1′-Acetoxychavicol acetate (ACA), extracted from rhizomes of the commonly used ethno-medicinal plant Languas galanga, has been found to suppress chemical- and virus-induced tumor initiation and promotion through a poorly understood mechanism. Because several genes that regulate cellular proliferation, carcinogenesis, metastasis, and survival are regulated by activation of the transcription factor NF-κB, we postulated that ACA might mediate its activity through modulation of NF-κB activation. For this report, we investigated the effect of ACA on NF-κB and NF-κB-regulated gene expression activated by various carcinogens. We found that ACA suppressed NF-κB activation induced by a wide variety of inflammatory and carcinogenic agents, including TNF, IL-1β, PMA, LPS, H2O2, doxorubicin, and cigarette smoke condensate. Suppression was not cell type specific, because both inducible and constitutive NF-κB activations were blocked by ACA. ACA did not interfere with the binding of NF-κB to the DNA, but, rather, inhibited IκBα kinase activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and subsequent p65 nuclear translocation. ACA also inhibited NF-κB-dependent reporter gene expression activated by TNF, TNFR1, TNFR-associated death domain protein, TNFR-associated factor-2, and IκBα kinase, but not that activated by p65. Consequently, ACA suppressed the expression of TNF-induced NF-κB-regulated proliferative (e.g., cyclin D1 and c-Myc), antiapoptotic (survivin, inhibitor of apoptosis protein-1 (IAP1), IAP2, X-chromosome-linked IAP, Bcl-2, Bcl-xL, Bfl-1/A1, and FLIP), and metastatic (cyclooxygenase-2, ICAM-1, vascular endothelial growth factor, and matrix metalloprotease-9) gene products. ACA also enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed invasion. Overall, our results indicate that ACA inhibits activation of NF-κB and NF-κB-regulated gene expression, which may explain the ability of ACA to enhance apoptosis and inhibit invasion.

Between 1981 and 2002, 48 of 65 drugs (74%) that were approved for cancer treatment were natural products, based on natural products, or mimics of natural products (1). One of these, 1′-acetoxychavicol acetate (ACA),3 derived from the rhizomes of a subtropical ginger, Languas galanga Stuntz (Zingiberaceae), has been shown to exhibit antitumor properties against a wide variety of cancers (2, 3, 4, 5, 6, 7, 8, 9, 10). For instance, ACA has been shown to inhibit phorbol ester-induced skin tumor promotion (8), azoxymethane-induced colonic aberrant crypt foci (7), estrogen-related endometrial carcinogenesis (6), hepatic focal lesions (5), rat oral carcinogenesis (4), and N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis (3). How ACA suppresses tumorigenesis is not well understood. It is known to induce apoptosis of tumor cells through activation of caspases (10) and through a dual mitochondrial- and Fas-mediated mechanism (11). Protein tyrosine phosphorylation and reduction of cellular sulfhydryl groups have been implicated in ACA-induced apoptosis (12). Still other reports have indicated that ACA has antioxidant and anti-inflammatory activities (13, 14), inhibits xanthine oxidase (4), and suppresses inducible NO synthase gene expression (14). How ACA mediates antitumor activities is not well understood.

For reasons detailed below, we postulated that ACA mediates its various activities through suppression of the transcription factor NF-κB. First, NF-κB is activated by various carcinogens, tumor promoters, and tumor microenvironment (hypoxia and acidic pH). Second, most inflammatory agents activate NF-κB. Third, NF-κB regulates the expression of genes that regulate transformation, tumor promotion, tumor invasion, angiogenesis, and metastasis. Fourth, suppression of apoptosis is regulated by NF-κB. And fifth, chemopreventive agents have been shown to suppress NF-κB activation (15).

NF-κB is a heterodimeric protein complex of members of the Rel (p50)/NF-κB (p60) protein family. NF-κB is primarily composed of proteins with molecular masses of 50 kDa (p50) and 65 kDa (p65) and is retained in the cytoplasm by inhibitory subunit, IκBα (16). In its unstimulated form, NF-κB is activated by a wide variety of inflammatory stimuli, including TNF, IL-1, PMA, H2O2, endotoxin, and gamma irradiation. Most of these agents induce the phosphorylation-dependent degradation of IκBα proteins, allowing active NF-κB to translocate to the nucleus, where it regulates gene expression. The phosphorylation of IκBα is mediated through the activation of the IκBα kinase (IKK) complex consisting of IKK-α, IKK-β, IKK-γ (also called NEMO), IKK-associated protein-1, 14,700-kDa-interacting protein-3 (FIP-3) (type 2 adenovirus E3–14.7kDa interacting protein), 90-kDa heat shock protein, and glutamic acid (E), leucine (L), lysine (K), and serine (S)-abundant protein (ELKS) (17).

Because of the critical role of NF-κB in proliferative and inflammatory diseases, we investigated the effect of ACA on NF-κB activation induced by carcinogens, tumor promoters, and inflammatory agents. The results described below strongly suggest that ACA is a potent suppressor of NF-κB activation induced by various agents and that this suppression is mediated through inhibition of IKK. As a result, the expression of gene products that regulate apoptosis, proliferation, angiogenesis, and invasion is suppressed.

ACA was synthesized as previously described (18). A 50-mM solution of ACA was prepared with DMSO, stored as small aliquots at −20°C, and then diluted as needed in cell culture medium. Bacteria-derived human recombinant human TNF, purified to homogeneity with a specific activity of 5 × 107 U/mg, was provided by Genentech. Cigarette smoke condensate, prepared as previously described (19), was supplied by Dr. G. Gairola (University of Kentucky, Lexington, KY). Penicillin, streptomycin, RPMI 1640 medium, and FBS were obtained from Invitrogen Life Technologies. PMA, okadaic acid, H2O2, and anti-β-actin Ab were obtained from Sigma-Aldrich. Abs anti-p65, anti-p50, anti-IκBα, anti-cyclin D1, anti-matrix metalloprotease-9 (anti-MMP-9), anti-c-Myc, anti- poly(ADP-ribose) polymerase (anti-PARP), anti-inhibitor of apoptosis protein-1 (anti-IAP1), anti-IAP2, anti-Bcl-2, anti-Bcl-xL, and anti-Bfl-1/A1 were obtained from Santa Cruz Biotechnology. Anti-cyclooxygenase-2 (anti-COX-2) and anti-X-chromosome-linked IAP (XIAP) Abs were obtained from BD Biosciences. Phosphospecific anti-IκBα (Ser32), phosphospecific anti-p65 (Ser536), and anti-acetyl-lysine Abs were purchased from Cell Signaling. Anti-IKK-α, anti-IKK-β, and anti-FLIP Abs were provided by Imgenex.

KBM-5 (human chronic myeloid leukemia), H1299 (lung adenocarcinoma), Jurkat (human T cell lymphoma), A293 (human embryonic kidney carcinoma), and MCF-7 (human breast adenocarcinoma) cells were obtained from American Type Culture Collection. LICR-LON-HN5 and SCC4 (both human squamous cell carcinoma) cells were obtained from Dr. M. J. O’Hare (Haddow Laboratories, Institute of Cancer Research, Sutton, U.K.). KBM-5 cells were cultured in IMDM with 15% FBS. Jurkat, H1299, MM1, and U266 cells were cultured in RPMI 1640 medium, and A293 cells were cultured in DMEM supplemented with 10% FBS. LICR-LON-HN5 and SCC4 cells were cultured in DMEM containing 10% FBS, 100 μM nonessential amino acids, 1 mM pyruvate, 6 mM l-glutamine, and 1× vitamins. Culture media were also supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

To determine NF-κB activation, we performed EMSA as described previously (20). Briefly, nuclear extracts prepared from TNF-treated cells were incubated with 32P-end-labeled, 45-mer, double-stranded NF-κB oligonucleotide (15 μg of protein with 16 fmol of DNA) from the HIV long terminal repeat, 5′-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG-3′ (underlining indicates NF-κB binding sites) for 30 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′-TTGTTACAA CTCACTTTC CGCTG CTCACTTTC CAGGGAGGCGTGG-3′, was used to examine the specificity of binding of NF-κB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with Abs against either p50 or p65 of NF-κB for 15 min at 37°C before the complex was analyzed by EMSA. Abs against preimmune serum (PIS) was included as negative controls. The dried gels were visualized, and radioactive bands were quantitated by a PhosphorImager (Molecular Dynamics) using ImageQuant software.

To determine the levels of protein expression in the cytoplasm or nucleus, we prepared extracts (21) and fractionated them by SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with each Ab, and detected by ECL regent (Amersham Biosciences). The bands obtained were quantitated using National Institutes of Health imaging software.

To determine the effect of ACA on TNF-induced IKK activation, an IKK assay was performed by a method described previously (22). To determine the total amounts of IKK-α and IKK-β in each sample, 50 μg of the whole-cell protein was resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-α or anti-IKK-β Abs.

NF-κB-dependent reporter gene expression was performed as previously described (23). The effects of ACA on TNF-, TNFR-, TNFR-associated death domain protein (TRADD)-, TNFR-associated factor-2 (TRAF2)-, NF-κB-inducing kinase (NIK)-, IKK-β-, and p65-induced NF-κB-dependent reporter gene transcription were analyzed by secretory alkaline phosphatase (SEAP) assay as previously described (23). Briefly, A293 cells (5 × 105 cells/well) were plated in six-well plates and transiently transfected by FuGene6 (Roche) with pNF-κB-SEAP (0.25 μg). To examine TNF-induced reporter gene expression, we transfected the cells with 0.25 μg of the SEAP expression plasmid for 24 h. Thereafter we coincubated the cells for 24 h with 10 μM ACA and 1 nM TNF for an additional 24 h. The cell culture medium was harvested after 24 h of TNF treatment and analyzed for SEAP activity essentially according to the protocol described by the manufacturer (BD Clontech) using a 96-well fluorescence plate reader (Fluoroscan II; Labsystems) with excitation set at 360 nm and emission at 460 nm.

The effect of ACA on the nuclear translocation of p65 was examined by immunocytochemistry as previously described (21).

To measure apoptosis, we also used the live and dead assay (Molecular Probes), which determines intracellular esterase activity and plasma membrane integrity. This assay was performed as previously described (24).

The effect of ACA on the cytotoxic effects of TNF and chemotherapeutic agents was determined by the MTT uptake method as previously described (25).

An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface to the extracellular surface. This loss of membrane asymmetry can be detected using the binding properties of annexin V. To identify apoptosis, we stained cells with annexin V Ab conjugated with FITC dye. Briefly, 5 × 105 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h at 37°C and then stained. Cells were washed in PBS, resuspended in 100 μl of binding buffer containing FITC-conjugated anti-annexin V Ab, and then analyzed by flow cytometer (FACSCalibur; BD Biosciences).

We also assayed cytotoxicity by the TUNEL method, which examines DNA strand breaks during apoptosis, using an in situ cell death detection reagent (Roche). Briefly, 5 × 105 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h at 37°C. Thereafter, cells were incubated with reaction mixture for 60 min at 37°C. Stained cells were analyzed by flow cytometer (FACSCalibur; BD Biosciences).

Invasion through the extracellular matrix is a crucial step in tumor metastasis. We used Matrigel basement membrane matrix extracted from the Englebreth-Holm-Swarm mouse tumor as a reconstituted basement membrane for in vitro invasion assays. The BD BioCoat tumor invasion system we used has a chamber with a light-tight polyethelyene terephlate membrane with 8-μm pores coated with a reconstituted basement membrane gel (BD Biosciences). We resuspended 2.5 × 104 H1299 cells in serum-free medium and seeded the suspension into the upper wells. After incubation overnight, cells were coincubated with 10 μM ACA and TNF for an additional 24 h in the presence of 1% FBS. The cells that passed through the Matrigel were labeled with 4 μg/ml calcein AM (Molecular Probes) in PBS for 30 min at 37°C and subjected to scan fluorescence by a Vector 3 luminometer (PerkinElmer).

The aim of the current study was to investigate the effects of ACA on the NF-κB activation pathway induced by various carcinogens and inflammatory stimuli and on NF-κB-regulated gene expression. Because the TNF-induced NF-κB activation pathway has been well characterized, we investigated in detail the effects of ACA on TNF-induced NF-κB activation. The structure of ACA is shown in Fig. 1 A.

FIGURE 1.

Effect of ACA on activator-induced NF-κB activation. A, The structure of ACA. B, ACA blocks NF-κB activation induced by TNF, LPS, IL-1β, PMA, H2O2, CSC, and DOX. KBM-5 cells were coincubated with 50 μM ACA, 0.1 nM TNF, 100 ng/ml IL-1β, and LPS for 30 min; with 15 ng/ml PMA and 1 μg/ml CSC for 1 h; with 500 μM H2O2 for 2 h; and with 1 μg/ml DOX for 6 h, then analyzed for NF-κB activation as described in Materials and Methods. The results shown are representative of three independent experiments.

FIGURE 1.

Effect of ACA on activator-induced NF-κB activation. A, The structure of ACA. B, ACA blocks NF-κB activation induced by TNF, LPS, IL-1β, PMA, H2O2, CSC, and DOX. KBM-5 cells were coincubated with 50 μM ACA, 0.1 nM TNF, 100 ng/ml IL-1β, and LPS for 30 min; with 15 ng/ml PMA and 1 μg/ml CSC for 1 h; with 500 μM H2O2 for 2 h; and with 1 μg/ml DOX for 6 h, then analyzed for NF-κB activation as described in Materials and Methods. The results shown are representative of three independent experiments.

Close modal

Because TNF, PMA, LPS, IL-1β, doxorubicin (DOX), H2O2, and cigarette smoke condensate (CSC) are potent activators of NF-κB (19, 25, 26, 27), we examined the effect of ACA on the activation of NF-κB by these agents. Coincubation of cells with 50 μM ACA suppressed the activation of NF-κB induced by all seven agents (Fig. 1 B). The concentration of ACA and NF-κB activators used and the time of exposure had minimal effect on cell viability. These results suggest that ACA acts at a step in the NF-κB activation pathway that is common to all seven agents.

Because TNF is one of the most potent activator of NF-κB, and the mechanism of activation of NF-κB is relatively well established (28), we examined the effects of different doses of ACA on TNF-induced NF-κB activation in human myeloid KBM5 cells. Cells were exposed to different concentrations of ACA together with TNF for 30 min and then examined for NF-κB activation. These studies indicated that ACA suppressed TNF-induced NF-κB activation in a dose-dependent manner, with 60% inhibition at 10 μM and almost 100% inhibition at 50 μM (Fig. 2 A).

FIGURE 2.

ACA suppresses TNF-induced NF-κB in a dose-dependent manner and in different cell lines. A, Human myeloid leukemia KBM-5 cells were coincubated with the indicated concentrations of ACA and 0.1 nM TNF for 30 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. B, Human breast cancer MCF-7, human T cell lymphoma Jurkat, and human lung carcinoma H1299 cells were coincubated with 50 μM ACA and 0.1 nM TNF for 30 min. C, Squamous cell carcinoma LICR-LON-HN5 (HN5) and SCC4 cells and human multiple myeloma MM1 and U266 cells were coincubated with 50 μM ACA and 0.1 nM TNF for 30 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. The results shown are representative of three independent experiments.

FIGURE 2.

ACA suppresses TNF-induced NF-κB in a dose-dependent manner and in different cell lines. A, Human myeloid leukemia KBM-5 cells were coincubated with the indicated concentrations of ACA and 0.1 nM TNF for 30 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. B, Human breast cancer MCF-7, human T cell lymphoma Jurkat, and human lung carcinoma H1299 cells were coincubated with 50 μM ACA and 0.1 nM TNF for 30 min. C, Squamous cell carcinoma LICR-LON-HN5 (HN5) and SCC4 cells and human multiple myeloma MM1 and U266 cells were coincubated with 50 μM ACA and 0.1 nM TNF for 30 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. The results shown are representative of three independent experiments.

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Because the signal transduction pathway mediated by NF-κB may be distinct in different cell types (29, 30), we investigated whether ACA could block TNF-induced NF-κB activation in breast adenocarcinoma MCF-7 (Fig. 2,B), human T cell lymphoma Jurkat (Fig. 2,B), and human lung carcinoma H1299 cells (Fig. 2 B). These cells were exposed to TNF in the presence or the absence of ACA for 30 min and then examined for NF-κB activation. TNF activated NF-κB in every cell type, and ACA completely inhibited most of this activation, indicating that ACA-induced suppression of NF-κB activation was not cell type specific.

Most tumor cells express constitutively active NF-κB (26, 27), although the mechanism is not well understood. We showed that ACA suppresses constitutive activation of NF-κB in human multiple myeloma (MM1 and U266) and head and neck squamous cell carcinoma (SCC4 and HN5) cells, which are known to express constitutive active NF-κB (31, 32) (Fig. 2 C).

The suppression of NF-κB by most agents, including TNF, requires that they be applied before the NF-κB-activating agent (21, 24). However, treatment with ACA 5 min before TNF treatment,at the same time as TNF treatment, or 5 or 10 min after TNF treatment all suppressed TNF-induced NF-κB activation (Fig. 3 A), suggesting that ACA is a fast-acting inhibitor of NF-κB activation.

FIGURE 3.

ACA suppresses TNF-induced NF-κB activation in human myeloid KBM-5 cells. A, KBM-5 cells were preincubated with 50 μM ACA for the indicated times and then tested for the NF-κB activation at 37°C for 30 min with 0.1 nM TNF. −, Incubation with TNF; +, time ACA was added after TNF. After these treatments, nuclear extracts were prepared and assayed for NF-κB activation by EMSA. B, NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated Abs, PIS, unlabeled NF-κB oligo-probe, or mutant oligo-probe and then assayed for NF-κB activation by EMSA. C, Direct effect of ACA on NF-κB complex. Nuclear extracts were prepared from untreated or 0.1 nM TNF-treated KBM-5 cells, incubated for 30 min with the indicated concentrations of ACA, and then assayed for NF-κB activation by EMSA. D, KBM-5 cells were coincubated with 50 μM ACA and the indicated concentrations of TNF for 30 min and then subjected to EMSA for NF-κB activation. The results shown are representative of three independent experiments.

FIGURE 3.

ACA suppresses TNF-induced NF-κB activation in human myeloid KBM-5 cells. A, KBM-5 cells were preincubated with 50 μM ACA for the indicated times and then tested for the NF-κB activation at 37°C for 30 min with 0.1 nM TNF. −, Incubation with TNF; +, time ACA was added after TNF. After these treatments, nuclear extracts were prepared and assayed for NF-κB activation by EMSA. B, NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated Abs, PIS, unlabeled NF-κB oligo-probe, or mutant oligo-probe and then assayed for NF-κB activation by EMSA. C, Direct effect of ACA on NF-κB complex. Nuclear extracts were prepared from untreated or 0.1 nM TNF-treated KBM-5 cells, incubated for 30 min with the indicated concentrations of ACA, and then assayed for NF-κB activation by EMSA. D, KBM-5 cells were coincubated with 50 μM ACA and the indicated concentrations of TNF for 30 min and then subjected to EMSA for NF-κB activation. The results shown are representative of three independent experiments.

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Various combinations of Rel/NF-κB protein constitute active NF-κB heterodimers, p50 and p65, that bind to a specific DNA sequence (16). When we incubated nuclear extracts from TNF-stimulated cells with Abs to either the p50 (NF-κB1) or the p65 (RelA) subunit of NF-κB, each shifted the band to a higher molecular mass (Fig. 3 B). PIS had no effect on the binding. Abs alone did not directly interact with the labeled probe (data not shown). Thus, the TNF-activated complex consisted of p50 and p65 subunits. Additionally, excess unlabeled NF-κB caused complete disappearance of the band, but a mutant oligonucleotide of NF-κB did not affect NF-κB binding activity.

Several NF-κB inhibitors have been shown to suppress NF-κB activation by directly blocking the binding of NF-κB to the DNA (33, 34, 35). When we incubated nuclear extracts from TNF-treated cells with ACA, EMSA showed that ACA had no direct effect on NF-κB binding to the DNA (Fig. 3 C). Thus, ACA must inhibit NF-κB activation through an indirect mechanism.

To determine the effect of ACA on NF-κB activation at higher concentrations, cells were treated with the indicated concentrations of TNF for 30 min in the absence or the presence of ACA and then analyzed NF-κB activation by EMSA (Fig. 3 D). TNF at a concentration of 10 nM activated NF-κB activity strongly, and ACA abolished TNF-induced NF-κB activation. These results show that ACA is a very potent inhibitor of TNF-induced NF-κB activation.

We examined ACA- and TNF-treated cells for NF-κB by EMSA and for IκBα by Western blot analysis. TNF-induced NF-κB activation was completely suppressed by ACA (Fig. 4,A). ACA also suppressed TNF-induced IκBα degradation, although not completely (Fig. 4 B). These results indicate that ACA inhibits both TNF-induced NF-κB activation and IκBα degradation.

FIGURE 4.

Effect of ACA on IκBα phosphorylation and degradation induced by TNF. A, ACA inhibits TNF-induced activation of NF-κB in a time-dependent manner. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times and then analyzed for NF-κB activation by EMSA. B, Effect of ACA on TNF-induced degradation of IκBα. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-IκB. C, Effect of ACA on TNF-induced translocalization of p65. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times. Nuclear extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-p65 Abs. D, Immunocytochemical analysis of p65 localization. KBM-5 cells were coincubated with 50 μM ACA and 1 nM TNF for 15 min and then subjected to immunocytochemistry as described in Materials and Methods. The results shown are representative of three independent experiments.

FIGURE 4.

Effect of ACA on IκBα phosphorylation and degradation induced by TNF. A, ACA inhibits TNF-induced activation of NF-κB in a time-dependent manner. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times and then analyzed for NF-κB activation by EMSA. B, Effect of ACA on TNF-induced degradation of IκBα. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-IκB. C, Effect of ACA on TNF-induced translocalization of p65. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times. Nuclear extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-p65 Abs. D, Immunocytochemical analysis of p65 localization. KBM-5 cells were coincubated with 50 μM ACA and 1 nM TNF for 15 min and then subjected to immunocytochemistry as described in Materials and Methods. The results shown are representative of three independent experiments.

Close modal

As shown in Fig. 4,C, Western blot analysis indicated that ACA significantly inhibited TNF-induced nuclear translocation of p65. Immunocytochemistry appeared to confirm this (Fig. 4 D).

Because IκBα phosphorylation is needed for IκBα degradation, we determined whether ACA modulated IκBα phosphorylation. Because TNF-induced phosphorylation of IκBα leads to its rapid degradation, we blocked IκBα phosphorylation and degradation with the proteasome inhibitor N-Ac-leu-leu-norleucinal (ALLN). Western blot analysis using an Ab specific for the serine-phosphorylated form of IκBα showed that ACA suppressed TNF-induced phosphorylation of IκBα (Fig. 5 A).

FIGURE 5.

Effects of ACA on TNF-induced phosphorylation and degradation of IκBα. A, KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for 15 min after ALLN was added. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-phospho-specific-IκBα and anti-IκBα. B, Effect of ACA on TNF-induced phosphorylation of p65. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times. Nuclear extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with phospho-specific p65. C, Effect of ACA on the TNF-induced activation of IKK. KBM-5 cells (2 × 106 cells/ml) were pretreated with 100 μg/ml ALLN for 1 h and then coincubated with 50 μM ACA and 1 nM TNF for the indicated times. Whole-cell extracts were immunoprecipitated with Ab against IKK-α and analyzed by immune complex kinase assay as described in Materials and Methods. To examine the effect of ACA on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β Abs. D, Direct effect of ACA on the activation of IKK induced by TNF. Whole-cell extracts were prepared from 1 nM TNF-treated KBM-5 cells and immunoprecipitated with IKK-α Ab. The immune complex kinase assay was then performed in the absence or the presence of the indicated concentration of ACA. The results shown are representative of three independent experiments.

FIGURE 5.

Effects of ACA on TNF-induced phosphorylation and degradation of IκBα. A, KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for 15 min after ALLN was added. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-phospho-specific-IκBα and anti-IκBα. B, Effect of ACA on TNF-induced phosphorylation of p65. KBM-5 cells were coincubated with 50 μM ACA and 0.1 nM TNF for the indicated times. Nuclear extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with phospho-specific p65. C, Effect of ACA on the TNF-induced activation of IKK. KBM-5 cells (2 × 106 cells/ml) were pretreated with 100 μg/ml ALLN for 1 h and then coincubated with 50 μM ACA and 1 nM TNF for the indicated times. Whole-cell extracts were immunoprecipitated with Ab against IKK-α and analyzed by immune complex kinase assay as described in Materials and Methods. To examine the effect of ACA on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β Abs. D, Direct effect of ACA on the activation of IKK induced by TNF. Whole-cell extracts were prepared from 1 nM TNF-treated KBM-5 cells and immunoprecipitated with IKK-α Ab. The immune complex kinase assay was then performed in the absence or the presence of the indicated concentration of ACA. The results shown are representative of three independent experiments.

Close modal

TNF also induces the phosphorylation of p65, which is required for its transcriptional activity (16). As shown in Fig. 5 B, the coincubation of cells with ACA consistently inhibited TNF-induced phosphorylation of p65.

IKK is required for TNF-induced phosphorylation of IκBα (17), and the phosphorylation of p65 requires IKK activation (36). Because ACA inhibited the phosphorylation of both IκBα and p65, we determined its effect on TNF-induced IKK activation. Immune complex kinase assays show that ACA suppressed the activation of IKK by TNF (Fig. 5,C). Neither TNF nor ACA had any effect on the expression of IKK-α or IKK-β proteins. To evaluate whether ACA suppresses IKK activity directly by binding to the IKK protein or by suppressing the activation of IKK, we incubated whole-cell extracts from untreated and TNF-treated cells with various concentrations of ACA. An immune complex kinase assay showed that ACA did not directly affect the activity of IKK, suggesting that ACA modulates TNF-induced IKK activation (Fig. 5 D).

Because DNA binding does not always correlate with NF-κB-dependent gene transcription (37), we investigated the effect of ACA on TNF-induced reporter activity. Cells transiently transfected with the NF-κB-regulated SEAP reporter construct, incubated with ACA, and then stimulated with TNF had significantly diminished reporter gene expression (Fig. 6). These results suggest that ACA inhibited TNF-induced gene expression.

FIGURE 6.

ACA inhibits the TNF-induced expression of the NF-κB-dependent genes, TNFR1, TRADD, TRAF2, NIK, and IKK-β, but not p65. A293 cells were transiently transfected with an NF-κB-containing SEAP reporter gene plasmid alone or with the indicated plasmids for 24 h. After transfection, cells were washed and treated with 50 μM ACA for 24 h. For TNF-treated cells, cells were treated with 1 nM TNF for an additional 24 h. The supernatants of the culture medium were assayed for SEAP activity as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.1; b, p < 0.01; c, p < 0.005.

FIGURE 6.

ACA inhibits the TNF-induced expression of the NF-κB-dependent genes, TNFR1, TRADD, TRAF2, NIK, and IKK-β, but not p65. A293 cells were transiently transfected with an NF-κB-containing SEAP reporter gene plasmid alone or with the indicated plasmids for 24 h. After transfection, cells were washed and treated with 50 μM ACA for 24 h. For TNF-treated cells, cells were treated with 1 nM TNF for an additional 24 h. The supernatants of the culture medium were assayed for SEAP activity as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.1; b, p < 0.01; c, p < 0.005.

Close modal

TNF-induced NF-κB activation is mediated through sequential interaction of the TNFR with TRADD, TRAF2, NIK, and IKK, resulting in phosphorylation of IκBα (38, 39). When we transiently transfected cells with the NF-κB-regulated SEAP reporter construct along with TNFR1-, TRADD-, TRAF2-, NIK-, IKK-β-, or p65-expressing plasmids; treated them with ACA; and then monitored NF-κB-dependent SEAP expression, we found that ACA suppressed NF-κB activation induced by TNFR1, TRADD, TRAF2, NIK, and IKK-β, but not that induced by p65 (Fig. 6). These results suggested that ACA acts at a step upstream from p65.

Cyclin D1 is overexpressed in a wide variety of tumors and mediates the progress of cells from G1 to S phase (40). Similarly, COX-2 is overexpressed in tumor cells and mediates proliferation (41). The role of c-Myc in the proliferation of tumor is well established (42). The expression of all three genes is regulated by NF-κB (43, 44, 45). We found that ACA also blocked the expression of these genes (Fig. 7 A). These results further strengthen our postulate that ACA blocks TNF-induced, NF-κB-regulated gene products.

FIGURE 7.

ACA inhibits the TNF-induced expression of NF-κB-dependent antiproliferation, antimetastatic, and antiapoptotic proteins. A, ACA inhibits the expression of TNF-induced Bcl-2, IAP1, IAP2, survivin, and antiapoptotic protein. B, ACA inhibits the expression of TNF-induced cyclin D1 and c-Myc. C, ACA inhibits the expression of TNF-induced ICAM-1, COX-2, and MMP-9. KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using the indicated Abs. The results shown are representative of three independent experiments.

FIGURE 7.

ACA inhibits the TNF-induced expression of NF-κB-dependent antiproliferation, antimetastatic, and antiapoptotic proteins. A, ACA inhibits the expression of TNF-induced Bcl-2, IAP1, IAP2, survivin, and antiapoptotic protein. B, ACA inhibits the expression of TNF-induced cyclin D1 and c-Myc. C, ACA inhibits the expression of TNF-induced ICAM-1, COX-2, and MMP-9. KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using the indicated Abs. The results shown are representative of three independent experiments.

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The roles of vascular endothelial growth factor (VEGF), MMP-9, and ICAM-1 in angiogenesis and metastasis of tumors are well established. All three gene products are also regulated by NF-κB (46, 47, 48), so we investigated the effect of ACA on this regulation. Western blot analysis (Fig. 7 B) showed that ACA blocked TNF-induced VEGF, ICAM-1. and MMP-9 protein expression in a time-dependent manner. These results suggest that ACA plays a role in suppressing angiogenesis and metastasis.

NF-κB regulates the expression of the antiapoptotic proteins, survivin (49), IAP1/2 (50, 51), XIAP (52), Bcl-2 (53, 54, 55), Bcl-xL (56), Bfl-1/A1 (57, 58), and FLIP (59), so we examined whether ACA can modulate the expression of these antiapoptotic gene products induced by TNF. As shown in Fig. 7 C, ACA blocked the expression of these TNF-induced, antiapoptotic proteins.

The activation of NF-κB can inhibit TNF-induced apoptosis (60, 61, 62, 63, 64), so we determined the potential of ACA to enhance apoptosis induced by TNF and other cytotoxic agents. We used the live and dead assay, MTT, PARP cleavage, annexin V staining, and TUNEL staining methods. We first established that ACA enhanced the cytotoxicity induced by TNF (Fig. 8,A1), cisplatin (Fig. 8,A2), DOX (Fig. 8,A3), and taxol (Fig. 8,A4). ACA by itself had little cytotoxic effect. Next, we showed that ACA enhanced cytotoxicity by potentiating TNF-induced apoptosis. As shown in Fig. 8,B, ACA potentiated the TNF activation of caspases, as indicated by the PARP cleavage assay. The live and dead assay indicated that ACA up-regulated TNF-induced cytotoxicity from 2 to 51% (Fig. 8,C), and annexin V staining indicated that ACA up-regulated TNF-induced early apoptosis (Fig. 8,D). TUNEL staining showed that TNF-induced apoptosis was enhanced by incubation with ACA (Fig. 8 E). In this assay, ACA alone exhibited slight toxicity. The results of all the assays taken together suggest that ACA enhanced cytotoxicity by enhancing the apoptotic effects of TNF, cisplatin, taxol, and DOX.

FIGURE 8.

ACA enhances TNF-induced cytotoxicity. A, ACA enhances TNF-induced cytotoxicity. Ten thousand KBM-5 cells were seeded in triplicate in 96-well plates. Cells were coincubated with 5 μM ACA and the indicated concentrations of TNF for 72 h (A1). KBM-5 cells were pretreated with 5 μM ACA and then incubated with the indicated concentrations of cisplatin (A2), DOX (A3), or taxol (A4) for 24 h. Thereafter, cell viability was analyzed by the MTT method as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.005; b, p < 0.01; c, p < 0.05. B, KBM-5 cells were coincubated with 50 μM ACA and 1 nM TNF for the indicated times. Whole-cell extracts were prepared, subjected to SDS-PAGE, and blotted with anti-PARP Ab. C, KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h. Cells were stained with live and dead assay reagent for 30 min and then analyzed under a fluorescence microscope as described in Materials and Methods. D, KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h. Cells were incubated with FITC-conjugated annexin V Ab and then analyzed by flow cytometer as described in Materials and Methods. E, KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h. Cells were fixed, stained with TUNEL assay reagent, and then analyzed by flow cytometer as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.005.

FIGURE 8.

ACA enhances TNF-induced cytotoxicity. A, ACA enhances TNF-induced cytotoxicity. Ten thousand KBM-5 cells were seeded in triplicate in 96-well plates. Cells were coincubated with 5 μM ACA and the indicated concentrations of TNF for 72 h (A1). KBM-5 cells were pretreated with 5 μM ACA and then incubated with the indicated concentrations of cisplatin (A2), DOX (A3), or taxol (A4) for 24 h. Thereafter, cell viability was analyzed by the MTT method as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.005; b, p < 0.01; c, p < 0.05. B, KBM-5 cells were coincubated with 50 μM ACA and 1 nM TNF for the indicated times. Whole-cell extracts were prepared, subjected to SDS-PAGE, and blotted with anti-PARP Ab. C, KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h. Cells were stained with live and dead assay reagent for 30 min and then analyzed under a fluorescence microscope as described in Materials and Methods. D, KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h. Cells were incubated with FITC-conjugated annexin V Ab and then analyzed by flow cytometer as described in Materials and Methods. E, KBM-5 cells were coincubated with 10 μM ACA and 1 nM TNF for 16 h. Cells were fixed, stained with TUNEL assay reagent, and then analyzed by flow cytometer as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.005.

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MMPs, COXs, and adhesion molecules that are regulated by NF-κB have been shown to mediate tumor invasion (65), and TNF can induce the expression of genes involved in tumor metastasis (66). Whether ACA modulates TNF-induced invasion activity in vitro was examined. For this study, we used H1299 cells seeded in the top chamber of a Matrigel invasion chamber in the absence of serum. Cells were coincubated with TNF in the presence or the absence of ACA for 24 h. As shown in Fig. 9, TNF induced cell invasion activity, and ACA suppressed it.

FIGURE 9.

ACA suppresses TNF-induced invasion activity. H1299 (2.5 × 104 cells) were seeded to the top chamber of a Matrigel invasion chamber overnight in the absence of serum, coincubated with 10 μM ACA and 1 nM TNF for 24 h in the presence of 1% serum, and then subjected to invasion assay as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.01

FIGURE 9.

ACA suppresses TNF-induced invasion activity. H1299 (2.5 × 104 cells) were seeded to the top chamber of a Matrigel invasion chamber overnight in the absence of serum, coincubated with 10 μM ACA and 1 nM TNF for 24 h in the presence of 1% serum, and then subjected to invasion assay as described in Materials and Methods. Determinations were made in triplicate. Data represent the mean of three measurements ± SD. a, p < 0.01

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The anticarcinogenic, apoptotic, anti-inflammatory, and immunomodulatory activities of ACA suggest that it must mediate its effects by suppressing NF-κB activation. In the present study we found that ACA did indeed inhibit NF-κB activated by a variety of agents and in a variety of cell lines. In detail, NF-κB activity was inhibited because ACA suppressed IKK activation, thus resulting in inhibition of IκBα phosphorylation and degradation. As a result, ACA also blocked p65 phosphorylation, p65 nuclear translocation, and NF-κB-dependent reporter gene transcription. It suppressed NF-κB-regulated reporter gene transcription and gene products involved in cell proliferation (e.g., cyclin D1, COX-2, and c-Myc), antiapoptosis (e.g., survivin, IAP1, IAP2, XIAP, Bcl-2, Bcl-xL, Bfl-1/A1, and FLIP), angiogenesis (e.g., VEGF), and invasion (e.g., MMP-9 and ICAM-1). Suppression of NF-κB by ACA enhanced the apoptosis induced by TNF and chemotherapeutic agents.

Our results indicate that ACA inhibits NF-κB activation instantly, because suppression was noted even when it was added after initiation of NF-κB activation by TNF. In this respect, ACA-induced suppression of NF-κB activation differs from that induced by curcumin (67), flavopiridol (36), and farnesyl transferase inhibitors (68). The latter requires preincubation for several hours before activating the cells for NF-κB. It is unlikely that the rapid mode of action of ACA is due to its solubility in organic solvents. Whether the acetyl group in ACA has any role in the speed of its action is unclear at present.

We found that ACA inhibited NF-κB activation induced by highly diverse stimuli, including inflammatory stimuli (TNF, LPS, IL-1β, and H2O2), tumor promoters (PMA), chemotherapeutic agents (e.g., DOX), and carcinogens (e.g., CSC). Most of these agents activate NF-κB through different pathways (15, 16, 17). For instance, we have reported that pathway for H2O2-induced NF-κB activation differs from that of TNF (21). Because NF-κB activated by all the agents tested was inhibited, ACA must suppress activation at a step common to all these activators. Various tumor cells express a constitutively activated form of NF-κB through a mechanism that is not fully understood (15). ACA also suppressed constitutive activation. Unlike some other inhibitors (33, 34, 35), however, ACA did not modify the NF-κB proteins to prevent their binding to DNA. Because TNF-induced phosphorylation and degradation of IκBα were also inhibited by ACA, it suggested that this agent mediates its effect through IKK, the kinase needed for IκBα phosphorylation. We found that ACA indeed inhibited TNF-induced activation of IKK. These results are consistent with our previous report that ACA inhibits LPS- plus IFN-γ-induced IκΒα degradation in RAW264.7 mouse macrophages (69). However, we show that ACA does not directly inhibit IKK activity. It is possible that this inhibition is the result of inhibition of an upstream kinase. Previous studies (70) have reported that Akt can associate with and activate IKK-α. Thus, it is possible that ACA suppresses TNF-induced Akt activation.

TNF-induced NF-κB activation involves the sequential interaction of TNFR with TRADD and TRAF2, which then activate IKK, leading to NF-κB activation. ACA suppressed NF-κB activation induced by TNFR1, TRADD, TRAF2, NIK, and IKK-β, but not that activated by p65. This suggests that ACA acts at a step downstream from IKK and upstream from p65, consistent with above findings that ACA may modulate IKK.

In our study, ACA down-regulated the expression of NF-κB-regulated gene products involved in cell proliferation (e.g., cyclin D1 and c-Myc) antiapoptosis (e.g., survivin, IAP1, IAP2, XIAP, Bcl-2, Bcl-xL, Bfl-1/A1, and FLIP) and invasion (MMP-9, COX-2, and ICAM-1). The down-regulation of COX-2 by ACA is consistent with a previous report that showed suppression of COX-2 expression induced by LPS/IFN-γ in mouse macrophages (69). Our results may also explain the down-regulation of inducible NO synthase expression (14), which is also regulated by NF-κB.

We found that ACA potentiates the apoptotic effects of TNF and chemotherapeutic agents. It is very likely that this potentiation is mediated through the suppression of antiapoptotic gene products regulated by NF-κB.

ACA alone has been shown to induce apoptosis in different cell types (10, 11, 12), and this may also be linked to the suppression of NF-κB. ACA suppressed TNF-induced tumor invasion. Invasion and metastasis require the expression of MMP-9, COX-2, and ICAM-1, all of which are modulated by ACA. VEGF, a potent angiogenic factor, is also down-regulated by ACA. These results thus suggest that ACA may be effective not only as a chemopreventive agent, but also as a therapeutic agent, through regulation of various mechanisms, as indicated above.

Overall, our results demonstrated that ACA has potent antiproliferative, proapoptotic, antimetastatic, anti-inflammatory, and immunomodulatory effects, all mediated through NF-κB activation (Fig. 10). They set the stage for preclinical studies to establish the potential of ACA for clinical trial.

FIGURE 10.

Proposed mechanism by which ACA inhibits NF-κB activation and NF-κB-regulated gene expression involved in cell proliferation, invasion, angiogenesis, and metastasis.

FIGURE 10.

Proposed mechanism by which ACA inhibits NF-κB activation and NF-κB-regulated gene expression involved in cell proliferation, invasion, angiogenesis, and metastasis.

Close modal

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Clayton Foundation for Research (to B.B.A.), Department of Defense U.S. Army Breast Cancer Research Program Grant BC010610 (to B.B.A.), PO1 Grant CA91844 from the National Institutes of Health on lung chemoprevention (to B.B.A.), a P50 Head and Neck SPORE grant from the National Institutes of Health (P50CA97007 to B.B.A.).

3

Abbreviations used in this paper: ACA, 1′-acetoxychavicol acetate; ALLN, N-Ac-leu-leu-norleucinal; COX, cyclooxygenase; CSC, cigarette smoke condensate; DOX, doxorubicin; IAP, inhibitor of apoptosis protein; IKK, IκBα kinase; MMP, matrix metalloproteinase; PARP, poly(ADP-ribose) polymerase; PIS, preimmune serum; SEAP, secretory alkaline phosphatase; TRADD, TNFR-associated death domain protein; TRAF, TNFR-associated factor; VEGF, vascular endothelial growth factor; XIAP, X-chromosome-linked IAP; NIK, NF-κB-inducing kinase.

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