0.1% trypsin at 37°C for 1 hour. Lipids in MCF-7 CM were extracted by the method of Bligh and Dyer’s,11 as described in Materials and Methods. MCF-7 CM was incubated at 95°C for 5 min. In the respective experiments, the amount of sample was adjusted to be equivalent to that of the untreated, control experiment. The activity of CPAE-cell retraction is expressed as the percent activity of untreated CM.
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2. Effects of acetyl salicylic acid (ASA) and nordihydroguaiaretic acid (NDGA) on CPAE cell retraction by crude MCF-7 cell CM.
Treatment with ASA (final concentration: 50 µM), a cyclooxygenase inhibitor did not affect the secretion of CPAE-cell retraction activity from MCF-7 cells. In contrast, NDGA (final concentration: 50 µM), a lypooxgenase inhibitor markedly suppressed its activity (Fig. 4).
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FIG. 4. Effect of acetyl salicylic acid (ASA) and nordihydroguaiaretic acid (NDGA) on CPAE cell retraction activity of MCF-7 CM. After incubation for 3 to 4 days to form a confluent monolayer, MCF-7 cells were washed three times with PBS, and incubated with 20ml serum free DMEN/F12, which added ASA (50 µM in final concentration) and NDGA (50 µM in final concentration) each. After incubation at 37°C for 48 hours, CM was made as same methods. Treated CM was added to the upper chamber of the CPAE-cell retraction assay system, and the vEC retraction activity was measured as in Materials and Methods.
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3. Separation of lipids extracted from MCF-7 CM by thin layer chromatography.
Lipids extracted from MCF-7 CM preserved about 64 ± 8% of the whole activity of crude CM (Fig. 6). These lipids were separated into three bands by thin layer chromatography which appeared at 0.78, 0.73, and 0.45 of Rf value (Fig. 5). The most activity (66 ± 7%) of CPAE-cell retraction was recovered in the 0.73 Rf band, corresponding to 12HETE (Fig. 6) while a total activity of 24 ± 10% was observed in the remaining two bands. To further confirm the activity of 12HETE, the stereoisomers of 12HETE, 12(S)HETE (0.3 µM: final concentration) and 12(R)HETE (0.3 µM: final concentration) were subject to endothelial-cell retraction assay. About 80% of crude MCF-7 CM activity was observed in 12(S)-HETE, but only 7% in 12(R)-HETE. (Fig. 7).
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FIG. 6. Separation of lipids extracted from MCF-7 CM by thin-layer chromatography. CPAE cell retraction activity of separated bands: The lipids of these bands were extracted and subjected to CPAE, cell retraction activity was measured. The CPAE cell retraction activity of each sample is expressed as the percentage activity of crude MCF-7 CM.
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FIG. 5. Separation of lipids extracted from MCF-7 CM by thin-layer chromatography. Lipids extracted from MCF-7 CM were separated by thin-layer chromatography into three bands (0.78, 0.73, and 0.45 of Rf value). The band Rf value 0.73 was equivalent to 12 HETE.
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FIG. 7. 12(R)HETE and 12(S)HETE (0.3µM: final concentration) were subjected to endothelial-cell retraction assay. CPAE cell retraction activity was expressed as the percent activity of crude MCF-7 CM.
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4. Localization of CPAE cell retraction activity in MCF-7 CM.
To determine the localization of CPAE cell retraction activity, crude MCF-7 was separated into the soluble, supernatant fraction and the pellet (microparticle) fraction by ultracentrifugation and subject to endothelial-cell retraction assay. Almost all CPAE cell retraction activity was obtained in the pellet fraction (Fig. 8). Then, flowcytometric analysis of the pellet fraction was performed using monoclonal antibody against 12(S)HETE to further investigate the expression of CPAE cell retraction activity. The expression of 12(S)HETE in the surface of microparticles, as shown in Fig. 8.
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FIG. 8. The localization of CPAE-cell retraction activity in MCF-7 CM. The supernatant fraction and pellet fraction were separated from crude MCF-7 CM by ultracentrifuge (105000g, 150 min at 4°C). Lipids of the respective fraction were extracted, and subjected to endothelial-cell retraction assay as described in Materials and Methods. CPAE cell retraction activity of the respective fraction was expressed as the percent activity of untreated MCF-7 CM.
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DISCUSSION
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It is well known that cancer cell causes the retraction of vascular endothelial cell, one of the key steps for the establishment of tumor metastasis.3 Several molecules, such as vasoactive substance,14 lipid mediator,9,15 thrombin,16 or cytokines,17–20 have been reported to increase the permeability of vasculature, but the molecular mechanisms of vEC retraction by cancer cells has not been fully understood. One of coauthors, Nakamori, previously reported a cancer cell derived, heat resistant glycoprotein with MW 10–15kDa as vEC retracting factor.7 In this study, we demonstrated that approximately 60% of heat labile, vEC retracting activity of MCF-7 CM was derived from the lipid fraction, where 12(s)HETE played a major role in this process.
Since culture medium of cancer cells were considered to contain multiple vEC retracting factors, we first estimated what component was responsible for the vEC retracting activity of MCF-7 CM. As shown in Fig. 3, vEC retracting activity was heat labile. Trypsin digestion of CM abolished approximately 40% of vEC retracting activity, suggesting the some involvement of peptide molecules. By lipid extraction, approximately 60% of vEC retracting activity was recovered. These results suggest that a major vEC retracting activity in MCF-7 CM belonged to lipid component. Since several arachidonate metabolites have been reported to show vasoactive activity,21 we next tested the effects of inhibitors of arachidonate metabolism. As shown in Fig. 4, treatment of cancer cells with a cyclooxygenase inhibitor, acetylsalicylic acid (ASA), did not affect the secretion of vEC retracting activity, whereas, a lypooxgenase inhibitor, nordihydroguaiaretic acid (NDGA), markedly suppressed the generation of vEC retracting activity from cancer cells. These findings strongly suggested that vEC retracting activity of lipid component resulted from arachidonate metabolites via lipoxygenase.
To further investigate the lipid component responsible for vEC retracting activity, we separated the lipids extract from MCF-7 CM by thin layer chromatography. As shown in Fig. 6, most, 60% of vEC retracting activity was recovered from the lipid at Rf value 0.73, which corresponded to 12-HETE. There have been several reports describing the natural existence of two isoforms of 12HETE,22 12(S)-HETE and 12(R)-HETE. Kenneth et al. reported the roles of 12(S)-HETE in the signal transduction system of vECs integral to retraction.21 Therefore, we tested vEC retracting activity of these isomers. Consistent with the previous report by Kenneth et al., 12(S)-HETE, but not 12(R)-HETE showed any vEC retracting activity, as shown in Fig. 7. From these observations, it is suggested that major vEC retracting activity derived from cancer cells is due to 12(S)-HETE.
It is quite interesting to know how cancer cells generate and secrete hydrophobic 12(S)-HETE, which was demonstrated to be present in culture medium of cancer cells. One possibility is that 12(S)-HETE might be present in microparticles, formed by shedding from cancer cells. Actually, several reports including ours23 demonstrated the important role of micropartcles as a carrier for hydrophobic lipids in blood stream. In order to test this hypothesis, we separated vEC retracting activity of MCF-7 CM by ultracentrifugation. As shown in Fig. 8, almost all vEC retracting activity was recovered from pellet fraction. The association of 12(s)-HETE with microparticles was further confirmed by flow cytometric analysis with monoclonal antibody against 12(S)HETE. As shown in Fig. 9, the expression of 12(S)HETE was confirmed in MCF-7 cells derived microparticles.
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FIG. 9. Flow cytometric histogram of 12(S)HETE expression on ultracentrifugated pellet of CM. The pellet fraction of MCF-7 CM (1 ml) was incubated with 1µg of FITC-labeled mAb against 12(S)HETE or control IgG for 30min at 4°C, as described in Materials and Methods. Then, samples were analyzed on a fluorescence-activated cell sorter (FACScan). Dotted line: second antibody alone; Solid line: anti-12(S)HETE antibody plus second antibody.
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Cancer cells are activated and shed microparticles following various humoral or mechanical stimuli. 12(S)-HETE is generated during this process because intracellular biochemical pathways including arachidonate metabolism are also activated. Therefore, it is quite possible that 12HETE generated and present in microparticles plays an important role in vEC retraction and the subsequent establishment of cancer metastasis.
In summary, cancer cells secrete vEC retraction activity. 12(S)-HETE generated and present in microparticles play a major role in cancer cells induced vEC retraction.
Received for publication August 10, 2006.
Accepted for publication August 10, 2006.
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ABSTRACT
INTRODUCTION
