Inhibition of ICAM-1 gene expression, monocyte adhesion and cancer cell invasion by targeting IKK complex: molecular and functional study of novel
-methylene-
-butyrolactone derivatives
Wei-Chien Huang1,
Shu-Ting Chan1,
Tzu-Lin Yang1,
Cherng-Chyi Tzeng2 and
Ching-Chow Chen1,3
1 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan and 2 School of Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
3 To whom correspondence should be addressed Email: ccchen{at}ha.mc.ntu.edu.tw
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Abstract
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The transcription factor nuclear factor-kappaB (NF-
B) is a regulator related to cellular inflammation, immune responses and carcinogenesis. Therefore, components of the NF-
B-activating singnaling pathways are frequent targets for the anti-inflammatory and anticancer agents. In this study, CYL-19 s and CYL-26z, two synthetic
-methylene-
-butyrolactone derivatives, were shown to inhibit the tumor necrosis factor-alpha (TNF-
)-induced intercellular adhesion molecule-1 (ICAM-1) expression in human A549 alveolar epithelial cells and the adhesion of U937 cells to these cells. RTPCR analysis also demonstrated their inhibitory effects on TNF-
-induced ICAM-1 mRNA expression. TNF-
-induced ICAM-1 and NF-
B-dependent promoter activities were attenuated by CYL-19 s and CYL-26z. ICAM-1 promoter activities induced by the over-expression of wild-type NF-
B-inducing kinase and I
B kinase ß (IKKß) were also inhibited by both compounds. Furthermore, CYL-19 s and CYL-26z inhibited the TNF-
-induced phosphorylation and degradation of I
B
and NF-
B-specific DNAprotein binding activity via targeting IKK complex directly, without any effect on the activations of other kinases such as ERK1/2 and p38. In addition to ICAM-1 expression, CYL-19 s and CYL-26z also suppressed other NF-
B-mediated gene expressions such as matrix metalloproteinase-9 (MMP-9) mRNA and cyclooxygnease-2 (COX-2) protein. In Matrigel assays, ICAM-1 and COX-2 expressions induced by TNF-
elicited A549 and NCI-H292 cell invasion, respectively, and these effects were inhibited by both compounds. In summary, our data demonstrated that CYL-19 s and CYL-26z down-regulate the TNF-
-induced inflammatory genes expression through suppression of IKK activity and NF-
B activation. These agents may be effective in the anti-inflammatory and anticancer therapy.
Abbreviations: COX-2, cyclooxygenase-2; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; ICAM-1, intercellular adhesion molecule-1; I
B, inhibitor of NF-
B; IKK, I
B kinase; MMP-9, matrix metalloproteinase-9; NF-
B, nuclear factor-kappaB; NIK, NF-
B-inducing kinase; TNF-
, tumor necrosis factor-alpha
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Introduction
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Intercellular adhesion molecule-1 (ICAM-1), also referred to as CD45, is an 80114 kDa inducible surface glycoprotein belonging to the immunoglobulin superfamily. It is associated with a wide range of inflammatory and immune response, including asthma, atherosclerosis, inflammatory disease, acute respiratory distress syndrome, ischemia reperfusion injury and autoimmune disease (15). ICAM-1 is expressed in both hematopoietic and non-hematopoietic cells and functions in cellcell and cellmatrix adhesive interactions by binding to two integrins belonging to the ß2 subfamily, i.e. CDlla/CD18 (LFA-1) and CD11b/CD18 (Mac-1) (6). Cell adhesion mediated by ICAM-1 is critical for the transendothelial migration of leukocytes and the activation of T cells, where ICAM-1 binding functions as a co-activation signal (7). ICAM-1 is present low on the cell surface of a variety of cell types including fibroblasts, leukocytes, keratinocytes, endothelial cells and epithelial cells, and it is up-regulated in response to a number of inflammatory mediators, including retinoic acid, virus infection, oxidant stresses such as H2O2, and the pro-inflammatory cytokines, IL-1ß, tumor necrosis factor-alpha (TNF-
) and IFN-
(6,811). The promoter region of the human ICAM-1 gene has been cloned and sequenced, and it was shown to contain the putative recognition sequence for a variety of transcriptional factors including nuclear factor-
B (NF-
B), activator protein-1 (AP-1), AP-2 and the interferon-stimulated response (12). Of these, NF-
B family proteins are the essential components for the enhanced ICAM-1 expression in response to cytokines in the human alveolar epithelial cells (10,11). NF-
B activation is tightly regulated by its endogenous inhibitor I
B, which complexes with and sequesters NF-
B into the cytoplasm.
Since NF-
B plays a central role in regulating the genes involved in the initiation of immune and inflammatory responses, inhibitors targeting the components of NF-
B activation pathways should be useful as the anti-inflammatory and anticancer agents. Sesquisterpene lactones, the most widely published class of natural products cited as inhibitors of NF-
B, contain a conjugated exomethylene group (
-methylene-
-butyrolactone) and an
,ß-unsaturated cyclopentenone (review in ref. 13). Accordingly, we synthesized two
-methylene-
-butyrolactone compounds, CYL-19 s and CYL-26z (Figure 1), to examine their inhibitory effects on the TNF-
-induced ICAM-1 expression on A549 epithelial cells, in which the molecular mechanism for this event has been explored to involve the protein kinase C (PKC)/c-Src-dependent activation of I
B kinase (IKK) and NF-
B (10,11,14). Our results demonstrated that CYL-19 s and CYL-26z blocked the TNF-
-induced ICAM-1 expression through NF-
B inhibition by targeting the IKK complex. NF-
B also regulates gene expressions involved in the turmorigenesis, angiogenesis, extravasation and degradation of extracellular matrix, hence plays a pivotal role in the promotion of cancer (15). For example, the expression of matrix metalloproteinase-9 (MMP-9) has been implicated in proteolytic turnover of the extracellular matrix associated with biologic processes including wound healing, inflammation and angiogenesis (16). The TNF-
-induced cyclooxygenase-2 (COX-2) expression has also been demonstrated to be regulated by NF-
B (17). Therefore, the inhibitory effects of CYL-19 s and CYL-26z on the expression of MMP-9 and COX-2 were also examined in this study to evaluate their potential anti-inflammatory and anticancer effect.
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Materials and methods
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Materials
Mouse monoclonal anti-human ICAM-1 antibody and recombinant human TNF-
were purchased from R&D System (Minneapolis, MN). The goat polyclonal antibodies specific for COX-2 and rabbit polyclonal antibodies specific for I
B
, IKK, ERK1/2 and p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). T4 polynucleotide kinase and rabbit polyclonal antibody specific for the phosphorylated form of I
B
[serine (Ser)-32], ERK1/2 and p38 were from New England Biolabs (Beverly, MA). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, fetal calf serum (FCS), penicillin and streptomycin were from Gibco BRL (Gaithersburg, MD). 2',7'-Bis-(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was from Molecule Probe (Eugene, OR). Reagents for SDSPAGE were from Bio-Rad (Hercules, CA). [
-32P]ATP (3000 Ci/mmol) was from Dupont-New England Nuclear (Beverly, MA). Poly (dIdC) and horseradish peroxidase-labeled donkey anti-rabbit second antibody and the ECL detecting reagent were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The ICAM-1 promoter constructs, pIC339, were gifts from Dr P.T.van der Saag (Hubrecht Laboratory, Utrecht, the Netherlands). The NF-
B-Luc expression plasmids were purchased from Stratagene (La Jolla, CA). Tfx-50 and the luciferase assay kit were from Promega (Madison, WI). CYL-19 s and CYL-26z were synthesized (C.-C.Tzeng, unpublished observation) and dissolved as stock solution (50 mM) in dimethyl sulfoxide.
Cell cultures
A549 cells and NCI-H292 cells, two alveolar epithelial carcinoma cell lines, were obtained from ATCC, and respectively cultured in DMEM and RPMI 1640 supplemented with 10% FCS, 100 U/ml of penicillin and 100 µg/ml of streptomycin in 96-well plates (ICAM-1 expression), in 6-well plates (transfection and COX-2 expression), in 6-cm dishes (IKK activity assay and RTPCR) or in 10-cm dishes (NF-
B gel shift assay).
U937 cells, a human monocytic leukemia cell, were obtained from the Department of Microbiology, College of Medicine, National Taiwan University, and cultured in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml of penicillin and 100 µg/ml of streptomycin. Cells split and fed every 34 days.
Quantification of ICAM-1 expression
The level of cell surface ICAM-1 expression was determined by ELISA as described previously (10). Following pre-treatment with CYL-19 s or CYL-26z for 30 min before challenge with TNF-
for 4.5 h at 37°C, cells were washed twice with phosphate-buffered saline (PBS) and fixed at room temperature with 3% paraformaldehyde for 10 min. After washing with PBS, they were then blocked with 1% BSA in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 15 min before being incubated successively with anti-ICAM-1 antibody (1:100) for 1 h and horseradish peroxidase-labeled anti-mouse antibody (1:1000) for 30 min. After each incubation, the cells were washed two times with PBS. OPD substrate (0.4 mg/ml in phosphate-citrate buffer, pH 5.0; 24.3 mM citric acid, 51.4 mM Na2HPO4·12H2O, 12% H2O2 v/v) was then applied to the cells for 30 min and 3 M sulfuric acid added to stop the reaction. Each assay was performed in triplicate and the absorbance was measured at 450 nm on an ELISA reader (Bio-Tek).
Cell adhesion assay
A549 cells, grown in 96-well plates, were treated at 37°C with TNF-
for 4.5 h after pre-treatment with CYL-19 s or CYL-26z for 30 min, then washed twice with PBS. U937 cells were labeled for 30 min at 37°C with 10 ng/ml of BCECF and washed twice with growth medium, then 2.5 x 105 of the labeled cells were added to the A549 monolayer in a final volume of 100 µl and incubated in a CO2 incubator for 1 h. Non-adherent cells were removed from the plate by gentle washing with PBS and the number of adherent cells determined by measuring the fluorescent intensity using a CytoFlour 2300 (Millipore, Bedford, MA).
RTPCR
Total RNA was isolated from A549 cell using TrizolTM Reagent (Life Technology). Reverse transcription reaction was performed using 2 µg of total RNA and reverse transcribed into cDNA using oligo dT primer, then amplified 30 cycles using two oligonucleotide primers derived from published ICAM-1, MMP-9 or ß-actin sequence, including 5'-TGCGGCTGCTACCACAGTGATGAT-3' and 5'-CCATCTACAGCTTTCGGCGCCCA-3' (ICAM-1), 5'-CAACATCACCTATTGGATCC-3' and 5'-CGGGTGTAGAGTCTCTCGCT-3' (MMP-9), or 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTTGCGGGGACGATGGAGGG-3' (ß-actin). PCR was carried out at 94°C for 30 s, at 55°C for 30 s and 1 min at 70°C for 34 cycles. The PCR products were subjected to 1% agarose gel electrophoresis. Quantitative data were obtained using a computing densitometer and ImageQuant Software (Molecular Dynamics).
Immunoprecipitation and in vitro IKK activity assay
Following 10 min treatment with TNF-
or 30 min pre-treatment with CYL-19 s or CYL-26z before addition of TNF-
, cells were rapidly washed with PBS, then lysed with ice-cold lysis buffer and the IKK proteins were immunoprecipitated. Fifty micrograms of total cell extract was incubated for 1 h at 4°C with 0.5 µg of anti-IKKß antibody and collected using protein ASepharose Cl-4B beads (Sigma). The beads were then washed three times with lysis buffer without Triton X-100 and incubated for 30 min at 30°C in 20 µl of kinase reaction mixture containing 20 mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3VO4, 1 mM DTT, 1 µg of bacterially expressed GSTI
B
(1-100) and 10 µM [
-32P]ATP. The reaction was stopped by the addition of Laemmli buffer and subjected to 10% SDSPAGE, phosphorylated GSTI
B
(1100) being visualized by autoradiography.
Preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA)
Control cells or cells pre-treated with CYL-19 s or CYL-26z were treated with TNF-
for 1 h, then nuclear extracts were prepared as described previously (10). Oligonucleotides corresponding to the downstream NF-
B binding sequences (5'-AGCTTGGAAATTCCGGA-3') in human ICAM-1 promoter were synthesized, annealed and end-labeled with [
-32P]ATP using T4 polynucleotide kinase, and EMSA was performed as described previously (10).
Transient transfection and luciferase assay
A549 cells were grown in 6-well plates. The human ICAM-1 firefly luciferase (LUC) plasmids (pIC339) or NF-
B luciferase reporter were transfected using Tfx-50 (Promega) according to the manufacturer's recommendations. Briefly, reporter DNA (0.4 µg) and ß-galactosidase DNA (0.2 µg) were mixed with 0.6 µl of Tfx-50 in 1 ml of serum-free DMEM. We used the plasmid PRK containing the ß-galactosidase gene driven by the constitutively active SV30 promoter to normalize the transfection efficiency. After a 1015-min incubation at room temperature, the mixture was applied onto the cells. One hour later, 1 ml DMEM containing 20% FCS was added and the cells were grown in medium containing 10% FCS. The following day, cells were exposed to 10 ng/ml of TNF-
or treated with CYL-19 s or CYL-26z for 30 min before challenge with TNF-
for 4.5 h, then cell extract was prepared, and luciferase (Promega Biotech System) and ß-galactosidase activity were measured. The luciferase activity of each well is normalized to ß-galactosidase activity. In over-expression experiments, cells were co-transfected with reporter (0.2 µg) and galactosidase (0.1 µg) and either the wild-type NF-
B-inducing kinase (NIK), IKKß or the empty vector (0.4 µg).
Preparation of cell extracts and western blot analysis
Following treatment with TNF-
or with various concentrations of CYL-19 s or CYL-26z before challenge with TNF-
for 10 min, the cells were rapidly washed with PBS, then lysed with ice-cold lysis buffer (50 mM TrisHCl, pH 7.4, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 5 µg/ml of leupeptin, 20 µg/ml of aprotinin, 1 mM NaF and 1 mM Na3VO4) as described previously (10). The lysates were subjected to SDSPAGE using a 10% running gel and the proteins were transferred to nitrocellulose paper. Immunoblot analysis was performed as described previously (10).
In vitro invasion assay
The invasion assay was carried out using Transwell cell culture chambers (Corning Costar No. 3422, MA) according to manufacturer's recommendation with some modifications. Briefly, polyvinylpyrrolidone-free polycarbonate filters (8.0 µm pore size, Nuclepore, CA) were pre-coated with 5 µg of Matrigel (BD Biosciences, MA) on the upper surface. A549 or NCI-H292 cells were harvested with 1 mM EDTA and then re-suspended in DMEM supplemented with 0.1% FBS. Cell suspensions (104 cells) were added to the upper compartment of the chamber. Cells were challenged with TNF-
following pre-treatment with CYL-19 s and CYL-26z for 30 min or in the presence of 10 µg/ml anti-ICAM-1 antibody. After a 24-h incubation, the top side of the insert membrane was scrubbed free of cells with a cotton swab and the bottom side was fixed with 3.7% paraformaldehyde, stained with 0.5% crystal violet in 20% methanol. The crystal violet dye retained on the filters was extracted with DMSO and colorimetrically assessed by measuring its absorbance at 590 nm on an ELISA reader (Bio-Tek).
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Results
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Inhibitory effects of CYL-19 s and CYL-26z on TNF-
-induced ICAM-1 protein expression in A549 cells, and U937 adhesion to A549 cells
In A549 cells, TNF-
has been shown to induce a dose- and time-dependent increase in ICAM-1 protein expression and U937 adhesion to A549 cells (11). To determine the effects of CYL19 s and CYL-26z on TNF-
-induced ICAM-1 protein expression, cells were pre-treated with 0.0330 µM of these two compounds for 30 min before incubation with TNF-
(10 ng/ml). As shown in Figure 2A, TNF-
-induced ICAM-1 expression was inhibited by CYL-19 s and CYL-26z in a dose-dependent manner with respective IC50 of 1.1 and 1.5 µM. At a concentration of 10 µM, both compounds did not cause toxicity on A549 and NCI-H292 cells. Since ICAM-1 expression mediated the adherence of U937 cells to A549 cells being demonstrated (11), the inhibitory effects of CYL-19 s and CYL-26z on U937 cell adhesion to A549 cells were examined. In parallel with the inhibitory effect on TNF-
-induced ICAM-1 expression, both compounds inhibited TNF-
-induced U937 adhesion to A549 cells (Figure 2B).
Inhibitory effects of CYL-19 s and CYL-26z on TNF-
-induced ICAM-1 mRNA expression and ICAM-1 promoter activity in A549 cells
To further confirm the inhibitory effect of CYL-19 s and CYL-26z on TNF-
-induced ICAM-1 gene expression, the mRNA level of ICAM-1 was analyzed by RTPCR. TNF-
increased the expression of ICAM-1 mRNA in a time-dependent manner; this effect was significant and maximal at 1 h, sustained at 3 h and declined after 6 h (Figure 3A). To determine the effects of CYL-19 s and CYL-26z on TNF-
-induced ICAM-1 gene expression, cells were pre-treated with each of these compounds at concentrations of 1, 5 or 10 µM for 30 min before stimulation with TNF-
for 3 h. As shown in Figure 3B, CYL-19 s and CYL-26z inhibited TNF-
-induced ICAM-1 mRNA expression in a dose-dependent manner (Figure 3B, lanes 49). To elucidate the mechanism involved in CYL-19 s- and CYL-26z-mediated inhibitions of ICAM-1 expression, transient transfections were performed using a human ICAM-1 promoter-luciferase construct, pIC339. As reported previously (11), treatment with TNF-
led to a 2.3-fold increase in ICAM-1 promoter activity, and this effect was inhibited by CYL-19 s and CYL-26z in a dose-dependent manner (Figure 4A). Since NF-
B was found to be critical for TNF-
-induced ICAM-1 expression (11), the effects of both compounds on NF-
B activation were examined using
B-Luc construct. The results showed that CYL-19 s and CYL-26z at 10 µM almost blocked TNF-
-induced NF-
B-dependent luciferase activity (Figure 4B). Cytokine-mediated I
B phosphorylation/degradation and NF-
B activation involve the activation of at least two sequential proximal kinases, NIK and IKK (18). IKK
/ß binds to NIK, a member of the mitogen-activated protein kinase kinase kinase family, to link I
B degradation and NF-
B activation. To further investigate the inhibitory effect of CYL-19 s and CYL-26z on NF-
B activation-induced ICAM-1 expression, co-transfection of pIC339 with wild-type NIK or IKKß was performed. Over-expression of wild-type NIK or IKKß resulted in a 3.2- or 2.4-fold increase, respectively, in ICAM-1 promoter activity, and these effects were suppressed by CYL-19 s and CYL-26z in a dose-dependent manner (Figure 5).

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Fig. 5. Inhibitory effects of CYL-19 s and CYL-26z on wild-type NIK- or IKKß-induced ICAM-1 promoter activity in A549 epithelial cells. Cells were co-transfected with wild-type NIK or IKKß or empty vector and the pIC339 luciferase construct, then treated with 1, 5 or 10 µM CYL-19 s or CYL-26z for 6 h. Luciferase activity was assayed as described in the Materials and methods. The results were normalized with ß-galactosidase activity and expressed as the mean ± SEM of three independent experiments performed in triplicate. *P < 0.05, **P < 0.01 as compared with vehicle.
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CYL-19 s and CYL-26z inhibit TNF-
-induced NF-
B activation via directly targeting an IKK complex
ICAM-1 promoter activity induced by TNF-
, wild-type NIK or IKKß was inhibited by CYL-19 s and CYL-26z, indicating that both compounds targeted IKKß to inhibited TNF-
-induced ICAM-1 gene expression. Therefore, the effects of these two compounds on TNF-
-induced IKK activity were examined. As reported previously (14), TNF-
induced IKK activation after 10 min of treatment. CYL-19 s and CYL-26z inhibited the TNF-
-induced IKK activation in a dose- dependent manner (Figure 6A, lanes 38). Phosphorylation of I
B
on Se-32 and -36 by IKK is necessary for its degradation and the subsequent NF-
B activation (19). Because TNF-
-induced IKK activity was inhibited by CYL-19 s and CYL-26z, we next examined their inhibitory effects on TNF-
-induced I
B
phosphorylation and degradation. Endogenous I
B phosphorylation was assessed using western blot with a specific I
B
phospho-Ser-32 antibody. As shown in Figure 6B, when cells were stimulated with TNF-
for 5, 10, 30 or 60 min, phosphorylation of I
B
was seen at 5 min of treatment (Figure 6B, lane 2), whereas it disappeared at 10 min and reappeared at 30 or 60 min (Figure 6B, lanes 35). Partial degradation of I
B
was seen after 5 min treatment with TNF-
(Figure 6B, lane 2) and almost complete degradation was seen after 10 min treatment (Figure 6B, lane 3). Partial and full restoration of I
B
level was seen at 30 and 60 min, respectively (Figure 6B, lanes 4 and 5) as reported previously (14). CYL-19 s and CYL-26z, but not MG132 (a proteasome inhibitor), inhibited the I
B
phosphorylation seen at 5 min treatment with TNF-
, and in turn prevented the partial degradation of I
B
induced by TNF-
(Figure 6C, compare lanes 3 and 4 with lane 2). The complete degradation of I
B
induced by 10 min treatment with TNF-
was prevented by the two compounds in a dose-dependent manner (Figure 6D, lanes 38). To test whether the inhibitory effects of CYL-19 s and CYL-26z on IKK activity and I
B
degradation led to NF-
B inhibition, the effects of both compounds on TNF-
-stimulated NF-
B-specific DNAprotein binding activity were analyzed. As shown in Figure 6E, CYL-19 s and CYL-26z inhibited TNF-
-induced NF-
B-specific DNAprotein binding in a dose-dependent manner (Figure 6E, lanes 36). These results reveal that CYL-19 s and CYL-26z are effective in the inhibition of NF-
B activation.

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Fig. 6. CYL-19 s and CYL-26z inhibited TNF- -induced NF- B activation via direct targeting on IKK complex in A549 epithelial cells. (A) Cells were pre-treated with 1, 5 or 10 µM CYL-19 s or CYL-26z for 30 min before incubation with 10 ng/ml of TNF- for 10 min. Whole cell lysates were immunoprecipitated with anti-IKKß antibody, kinase assay (KA) was performed and autoradiography of phosphorylated GSTI B (1-100) was detected as described in the Materials and methods. The amount of IKKß in immunoprecipitates was determined by western blot (WB) using anti-IKKß antibody. (BD) After treatment with TNF- for 5, 10, 30 or 60 min (B) or pre-treatment with 10 µM CYL-19 s, CYL-26z, or MG132 (C) or indicated concentrations of CYL-19 s or CYL-26z (D) for 30 min before incubation with 10 ng/ml of TNF- for 5 min (C) or 10 min (D), the cytosolic levels of phosphorylated I B or I B were immunodetected using I B phospho-Ser-32 or I B -specific antibody as described in the Materials and methods. Actin was used as a loading control. In (E), cells were pre-treated with 5 or 10 µM CYL-19 s or CYL-26z for 30 min before incubation with 10 ng/ml of TNF- for 30 min, then nuclear extracts were prepared and NF- B DNAprotein binding activity was determined by EMSA as described in the Materials and methods. (F) Cells were incubated with 10 ng/ml of TNF- for 10 min, and then whole cell lysates were prepared. IKK was immunoprecipitated using anti-IKKß antibody from the lyastes and incubated with GSTI B (1-100), [ -32P]ATP and 10 µM CYL-19 s or CYL-26z in the kinase assay buffer for 30 min. Kinase assay (KA) was performed as described in the Materials and methods. (G) Cells were pre-treated with 10 µM CYL-19 s, 10 µM CYL-26z, 50 µM PD98059 or 30 µM SB203580 for 30 min before incubation with 10 ng/ml of TNF- for 30 min, the cytosolic levels of activated ERK1/2 (p-ERK1/2) and activated p38 (p-p38) or expression level of ERK1/2 and p38 were immunodetected using ERK1/2 phospho-Thr202/Tyr204 and p38 phopsho-Thr180/Tyr182 or ERK1/2 and p38-specific antibodies as described in the Materials and methods.
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CYL-19 s and CYL-26z directly and specifically interfere with the activity of IKK
The inhibitory effects of CYL-19 s and CYL-26z on the kinase activity of IKK might be due to direct modification of IKK or inhibition of upstream events leading to IKK activation. Thus, we performed the in vitro kinase assay to examine whether CYL-19 s and CYL-26z inhibit IKK activity directly. A549 cells were treated with TNF-
for 10 min followed by immunoprecipitation of the endogenous IKK. The immune complexes were incubated with 10 µM CYL-19 s or CYL-26z for 30 min. As shown in Figure 6F, in vitro treatment with these compounds caused a dramatic inhibition of IKK activity (Figure 6F, lanes 36), demonstrating their direct interference with the IKK activity.
The TNF-
-derived signals led to the activation of several parallel and interconnected signaling cascades, resulting in the activations of NF-
B and the MAP kinases ERK1/2, p38 and JNK (11). To test whether CYL-19 s and CYL-26z target mitogen-activated protein kinase signaling cascades, their effect on the activations of ERK1/2 and p38 were examined. In comparison with PD98059 (a specific MEK1/2 inhibitor) and SB203580 (a specific p38 inhibitor), the activation of ERK/12 or p38 induced by TNF-
was unchanged in the presence of CYL-19 s or CYK-26z. These results demonstrate that these two compounds could directly and specifically target the IKK complex.
Inhibitory effects of CYL-19 s and CYL-26z on TNF-
-induced ICAM-1-dependent cell invasion and MMP-9 mRNA expression
To evaluate the involvement of ICAM-1 in the invasion of malignant alveolar epithelial cells, a Matrigel assay was performed. TNF-
-induced A549 cell invasion was completely inhibited by the pre-treatment with anti-ICAM-1 antibody (Figure 7A), indicating the critical role of ICAM-1 in mediating TNF-
-induced A549 cell invasion. Both CYL-19 s and CYL-26z at 5 µM inhibited TNF-
-induced A549 cell invasion (Figure 7A). In addition to ICAM-1, MMP-9 mRNA expression induced by TNF-
was attenuated by CYL-19 s and CYL-26z in a dose-dependent manner (Figure 7B).
Inhibitory effects of CYL-19 s and CYL-26z on TNF-
-induced COX-2 expression and on PGE2-dependent cell invasion
Our previous studies demonstrated that TNF-
-induced COX-2 expression and PGE2 release in NCI-H292 cells, another human alveolar epithelial carcinoma cell line (17). COX-2 is the rate-limiting enzyme in the conversion of arachidonic acid into prostaglandin G2/H2 (PGG2/H2) and involved in a wide range of inflammatory responses. We further examined whether CYL-19 s and CYL-26z had inhibitory effects on TNF-
-induced COX-2 expression. As shown in Figure 8A, CYL-19 s or CYL-26z inhibited the induced COX-2 expression in a doseresponse manner (Figure 8A, lanes 38).
To evaluate the role of COX-2 in the invasion of malignant alveolar epithelial cells, a Matrigel assay was performed. In NCI-H292 cells, TNF-
-induced cell invasion was blocked by 10 µM NS-398, a specific COX-2 inhibitor. Addition of exogenous 100 nM PGE2 reversed the inhibition of NCI-H292 cell invasion by NS-398, and PGE2 itself enhanced Matrigel invasion (Figure 8B). Both CYL-19 s and CYL-26z also inhibited TNF-
-induced NCI-H292 cell invasion (Figure 8C).
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Discussion
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We have identified two structurally related
-metheylene-
-lactone compounds, CYL-19 s and CYL-26z, which inhibit the IKK activity, the I
B
phosphorylation and degradation, and the NF-
B activation with concomitant down-regulation of ICAM-1 gene expression in the alveolar epithelial cells, resulting in the inhibition of ICAM-1-dependent monocyte adhesion to epithelial cells and the ICAM-1-dependent tumor cell invasion. In addition, they also inhibit MMP-9 mRNA expression, inducible COX-2 expression and PGE2-dependent tumor cell invasion. The phosphorylation of I
B is a key regulatory step that dictates NF-
B activation (20). IKK is a large 500900 kDa protein complex with three subunits. Two of the subunits, IKK
and IKKß, are serine kinase (21). A third subunit, IKK
interacts preferentially with IKKß and seems necessary for the activation (22). IKK directly phosphorylates two serine residues at the N-termini of I
B
(Ser-32 and Ser-36) and I
Bß (Ser-19 and Ser-23) (23). This phosphorylation event triggers the polyubiquitination of I
B and the subsequent degradation by the 26S proteasome (24). Of the three subunits, the phosphorylation of IKKß is necessary for the IKK activation, and IKKß seems to be responsible for the phosphorylation of I
B (25). Using molecular biological approaches, ectopic expression of NIK or IKKß allowed us to bypass the TNF-
receptor and specifically address the effect of these two compounds on more proximal NF-
B-inducing signals. Using this approach, we found that CYL-19 s and CYL-26z interfere with NIK- or IKKß-induced ICAM-1 promoter activity, indicating the action site of these two compounds at the level of IKKß in TNF-
-mediated NF-
B activation, leading to the inhibition of ICAM-1 expression. Among the parameters involved in the IKKß/NF-
B activation, the IKKß activity, the I
B
phosphorylation and degradation, NF-
B-specific DNAprotein complex formation, and NF-
B-luc activity were examined. TNF-
-induced IKK activity was inhibited by these two compounds. The two
-methylene-
-butyrolactone derivatives may directly interfere with the IKK complex to inhibit the signal leading to IKK activation, and then attenuate NF-
B activation. Because the phosphorylated I
B
protein was seen at 5 min after treatment with TNF-
and disappeared at 10 min due to I
B
degradation (Figure 4B), cells were treated with TNF-
for 5 min to examine the phosphorylation of I
B
and 10 min to examine the degradation of I
B
. Accumulation of phosphorylated I
B
protein was inhibited by CYL-19 s and CYL-26z, not by MG132 (Figure 4C), confirming that these two compounds inhibited IKK activity, but not the proteasome complex. The ability to directly inhibit IKK activity without any effect on the activations of ERK1/2 and p38 further demonstrated their specific targeting on the IKK complex.
ICAM-1 has been reported to play a pivotal role in transducing signals in the endothelial cells following the adhesion of lymphocytes, an essential step in the endothelial cell signal cascade responsible for facilitating lymphocyte transendothelial migration across the brain endothelial cells (26). The intracellular domain of ICAM-1 is essential for the transendothelial migration of lymphocytes (27). Targeted disruption of ICAM-1 gene inhibits the choroidal neovascularization (28), and protection from lymphoma cell metastasis has been reported in ICAM-1 mutant mice (29). In addition to the increase in monocyte adhesion to the A549 epithelial cells, TNF-
-induced ICAM-1 over-expression also elicited A549 cell invasion across the Matrigel. Anti-ICAM-1 antibody inhibited this invasion, demonstrating the critical role of ICAM-1 in tumor cell invasion. In addition, MMP-9 is thought to be a key enzyme for the degradation of type IV collagen in the extracellular matrix and basement membrane that facilitates local invasion of various types of human lung adenocarcinoma cell lines (30). Mutation of NF-
B on the MMP-9 promoter abrogated the TNF-
-mediated MMP-9 activity, suggesting the essential role of NF-
B in the expression of MMP-9 (31). TNF-
also induced MMP-9 mRNA expression in A549 cells, and this effect was inhibited by CYL-19 s and CYL-26z. This inhibition of MMP-9 mRNA expression may also contribute to the effectiveness of CYL-19 s and CYL-26z on the inhibition of A549 alveolar carcinoma cell invasion in vitro.
The increased COX-2 expression has been reported to correlate with the malignant changes observed in a variety of human cancers, including colorectal, gastric, pancreatic, esophageal, brain and lung tumors (3236). Its role has been extensively studied in colorectal cancer, where this enzyme is expressed in adenomas (35,37). COX-2 may function as a survival factor by protecting cells from apoptosis (38). Increasing tumorigenic potential by the COX-2 over-expression has been suggested to be associated with the resistance to apoptosis (39). In addition to the inhibition of apoptosis, other functions of inducible COX-2 have been described in the biology of various carcinomas: increased cell proliferation (40), induced tumor invasion (39), stimulation of angiogenesis (41) and the inhibition of immunosurveillance (42). In addition to the inhibitory effects on inducible ICAM-1 expression, CYL-19 s and CYL-26z also suppressed the TNF-
-induced COX-2 expression in NCI-H292 cells, in which the molecular mechanism has been explored to involve the PKC/c-Src-dependent activation of IKK and NF-
B as well (17,43). TNF-
-induced COX-2 expression increased Matrigel invasion. The highly specific COX-2 inhibitor NS-398 inhibited Matrigel invasion, and the inhibitory effect of NS-398 was reversed by exogenous PGE2. PGE2 itself also stimulates the invasiveness of alveolar cancer cells. This, in addition to the invasion inhibition/reversal data, indicated that PGE2 is a necessary invasive/permissive factor and is sufficient to induce the invasiveness by itself. Therefore, TNF-
-induced COX-2 expression releases PGE2 in the NCI-H292 cells (17) and mediates the cancer cell invasion. The effectiveness of NS-398, CYL-19 s and CYL-26z on the inhibition of NCI-H292 alveolar carcinoma cell invasion in vitro point to a very promising therapeutic target in the lung cancer prevention and treatment, in view of the great success of COX-2 inhibitors demonstrated in the colon cancer prevention and treatment (44).
A number of natural products with different chemical structures, including isoprenoids, phenolics, triterpenoids and sesquiterpene lactones, have been demonstrated to possess the NF-
B-inhibitory activity. The kaurene diterpenoids, most notably isoprenoids, were found to inhibit the iNOS expression and the NF-
B activation in LPS-stimulated J744 macrophages via diminishing IKK activity (45). Similar interference at the IKK
/ß level, leading to the inhibition of NF-
B activation and COX-2 expression, is seen with curcumin, which is one of the phenolic compounds from Curcuma longa (46). Oleandrin, belonging to triterpenoids, was also reported to inhibit the NF-
B activation by targeting the IKK complex in U937 cells (47). The sesquiterpene lactones, such as helenalin, parthenolide and ergolide, are natural products and the most widely published NF-
B inhibitors. Although the majority of sesquiterpene lactones has been suggested to inhibit NF-
B activity via alkylating IKK and p65 by two reactive centers of an
-methylene-
-lactone group and an
,ß-unsaturated cyclopentenone (4851), the exact mechanism of action remains to be found. Those studies were less to examine their anti-inflammatory functions. In our study, we showed that CYL-19 s and CYL-26z, two synthetic
-methylene-
-lactone derivates, are more potent NF-
B inhibitors (IC50 1.1 and 1.5 µM, respectively) compared with sesquiterpene lactones (IC50 1050 µM). CYL-19 s and CYL-26z possess
-methylene-
-lactone and acridine but not
,ß-unsaturated cyclopentenone group to target IKK complex at a lower concentration, indicating the important role of
-methylene-
-lactone moiety in NF-
B inhibition. The inhibition of ICAM-1 and COX-2 expression and monocyte adhesion further confirms their anti-inflammatory effects. Moreover, their inhibitory effects on the ICAM-1- or COX-2-mediated tumor cell invasion were also demonstrated in this study. Several synthetic chemicals with different structures are found to possess the characteristics of NF-
B inhibition independent of I
B degradation. We found that GF-015 and GF-90, two conjugated polyhydroxybenzene derivatives, inhibited the TNF-
-induced COX-2 expression by targeting IKK activity and I
B phosphorylation without preventing the I
B degradation (52). Dehydroxymethylepoxyquinomicin based on the epoxydone structures inhibited the TNF-
-induced nuclear translocation and DNA binding of NF-
B without affecting the phosphorylation and degradation of I
B (53). The continual identification of new selective inhibitors of NF-
B may lead to a more effective treatment of inflammatory disorders and cancers. Our identification of CYL-19 s and CYL-26z as inhibitors of IKK should provide insight into the IKK complex and potentially lead to the development of new IKK inhibitors that are more potent and hopefully more selective.
 |
Acknowledgments
|
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This work was supported by a research grant from the National Science Council of Taiwan (NSC-92-2320-B002-074).
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Received March 4, 2004;
revised May 20, 2004;
accepted June 6, 2004.