- Email: [email protected]
Secretory Phospholipase A2 Inhibition Attenuates Intercellular Adhesion Molecule-1 Expression in Human Esophageal Adenocarcinoma Cells
Secretory Phospholipase A2 Inhibition Attenuates Intercellular Adhesion Molecule-1 Expression in Human Esophageal Adenocarcinoma Cells
Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado
Background. Esophageal adenocarcinoma is an aggressive malignancy, with most patients succumbing to metastatic disease. The presence of intercellular adhesion molecule-1 (ICAM-1) in these cancer cells contributes to their metastatic potential. The ICAM-1 production in other cell types is stimulated by the actions of phospholipase enzymes. We hypothesize that inhibition of the enzyme secretory phospholipase A2 (sPLA2), which contributes to the growth potential of normal esophageal mucosa and esophageal cancer cells, may attenuate ICAM-1 production and nuclear factor-kappa beta activation in human esophageal adenocarcinoma cells. Methods. The FLO-1 verified human esophageal adenocarcinoma cells were treated with 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino) pentanoic acid, a specific inhibitor of group IIa sPLA2 (5 M, 10 M, and 15 M doses), followed by tumor necrosis factor-alpha stimulation (20 ng/mL). Cells and medium were collected and analyzed by immunoblotting, flow cytometry, and enzyme-linked immunosorbent assay. Statistical analysis was performed using analysis of variance with the Fisher’s least significant difference post-hoc test.
Results. Treatment with sPLA2 inhibitor attenuated total cellular ICAM-1 expression in a dose-dependent manner (p < 0.005). Cell-surface and secreted ICAM-1 expression decreased significantly with sPLA2 inhibitor treatment (p < 0.001 and p < 0.05, respectively). sPLA2 inhibition attenuated nuclear factor-kappa beta activation dose-dependently (p < 0.05). Conclusions. Esophageal adenocarcinoma has significant metastatic potential, and inhibiting its metastasis would significantly advance the treatment of this disease. We demonstrate here that treatment of human esophageal adenocarcinoma cells with sPLA2 inhibitor attenuates the expression of ICAM-1, a marker of metastatic potential, and nuclear factor-kappa beta activation, suggesting a common pathway between the two. These findings identify inhibition of sPLA2 as a potential therapeutic target for esophageal adenocarcinoma.
E
The expression of intercellular adhesion molecule-1 (ICAM-1) in tumor cells is thought to contribute to their metastatic potential and ultimately cancer lethality by permitting transendothelial tumor cell migration [6]. Although normal esophageal tissue does not express ICAM-1, the presence of ICAM-1 has been demonstrated in primary esophageal adenocarcinoma as well as its metastatic tissue, contributing to the metastatic potential of these tumor cells [7, 8]. In addition the expression of ICAM-1 in human esophageal cancer adversely affects prognosis. ICAM-1 expression is primarily modulated by translocation of nuclear factor-kappa beta (NF-B) to the nucleus of the cell, which is promoted by a variety of inflammatory stimuli including tumor necrosis factoralpha (TNF-␣) [9 –12]. Secretory phospholipase A2 (sPLA2), a subclass of phospholipase A2 enzymes, catalyzes the hydrolysis of membrane phospholipids leading to the production of various inflammatory mediators, such as prostaglandins, leukotrienes, and thromboxane [13, 14]. We have
sophageal adenocarcinoma is an aggressive malignancy with a rapidly rising incidence compared with other malignancies in the United States. In certain populations, the incidence has increased greater than 800% over the last 30 years [1, 2]. Despite advancements made in both surgery and adjuvant therapies, the overall prognosis remains bleak [3]. Early diagnosis is necessary for improved survival, but most patients present late in the disease process and often with multiple sites of metastasis [4, 5]. Given the high percentage of patients presenting with metastatic disease, it is critical to understand the mechanisms of dissemination of tumor cells to more effectively treat the disease.
Accepted for publication Jan 10, 2011. Presented at the Basic Science Forum of the Fifty-seventh Annual Meeting of the Southern Thoracic Surgical Association, Orlando, FL, Nov 3– 6, 2010. Address correspondence to Dr Weyant, Division of Cardiothoracic Surgery, University of Colorado at Denver and Health Sciences Center, Academic Office One, Room 6602, 12631 E 17th Ave, C310, Aurora, CO 80045; e-mail: [email protected].
© 2011 by The Society of Thoracic Surgeons Published by Elsevier Inc
(Ann Thorac Surg 2011;91:1539 – 45) © 2011 by The Society of Thoracic Surgeons
0003-4975/$36.00 doi:10.1016/j.athoracsur.2011.01.017
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Miral R. Sadaria, MD, Xianzhong Meng, MD, PhD, David A. Fullerton, MD, T. Brett Reece, MD, Roopali R. Shah, BA, Frederick L. Grover, MD, and Michael J. Weyant, MD
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Abbreviations DMSO ⫽ ELISA ⫽ FACS ⫽ FLO-1 ⫽ GAPDH ⫽
GENERAL THORACIC
ICAM-1 IgG NF-B PBS PE sPLA2 TNF-␣
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
and Acronyms dimethyl sulfoxide enzyme-linked immunosorbent assay fluorescence-activated cell sorting verified human esophageal adenocarcinoma cell line glyceraldehyde-3-phosphate dehydrogenase intercellular adhesion molecule-1 immunoglobulin G nuclear factor-kappa beta phosphate buffered saline phycoerythrin secretory phospholipase A2 tumor necrosis factor-alpha
previously identified sPLA2 to be an essential mediator of esophageal hyperplasia in vivo in response to gastroesophageal reflux [15–17]. In addition, we recently demonstrated that sPLA2 is not only expressed at baseline in human esophageal adenocarcinoma cells (FLO-1 and OE33 cell lines) but also regulates the growth and proliferation of these cells in vitro. Treatment of both of these cell lines with the specific group IIa sPLA2 inhibitor significantly attenuated growth and cell number. Furthermore, overexpression of the gene for group IIa sPLA2 significantly enhanced esophageal cancer cell growth, while gene knockdown significantly diminished growth [18]. In nonmalignant cells, inhibitors of sPLA2 have been shown to attenuate ICAM-1 production in response to inflammatory stimuli such as TNF-␣ [9]. We, therefore, hypothesized that specific inhibition of the sPLA2 enzyme, which contributes to the growth potential of both normal esophageal mucosa and esophageal cancer cells, may attenuate ICAM-1 production and NF-B activation in human esophageal adenocarcinoma cells to suggest a common pathway between the two.
Material and Methods Cell Line and Drug Preparation An institutional review board waiver was obtained for the use of FLO-1 cells, a verified human esophageal adenocarcinoma cell line, in this study. The FLO-1 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 g/mL), and fungizone (1.8 g/mL). Cells were cultured incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino) pentanoic acid (Sigma-Aldrich, St. Louis, MO), a specific inhibitor of group IIa sPLA2, was dissolved in dimethyl sulfoxide (DMSO). Prior to each experiment, stock solution of the drug was diluted in serum-reduced medium (0.5% fetal bovine serum) such that the concentration of DMSO never exceeded 0.03%. Recombinant human TNF-␣ (Sigma-Aldrich, St. Louis, MO) was reconstituted in sterile-filtered distilled water
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for a final concentration of 100 ng/L. This was aliquoted appropriately and stored at ⫺20°C until future use. At the time of use, the stock solution was thawed and dissolved in phosphate buffered saline (PBS) to a working concentration of 4 ng/L.
Cell Treatment The FLO-1 cells were cultured in full growth medium (10% fetal bovine serum) for 48 hours on a 6-well plate at a density of 3 ⫻ 105 cells per well for immunoblotting and 5 ⫻ 105 cells per well for flow cytometry. The cells were serum-reduced for 24 hours and then treated with either vehicle control (DMSO 0.03%) or sPLA2 inhibitor (5 M, 10 M, 15 M doses) such that all wells contained equivalent final DMSO concentrations of 0.03%. Cells were stimulated one hour later with 20 ng/mL of TNF-␣. In experiments evaluating the effect of TNF-␣ on ICAM-1 expression, cells were treated with either vehicle control (PBS), 20 ng/mL, or 40 ng/mL of TNF-␣. In experiments evaluating ICAM-1 expression, medium and cells were collected 12 hours after vehicle control or TNF-␣ treatment. In experiments evaluating NF-B expression, medium and cells were collected 10 minutes after TNF-␣ treatment. The medium was stored in ⫺80 °C for future use, and cells were collected appropriately for either immunoblotting or flow cytometry.
Immunoblotting Immunoblotting was used to detect ICAM-1 and NF-B (phosphorylated and total) with glyceraldehyde-3phosphate dehydrogenase (GAPDH) used as a housekeeping gene. After treatment was completed, cells were washed twice with PBS and lysed using Laemmli sample buffer (Bio-Rad Laboratories, Inc, Hercules, CA). Lysates were collected and stored in ⫺80 °C for future use. At the time of use, lysates were heated and loaded in a 4% to 20% linear gradient polyacrylamide gel (Bio-Rad Laboratories, Inc). The protein was transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Inc). The membrane was blocked in 5% non-fat dry milk for 1 hour at room temperature and then probed for ICAM-1 (1:200 rabbit anti-human ICAM-1 primary antibody; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), total NF-B (1:1000 rabbit anti-human total NF-B antibody; Cell Signaling Technology, Inc, Danvers, MA), or phosphorylated NF-B (1:1000 rabbit anti-human phospho-NF-B antibody; Cell Signaling Technology, Inc) overnight at 4°C. After incubation in primary antibodies, the membrane was washed 3 times for 5 minutes each in 0.1% PBSpolysorbate and incubated in 1:5000 horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, Inc) for 1 hour at room temperature. The membrane was washed again 3 times for 5 minutes each in 0.1% PBS-polysorbate. The membrane was developed with enhanced chemiluminescence (Pierce Protein Research Products, Thermo Scientific, Rockford, IL) and exposed on film. The GAPDH was probed for on the same membranes (1:1000 rabbit anti-human GAPDH antibody; Cell Signaling Technology, Inc) using the protocol described above. ImageJ (National Institute of Health,
Bethesda, MD) was used to determine densitometry of the protein band of interest.
Flow Cytometry Flow cytometry was used to detect cell-surface ICAM-1 expression with mouse immunoglobulin G (IgG) isotype used as a control. After treatment was completed, cells were washed twice with PBS and harvested with trypsin. Cells were centrifuged, and supernatant was aspirated off. Cells were resuspended in fluorescence-activated cell sorting (FACS) buffer, which consists of 1% bovine serum albumin and 0.05% sodium azide dissolved in Dulbecco’s phosphate buffered solution. Cells were centrifuged, and supernatant was aspirated off. Cells were resuspended in FACS buffer. Each sample was divided in half and aliquoted into 2 separate wells of a 96-well U-bottom plate, one for ICAM-1 staining and the other for isotype control staining. The 96-well plate was centrifuged, and FACS buffer was aspirated off. Cells were washed in FACS buffer twice. Cells were stained with either phycoerythrin (PE)conjugated mouse anti-human ICAM-1 (CD54) (eBioscience, Inc, San Diego, CA) or PE-conjugated mouse IgG1 isotype control (eBioscience, Inc) and washed twice with FACS buffer. Cells were fixed with 4% paraformaldehyde (USB Corporation, Cleveland, OH) and washed twice with FACS buffer. Cells were collected in FACS buffer and stored overnight in 4°C. The following morning, cell-surface ICAM-1 expression was determined using flow cytometry (CyAn ADP by Beckman Coulter, Brea, CA). Mean intensity fluorescence was determined using Summit software (Beckman Coulter).
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Phosphorylated and total NF-B densities were normalized to the GAPDH on their respective membranes, accounting for any variation in protein loading. The normalized phosphorylated NF-B density ratio was then divided by the normalized total NF-B density ratio. This adjusted unit was then normalized to the vehicle control to compare results between experimental groups, giving the vehicle control an adjusted unit value of 1.0. For all flow cytometry results, mean intensity fluorescence of each sample was divided by the vehicle control on the same experimental plate, giving the vehicle control an adjusted unit value of 1.0. This again allowed for comparison of results between experiments and for statistical analysis. For all ELISA results, raw data was used to perform statistical analysis. Data are presented as mean ⫾ standard error. Statistical analysis was performed using analysis of variance with the Fisher’s least significant difference post-hoc test (StatView by SAS Institute Inc, Cary, NC). For all statistical comparisons, a p value less than 0.05 was considered significant.
Results ICAM-1 Expression in FLO-1 Human Esophageal Adenocarcinoma Cells Stimulated With TNF-␣ In FLO-1 human esophageal adenocarcinoma cells, the expression of ICAM-1 peaked 12 hours after TNF-␣ administration (data not shown). Using immunoblotting, flow cytometry, and ELISA, 20 ng/mL of TNF-␣ stimulation increased total cellular, cell-surface, and secreted
Enzyme-Linked Immunosorbent Assay (ELISA) The medium from cells plated and treated in 6-well plates at 3 ⫻ 105 cells per well was used to perform ELISA and determine secreted ICAM-1 expression. At the time of use, the medium was thawed at room temperature. Secreted ICAM-1 expression was quantified using a commercially available ICAM-1 (CD54) ELISA kit according to manufacturer’s instructions (R&D Systems, Inc, Minneapolis, MN). Recombinant human soluble ICAM-1 was used as a standard. Absorbance of standards and samples were determined spectrophotometrically at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc). Results were plotted against the linear portion of the standard curve, and the concentration of secreted ICAM-1 was expressed as picogram/milliliter of sample.
Statistical Analysis For all immunoblotting results, a density ratio was created by dividing density of the primary band by density of the GAPDH band. The density ratio of each sample was then divided by the vehicle control on the same experimental plate, giving the vehicle control an adjusted unit value of 1.0. By normalizing to the vehicle control, results were able to be compared between experiments, allowing for statistical analysis.
Fig 1. The ICAM-1 expression in human esophageal adenocarcinoma cells (FLO-1) stimulated with TNF-␣. Stimulating FLO-1 cells with 20 ng/mL of TNF-␣ increased total cellular (immunoblotting), cell-surface (flow cytometry), and secreted (ELISA) ICAM-1 expression 2.6-fold, 2.0-fold, and 2.3-fold, respectively, compared with vehicle control. (*p ⬍ 0.05 compared with vehicle control, immunoblotting n ⫽ 5, flow cytometry n ⫽ 7, ELISA n ⫽ 6.) (ICAM-1 ⫽ intercellular adhesion molecule-1; ELISA ⫽ enzyme-linked immunosorbent assay; TNF-␣ ⫽ tumor necrosis factor-alpha.)
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ICAM-1 expression 2.6-fold, 2.0-fold, and 2.3-fold, respectively, compared to vehicle control (p ⬍ 0.05) (Fig 1).
Total Cellular ICAM-1 Expression Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells
GENERAL THORACIC
Immunoblotting demonstrated treatment of FLO-1 human esophageal adenocarcinoma cells with specific group IIa sPLA2 inhibitor decreased total cellular ICAM-1 expression in a dose-dependent fashion (Fig 2A). The 5 M, 10 M, and 15 M doses produced a 16.2%, 28.0%, and 52.1% reduction in total cellular ICAM-1 expression, respectively, compared with the vehicle control (p ⬍ 0.05) (Fig 2B).
Cell-Surface ICAM-1 Expression Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells Flow cytometry demonstrated treatment of FLO-1 human esophageal adenocarcinoma cells with specific sPLA2
Fig 3. Cell-surface ICAM-1 expression attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO1). A representative flow cytometry histogram demonstrates a reduction in mean intensity fluorescence and, thus, cell-surface ICAM-1 expression after treatment of FLO-1 cells with specific group IIa. sPLA2 inhibitor (A). The mean intensity fluorescence of each sample was normalized to the vehicle control, giving the vehicle control an adjusted unit of 1.0. Cell-surface ICAM-1 expression decreased at the 15 M dose (B). (*p ⬍ 0.001 compared with vehicle control and all doses, n ⫽ 7.) (ICAM-1 ⫽ intercellular adhesion molecule-1; PE ⫽ phycoerythrin; sPLA2 ⫽ secretory phospholipase A2.)
group IIa inhibitor at 15 M concentration decreased cell-surface ICAM-1 expression 32.7% compared with the vehicle control (p ⬍ 0.001) (Figs 3A;B). Fig 2. Total cellular ICAM-1 expression attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO1). A representative Western blot demonstrates a dose-dependent attenuation of total cellular ICAM-1 expression in FLO-1 cells when treated with specific group IIa sPLA2 inhibitor (A). A density ratio was created by dividing the density of the ICAM-1 by the density of the GAPDH band. The density ratio of each sample was then divided by the vehicle control, giving the vehicle control an adjusted unit value of 1.0. All doses of sPLA2 inhibitor reduced total cellular ICAM-1 expression compared with control (B). (*p ⬍ 0.05 compared with vehicle control and 15 M dose; **p ⬍ 0.005 compared with vehicle control and all doses, n ⫽ 5.) (GAPDH ⫽ glyceraldehyde-3phosphate dehydrogenase; ICAM-1 ⫽ intercellular adhesion molecule-1; sPLA2 ⫽ secretory phospholipase A2.)
Secreted ICAM-1 Expression Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells Using ELISA, the medium collected after treatment of FLO-1 human esophageal adenocarcinoma cells was analyzed for soluble ICAM-1 expression. Treatment with specific group IIa sPLA2 inhibitor at the 10 M and 15 M doses attenuated soluble ICAM-1 expression 16.6% and 27.5%, respectively, compared with the vehicle control, indicating that secretion of the ICAM-1 adhesion molecule was attenuated by treatment with the inhibitor (p ⬍ 0.05) (Fig 4).
Fig 4. Secreted ICAM-1 expression attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO-1). Specific group IIa sPLA2 inhibitor treatment attenuated ICAM-1 secretion by FLO-1 cells at the 10 M and 15 M dose. (*p ⬍ 0.05 compared with vehicle control; **p ⬍ 0.05 compared with vehicle control and 5 M dose, n ⫽ 6.) (ICAM-1 ⫽ intercellular adhesion molecule-1.)
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In addition, recognizing the similarity between inflammation and tumorigenesis may help define a beneficial role of antiinflammatory pharmacotherapy in cancer treatment [19]. The tumor microenvironment contains many components of inflammation that are often associated with poor prognosis, such as TNF-␣ and interferon-gamma [20]. These inflammatory mediators, in turn, activate NF-B, which translocates to the nucleus to stimulate production of numerous proteins, including ICAM-1. TNF-␣, in particular, has been demonstrated to widely generate an ICAM-1 response in numerous cancer cell lines [11, 12, 21, 22]. The presence of ICAM-1 in these cancer cells clinically correlates with a higher propensity to metastasize and has been shown to be an adverse prognostic indicator in various human cancers [7, 11, 12, 23]. ICAM-1 can be expressed in several forms, such as intracellular, cell-surface, and secreted. Although this study demonstrates that sPLA2 mediates expression of all of these forms of ICAM-1, cell-surface ICAM-1 has been implicated as the major culprit in metastasis. The tumor cell is thought to bind to ICAM-1 on the surface of endothelial
NF-B Activation Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells In FLO-1 human esophageal adenocarcinoma cells, NF-B phosphorylation (activation) peaked 10 minutes after TNF-␣ administration (data not shown). When compared with the vehicle control, immunoblotting demonstrated an attenuation of NF-B activation by 25.8% after treatment of FLO-1 cells with specific group IIa sPLA2 inhibitor at the 15 M dose (p ⬍ 0.05) (Figs 5A;B).
Comment The aim of this study was to determine the effect of sPLA2 on ICAM-1 expression in human esophageal adenocarcinoma cells and the mechanism by which this occurs. The results of the study demonstrate that inhibition of sPLA2 in stimulated human esophageal adenocarcinoma cells (FLO-1 cells) attenuated expression of total cellular, cell-surface, and secreted ICAM-1 expression and activation of NF-B, suggesting a common pathway between the inhibitor’s effect on ICAM-1 expression and NF-B activation. Intercellular adhesion molecule-1 is a member of a family of adhesion molecules that have been extensively studied in their relationship to mechanisms of inflammation. During inflammation these molecules are responsible for recruiting leukocytes onto the vascular endothelium prior to circulating to the injured tissue. Cancer cells have been demonstrated to act analogous to these inflammatory cells by extravasating to sites beyond the primary tumor. This shared process between inflammatory cells and cancer cells may help explain the link between inflammation and the development of tumors.
Fig 5. NF-B activation attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO-1). A representative Western blot demonstrates downregulation of NF-B activation in FLO-1 cells when treated with specific group IIa sPLA2 inhibitor (A). The phosphorylated and total NF-B densities were normalized to the glyceraldehyde-3-phosphate dehydrogenase on their respective membranes. The normalized phosphorylated NF-B density ratio was then divided by the normalized total NF-B density ratio. This adjusted unit was then normalized to the vehicle control, giving the vehicle control an adjusted unit value of 1.0. A decrease in NF-B activation was demonstrated specifically at the 15 M dose of sPLA2 inhibitor (B). (*p ⬍ 0.05 compared with vehicle control and 5 M dose, n ⫽ 5.) (NF-B ⫽ nuclear factor-kappa beta).
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cells, inducing both the release of chemoattractant cytokines and the upregulation of ICAM-1 expression by the tumor cell itself. This leads to neutrophils binding to the tumor’s cell-surface ICAM-1, which promotes degranulation of the neutrophil and finally transendothelial tumor cell migration [6]. By inhibiting cell-surface ICAM-1 expression as was done in this study, we hypothesize that the ability of the tumor cell to migrate and form metastases is impeded. TNF-␣ has also been demonstrated to induce the synthesis and secretion of an enzyme known as phospholipase A2 in various human cells, such as keratinocytes, synovial cells, and intestinal epithelial cells [9, 10]. sPLA2, a subclass of phospholipase A2, catalyzes the hydrolysis of membrane phospholipids to generate free fatty acids and lysophospholipids, such as arachidonic acid, the rate-limiting step in eicosanoid production [13, 14]. These lipid mediators generated by sPLA2 activate protein kinase C, which has been suggested to be involved in TNF-␣-mediated NF-B activation (Fig 6) [9, 24]. Furthermore, upregulation of sPLA2 has been implicated as a pathogenic factor not only in a variety of inflammatory processes, such as ulcerative colitis, Crohn’s disease, acute pancreatitis, septic shock, and bronchial asthma, but also in multiple human tumors, such as prostate, ovarian, gastric, and intestinal cancer [13, 14]. We re-
Fig 6. Schematic illustrating the proposed relationship between sPLA2 and the TNF-␣ signaling pathway and the proposed mechanism of action of the specific sPLA2 inhibitor. sPLA2 catalyzes the hydrolysis of membrane phospholipids to generate free fatty acids such as arachidonic acid. These lipid mediators activate protein kinase C, which has been suggested to be involved in TNF-␣-mediated NF-B activation and, ultimately, ICAM-1 expression. The specific sPLA2 inhibitor functions by inhibiting the hydrolysis of membrane phospholipids as denoted by the X in the figure, thus preventing protein kinase C activation and leading to attenuated TNF␣-mediated NF-B activation and ICAM-1 expression. (ICAM-1 ⫽ intercellular adhesion molecule-1; NF-B ⫽ nuclear factor-kappa beta; sPLA2 ⫽ secretory phospholipase A2; TNF-␣—tumor necrosis factor-alpha.)
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cently demonstrated that sPLA2 regulates the growth and proliferation of human esophageal adenocarcinoma cells in vitro [18]. Given the available data regarding sPLA2 and NF-B-induced ICAM-1 production, we hypothesized that inhibition of this enzyme in esophageal cancer cells would lead to decreased ICAM-1 production. These data demonstrate that sPLA2 may be a potential chemotherapeutic target in patients with esophageal adenocarcinoma. Inhibition of sPLA2, through its secondary effects of reducing ICAM-1 expression, has the potential to act in conjunction with standard chemotherapeutic regimens to prevent or treat metastatic disease. Such advances in the medical management of esophageal adenocarcinoma could improve patient survival and overall prognosis. Furthermore, inhibition of sPLA2 can be used as a prophylactic measure for those at risk of developing esophageal adenocarcinoma. Both Barrett’s esophagus, a well-established precursor lesion to esophageal adenocarcinoma, and inflamed esophageal tissue have been shown to express ICAM-1 as well [8, 25, 26]. By targeting sPLA2 and attenuating ICAM-1 expression, the transformation to esophageal adenocarcinoma may be prevented. Thus, inhibition of sPLA2 could be used not only therapeutically in those suffering from esophageal adenocarcinoma, but also prophylactically in high-risk patients with Barrett’s esophagus or severe gastroesophageal reflux disease. In summary, the results of the present study demonstrate that treatment of human esophageal adenocarcinoma cells with a sPLA2 inhibitor attenuates the expression of total cellular, cell-surface, and secreted ICAM-1 as well as the activation of NF-B, suggesting a common pathway between the mechanism of action of specific sPLA2 inhibition on ICAM-1 expression and NF-B activation. To our knowledge, this is the first report not only of the abundant expression of ICAM-1 at baseline in FLO-1 cells, but also of the role of sPLA2, TNF-␣, and NF-B in ICAM-1 expression of these cells. Although this study demonstrates a clear association between sPLA2 and ICAM-1 expression as well as sPLA2 and NF-B activation, it is unclear if these results would be comparable in an in vivo setting. These findings could identify inhibition of sPLA2 as a potential mode of therapy and target for prevention of esophageal adenocarcinoma. We would like to thank Dr David Beer (Department of Surgery, University of Michigan, Ann Arbor, MI) for his generous gift of the FLO-1 human esophageal adenocarcinoma cell line. Our study was supported by the University of Colorado Academic Enrichment Funds.
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15. Mauchley D, Meng X, Babu A, et al. Gastroduodenal reflux induces group IIa secretory phospholipase A2 expression and activity in murine esophagus. Dis Esophagus 2010;23: 430 – 6. 16. Babu A, Mauchley D, Meng X, et al. The secretory phospholipase A2 gene is required for gastroesophageal refluxrelated changes in murine esophagus. J Gastrointest Surg 2009;13:2212– 8. 17. Babu A, Meng X, Banerjee AM, et al. Secretory phospholipase A2 is required to produce histologic changes associated with gastroduodenal reflux in a murine model. J Thorac Cardiovasc Surg 2008;135:1220 –7. 18. Mauchley D, Meng X, Johnson T, Fullerton DA, Weyant MJ. Modulation of growth in human esophageal adenocarcinoma cells by group IIa secretory phospholipase A2. J Thorac Cardiovasc Surg 2010;139:591–9. 19. Kobayashi H, Boelte KC, Lin PC. Endothelial cell adhesion molecules and cancer progression. Curr Med Chem 2007;14: 377– 86. 20. Wu Y, Zhou BP. TNF-␣/NF-B/Snail pathway in cancer cell migration and invasion. Br J Cancer 2010;102:639 – 44. 21. Mueller L, von Seggern L, Schumacher J, et al. TNF-␣ similarly induces IL-6 and MCP-1 in fibroblasts from colorectal liver metastases and normal liver fibroblasts. Biochem Biophys Res Commun 2010;397:586 –91. 22. Katerinaki E, Haycock JW, Lalla R, et al. Sodium salicylate inhibits TNF-␣-induced NF-B activation, cell migration, invasion and ICAM-1 expression in human melanoma cells. Melanoma Res 2006;16:11–22. 23. Rosette C, Roth RB, Oeth P, et al. Role of ICAM1 in invasion of human breast cancer cells. Carcinogenesis 2005;26:943–50. 24. Lozano J, Berra E, Municio MM, et al. Protein kinase C isoform is critical for B-dependent promoter activation by sphingomyelinase. J Biol Chem 1994;269:19200 –2. 25. Tozzi A, Staiano A, Paparo F, et al. Characterization of the inflammatory infiltrate in peptic oesophagitis. Dig Liver Dis 2001;33:452– 8. 26. Hamaguchi M, Fujiwara Y, Takashima T, et al. Increased expression of cytokines and adhesion molecules in rat chronic esophagitis. Digestion 2003;68:189 –97.
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Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado
Background. Esophageal adenocarcinoma is an aggressive malignancy, with most patients succumbing to metastatic disease. The presence of intercellular adhesion molecule-1 (ICAM-1) in these cancer cells contributes to their metastatic potential. The ICAM-1 production in other cell types is stimulated by the actions of phospholipase enzymes. We hypothesize that inhibition of the enzyme secretory phospholipase A2 (sPLA2), which contributes to the growth potential of normal esophageal mucosa and esophageal cancer cells, may attenuate ICAM-1 production and nuclear factor-kappa beta activation in human esophageal adenocarcinoma cells. Methods. The FLO-1 verified human esophageal adenocarcinoma cells were treated with 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino) pentanoic acid, a specific inhibitor of group IIa sPLA2 (5 M, 10 M, and 15 M doses), followed by tumor necrosis factor-alpha stimulation (20 ng/mL). Cells and medium were collected and analyzed by immunoblotting, flow cytometry, and enzyme-linked immunosorbent assay. Statistical analysis was performed using analysis of variance with the Fisher’s least significant difference post-hoc test.
Results. Treatment with sPLA2 inhibitor attenuated total cellular ICAM-1 expression in a dose-dependent manner (p < 0.005). Cell-surface and secreted ICAM-1 expression decreased significantly with sPLA2 inhibitor treatment (p < 0.001 and p < 0.05, respectively). sPLA2 inhibition attenuated nuclear factor-kappa beta activation dose-dependently (p < 0.05). Conclusions. Esophageal adenocarcinoma has significant metastatic potential, and inhibiting its metastasis would significantly advance the treatment of this disease. We demonstrate here that treatment of human esophageal adenocarcinoma cells with sPLA2 inhibitor attenuates the expression of ICAM-1, a marker of metastatic potential, and nuclear factor-kappa beta activation, suggesting a common pathway between the two. These findings identify inhibition of sPLA2 as a potential therapeutic target for esophageal adenocarcinoma.
E
The expression of intercellular adhesion molecule-1 (ICAM-1) in tumor cells is thought to contribute to their metastatic potential and ultimately cancer lethality by permitting transendothelial tumor cell migration [6]. Although normal esophageal tissue does not express ICAM-1, the presence of ICAM-1 has been demonstrated in primary esophageal adenocarcinoma as well as its metastatic tissue, contributing to the metastatic potential of these tumor cells [7, 8]. In addition the expression of ICAM-1 in human esophageal cancer adversely affects prognosis. ICAM-1 expression is primarily modulated by translocation of nuclear factor-kappa beta (NF-B) to the nucleus of the cell, which is promoted by a variety of inflammatory stimuli including tumor necrosis factoralpha (TNF-␣) [9 –12]. Secretory phospholipase A2 (sPLA2), a subclass of phospholipase A2 enzymes, catalyzes the hydrolysis of membrane phospholipids leading to the production of various inflammatory mediators, such as prostaglandins, leukotrienes, and thromboxane [13, 14]. We have
sophageal adenocarcinoma is an aggressive malignancy with a rapidly rising incidence compared with other malignancies in the United States. In certain populations, the incidence has increased greater than 800% over the last 30 years [1, 2]. Despite advancements made in both surgery and adjuvant therapies, the overall prognosis remains bleak [3]. Early diagnosis is necessary for improved survival, but most patients present late in the disease process and often with multiple sites of metastasis [4, 5]. Given the high percentage of patients presenting with metastatic disease, it is critical to understand the mechanisms of dissemination of tumor cells to more effectively treat the disease.
Accepted for publication Jan 10, 2011. Presented at the Basic Science Forum of the Fifty-seventh Annual Meeting of the Southern Thoracic Surgical Association, Orlando, FL, Nov 3– 6, 2010. Address correspondence to Dr Weyant, Division of Cardiothoracic Surgery, University of Colorado at Denver and Health Sciences Center, Academic Office One, Room 6602, 12631 E 17th Ave, C310, Aurora, CO 80045; e-mail: [email protected].
© 2011 by The Society of Thoracic Surgeons Published by Elsevier Inc
(Ann Thorac Surg 2011;91:1539 – 45) © 2011 by The Society of Thoracic Surgeons
0003-4975/$36.00 doi:10.1016/j.athoracsur.2011.01.017
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Miral R. Sadaria, MD, Xianzhong Meng, MD, PhD, David A. Fullerton, MD, T. Brett Reece, MD, Roopali R. Shah, BA, Frederick L. Grover, MD, and Michael J. Weyant, MD
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Abbreviations DMSO ⫽ ELISA ⫽ FACS ⫽ FLO-1 ⫽ GAPDH ⫽
GENERAL THORACIC
ICAM-1 IgG NF-B PBS PE sPLA2 TNF-␣
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
and Acronyms dimethyl sulfoxide enzyme-linked immunosorbent assay fluorescence-activated cell sorting verified human esophageal adenocarcinoma cell line glyceraldehyde-3-phosphate dehydrogenase intercellular adhesion molecule-1 immunoglobulin G nuclear factor-kappa beta phosphate buffered saline phycoerythrin secretory phospholipase A2 tumor necrosis factor-alpha
previously identified sPLA2 to be an essential mediator of esophageal hyperplasia in vivo in response to gastroesophageal reflux [15–17]. In addition, we recently demonstrated that sPLA2 is not only expressed at baseline in human esophageal adenocarcinoma cells (FLO-1 and OE33 cell lines) but also regulates the growth and proliferation of these cells in vitro. Treatment of both of these cell lines with the specific group IIa sPLA2 inhibitor significantly attenuated growth and cell number. Furthermore, overexpression of the gene for group IIa sPLA2 significantly enhanced esophageal cancer cell growth, while gene knockdown significantly diminished growth [18]. In nonmalignant cells, inhibitors of sPLA2 have been shown to attenuate ICAM-1 production in response to inflammatory stimuli such as TNF-␣ [9]. We, therefore, hypothesized that specific inhibition of the sPLA2 enzyme, which contributes to the growth potential of both normal esophageal mucosa and esophageal cancer cells, may attenuate ICAM-1 production and NF-B activation in human esophageal adenocarcinoma cells to suggest a common pathway between the two.
Material and Methods Cell Line and Drug Preparation An institutional review board waiver was obtained for the use of FLO-1 cells, a verified human esophageal adenocarcinoma cell line, in this study. The FLO-1 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 g/mL), and fungizone (1.8 g/mL). Cells were cultured incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino) pentanoic acid (Sigma-Aldrich, St. Louis, MO), a specific inhibitor of group IIa sPLA2, was dissolved in dimethyl sulfoxide (DMSO). Prior to each experiment, stock solution of the drug was diluted in serum-reduced medium (0.5% fetal bovine serum) such that the concentration of DMSO never exceeded 0.03%. Recombinant human TNF-␣ (Sigma-Aldrich, St. Louis, MO) was reconstituted in sterile-filtered distilled water
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for a final concentration of 100 ng/L. This was aliquoted appropriately and stored at ⫺20°C until future use. At the time of use, the stock solution was thawed and dissolved in phosphate buffered saline (PBS) to a working concentration of 4 ng/L.
Cell Treatment The FLO-1 cells were cultured in full growth medium (10% fetal bovine serum) for 48 hours on a 6-well plate at a density of 3 ⫻ 105 cells per well for immunoblotting and 5 ⫻ 105 cells per well for flow cytometry. The cells were serum-reduced for 24 hours and then treated with either vehicle control (DMSO 0.03%) or sPLA2 inhibitor (5 M, 10 M, 15 M doses) such that all wells contained equivalent final DMSO concentrations of 0.03%. Cells were stimulated one hour later with 20 ng/mL of TNF-␣. In experiments evaluating the effect of TNF-␣ on ICAM-1 expression, cells were treated with either vehicle control (PBS), 20 ng/mL, or 40 ng/mL of TNF-␣. In experiments evaluating ICAM-1 expression, medium and cells were collected 12 hours after vehicle control or TNF-␣ treatment. In experiments evaluating NF-B expression, medium and cells were collected 10 minutes after TNF-␣ treatment. The medium was stored in ⫺80 °C for future use, and cells were collected appropriately for either immunoblotting or flow cytometry.
Immunoblotting Immunoblotting was used to detect ICAM-1 and NF-B (phosphorylated and total) with glyceraldehyde-3phosphate dehydrogenase (GAPDH) used as a housekeeping gene. After treatment was completed, cells were washed twice with PBS and lysed using Laemmli sample buffer (Bio-Rad Laboratories, Inc, Hercules, CA). Lysates were collected and stored in ⫺80 °C for future use. At the time of use, lysates were heated and loaded in a 4% to 20% linear gradient polyacrylamide gel (Bio-Rad Laboratories, Inc). The protein was transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Inc). The membrane was blocked in 5% non-fat dry milk for 1 hour at room temperature and then probed for ICAM-1 (1:200 rabbit anti-human ICAM-1 primary antibody; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), total NF-B (1:1000 rabbit anti-human total NF-B antibody; Cell Signaling Technology, Inc, Danvers, MA), or phosphorylated NF-B (1:1000 rabbit anti-human phospho-NF-B antibody; Cell Signaling Technology, Inc) overnight at 4°C. After incubation in primary antibodies, the membrane was washed 3 times for 5 minutes each in 0.1% PBSpolysorbate and incubated in 1:5000 horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, Inc) for 1 hour at room temperature. The membrane was washed again 3 times for 5 minutes each in 0.1% PBS-polysorbate. The membrane was developed with enhanced chemiluminescence (Pierce Protein Research Products, Thermo Scientific, Rockford, IL) and exposed on film. The GAPDH was probed for on the same membranes (1:1000 rabbit anti-human GAPDH antibody; Cell Signaling Technology, Inc) using the protocol described above. ImageJ (National Institute of Health,
Bethesda, MD) was used to determine densitometry of the protein band of interest.
Flow Cytometry Flow cytometry was used to detect cell-surface ICAM-1 expression with mouse immunoglobulin G (IgG) isotype used as a control. After treatment was completed, cells were washed twice with PBS and harvested with trypsin. Cells were centrifuged, and supernatant was aspirated off. Cells were resuspended in fluorescence-activated cell sorting (FACS) buffer, which consists of 1% bovine serum albumin and 0.05% sodium azide dissolved in Dulbecco’s phosphate buffered solution. Cells were centrifuged, and supernatant was aspirated off. Cells were resuspended in FACS buffer. Each sample was divided in half and aliquoted into 2 separate wells of a 96-well U-bottom plate, one for ICAM-1 staining and the other for isotype control staining. The 96-well plate was centrifuged, and FACS buffer was aspirated off. Cells were washed in FACS buffer twice. Cells were stained with either phycoerythrin (PE)conjugated mouse anti-human ICAM-1 (CD54) (eBioscience, Inc, San Diego, CA) or PE-conjugated mouse IgG1 isotype control (eBioscience, Inc) and washed twice with FACS buffer. Cells were fixed with 4% paraformaldehyde (USB Corporation, Cleveland, OH) and washed twice with FACS buffer. Cells were collected in FACS buffer and stored overnight in 4°C. The following morning, cell-surface ICAM-1 expression was determined using flow cytometry (CyAn ADP by Beckman Coulter, Brea, CA). Mean intensity fluorescence was determined using Summit software (Beckman Coulter).
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Phosphorylated and total NF-B densities were normalized to the GAPDH on their respective membranes, accounting for any variation in protein loading. The normalized phosphorylated NF-B density ratio was then divided by the normalized total NF-B density ratio. This adjusted unit was then normalized to the vehicle control to compare results between experimental groups, giving the vehicle control an adjusted unit value of 1.0. For all flow cytometry results, mean intensity fluorescence of each sample was divided by the vehicle control on the same experimental plate, giving the vehicle control an adjusted unit value of 1.0. This again allowed for comparison of results between experiments and for statistical analysis. For all ELISA results, raw data was used to perform statistical analysis. Data are presented as mean ⫾ standard error. Statistical analysis was performed using analysis of variance with the Fisher’s least significant difference post-hoc test (StatView by SAS Institute Inc, Cary, NC). For all statistical comparisons, a p value less than 0.05 was considered significant.
Results ICAM-1 Expression in FLO-1 Human Esophageal Adenocarcinoma Cells Stimulated With TNF-␣ In FLO-1 human esophageal adenocarcinoma cells, the expression of ICAM-1 peaked 12 hours after TNF-␣ administration (data not shown). Using immunoblotting, flow cytometry, and ELISA, 20 ng/mL of TNF-␣ stimulation increased total cellular, cell-surface, and secreted
Enzyme-Linked Immunosorbent Assay (ELISA) The medium from cells plated and treated in 6-well plates at 3 ⫻ 105 cells per well was used to perform ELISA and determine secreted ICAM-1 expression. At the time of use, the medium was thawed at room temperature. Secreted ICAM-1 expression was quantified using a commercially available ICAM-1 (CD54) ELISA kit according to manufacturer’s instructions (R&D Systems, Inc, Minneapolis, MN). Recombinant human soluble ICAM-1 was used as a standard. Absorbance of standards and samples were determined spectrophotometrically at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc). Results were plotted against the linear portion of the standard curve, and the concentration of secreted ICAM-1 was expressed as picogram/milliliter of sample.
Statistical Analysis For all immunoblotting results, a density ratio was created by dividing density of the primary band by density of the GAPDH band. The density ratio of each sample was then divided by the vehicle control on the same experimental plate, giving the vehicle control an adjusted unit value of 1.0. By normalizing to the vehicle control, results were able to be compared between experiments, allowing for statistical analysis.
Fig 1. The ICAM-1 expression in human esophageal adenocarcinoma cells (FLO-1) stimulated with TNF-␣. Stimulating FLO-1 cells with 20 ng/mL of TNF-␣ increased total cellular (immunoblotting), cell-surface (flow cytometry), and secreted (ELISA) ICAM-1 expression 2.6-fold, 2.0-fold, and 2.3-fold, respectively, compared with vehicle control. (*p ⬍ 0.05 compared with vehicle control, immunoblotting n ⫽ 5, flow cytometry n ⫽ 7, ELISA n ⫽ 6.) (ICAM-1 ⫽ intercellular adhesion molecule-1; ELISA ⫽ enzyme-linked immunosorbent assay; TNF-␣ ⫽ tumor necrosis factor-alpha.)
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ICAM-1 expression 2.6-fold, 2.0-fold, and 2.3-fold, respectively, compared to vehicle control (p ⬍ 0.05) (Fig 1).
Total Cellular ICAM-1 Expression Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells
GENERAL THORACIC
Immunoblotting demonstrated treatment of FLO-1 human esophageal adenocarcinoma cells with specific group IIa sPLA2 inhibitor decreased total cellular ICAM-1 expression in a dose-dependent fashion (Fig 2A). The 5 M, 10 M, and 15 M doses produced a 16.2%, 28.0%, and 52.1% reduction in total cellular ICAM-1 expression, respectively, compared with the vehicle control (p ⬍ 0.05) (Fig 2B).
Cell-Surface ICAM-1 Expression Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells Flow cytometry demonstrated treatment of FLO-1 human esophageal adenocarcinoma cells with specific sPLA2
Fig 3. Cell-surface ICAM-1 expression attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO1). A representative flow cytometry histogram demonstrates a reduction in mean intensity fluorescence and, thus, cell-surface ICAM-1 expression after treatment of FLO-1 cells with specific group IIa. sPLA2 inhibitor (A). The mean intensity fluorescence of each sample was normalized to the vehicle control, giving the vehicle control an adjusted unit of 1.0. Cell-surface ICAM-1 expression decreased at the 15 M dose (B). (*p ⬍ 0.001 compared with vehicle control and all doses, n ⫽ 7.) (ICAM-1 ⫽ intercellular adhesion molecule-1; PE ⫽ phycoerythrin; sPLA2 ⫽ secretory phospholipase A2.)
group IIa inhibitor at 15 M concentration decreased cell-surface ICAM-1 expression 32.7% compared with the vehicle control (p ⬍ 0.001) (Figs 3A;B). Fig 2. Total cellular ICAM-1 expression attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO1). A representative Western blot demonstrates a dose-dependent attenuation of total cellular ICAM-1 expression in FLO-1 cells when treated with specific group IIa sPLA2 inhibitor (A). A density ratio was created by dividing the density of the ICAM-1 by the density of the GAPDH band. The density ratio of each sample was then divided by the vehicle control, giving the vehicle control an adjusted unit value of 1.0. All doses of sPLA2 inhibitor reduced total cellular ICAM-1 expression compared with control (B). (*p ⬍ 0.05 compared with vehicle control and 15 M dose; **p ⬍ 0.005 compared with vehicle control and all doses, n ⫽ 5.) (GAPDH ⫽ glyceraldehyde-3phosphate dehydrogenase; ICAM-1 ⫽ intercellular adhesion molecule-1; sPLA2 ⫽ secretory phospholipase A2.)
Secreted ICAM-1 Expression Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells Using ELISA, the medium collected after treatment of FLO-1 human esophageal adenocarcinoma cells was analyzed for soluble ICAM-1 expression. Treatment with specific group IIa sPLA2 inhibitor at the 10 M and 15 M doses attenuated soluble ICAM-1 expression 16.6% and 27.5%, respectively, compared with the vehicle control, indicating that secretion of the ICAM-1 adhesion molecule was attenuated by treatment with the inhibitor (p ⬍ 0.05) (Fig 4).
Fig 4. Secreted ICAM-1 expression attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO-1). Specific group IIa sPLA2 inhibitor treatment attenuated ICAM-1 secretion by FLO-1 cells at the 10 M and 15 M dose. (*p ⬍ 0.05 compared with vehicle control; **p ⬍ 0.05 compared with vehicle control and 5 M dose, n ⫽ 6.) (ICAM-1 ⫽ intercellular adhesion molecule-1.)
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In addition, recognizing the similarity between inflammation and tumorigenesis may help define a beneficial role of antiinflammatory pharmacotherapy in cancer treatment [19]. The tumor microenvironment contains many components of inflammation that are often associated with poor prognosis, such as TNF-␣ and interferon-gamma [20]. These inflammatory mediators, in turn, activate NF-B, which translocates to the nucleus to stimulate production of numerous proteins, including ICAM-1. TNF-␣, in particular, has been demonstrated to widely generate an ICAM-1 response in numerous cancer cell lines [11, 12, 21, 22]. The presence of ICAM-1 in these cancer cells clinically correlates with a higher propensity to metastasize and has been shown to be an adverse prognostic indicator in various human cancers [7, 11, 12, 23]. ICAM-1 can be expressed in several forms, such as intracellular, cell-surface, and secreted. Although this study demonstrates that sPLA2 mediates expression of all of these forms of ICAM-1, cell-surface ICAM-1 has been implicated as the major culprit in metastasis. The tumor cell is thought to bind to ICAM-1 on the surface of endothelial
NF-B Activation Attenuated by Group IIa sPLA2 Inhibition in FLO-1 Human Esophageal Adenocarcinoma Cells In FLO-1 human esophageal adenocarcinoma cells, NF-B phosphorylation (activation) peaked 10 minutes after TNF-␣ administration (data not shown). When compared with the vehicle control, immunoblotting demonstrated an attenuation of NF-B activation by 25.8% after treatment of FLO-1 cells with specific group IIa sPLA2 inhibitor at the 15 M dose (p ⬍ 0.05) (Figs 5A;B).
Comment The aim of this study was to determine the effect of sPLA2 on ICAM-1 expression in human esophageal adenocarcinoma cells and the mechanism by which this occurs. The results of the study demonstrate that inhibition of sPLA2 in stimulated human esophageal adenocarcinoma cells (FLO-1 cells) attenuated expression of total cellular, cell-surface, and secreted ICAM-1 expression and activation of NF-B, suggesting a common pathway between the inhibitor’s effect on ICAM-1 expression and NF-B activation. Intercellular adhesion molecule-1 is a member of a family of adhesion molecules that have been extensively studied in their relationship to mechanisms of inflammation. During inflammation these molecules are responsible for recruiting leukocytes onto the vascular endothelium prior to circulating to the injured tissue. Cancer cells have been demonstrated to act analogous to these inflammatory cells by extravasating to sites beyond the primary tumor. This shared process between inflammatory cells and cancer cells may help explain the link between inflammation and the development of tumors.
Fig 5. NF-B activation attenuated by inhibition of group IIa sPLA2 in human esophageal adenocarcinoma cells (FLO-1). A representative Western blot demonstrates downregulation of NF-B activation in FLO-1 cells when treated with specific group IIa sPLA2 inhibitor (A). The phosphorylated and total NF-B densities were normalized to the glyceraldehyde-3-phosphate dehydrogenase on their respective membranes. The normalized phosphorylated NF-B density ratio was then divided by the normalized total NF-B density ratio. This adjusted unit was then normalized to the vehicle control, giving the vehicle control an adjusted unit value of 1.0. A decrease in NF-B activation was demonstrated specifically at the 15 M dose of sPLA2 inhibitor (B). (*p ⬍ 0.05 compared with vehicle control and 5 M dose, n ⫽ 5.) (NF-B ⫽ nuclear factor-kappa beta).
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cells, inducing both the release of chemoattractant cytokines and the upregulation of ICAM-1 expression by the tumor cell itself. This leads to neutrophils binding to the tumor’s cell-surface ICAM-1, which promotes degranulation of the neutrophil and finally transendothelial tumor cell migration [6]. By inhibiting cell-surface ICAM-1 expression as was done in this study, we hypothesize that the ability of the tumor cell to migrate and form metastases is impeded. TNF-␣ has also been demonstrated to induce the synthesis and secretion of an enzyme known as phospholipase A2 in various human cells, such as keratinocytes, synovial cells, and intestinal epithelial cells [9, 10]. sPLA2, a subclass of phospholipase A2, catalyzes the hydrolysis of membrane phospholipids to generate free fatty acids and lysophospholipids, such as arachidonic acid, the rate-limiting step in eicosanoid production [13, 14]. These lipid mediators generated by sPLA2 activate protein kinase C, which has been suggested to be involved in TNF-␣-mediated NF-B activation (Fig 6) [9, 24]. Furthermore, upregulation of sPLA2 has been implicated as a pathogenic factor not only in a variety of inflammatory processes, such as ulcerative colitis, Crohn’s disease, acute pancreatitis, septic shock, and bronchial asthma, but also in multiple human tumors, such as prostate, ovarian, gastric, and intestinal cancer [13, 14]. We re-
Fig 6. Schematic illustrating the proposed relationship between sPLA2 and the TNF-␣ signaling pathway and the proposed mechanism of action of the specific sPLA2 inhibitor. sPLA2 catalyzes the hydrolysis of membrane phospholipids to generate free fatty acids such as arachidonic acid. These lipid mediators activate protein kinase C, which has been suggested to be involved in TNF-␣-mediated NF-B activation and, ultimately, ICAM-1 expression. The specific sPLA2 inhibitor functions by inhibiting the hydrolysis of membrane phospholipids as denoted by the X in the figure, thus preventing protein kinase C activation and leading to attenuated TNF␣-mediated NF-B activation and ICAM-1 expression. (ICAM-1 ⫽ intercellular adhesion molecule-1; NF-B ⫽ nuclear factor-kappa beta; sPLA2 ⫽ secretory phospholipase A2; TNF-␣—tumor necrosis factor-alpha.)
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cently demonstrated that sPLA2 regulates the growth and proliferation of human esophageal adenocarcinoma cells in vitro [18]. Given the available data regarding sPLA2 and NF-B-induced ICAM-1 production, we hypothesized that inhibition of this enzyme in esophageal cancer cells would lead to decreased ICAM-1 production. These data demonstrate that sPLA2 may be a potential chemotherapeutic target in patients with esophageal adenocarcinoma. Inhibition of sPLA2, through its secondary effects of reducing ICAM-1 expression, has the potential to act in conjunction with standard chemotherapeutic regimens to prevent or treat metastatic disease. Such advances in the medical management of esophageal adenocarcinoma could improve patient survival and overall prognosis. Furthermore, inhibition of sPLA2 can be used as a prophylactic measure for those at risk of developing esophageal adenocarcinoma. Both Barrett’s esophagus, a well-established precursor lesion to esophageal adenocarcinoma, and inflamed esophageal tissue have been shown to express ICAM-1 as well [8, 25, 26]. By targeting sPLA2 and attenuating ICAM-1 expression, the transformation to esophageal adenocarcinoma may be prevented. Thus, inhibition of sPLA2 could be used not only therapeutically in those suffering from esophageal adenocarcinoma, but also prophylactically in high-risk patients with Barrett’s esophagus or severe gastroesophageal reflux disease. In summary, the results of the present study demonstrate that treatment of human esophageal adenocarcinoma cells with a sPLA2 inhibitor attenuates the expression of total cellular, cell-surface, and secreted ICAM-1 as well as the activation of NF-B, suggesting a common pathway between the mechanism of action of specific sPLA2 inhibition on ICAM-1 expression and NF-B activation. To our knowledge, this is the first report not only of the abundant expression of ICAM-1 at baseline in FLO-1 cells, but also of the role of sPLA2, TNF-␣, and NF-B in ICAM-1 expression of these cells. Although this study demonstrates a clear association between sPLA2 and ICAM-1 expression as well as sPLA2 and NF-B activation, it is unclear if these results would be comparable in an in vivo setting. These findings could identify inhibition of sPLA2 as a potential mode of therapy and target for prevention of esophageal adenocarcinoma. We would like to thank Dr David Beer (Department of Surgery, University of Michigan, Ann Arbor, MI) for his generous gift of the FLO-1 human esophageal adenocarcinoma cell line. Our study was supported by the University of Colorado Academic Enrichment Funds.
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