Identification of a Novel Blocker of IB Kinase That Enhances Cellular Apoptosis and Inhibits Cellular Invasion through Suppression of NF-B-R
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免疫学杂志 2005年第11期
Abstract
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-B, we postulated that ACA might mediate its activity through modulation of NF-B activation. For this report, we investigated the effect of ACA on NF-B and NF-B-regulated gene expression activated by various carcinogens. We found that ACA suppressed NF-B activation induced by a wide variety of inflammatory and carcinogenic agents, including TNF, IL-1, PMA, LPS, H2O2, doxorubicin, and cigarette smoke condensate. Suppression was not cell type specific, because both inducible and constitutive NF-B activations were blocked by ACA. ACA did not interfere with the binding of NF-B to the DNA, but, rather, inhibited IB kinase activation, IB phosphorylation, IB degradation, p65 phosphorylation, and subsequent p65 nuclear translocation. ACA also inhibited NF-B-dependent reporter gene expression activated by TNF, TNFR1, TNFR-associated death domain protein, TNFR-associated factor-2, and IB kinase, but not that activated by p65. Consequently, ACA suppressed the expression of TNF-induced NF-B-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-B and NF-B-regulated gene expression, which may explain the ability of ACA to enhance apoptosis and inhibit invasion.
Introduction
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-B. First, NF-B is activated by various carcinogens, tumor promoters, and tumor microenvironment (hypoxia and acidic pH). Second, most inflammatory agents activate NF-B. Third, NF-B regulates the expression of genes that regulate transformation, tumor promotion, tumor invasion, angiogenesis, and metastasis. Fourth, suppression of apoptosis is regulated by NF-B. And fifth, chemopreventive agents have been shown to suppress NF-B activation (15).
NF-B is a heterodimeric protein complex of members of the Rel (p50)/NF-B (p60) protein family. NF-B 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, IB (16). In its unstimulated form, NF-B 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 IB proteins, allowing active NF-B to translocate to the nucleus, where it regulates gene expression. The phosphorylation of IB is mediated through the activation of the IB kinase (IKK) complex consisting of IKK-, IKK-, IKK- (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-B in proliferative and inflammatory diseases, we investigated the effect of ACA on NF-B activation induced by carcinogens, tumor promoters, and inflammatory agents. The results described below strongly suggest that ACA is a potent suppressor of NF-B 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.
Materials and Methods
Invasion assay
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 x 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).
Results
The aim of the current study was to investigate the effects of ACA on the NF-B activation pathway induced by various carcinogens and inflammatory stimuli and on NF-B-regulated gene expression. Because the TNF-induced NF-B activation pathway has been well characterized, we investigated in detail the effects of ACA on TNF-induced NF-B activation. The structure of ACA is shown in Fig. 1A.
ACA inhibits NF-B activation induced by carcinogens-tumor promoters and inflammatory agents
Because TNF, PMA, LPS, IL-1, doxorubicin (DOX), H2O2, and cigarette smoke condensate (CSC) are potent activators of NF-B (19, 25, 26, 27), we examined the effect of ACA on the activation of NF-B by these agents. Coincubation of cells with 50 μM ACA suppressed the activation of NF-B induced by all seven agents (Fig. 1B). The concentration of ACA and NF-B 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-B activation pathway that is common to all seven agents.
Inhibition of NF-B activation by ACA is dose dependent
Because TNF is one of the most potent activator of NF-B, and the mechanism of activation of NF-B is relatively well established (28), we examined the effects of different doses of ACA on TNF-induced NF-B 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-B activation. These studies indicated that ACA suppressed TNF-induced NF-B activation in a dose-dependent manner, with 60% inhibition at 10 μM and almost 100% inhibition at 50 μM (Fig. 2A).
Inhibition of NF-B activation by ACA is not cell type specific
Because the signal transduction pathway mediated by NF-B may be distinct in different cell types (29, 30), we investigated whether ACA could block TNF-induced NF-B activation in breast adenocarcinoma MCF-7 (Fig. 2B), human T cell lymphoma Jurkat (Fig. 2B), and human lung carcinoma H1299 cells (Fig. 2B). These cells were exposed to TNF in the presence or the absence of ACA for 30 min and then examined for NF-B activation. TNF activated NF-B in every cell type, and ACA completely inhibited most of this activation, indicating that ACA-induced suppression of NF-B activation was not cell type specific.
ACA also suppresses constitutive NF-B activation
Most tumor cells express constitutively active NF-B (26, 27), although the mechanism is not well understood. We showed that ACA suppresses constitutive activation of NF-B 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-B (31, 32) (Fig. 2C).
ACA is a fast-acting inhibitor of NF-B activation
The suppression of NF-B by most agents, including TNF, requires that they be applied before the NF-B-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-B activation (Fig. 3A), suggesting that ACA is a fast-acting inhibitor of NF-B activation.
ACA inhibits TNF-induced nuclear translocation of p65
As shown in Fig. 4C, Western blot analysis indicated that ACA significantly inhibited TNF-induced nuclear translocation of p65. Immunocytochemistry appeared to confirm this (Fig. 4D).
ACA inhibits TNF-dependent IB phosphorylation
Because IB phosphorylation is needed for IB degradation, we determined whether ACA modulated IB phosphorylation. Because TNF-induced phosphorylation of IB leads to its rapid degradation, we blocked IB 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 IB showed that ACA suppressed TNF-induced phosphorylation of IB (Fig. 5A).
ACA inhibits TNF-induced phosphorylation of p65
TNF also induces the phosphorylation of p65, which is required for its transcriptional activity (16). As shown in Fig. 5B, the coincubation of cells with ACA consistently inhibited TNF-induced phosphorylation of p65.
ACA inhibits TNF-induced IKK activation
IKK is required for TNF-induced phosphorylation of IB (17), and the phosphorylation of p65 requires IKK activation (36). Because ACA inhibited the phosphorylation of both IB 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. 5C). Neither TNF nor ACA had any effect on the expression of IKK- or IKK- 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. 5D).
ACA represses TNF-induced NF-B-dependent reporter gene expression
Because DNA binding does not always correlate with NF-B-dependent gene transcription (37), we investigated the effect of ACA on TNF-induced reporter activity. Cells transiently transfected with the NF-B-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.
ACA represses the TNF-induced NF-B-dependent gene products involved in angiogenesis and metastasis
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-B (46, 47, 48), so we investigated the effect of ACA on this regulation. Western blot analysis (Fig. 7B) 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.
ACA represses TNF-induced, NF-B-dependent antiapoptotic gene products
NF-B 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. 7C, ACA blocked the expression of these TNF-induced, antiapoptotic proteins.
ACA potentiates apoptosis induced by TNF and chemotherapeutic agents
The activation of NF-B 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. 8A1), cisplatin (Fig. 8A2), DOX (Fig. 8A3), and taxol (Fig. 8A4). ACA by itself had little cytotoxic effect. Next, we showed that ACA enhanced cytotoxicity by potentiating TNF-induced apoptosis. As shown in Fig. 8B, 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. 8C), and annexin V staining indicated that ACA up-regulated TNF-induced early apoptosis (Fig. 8D). TUNEL staining showed that TNF-induced apoptosis was enhanced by incubation with ACA (Fig. 8E). 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.
ACA suppresses TNF-induced invasion activity
MMPs, COXs, and adhesion molecules that are regulated by NF-B 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.
Discussion
The anticarcinogenic, apoptotic, anti-inflammatory, and immunomodulatory activities of ACA suggest that it must mediate its effects by suppressing NF-B activation. In the present study we found that ACA did indeed inhibit NF-B activated by a variety of agents and in a variety of cell lines. In detail, NF-B activity was inhibited because ACA suppressed IKK activation, thus resulting in inhibition of IB phosphorylation and degradation. As a result, ACA also blocked p65 phosphorylation, p65 nuclear translocation, and NF-B-dependent reporter gene transcription. It suppressed NF-B-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-B by ACA enhanced the apoptosis induced by TNF and chemotherapeutic agents.
Our results indicate that ACA inhibits NF-B activation instantly, because suppression was noted even when it was added after initiation of NF-B activation by TNF. In this respect, ACA-induced suppression of NF-B 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-B. 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-B activation induced by highly diverse stimuli, including inflammatory stimuli (TNF, LPS, IL-1, and H2O2), tumor promoters (PMA), chemotherapeutic agents (e.g., DOX), and carcinogens (e.g., CSC). Most of these agents activate NF-B through different pathways (15, 16, 17). For instance, we have reported that pathway for H2O2-induced NF-B activation differs from that of TNF (21). Because NF-B 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-B 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-B proteins to prevent their binding to DNA. Because TNF-induced phosphorylation and degradation of IB were also inhibited by ACA, it suggested that this agent mediates its effect through IKK, the kinase needed for IB 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--induced IB 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-. Thus, it is possible that ACA suppresses TNF-induced Akt activation.
TNF-induced NF-B activation involves the sequential interaction of TNFR with TRADD and TRAF2, which then activate IKK, leading to NF-B activation. ACA suppressed NF-B activation induced by TNFR1, TRADD, TRAF2, NIK, and IKK-, 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-B-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- in mouse macrophages (69). Our results may also explain the down-regulation of inducible NO synthase expression (14), which is also regulated by NF-B.
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-B.
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-B. 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-B activation (Fig. 10). They set the stage for preclinical studies to establish the potential of ACA for clinical trial.
Footnotes
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.).
2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Section, Department of Experimental Therapeutics, Box 143, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address: aggarwal{at}mdanderson.org
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, IB 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-B-inducing kinase.
Received for publication November 5, 2004. Accepted for publication March 22, 2005.
References
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Ohnishi, M., T. Tanaka, H. Makita, T. Kawamori, H. Mori, K. Satoh, A. Hara, A. Murakami, H. Ohigashi, K. Koshimizu. 1996. Chemopreventive effect of a xanthine oxidase inhibitor, 1'-acetoxychavicol acetate, on rat oral carcinogenesis. Jpn. J. Cancer Res. 87: 349-356
Kobayashi, Y., D. Nakae, H. Akai, H. Kishida, E. Okajima, W. Kitayama, A. Denda, T. Tsujiuchi, A. Murakami, K. Koshimizu, et al 1998. Prevention by 1'-acetoxychavicol acetate of the induction but not growth of putative preneoplastic, glutathione S-transferase placental form-positive, focal lesions in the livers of rats fed a choline-deficient, L-amino acid-defined diet. Carcinogenesis 19: 1809-1814.(Haruyo Ichikawa*, Yasunar)
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-B, we postulated that ACA might mediate its activity through modulation of NF-B activation. For this report, we investigated the effect of ACA on NF-B and NF-B-regulated gene expression activated by various carcinogens. We found that ACA suppressed NF-B activation induced by a wide variety of inflammatory and carcinogenic agents, including TNF, IL-1, PMA, LPS, H2O2, doxorubicin, and cigarette smoke condensate. Suppression was not cell type specific, because both inducible and constitutive NF-B activations were blocked by ACA. ACA did not interfere with the binding of NF-B to the DNA, but, rather, inhibited IB kinase activation, IB phosphorylation, IB degradation, p65 phosphorylation, and subsequent p65 nuclear translocation. ACA also inhibited NF-B-dependent reporter gene expression activated by TNF, TNFR1, TNFR-associated death domain protein, TNFR-associated factor-2, and IB kinase, but not that activated by p65. Consequently, ACA suppressed the expression of TNF-induced NF-B-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-B and NF-B-regulated gene expression, which may explain the ability of ACA to enhance apoptosis and inhibit invasion.
Introduction
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-B. First, NF-B is activated by various carcinogens, tumor promoters, and tumor microenvironment (hypoxia and acidic pH). Second, most inflammatory agents activate NF-B. Third, NF-B regulates the expression of genes that regulate transformation, tumor promotion, tumor invasion, angiogenesis, and metastasis. Fourth, suppression of apoptosis is regulated by NF-B. And fifth, chemopreventive agents have been shown to suppress NF-B activation (15).
NF-B is a heterodimeric protein complex of members of the Rel (p50)/NF-B (p60) protein family. NF-B 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, IB (16). In its unstimulated form, NF-B 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 IB proteins, allowing active NF-B to translocate to the nucleus, where it regulates gene expression. The phosphorylation of IB is mediated through the activation of the IB kinase (IKK) complex consisting of IKK-, IKK-, IKK- (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-B in proliferative and inflammatory diseases, we investigated the effect of ACA on NF-B activation induced by carcinogens, tumor promoters, and inflammatory agents. The results described below strongly suggest that ACA is a potent suppressor of NF-B 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.
Materials and Methods
Invasion assay
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 x 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).
Results
The aim of the current study was to investigate the effects of ACA on the NF-B activation pathway induced by various carcinogens and inflammatory stimuli and on NF-B-regulated gene expression. Because the TNF-induced NF-B activation pathway has been well characterized, we investigated in detail the effects of ACA on TNF-induced NF-B activation. The structure of ACA is shown in Fig. 1A.
ACA inhibits NF-B activation induced by carcinogens-tumor promoters and inflammatory agents
Because TNF, PMA, LPS, IL-1, doxorubicin (DOX), H2O2, and cigarette smoke condensate (CSC) are potent activators of NF-B (19, 25, 26, 27), we examined the effect of ACA on the activation of NF-B by these agents. Coincubation of cells with 50 μM ACA suppressed the activation of NF-B induced by all seven agents (Fig. 1B). The concentration of ACA and NF-B 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-B activation pathway that is common to all seven agents.
Inhibition of NF-B activation by ACA is dose dependent
Because TNF is one of the most potent activator of NF-B, and the mechanism of activation of NF-B is relatively well established (28), we examined the effects of different doses of ACA on TNF-induced NF-B 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-B activation. These studies indicated that ACA suppressed TNF-induced NF-B activation in a dose-dependent manner, with 60% inhibition at 10 μM and almost 100% inhibition at 50 μM (Fig. 2A).
Inhibition of NF-B activation by ACA is not cell type specific
Because the signal transduction pathway mediated by NF-B may be distinct in different cell types (29, 30), we investigated whether ACA could block TNF-induced NF-B activation in breast adenocarcinoma MCF-7 (Fig. 2B), human T cell lymphoma Jurkat (Fig. 2B), and human lung carcinoma H1299 cells (Fig. 2B). These cells were exposed to TNF in the presence or the absence of ACA for 30 min and then examined for NF-B activation. TNF activated NF-B in every cell type, and ACA completely inhibited most of this activation, indicating that ACA-induced suppression of NF-B activation was not cell type specific.
ACA also suppresses constitutive NF-B activation
Most tumor cells express constitutively active NF-B (26, 27), although the mechanism is not well understood. We showed that ACA suppresses constitutive activation of NF-B 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-B (31, 32) (Fig. 2C).
ACA is a fast-acting inhibitor of NF-B activation
The suppression of NF-B by most agents, including TNF, requires that they be applied before the NF-B-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-B activation (Fig. 3A), suggesting that ACA is a fast-acting inhibitor of NF-B activation.
ACA inhibits TNF-induced nuclear translocation of p65
As shown in Fig. 4C, Western blot analysis indicated that ACA significantly inhibited TNF-induced nuclear translocation of p65. Immunocytochemistry appeared to confirm this (Fig. 4D).
ACA inhibits TNF-dependent IB phosphorylation
Because IB phosphorylation is needed for IB degradation, we determined whether ACA modulated IB phosphorylation. Because TNF-induced phosphorylation of IB leads to its rapid degradation, we blocked IB 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 IB showed that ACA suppressed TNF-induced phosphorylation of IB (Fig. 5A).
ACA inhibits TNF-induced phosphorylation of p65
TNF also induces the phosphorylation of p65, which is required for its transcriptional activity (16). As shown in Fig. 5B, the coincubation of cells with ACA consistently inhibited TNF-induced phosphorylation of p65.
ACA inhibits TNF-induced IKK activation
IKK is required for TNF-induced phosphorylation of IB (17), and the phosphorylation of p65 requires IKK activation (36). Because ACA inhibited the phosphorylation of both IB 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. 5C). Neither TNF nor ACA had any effect on the expression of IKK- or IKK- 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. 5D).
ACA represses TNF-induced NF-B-dependent reporter gene expression
Because DNA binding does not always correlate with NF-B-dependent gene transcription (37), we investigated the effect of ACA on TNF-induced reporter activity. Cells transiently transfected with the NF-B-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.
ACA represses the TNF-induced NF-B-dependent gene products involved in angiogenesis and metastasis
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-B (46, 47, 48), so we investigated the effect of ACA on this regulation. Western blot analysis (Fig. 7B) 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.
ACA represses TNF-induced, NF-B-dependent antiapoptotic gene products
NF-B 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. 7C, ACA blocked the expression of these TNF-induced, antiapoptotic proteins.
ACA potentiates apoptosis induced by TNF and chemotherapeutic agents
The activation of NF-B 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. 8A1), cisplatin (Fig. 8A2), DOX (Fig. 8A3), and taxol (Fig. 8A4). ACA by itself had little cytotoxic effect. Next, we showed that ACA enhanced cytotoxicity by potentiating TNF-induced apoptosis. As shown in Fig. 8B, 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. 8C), and annexin V staining indicated that ACA up-regulated TNF-induced early apoptosis (Fig. 8D). TUNEL staining showed that TNF-induced apoptosis was enhanced by incubation with ACA (Fig. 8E). 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.
ACA suppresses TNF-induced invasion activity
MMPs, COXs, and adhesion molecules that are regulated by NF-B 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.
Discussion
The anticarcinogenic, apoptotic, anti-inflammatory, and immunomodulatory activities of ACA suggest that it must mediate its effects by suppressing NF-B activation. In the present study we found that ACA did indeed inhibit NF-B activated by a variety of agents and in a variety of cell lines. In detail, NF-B activity was inhibited because ACA suppressed IKK activation, thus resulting in inhibition of IB phosphorylation and degradation. As a result, ACA also blocked p65 phosphorylation, p65 nuclear translocation, and NF-B-dependent reporter gene transcription. It suppressed NF-B-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-B by ACA enhanced the apoptosis induced by TNF and chemotherapeutic agents.
Our results indicate that ACA inhibits NF-B activation instantly, because suppression was noted even when it was added after initiation of NF-B activation by TNF. In this respect, ACA-induced suppression of NF-B 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-B. 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-B activation induced by highly diverse stimuli, including inflammatory stimuli (TNF, LPS, IL-1, and H2O2), tumor promoters (PMA), chemotherapeutic agents (e.g., DOX), and carcinogens (e.g., CSC). Most of these agents activate NF-B through different pathways (15, 16, 17). For instance, we have reported that pathway for H2O2-induced NF-B activation differs from that of TNF (21). Because NF-B 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-B 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-B proteins to prevent their binding to DNA. Because TNF-induced phosphorylation and degradation of IB were also inhibited by ACA, it suggested that this agent mediates its effect through IKK, the kinase needed for IB 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--induced IB 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-. Thus, it is possible that ACA suppresses TNF-induced Akt activation.
TNF-induced NF-B activation involves the sequential interaction of TNFR with TRADD and TRAF2, which then activate IKK, leading to NF-B activation. ACA suppressed NF-B activation induced by TNFR1, TRADD, TRAF2, NIK, and IKK-, 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-B-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- in mouse macrophages (69). Our results may also explain the down-regulation of inducible NO synthase expression (14), which is also regulated by NF-B.
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-B.
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-B. 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-B activation (Fig. 10). They set the stage for preclinical studies to establish the potential of ACA for clinical trial.
Footnotes
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.).
2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Section, Department of Experimental Therapeutics, Box 143, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address: aggarwal{at}mdanderson.org
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, IB 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-B-inducing kinase.
Received for publication November 5, 2004. Accepted for publication March 22, 2005.
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