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Endotoxin/Lipopolysaccharide Activates NF-B and Enhances Tumor Cell Adhesion and Invasion Through a ₁ Integrin-Dependent Mechanism

Jiang Huai Wang^1,*, Brian J. Manning^*, Qiong Di Wu^*, Siobhan Blankson^*, D. Bouchier-Hayes and H. Paul Redmond^*

^* Department of Academic Surgery, National University of Ireland, Cork University Hospital, Cork, Ireland; and Department of Academic Surgery, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
₁ integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a ₁ integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the ₁ subunit, whereas various low levels of ₁, ₂, ₄, and ₆ expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated ₁ integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-₁ blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-B activation in both SW480 and SW620 cells. Inhibition of the NF-B pathway significantly attenuated LPS-induced up-regulation of ₁ integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-B, up-regulate ₁ integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is ₁ subunit-dependent.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The establishment of metastases in distant organs is dependent upon the adherence of shed circulating tumor cells to the endothelium lining the microvessels in target organs, attachment to the subendothelial basement membrane, invasion into the extracellular matrix by local proteolysis, and, finally, proliferation and angiogenesis (1, 2). During these steps it is necessary for tumor cells to interact with endothelial cells and extracellular matrix in distant organs, and this interaction is dependent on the various adhesion molecules expressed on the tumor cell surface. Among these, the integrin-mediated interaction between tumor cells and the subendothelial extracellular matrix is one of the most important determinants for organ-specific metastasis (3, 4).

Integrins are the most important family of cell surface adhesion molecules that mediate interactions between cells and the extracellular matrix (5). They are heterodimeric transmembrane receptors consisting of and subunits. Each subunit is a glycoprotein with a large extracellular domain and a relatively small cytoplasmic domain. The different integrin subfamilies are determined by the subunit; for example, the ₁ subunit associates with different subunits to form the ₁ integrin subfamily. To date >20 different integrin heterodimers are known, which bind to specific ligands present in the extracellular matrix or expressed on target cells. Members of the ₁ integrin subfamily primarily bind to components of the extracellular matrix, such as fibronectin, collagens, and laminins, but some of them also participate in direct cell-to-cell adhesion (5, 6). Among the different integrin heterodimers, ₁ and ₃ integrins appear to play a crucial role in regulating tumor cell proliferation, differentiation, adhesion, motility, and invasion (7, 8). Increased expression of different members of the ₁ integrin has been found to be associated with tumor cell survival, invasion, and tumor progression. Up-regulation of ₂₁ integrin expression enhances tumor cell adhesion and prevents high dose epidermal growth factor-induced cell death (9). Overexpression of the ₃ subunit of ₁ integrin in the MDA-MB-231 breast carcinoma cell line is associated with the potent migratory and invasive properties of these cells, which is strongly inhibited by a specific function-blocking anti-₃ mAb (10). Using a targeted elimination technique, it has been found that the expression of integrin ₆₁ in MDA-MB-435 breast carcinoma cell line is essential for facilitating tumorigenesis and promoting tumor cell survival in distant organs in mice (11). Overexpression of ₆ subunit of the ₁ integrin has been found in human hepatocellular carcinoma with aggressive phenotypes (12). Recently, it has been shown that organ-specific sites of metastatic lesions are determined at least in part by ₁ integrin-mediated adhesion to and invasion into the subendothelial extracellular matrix, and furthermore, different metastatic behaviors of tumors correlate with ₁ integrin-mediated adhesive properties (4, 13).

LPS or endotoxin, a predominant glycolipid in the outer membrane of Gram-negative bacteria, provides a highly potent stimulus to cells of the immune system. LPS stimulates monocytes, macrophages, and neutrophils through the activation of transcription factors and protein kinases such as NF-B and p38 kinase, resulting in an increased production of proinflammatory cytokines and overexpression of cell adhesion molecules (14, 15). LPS-induced cellular activation is mediated by its complexing with circulating LPS-binding protein (LBP)² and subsequent binding to CD14, a 55-kDa glycosylphosphatidylinositol (GPI) membrane-anchored glycoprotein (16, 17), which, in turn, facilitates intracellular transduction of LPS signaling through Toll-like receptor 4 (TLR4) (18, 19). It has been noted that rapid growth of previously dormant metastases occurs following surgical removal of a primary tumor (20). The mechanisms underlying this phenomenon are not fully elucidated. Two recent studies have shown a crucial role for LPS contamination following surgery in tumor growth and metastases (21, 22). Both laparotomy and air laparoscopy result in endotoxin contamination of the peritoneal cavity and systemic endotoxemia via bacterial translocation across the gut (21). In a murine model of metastatic disease, animals subjected to laparotomy or air laparoscopy with high levels of circulating LPS had increased lung metastatic burdens, whereas animals subjected to CO₂ laparoscopy with very low levels of plasma LPS had metastatic tumor growth similar to that in controls. Furthermore, there were significantly increased lung metastases in animals that received an equivalent LPS injection (22). These results indicate that LPS entering the peritoneal cavity or systemic circulation during surgery is associated with enhanced growth of metastases following surgical trauma. However, the direct effect of LPS on invasive and metastatic behavior of tumor cells is unclear.

The transcription factor NF-B is involved in the regulation of multiple cellular processes, including proinflammatory cytokine gene expression, cellular adhesion, cell cycle activation, apoptosis, and oncogenesis. Currently known subunit members of the NF-B family in mammals are five proteins related by the Rel homology domain, namely p50, p65 (Rel A), the proto-oncogene c-rel (c-Rel), p52, and Rel B (23). In most cells the NF-B heterodimer is sequestered in the cytoplasm as an inactive form through interaction with an inhibitory B (IB) protein that inhibits nuclear translocation of NF-B. Several IB isoforms have been identified, but the most extensively characterized isoforms are IB-, IB-, and IB- (24). When IB is degraded through the phosphorylation of IB- induced by activated IB kinases, the NF-B heterodimer will enter the nucleus, bind to the promoter regions of the target genes, and stimulate transcription (23, 25). It has been shown that constitutive activation of NF-B is present in a number of tumor cells, including hepatocellular carcinoma cells (26), breast cancer cells (27), and non-small cell lung cancer cells (28). Elevated NF-B activity in tumor cells may be related to an overproduction of cytokines, such as vascular endothelial growth factor (VEGF), GM-CSF, and IL-8, by tumor cells (29, 30), which may provide continued positive growth stimuli. Constitutive activation of NF-B has been shown to protect tumor cells against apoptosis (31), and inhibition of NF-B can sensitize tumor cells to undergo chemotherapy-induced apoptosis (28, 32), indicating a role for NF-B in the development of tumor resistance to cancer treatment. A recent study has shown that blockade of NF-B signal in a murine lung alveolar carcinoma cell line by transfection of a dominant negative mutant form of IB- that cannot be phosphorylated prevents intravasation of tumor cells in an in vivo chick embryo metastasis model and lung metastasis in a murine model (33), suggesting that activation of NF-B plays a central and specific role in the regulation of tumor metastasis.

In the present study we examined the effects of LPS on ₁ integrin expression and subsequent tumor cell adhesion to and invasion into the extracellular matrix. We also investigated the effects of LPS on NF-B activation and its relation to tumor cell invasive behavior. Here we provide evidence for the first time that LPS directly enhances tumor cell invasive and metastatic potentials. Using two human colorectal tumor cell lines, SW480 and SW620, cultured in an in vitro serum-free culture system, this report shows that LPS in the presence of soluble CD14 (sCD14) can up-regulate ₁ integrin expression and promote tumor cell adhesion and invasion through a ₁ integrin-dependent mechanism. Furthermore, LPS can directly activate NF-B in SW480 and SW620 tumor cells. Blockade of NF-B activation prevents LPS-induced ₁ integrin overexpression and attenuates tumor cell adhesion and invasion, indicating a key role for NF-B activation in transducing LPS signaling and subsequent LPS-promoted tumor cell adhesion and invasion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and mAbs

Medium 199, medium L-15, HBSS, PBS without Ca²⁺ and Mg²⁺, FCS, penicillin, streptomycin sulfate, glutamine, and 0.05% trypsin/0.02% EDTA solution were purchased from Life Technologies (Paisley, Scotland). Human plasma fibronectin, collagen I from calf skin, laminin from human placenta, LPS (Escherichia coli O55B5), HEPES, MgCl₂, KCl, NaCl, EDTA, Tris-HCl, glycerol, nuclease-free BSA, Nonidet P-40, PMSF, and DTT were purchased from Life Technologies and Sigma-Aldrich (St. Louis, MO), respectively. [-³²P]ATP (3000 Ci/mmol) and poly(dI-dC) were obtained from Amersham International (Little Chalfont, U.K.) and Amersham Pharmacia Biotech (Milton Keynes, U.K.), respectively. SN50, a cell-permeable peptide inhibitor of NF-B, was purchased from Calbiochem (San Diego, CA). Recombinant human sCD14 was obtained from Biometec (Greifswald, Germany). Mouse anti-human CD14 mAb was obtained from BD Biosciences (Mountain View, CA). Mouse anti-human integrin ₁, 1, ₂, ₃, ₄, ₅, ₆, and _v3 mAbs were purchased from Chemicon (Temecula, CA), BD PharMingen (San Diego, CA), and Serotec (Oxford, U.K.), respectively. ₁ integrin-blocking mAbs 4B4 and 6S6 were purchased from Coulter Clone (Miami, FL) and Chemicon, respectively.

Cell culture

Human colorectal tumor cell lines SW480 and SW620 were obtained from American Type Culture Collection (Manassas, VA). SW480 and SW620 cells were grown in medium L-15 supplemented with 10% FCS, penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), and glutamine (2.0 mM). Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere and subcultured by trypsinization with 0.05% trypsin/0.02% EDTA when cells became confluent. After the second passage, SW480 and SW620 cells were incubated in an in vitro serum-free culture system in all experiments conducted in this study.

FACS analysis of immunofluorescence

SW480 and SW620 cells cultured in serum-free medium were exposed to LPS alone (0.1 µg/ml) or a combination of LPS (0.1 µg/ml) and sCD14 (0.2 µg/ml) for 4 h at 37°C in humidified 5% CO₂ conditions. The expression of different subunits of ₁ integrins, _v3, and membrane-bound CD14 (mCD14) on SW480 and SW620 cells was determined using direct and indirect immunofluorescent staining. For direct immunofluorescent staining, 20 µl of FITC-conjugated anti-integrin ₅ (anti-CD49e), FITC-conjugated anti-integrin _v3 (anti-CD51/61), FITC-conjugated anti-Leu M3 (anti-CD14), PE-conjugated anti-integrin ₄ (anti-CD49d), and PE-conjugated anti-integrin ₁ (anti-CD29) mAbs were added to 100 µl of cell suspension (1 x 10⁶ cells/ml) and incubated at 4°C for 30 min. FITC- and PE-conjugated isotype IgG1 and IgG2b mAbs were used as negative controls. For indirect immunofluorescent staining, 100 µl of cell suspension (1 x 10⁶ cells/ml) was incubated with 20 µl of pure mAbs against integrin ₁ (anti-CD49a), integrin ₂ (anti-CD49b), integrin ₃ (anti-CD49c), and integrin ₆ (anti-CD49f) at 4°C for 30 min and further stained with secondary FITC-conjugated mAb at 4°C for 30 min. Pure isotype IgG1 mAb was used as a negative control. Different ₁ integrins, _v3, and mCD14 expression on SW480 and SW620 cells were analyzed on a FACScan flow cytometer (BD Biosciences) for detecting the log of the mean channel fluorescence intensity with an acquisition of FL1 and FL2, respectively. A minimum of 10,000 events were collected and analyzed on CellQuest software (BD Biosciences).

Tumor cell adhesion

Tumor cell attachment to fibronectin, collagen I, and laminin was performed as previously described (34) with some modifications. Briefly, fibronectin (2.0 µg/ml), collagen I (1.5 µg/ml), and laminin (3.5 µg/ml) were coated onto 96-well, flat-bottom, microtiter plates (Falcon, Lincoln Park, NJ). SW480 and SW620 cells cultured in serum-free medium were treated with LPS alone (0.1 µg/ml) or LPS plus sCD14 (0.1 and 0.2 µg/ml) for 4 h at 37°C in humidified 5% CO₂ conditions. For ₁ integrin and NF-B blocking experiments, cells were treated with different mAbs or SN50 for 30 min before being exposed to LPS or LPS plus sCD14. Cells (5 x 10⁴ cells/ml) were then added to the fibronectin-, collagen I-, and laminin-coated 96-well plates and incubated at 37°C in humidified 5% CO₂ conditions for 1 h. The plate was washed twice with HBSS to remove unbound cells. Tumor cell adhesion to fibronectin, collagen I, and laminin was assessed using CellTite 96 Aqueous One Solution Assay (Promega, Madison, WI) on a Microtiter Plate Reader (Dynex Technologies, Chantilly, VA). The specific absorbance at 490 nm generated from CellTite 96 Aqueous One Solution Assay is directly proportional to the number of adherent cells in the plate. The ratio of the percentage of adherent tumor cells to that of total added tumor cells was calculated according to the following formula:

Background binding was <5% of the total.

Tumor cell invasion

In vitro tumor cell invasion was assessed using a Biocoat Matrigel invasion chamber (BD Biosciences) with cell culture inserts containing an 8-µm pore size positron emission tomography membrane with a thin layer of Matrigel basement membrane matrix as previously described (35, 36). Briefly, 0.5 ml of tumor cells (1 x 10⁵ cells/ml) resuspended in serum-free medium containing either LPS alone (0.1 µg/ml) or LPS plus sCD14 (0.1 and 0.2 µg/ml) was added to the cell culture insert of a Biocoat Matrigel invasion chamber. Fibronectin (20 µg/ml) was added in the outer chamber as a chemoattractant. The cells were then incubated at 37°C in humidified 5% CO₂ conditions for 18 h. To quantitative tumor cell invasion, noninvading cells were removed from the upper surface of the membrane by scrubbing gently with a cotton-tipped swab. The cells on the lower surface of the membrane were fixed with Rapi-Diff II (DiaChem, Lancashire, U.K.). Five random microscope fields of the lower surface of the membrane were counted for numbers of cells that had invaded through the Matrigel layer and the membrane. The results were expressed as numbers of invaded cells per microscope field. To examine whether ₁ integrins and NF-B activation are involved in LPS-induced tumor cell invasion, SW480 and SW620 cells were treated with different blocking mAbs or SN50 for 30 min before being exposed to LPS alone or LPS plus sCD14.

Transfection assays

Dual transfection of SW480 and SW620 cells was accomplished using 16 µl/ml of Lipofectamine 2000 reagent (Life Technologies) and 4.0 µg/ml of pNF-B-Luc vector DNA (Clontech Laboratories, Palo Alto, CA) that contains the firefly luciferase gene as the reporter. The pRL-CMV vector (Promega) containing the Renilla luciferase gene was used as an internal control. Briefly, SW480 and SW620 cells were plated in 96-well plates (Falcon; 2 x 10⁴ cells/well) to grow at 37°C in humidified 5% CO₂ conditions until they reached 90–95% confluence. Reporter DNA (0.2 µg) was mixed with 0.8 µl of Lipofectamine in 50 µl of serum-free Opti-MEM I (Life Technologies), and incubated at room temperature for 20 min to form Lipofectamine-DNA complexes. Cells in 96-well plates were transfected with the complexes for 6 h and cultured for an additional 18-h period after replacement of the medium. Each transfection was performed in duplicate. Transfected cells were exposed to LPS alone (0.1 µg/ml) or LPS plus sCD14 (0.1 and 0.2 µg/ml) for 6 h. Cell extract was prepared using the Passive lysis buffer (Promega), and protein content in each sample was determined using a Micro BCA protein assay reagent kit (Pierce, Rockford, IL). Firefly and Renilla luciferase activities were measured using the Dual luciferase reporter assay system (Promega) to assess promoter activity and transfection efficiency.

SW480 cells were also transfected with a dominant negative IB- vector or an empty expression vector pcDNA3.1 as a negative control (provided by Dr. A. G. Bowie, Trinity College, Dublin, Ireland) (37). Briefly, SW480 cells were plated in 24-well plates (Falcon; 1 x 10⁵ cells/well) and grown until they reached 90–95% confluence. The vector DNA-Plus Reagent-Lipofectamine complexes were produced by mixing dominant negative IB- vector DNA (0.12 µg) or empty vector DNA (0.12 µg/ml) with 4 µl of Plus Reagent (Life Technologies) and 1 µl of Lipofectamine (Life Technologies). Cells in 24-well plates were transfected with the complexes for 3 h and cultured for an additional 15-h period after replacement of the medium. Each transfection was performed in duplicate. Expression of ₁ integrin, cell adhesion, and invasion in response to LPS plus sCD14 stimulation were assessed in the transfected cells. Transfection efficiency, and inhibition of NF-B activation were assessed by dual transfection of dominant negative IB- vector (0.04–0.16 µg) in combination with pSV--galactosidase vector (Promega) or pNF-B-Luc vector (Clontech Laboratories).

Nuclear extract preparation

SW480 and SW620 cells were cultured in serum-free medium in six-well plates (Falcon; 1 x 10⁶ cells/well) and treated with LPS alone (0.1 µg/ml) or LPS plus sCD14 (0.1 and 0.2 µg/ml) for 30 min. Nuclear and cytoplasmic extracts were prepared as previously described (38). Briefly, cells were lysed in a hypotonic solution (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, and 0.1% Nonidet P-40, pH 7.9) on ice for 10 min and centrifuged at 13,000 rpm to pellet nuclei. Cytoplasmic supernatants were removed, and nuclei were resuspended in nuclear extract buffer (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, and 0.2 mM EDTA, pH 8.0) on ice for 15 min. The lysates were centrifuged at 13,000 rpm, and supernatants containing the nuclear proteins were collected. All buffers contained freshly added 0.5 mM DTT, 0.5 mM PMSF, and protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined using a Micro BCA protein assay reagent kit (Pierce). All extracts were stored at -70°C until analyzed.

Electrophoretic mobility shift assays

EMSAs were performed as previously described (39). Briefly, 2.0–4.0 µg of nuclear extracts were incubated with 30,000 cpm of double-stranded oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG C-3' containing the NF-B consensus sequence (underlined; Promega) that had been previously labeled with [-³²P]ATP (10 mCi/mmol) by T4 polynucleotide kinase (Promega). The DNA binding reactions were performed in the presence of 2.0 µg of poly(dI-dC) as a nonspecific competitor in binding buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1.0 mM EDTA, 5.0 mM DTT, 4% glycerol, and 100 µg/ml of nuclease-free BSA) at room temperature for 30 min. For competition experiments, unlabeled double-stranded oligonucleotide 5'-AGT TGA GGC GAC TTT CCC AGG C-3' containing the mutated NF-B consensus sequence (underlined; Promega) was added to the nuclear extracts 30 min before the addition of the radiolabeled probe. All reaction mixtures were subjected to electrophoresis on native 5% (w/v) polyacrylamide gels, which were subsequently dried and autoradiographed.

Statistical analysis

All data are presented as the mean ± SD. Statistical analysis was performed using ANOVA. Differences were judged statistically significant at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Constitutive expression of ₁ integrins, _v3 and mCD14

As determined by FACScan analysis, human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of integrin ₁ subunit, whereas various low levels of integrin ₁, ₂, and ₄ expression on SW480 and of ₂, ₄, and ₆ expression on SW620 were also detected (Fig. 1). However, the expressions of integrin ₃ and ₅ subunits, and integrin _v3 were almost absent in both SW480 and SW620 cells (data not shown). Furthermore, these two tumor cell lines did not contain mCD14 (Fig. 2), the cell surface receptor of a 55-kDa GPI membrane-anchored glycoprotein for LPS recognition and binding.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Constitutive expression of 1 integrins on SW480 and SW620 cells. The expression of different subunits of 1 integrin was analyzed by flow cytometry using either direct or indirect immunofluorescent staining as described in Materials and Methods. Filled histograms, Isotype-matched mAbs served as a negative control; open histograms, anti-1 integrin mAbs. Shown are data from one representative experiment from a total of three independent assays.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. FACScan analysis of mCD14 expression on SW480 and SW620 cells. The expression of mCD14 was assessed by flow cytometry using FITC-conjugated anti-Leu M3 (anti-CD14) mAb (open histogram) and isotype-matched mAb as a negative control (filled histogram). A representative of three separate assays is shown.

Up-regulation of ₁ integrin expression by LPS in the presence of sCD14

When tumor cells were incubated in a serum-free culture system, LPS alone at 0.1 µg/ml had no effect on modulating ₁ integrin expression. However, LPS (0.1 µg/ml) in the presence of sCD14 (0.2 µg/ml) significantly up-regulated the expression of integrin ₁, ₂, and ₄ subunits on SW480 cells and the expression of integrin ₁, ₄, and ₆ subunits on SW620 cells (Fig. 3). LPS plus sCD14 did not enhance integrin ₁ expression on SW480 and integrin ₂ expression on SW620 cells (Fig. 3). Soluble CD14 alone (0.2 µg/ml) did not affect the expression of different ₁ integrin subunits on these two tumor cell lines (data not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Up-regulation of 1 integrin expression by LPS in the presence of sCD14. SW480 and SW620 cells were incubated with serum-free culture medium (), LPS alone at 0.1 µg/ml (), or LPS plus sCD14 at 0.1 and 0.2 µg/ml () for 4 h. The expression of integrin 1, 1, 2, 4, and 6 subunits was analyzed by flow cytometry. Data are expressed as the mean ± SD and are representative of five separate experiments. The statistical significance was compared with the cells incubated with either culture medium or LPS alone (*, p < 0.05).

In the absence of exogenous stimulation, both SW480 and SW620 cells showed various levels of spontaneous adhesion to extracellular matrix proteins such as fibronectin, collagen I, and laminin (Fig. 4). Under an in vitro serum-free culture condition, SW480 and SW620 cells stimulated with LPS alone (0.1 µg/ml) did not significantly affect the attachment of tumor cells to extracellular matrix proteins (Fig. 4, A and B). When tumor cells were treated with a combination of LPS (0.1 µg/ml) and sCD14 (0.2 µg/ml), however, there were significant increases in SW480 and SW620 cell adhesion to fibronectin, collagen I, and laminin by 2- to 3-fold (p < 0.05 vs tumor cells treated with either culture medium or LPS alone; Fig. 4, A and B). To evaluate LPS-dependent tumor cell invasion in an in vitro model, we used Biocoat Matrigel chambers. As shown in Fig. 5, LPS (0.1 µg/ml) in the presence of sCD14 (0.2 µg/ml) significantly promoted SW480 and SW620 cell invasion through the Matrigel (p < 0.05 vs tumor cells treated with either culture medium or LPS alone). In contrast, when SW480 and SW620 cells were treated with LPS alone (0.1 µg/ml), no significant effects on tumor cell invasion were observed (Fig. 5).

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Effects of LPS on SW480 and SW620 cell attachment to extracellular matrix proteins. After the cells were incubated with serum-free culture medium (), LPS alone at 0.1 µg/ml (), or LPS plus sCD14 at 0.1 and 0.2 µg/ml () for 4 h, the adherence of SW480 (A) and SW620 (B) cells to fibronectin, collagen I, and laminin was assessed as described in Materials and Methods. Data are expressed as the mean ± SD and are representative of six separate experiments. Each experiment was conducted in triplicate. Statistical significance was compared with the cells incubated with either culture medium or LPS alone (*, p < 0.05).

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Effect of LPS on SW480 and SW620 cell invasion through Matrigel. Tumor cell invasion in Matrigel was assessed using Biocoat Matrigel invasion chambers. , Cells incubated with serum-free culture medium; , cells treated with LPS alone (0.1 µg/ml); , cells treated with LPS plus sCD14 (0.1 and 0.2 µg/ml). Data are expressed as the mean ± SD and are representative of six separate experiments. Statistical significance was compared with the cells incubated with either culture medium or LPS alone (*, p < 0.01).

Involvement of integrin ₁ in LPS-induced tumor cell adhesion and invasion

₁ integrins play a crucial role in supporting tumor cell attachment to, migration, and invasion into extracellular matrix proteins. To determine whether ₁ integrin is involved in LPS-induced tumor cell adhesion and invasion, we used anti-₁ integrin mAbs to selectively block integrin ₁ subunits. Pretreatment of SW480 and SW620 cells with 4B4 and 6S6, two specific integrin ₁ subunit blocking mAbs, almost totally prevented LPS plus sCD14-induced tumor cell adhesion to fibronectin (p < 0.05 vs tumor cells treated with LPS plus sCD14; Fig. 6). Furthermore, 4B4 and 6S6 significantly attenuated LPS plus sCD14-promoted SW480 and SW620 cell invasion (p < 0.05 vs tumor cells treated with LPS plus sCD14; Fig. 6). An isotype-matched mouse IgG1 used as the control for 4B4 and 6S6 had no effect on LPS- plus sCD14-induced tumor cell adhesion and invasion (Fig. 6). These results indicate that LPS-induced SW480 and SW620 cell adhesion and invasion through extracellular matrix proteins are ₁ integrin dependent.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Effects of 1 integrin blocking mAbs on LPS-induced SW480 and SW620 cell adhesion and invasion. Tumor cell attachment to fibronectin and invasion in Matrigel were assessed as described in Materials and Methods. , Cells incubated with serum-free culture medium (M); , cells treated with LPS plus sCD14 (0.1 and 0.2 µg/ml; LPS); , cells pretreated with 1 integrin function-blocking mAbs 4B4 (2.0 µg/ml) and 6S6 (1.5 µg/ml) or an isotype-matched mouse IgG1 (2.0 µg/ml) for 30 min before they were exposed to LPS plus sCD14. Results are expressed as the mean ± SD and are representative of five separate experiments. Each experiment was conducted in triplicate. Statistical significance was compared with cells incubated with either culture medium (*, p < 0.01) or LPS plus sCD14 (@, p < 0.01).

Activation of NF-B by LPS in the presence of sCD14

NF-B activation induced by a combination of LPS and sCD14 was assessed by transfection of SW480 cells with pNF-B-Luc reporter vector. As shown in Fig. 7A, LPS in the presence of sCD14 resulted in a significant increase in luciferase activity (p < 0.05), indicating the activation of NF-B following LPS and sCD14 stimulation. NF-B-DNA binding activity was also assayed by EMSA using nuclear extracts that were prepared from SW480 cells incubated with serum-free culture medium, LPS alone, sCD14 alone, or LPS plus sCD14. There was a low level of constitutive activation of NF-B in unstimulated cells; however, LPS in the presence of sCD14 caused a marked activation of NF-B (Fig. 7B). LPS alone or sCD14 alone had no effect on NF-B activation. Similar levels of NF-B activation were also found in SW620 cells treated with LPS plus sCD14 (data not shown).

View larger version (54K): [in this window] [in a new window]   FIGURE 7. Activation of NF-B by LPS in the presence of sCD14. After SW480 cells were treated with serum-free culture medium, LPS alone (0.1 µg/ml), sCD14 alone (0.2 µg/ml), or LPS plus sCD14 (0.1 and 0.2 µg/ml), NF-B activation was assessed by measuring luciferase activity (A) or by EMSA (B). A, results are expressed as the mean ± SD and are representative of three separate experiments. Each experiment was conducted in duplicate. Statistical significance was compared with the cells incubated with culture medium, LPS alone, or sCD14 alone (*, p < 0.01). B, Data presented are one representative from a total of three separate assays.

Attenuation of LPS-induced ₁ integrin overexpression, tumor cell adhesion and invasion by a NF-B inhibitor SN50 or in the dominant negative IB- transfected cells

SN50 is a synthetic peptide containing a cell membrane-permeable motif and nuclear trans-locating hydrophobic sequence that inhibits nuclear translocation of NF-B in a dose-dependent manner in cultured endothelial and monocytic cells stimulated with LPS or TNF- (40). In a dose-response experiment we found that inhibition of NF-B activation, as represented by luciferase activity in the transfected SW480 cells by SN50 was 18% at 25 µg/ml, 62% at 50 µg/ml, 94% at 100 µg/ml, and 92% at 200 µg/ml. As shown in Fig. 8, when SW480 cells were pretreated with SN50 at 100 µg/ml, there was a total abrogation of up-regulation of integrin ₁, ₂, and ₄ expression induced by LPS plus sCD14. Furthermore, pretreatment of SW480 cells with SN50 at 100 µg/ml significantly attenuated LPS-induced tumor cell adhesion and invasion (p < 0.05 vs tumor cells treated with LPS plus sCD14; Fig. 8). SN50 at 100 µg/ml was not toxic to the cells as determined by measurement of lactate dehydrogenase release and did not affect cell viability as determined by trypan blue exclusion and propidium iodide staining on flow cytometry (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Abrogation of LPS-induced up-regulation of 1 integrin expression and attenuation of LPS-induced tumor cell adhesion and invasion by SN50. The expression of integrin 1, 2, and 4 on SW480 cells was analyzed by flow cytometry. SW480 cell attachment to fibronectin and invasion in Matrigel were determined as described in Materials and Methods. , Cells incubated with serum-free culture medium; , cells treated with LPS plus sCD14 (0.1 and 0.2 µg/ml); , cells pretreated with SN50 (100 µg/ml) for 30 min before being exposed to LPS plus sCD14. Data are expressed as the mean ± SD and are representative of five or six separate experiments. Statistical significance was compared with the cells incubated with either culture medium (*, p < 0.05) or LPS plus sCD14 (@, p < 0.05).

View larger version (37K): [in this window] [in a new window]   FIGURE 9. Attenuation of LPS-induced 1 integrin overexpression, tumor cell adhesion, and invasion in dominant negative IB- transfected SW480 cells. The expression of integrin 1, 2 and 4, tumor cell adhesion to fibronectin, and invasion in Matrigel were determined as described. Medium, Nontransfected cells incubated with serum-free culture medium; EV+medium, empty vector-transfected cells incubated with serum-free culture medium; IB-+medium, dominant negative IB- vector-transfected cells incubated with serum-free culture medium; LPS, nontransfected cells treated with LPS plus sCD14 (0.1 and 0.2 µg/ml); EV+LPS, empty vector-transfected cells treated with LPS plus sCD14 (0.1 and 0.2 µg/ml); IB-+LPS, dominant negative IB- vector-transfected cells treated with LPS plus sCD14 (0.1 and 0.2 µg/ml). Data are expressed as the mean ± SD and are representative of three separate experiments. Statistical significance was compared with the nontransfected cells incubated with either culture medium (*, p < 0.05) or LPS plus sCD14 (@, p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study SW480 and SW620 cells did not express mCD14, as confirmed by FACScan analysis. To identify a critical role for CD14 in LPS-induced cellular responses in tumor cells, we have employed an in vitro serum-free culture system to eliminate the influences of sCD14 and other soluble factors, such as LBP, that are present in the serum. Results obtained under serum-free culture conditions demonstrated that SW480 and SW620 cells did not respond to LPS stimulation. In contrast, LPS in the presence of recombinant sCD14 activated NF-B, up-regulated ₁ integrin expression, and promoted tumor cell adhesion and invasion. Thus, the apparent function of sCD14 is to enable tumor cells that lack mCD14 to recognize and respond to LPS. Furthermore, LPS at 0.1 µg/ml, an optimal dose according to the preliminary dose-response experiments (data not shown), was sufficient to cause SW480 and SW620 cell activation. These results are consistent with previous studies in which endothelial cells can be activated by high concentrations of LPS (50–500 ng/ml) in the presence of sCD14, whereas low concentrations of LPS (5–10 ng/ml) require the presence of LBP for sCD14-dependent activation (45).

Different subunits of ₁ integrin and various levels of their expression have been found in different types of tumor cells. Forexample, human hepatocellular carcinoma cell lines, PLC/PRF/5, Hep3B, HepG2, HLE, HuH7, and C3A cells, constitutively express high levels of ₁ and ₆ subunits and various low levels of 1, ₂, ₃, and ₅ subunits, whereas expression of the ₄ subunit is absent in each cell line (50). We found that the predominant adhesion receptors in SW480 cells were the 1₁, ₂₁, and ₄₁ integrins; that ₂₁, ₄₁, and ₆₁ integrins were highly expressed in SW620 cells; and that the ₃₁ and ₅₁ integrins were undetectable in either cell line. It has been found that overexpression of integrin _v3 correlates with aggressive phenotypes in different malignancies and with tumor angiogenesis (51, 52). However, neither SW480 nor SW620 cells expressed _v3. The various levels of ₁ integrin expression on SW480 and SW620 cells may represent a low constitutive activity of different ₁ integrin subunits, which may account for spontaneous adhesion of tumor cells to fibronectin, collagen I, and laminin, and invasion in Matrigel as observed in the present study.

It is notable that LPS stimulation in the presence of sCD14 resulted in the up-regulation of ₁ integrin expression in SW480 and SW620 cells. LPS-modulated ₁ integrin expression appears to be both subunit and cell specific, as LPS significantly enhanced the expression of ₁, ₂, ₄, and ₆ subunits, but had no effect on ₁ expression on SW480 cells or ₂ expression on SW620 cells. In contrast, a stimulatory anti-₁ mAb TS2/16 has been found to induce a rapid activation of ₁ integrin as confirmed by an enhanced cell adhesion to collagen, but it does not change the expression of different ₁ integrin subunits (50, 53). Increased expression of ₁ integrin is thought to be associated with the ability of tumor cells to interact with the extracellular matrix and to subsequently form an organ-specific metastatic colonization through ₁ integrin-mediated cell adhesion and invasion. For example, TGF-₁ stimulates the hepatocellular carcinoma cell line SMMC-7721 cell adhesion to extracellular matrix through up-regulation of ₅₁ integrin expression (54). Furthermore, blockade of the ₃ subunit prevents breast carcinoma cell line MDA-MB-231 cell migration and invasion (10). ₄- CYT-transfected MDA-MB-435 cells that are deficient in forming the ₆₁ heterodimer are unable to establish metastatic foci in lungs (11).

Importantly, the present study has demonstrated that LPS stimulation significant enhances tumor cell attachment to extracellular matrix proteins and promotes tumor cell invasion, indicating a direct and distinct effect of LPS on the invasive and metastatic ability of tumor cells. By using ₁ integrin function-blocking mAbs, we further demonstrated that LPS-mediated tumor cell adhesion and invasion correlated with an increased expression of ₁ integrins, as 4B4 and 6S6 strongly inhibited LPS-induced tumor cell adhesion and invasion. The process of tumor cell invasion involves cell attachment to the subendothelial extracellular matrix and subsequent unidirectionally cell migration coupled with local proteolysis induced by a number of degradative enzymes, particularly matrix metalloproteinases (MMP). It has been shown that increased activity of ₁ integrin subunits results in MMP production and the activation of MMP proenzymes (10, 55). Although we did not measure MMP directly in the present study, the enhanced tumor cell invasion may correlate with the release and activation of MMP that could be mediated by increased ₁ integrin activity or by LPS stimulation directly. We have previously shown that endotoxin or LPS contamination of the peritoneal cavity and systemic endotoxemia are involved in surgically induced lung metastases of 4T1 mammary adenocarcinoma cells in mice, which correlates with an elevation in circulating levels of VEGF and an increased cell mitosis/apoptosis ratio in metastatic tumor burden (22). Results from this study provide further evidence for a direct effect of LPS on tumor cell adhesion to and invasion into the extracellular matrix.

Constitutive activation of NF-B has been described in a number of tumor cells, and appears to be associated with continued cytokine production, inhibition of apoptosis, activation of cell cycle, and possibly tumor progression (56). A low level of constitutive activation of NF-B was also found in SW480 and SW620 cells, which may account for an overproduction of VEGF by these cells in standardized culture conditions (data not shown). In the present study it is interesting to find that LPS stimulation of tumor cells resulted in a marked increase in NF-B-DNA binding activity. To examine the correlation between NF-B activation and LPS-induced tumor cell metastatic potential, SN50, a synthetic peptide that blocks NF-B signaling by inhibition of nuclear translocation of NF-B (40), and a dominant negative IB- vector, which prevents NF-B activation in transfected cells (41), were used in additional experiments. Blockade of NF-B activation by either SN50 or IB- transfection abrogated LPS-induced ₁ integrin overexpression and attenuated tumor cell adhesion and invasion, indicating that NF-B activation is a prerequisite not only for the transduction of LPS signals in tumor cells, but also for the enhanced tumor cell metastatic ability induced by LPS stimulation. LPS-mediated NF-B activation in the tumor cells switches the target gene to transcribe and synthesize new adhesion molecules, including ₁ integrins. Increased surface expression of ₁ integrins mediates tumor cell adhesion to ligands in the extracellular matrix, which elicits a variety of intracellular signals, including NF-B activation (57, 58), a positive feedback loop that could occur to sustain enhanced levels of NF-B activity. Activated NF-B has been found to stimulate the production of a number of degradative enzymes, such as MMP and urokinase-like plasminogen activator (59, 60), which, together with ₁ integrin, results in tumor cell invasion. Blockade of NF-B signaling has been shown to result in the down-regulation of MMP9 and heparanase and the up-regulation of tissue inhibitors of matrix metalloproteinases, thus preventing tumor cell intravasation and lung metastases (33). Taken together, these reports suggest NF-B to be an important regulator of the metastatic phenotype, and the up-regulation of NF-B in tumor cells induced by LPS in this study could have significant effects on metastatic potential.

Two recent studies have reported that commercially available LPS is contaminated by microbial proteins such as bacterial lipoprotein, as repurification of commercial preparations of LPS results in TLR4-mediated, but not TLR2-mediated, cellular activation (61, 62). Although we cannot completely exclude the possibility that LPS used in this study was contaminated with non-LPS bacterial cell wall components, it must be pointed out that LPS from any bacterial species is a mixture of different LPS, which may elicit distinct biological effects (63, 64), and that repurification of LPS by repeated phenol extraction may preferentially concentrate certain LPS subsets more than others. Further work will be required to confirm the effect of pure LPS on tumor cell activation. Furthermore, although this study has demonstrated the activation of NF-B by LPS through a CD14-dependent mechanism, the signal transduction pathways of LPS in tumor cells are largely unexplored.

In conclusion, LPS or endotoxin released by Gram-negative bacteria may have a direct effect on tumor progression by promoting tumor cell adhesion and invasion. This effect is mediated by LPS-induced up-regulation of ₁ integrin and activation of NF-B. These findings further implicate LPS, introduced by surgical procedures such as laparotomy, as a causative factor in surgically induced tumor metastatic growth. Therefore, neutralization of LPS and modulation of NF-B may be considered therapeutic strategies for the prevention of tumor relapse and metastasis in perioperative surgical practice.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Jiang Huai Wang, Department of Academic Surgery, National University of Ireland, Cork University Hospital, Cork, Ireland. E-mail address: jh.wang{at}ucc.i.e

² Abbreviations used in this paper: LBP, LPS-binding protein; mCD14, membrane-bound CD14; MMP, matrix metalloproteinase; sCD14, soluble CD14; TLR, Toll-like receptor; VEGF, vascular endothelial growth factor.

Received for publication May 15, 2002. Accepted for publication November 26, 2002.

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