Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced NF-
B activation by tea polyphenols, ()-epigallocatechin gallate and theaflavins
Masaaki Nomura,
Wei-ya Ma,
Nanyue Chen,
Ann M. Bode and
Zigang Dong1
The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
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Abstract
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()-Epigallocatechin gallate (EGCG) and theaflavins are believed to be the key active components in tea for the chemoprevention of cancer. However, the molecular mechanisms by which EGCG and theaflavins block carcinogenesis are not clear. In the JB6 mouse epidermal cell line a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which causes cell transformation at high frequency, markedly induced NF-
B activation. We found that EGCG and theaflavins inhibited TPA-induced NF-
B activity in a concentration-dependent manner. These polyphenols blocked TPA-induced phosphorylation of I
B
at Ser32 in the same concentration range. Moreover, the NF-
B sequence-specific DNA-binding activity induced by TPA was also inhibited by these polyphenols. These results suggest that inhibition of NF-
B activation is also important in accounting for the anti-tumor promotion effects of EGCG and theaflavins.
Abbreviations: AP-1, activator protein-1; EGF, epidermal growth factor; EGCG, ()-epigallocatechin gallate; FBS, fetal bovine serum; MEM, minimum essential medium; NF-
B, nuclear factor
B, P+, tumor promoter sensitive; P, tumor promoter resistant; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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Prevention of carcinogenesis is one of the major strategies for cancer control. Many studies have shown that green tea, black tea and tea polyphenol preparations have inhibitory effects on carcinogenesis in rodent models (1,2). The anti-promoting effect of a green tea constituent, ()-epigallocatechin 3-gallate (EGCG), has been demonstrated (3). The molecular mechanism of its anti-promoting effect on growth might be through blocking of the signal transduction pathways leading to the activation of important transcription factors, such as activator protein-1 (AP-1) and nuclear factor
B (NF-
B) (46). On the other hand, black tea, the major tea beverage in Western nations, is reported to significantly inhibit proliferation and enhance apoptosis of skin tumors in mice (7). Among the black tea components, theaflavins (a mixture of theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, and theaflavin 3,3'-gallate) are generally considered to be the most effective components for inhibition of carcinogenesis (8,9). We have shown that EGCG and theaflavins inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)- or epidermal growth factor (EGF)-induced AP-1 activation and cell transformation in the JB6 mouse epidermal cell line (6). Thus, the inhibitory activities of theaflavins on carcinogenesis are suggested to be due to their ability to inhibit growth-related signal transduction pathways.
The JB6 cell system of clonal genetic variants that are promotion sensitive (P+) or promotion resistant (P) allows the study of genetic susceptibility to transformation, promotion and progression at the molecular level (1012). The P+, P and transformed variants are a series of cell lines representing `earlier-to-later' stages of preneoplastic-to-neoplastic progression (11,13,14). Through a comparison of P+ and P cells we found that transcriptional factor AP-1 plays a critical role in tumor promotion (1012). On the other hand, Li et al. (15) showed that both NF-
B and AP-1 may be involved in signaling tumor promoter-induced transformation in JB6 P+ cells. In addition, we found that transactivation of NF-
B as well as of AP-1 occurred during progression in mouse keratinocytes and that a dominant negative mutant of Jun inhibited activation of both transcription factors (16). These findings suggested that NF-
B activation is required in addition to AP-1 activation in tumor progression.
A skin carcinogenesis model induced by TPA is one of the most extensively utilized systems for studying tumor development (17,18). The mechanism by which TPA provides a selective growth and mitotic advantage to a tumor cell over a non-tumor cell has been suggested to be related to the expression of activated p21 ras and protein kinase C (PKC) (19). In addition, TPA induces its interaction with the cell membrane followed by an altered program of gene expression and stimulation of cell differentiation and growth, e.g. activation of the mitogen-responsive phospholipase C pathway, direct PKC activation, increased expression of the Jun and Fos family and subsequent binding to the AP-1 binding site (2022). In JB6 P+ cells TPA induces the formation in soft agar of large, tumorigenic, anchorage-independent colonies at a high frequency (1214). EGCG and theaflavins block TPA-induced cell transformation as well as inhibition of AP-1 activation in JB6 P+ cells (6). Therefore, the inhibition of AP-1 activation seems to be important in the anti-tumor promotion effects of these polyphenols. However, Ahmad et al. (23) suggested that cell cycle deregulation and apoptosis caused by EGCG in cancer cells is mediated through NF-
B. Furthermore, several studies showed that EGCG also blocked the signaling pathway leading to NF-
B activation (5,24) and theaflavins were shown to inhibit lipopolysaccharide-induced NF-
B activity in macrophages (25,26). AP-1 and NF-
B signal transduction pathways are considered to be important in tumor promoter-induced transformation and tumor promotion. TPA induces not only AP-1 activation but also activates NF-
B (27). Therefore, the inhibitory effect of EGCG and theaflavins on TPA-induced cell transformation may occur through inhibition of NF-
B as well as AP-1. To test this hypothesis, we investigated the effect of EGCG and theaflavins on TPA-induced NF-
B transactivation.
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Materials and methods
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Materials
Eagle's minimum essential medium (MEM), fetal bovine serum (FBS) and gentamycin were from Bio Whittaker (Walkersville, MD), L-glutamine was from Life Technologies (Grand Island, NY) and luciferase assay substrate was from Promega (Madison, WI). EGCG (purity >98%) was a gift from Dr Yukihiko Hara (Mitsui Norin Co., Fujieda, Japan). Theaflavins (a mixture of theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, theaflavin 3,3'-gallate and unknowns, accounting for 21, 30, 15, 28 and 6%, respectively) were gifts from Thomas J.Lipton Co. (Englewood Cliffs, NJ). TPA was obtained from Sigma Chemical Co. (St Louis, MO). The antibody and phospho-specific antibody against phosphorylated sites of I
B
were from New England Biolabs (Beverly, MA). An antibody against the NF-
B p65 subunit was from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
The JB6 mouse epidermal cell line Cl 41 and its NF-
B luciferase reporter transfectant cell line were cultured in monolayers at 37°C, 5% CO2 using MEM containing 5% FBS, 2 mM L-glutamine and 25 µg/ml gentamycin.
Assay for NF-
B activity
JB6 NF-
Bluciferase stable transfectant cells were used to assay NF-
B activity (28). Viable cells (8x103) suspended in 100 µl of 5% FBS MEM were added to each well of a 96-well plate. Plates were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Twenty hours later cells were starved by being cultured in 0.1% FBS MEM for 24 h. The cells were treated with or without EGCG or theaflavins for 30 min, after which they were exposed to TPA (20 ng/ml) and incubated in the presence or absence of EGCG or theaflavins for different time periods. The cells were extracted with lysis buffer and the luciferase activity was measured as described previously using a luminometer (Monolight 2010) (28).
Nuclear protein analysis
Gel shift assays were used to detect NF-
B binding activity after exposure to the tumor promoter TPA with or without EGCG or theaflavins. Nuclear extracts were prepared as described previously (6). In brief, cells were lysed with 500 µl of lysis buffer (50 mM KCl, 0.5% NP40, 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin and 100 µM DL-DTT). After centrifugation at 14 000 r.p.m. for 1 min in a microcentrifuge, the nuclei were washed with 500 µl of the same buffer without NP40, then placed into 300 µl of extraction buffer (500 mM KCl and 10% glycerol with the same concentration of HEPES, phenylmethylsulfonyl fluoride, leupeptin, aprotinin and DL-DTT as the lysis buffer). After centrifugation at 14 000 r.p.m. for 5 min, the supernatant fraction was harvested as the nuclear protein extract and stored at 70°C. The NF-
B-binding oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGC C-3' was from Promega and was labeled with [32P]dCTP using the Klenow fragment (Life Science Co., Gaithersburg, MD). Three micrograms of nuclear protein extracted from cells exposed to TPA for the indicated time intervals were added to the DNA-binding buffer, which contained 5x104 c.p.m. 32P-labeled oligonucleotide probe, 1.5 µg poly(dI·dC) and 3 µg BSA. The reaction mixture was incubated on ice for 10 min followed by incubation at room temperature for 20 min. The DNAprotein complexes were resolved on a 6% non-denaturing acrylamide gel. The gel was dried and exposed to X-ray film at 70°C overnight or analyzed using a Storm 840 Image Analyzer (Molecular Dynamics).
I
B
phosphorylation assay
Immunoblotting for I
B
and its phosphorylated protein was carried out as described previously (28). In brief, JB6 Cl 41 cells were cultured in monolayers in 6-well plates. The cells were starved in 0.1% FBS MEM for 48 h at 37°C and then the medium was changed to fresh 0.1% FBS MEM for another 24 h at 37°C. Before the cells were treated with TPA they were treated with or without EGCG or theaflavins for 30 min. Then, TPA (20 ng/ml) was added and the cells were incubated for different time periods in the presence or absence of EGCG or theaflavins at 37°C. The cells were then lysed and immunoblot analysis performed using an antibody against I
B
or a phospho-specific antibody against the phosphorylated protein. Antibody-bound proteins were detected by chemiluminescence (ECF; Amersham Pharmacia Biotech) and analyzed using a Storm 840 Image Analyzer (Molecular Dynamics).
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Results
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EGCG and theaflavins inhibit TPA-induced NF-
B activation
Activation of NF-
B is known to be induced by a variety of stimuli, including TPA and UV irradiation (29,30). Treatment of JB6 cells with TPA (20 ng/ml) markedly induced NF-
B activation (Figure 1A
). The maximal NF-
B activation by TPA was 8.5-fold at 24 h (Figure 1A
). We showed previously that EGCG and theaflavins significantly inhibit TPA-induced cell transformation in the concentration range 520 µM (6). As shown in Figure 1B
, EGCG and theaflavins also inhibited TPA-induced NF-
B activation in a concentration-dependent manner. The concentration range of EGCG and theaflavins that blocked NF-
B activation was similar to that which inhibited cell transformation (6). The inhibitory effects were not due to cytotoxic effects because [3H]thymidine incorporation in the drug-treated cells was not affected at the doses used in this study (data not shown). These results indicate that inhibition of NF-
B activation by EGCG and theaflavins may be important in preventing tumor promoter-induced cell transformation.
EGCG and theaflavins inhibit NF-
B sequence-specific DNA-binding activity
To study the molecular basis of the inhibition of NF-
B activation by EGCG and theaflavins, we considered the possibility that NF-
B transactivation activity might be altered by these tea components. The NF-
B DNA-binding activity was analyzed by gel shift assay. As shown in Figure 2A
, EGCG and theaflavins inhibited TPA-induced NF-
B DNA-binding activity in a similar dose range to that affecting inhibition of NF-
B activation. By a competition experiment with unlabeled NF-
B-binding oligonucleotides, these bands were shown to be compatible, whereas added unlabeled AP-1-binding oligonucleotides did not affect the binding activity (Figure 2B
). Furthermore, anti-p65 subunit antibody impaired formation of the proteinDNA complex (Figure 2B
). Therefore, the inhibition of NF-
B activation by these tea constituents appears to be, at least in part, due to inhibition of NF-
B DNA-binding activity.
EGCG and theaflavins inhibit TPA-induced I
B
phosphorylation
Treatment of cells with a variety of diverse stimuli such as cytokines, TPA, lipopolysaccharide, UV light and several mitogens activates the I
B kinase complex, leading to phosphorylation of Ser32 and Ser36 of I
B
or Ser19 and Ser23 of I
Bß (3134). This phosphorylation event targets I
B for ubiquitin, resulting in the release and nuclear translocation of NF-
B (35,36). To test whether phosphorylation of I
B is affected in the inhibition of NF-
B activation by EGCG and theaflavins, we used a phospho-specific antibody for phosphorylated Ser32 of I
B
. The result indicates that I
B
phosphorylation at Ser32 was stimulated by treatment of cells with TPA for 5 min (Figure 3A and B
). EGCG or theaflavins inhibited TPA-induced I
B
phosphorylation at Ser32 in a concentration-dependent manner (Figure 4A and B
). These results suggest that inhibition of NF-
B activation by EGCG and theaflavins is through the attenuation of I
B
phosphorylation.
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Discussion
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Green tea is widely used as a beverage in China, Japan and other Asian countries, whereas black tea is more popular in Western countries (1,2). In recent years many animal studies and several epidemiological studies have reported the anti-carcinogenic effects of tea (1,2). The polyphenols from green tea and black tea, EGCG and theaflavins, are generally considered to be the most effective components for inhibition of carcinogenesis (13,6,23). The most widely recognized biological properties of tea polyphenols include their anti-oxidant capacity to scavenge reactive oxygen or nitrogen species and to sequester metal ions (37). These polyphenols are known to bind to proteins, as exemplified by their binding to the proline-rich salivary proteins (38). At low concentrations this binding property may specifically affect certain enzymes and membrane receptors resulting in distinct biological activities. On the other hand, we previously reported that EGCG and theaflavins inhibit tumor promoter TPA- or EGF-induced cell transformation as well as AP-1 activity (6). Furthermore, we have shown that EGCG and theaflavins also inhibit UVB-induced AP-1 activity (39). EGCG and theaflavins have been shown to block the induction of nitric oxide synthase through inhibition of NF-
B activity (5,26). The AP-1 and NF-
B signal transduction pathways are considered to be important in tumor progression and both AP-1 and NF-
B are activated by various tumor promoters. Therefore, inhibition of signal transduction to transcription factors, such as AP-1 and NF-
B, may be functionally linked to the anti-tumor promotion effects of EGCG and theaflavins.
In this study we found that EGCG and theaflavins inhibit TPA-induced NF-
B activity. NF-
B activity is regulated by an inhibitory binding protein, I
B, found in the cytoplasm complexed with NF-
B proteins. I
B family members include I
B
, I
Bß, p105/I
B
(precursor of p50), p100 (precursor of p52) and I
B
. Each has in common a series of ankyrin repeats that interact with the DNA-binding domain and the nuclear localization signal of NF-
B, thus maintaining the transcription factor as an inactive complex in the cytoplasm. Activation of NF-
B is induced by a variety of diverse stimuli, including inflammatory cytokines, phorbol esters, bacterial toxins (such as lipopolysaccharide), viruses, UV light and several mitogens (29,30). Treatment of cells with any of these stimuli activates the I
B kinase complex, leading to phosphorylation of Ser32 and Ser36 of I
B
or Ser19 and Ser23 of I
Bß (3134). This phosphorylation event targets I
B for ubiquitination, resulting in release and nuclear translocation of NF-
B (35,36). We found that EGCG and theaflavins blocked TPA-induced phosphorylation of I
B
at Ser32. Much research has been directed at elucidating the signal transduction pathways involved in regulating NF-
B activation. Studies have implicated PKC as playing a critical role in NF-
B activation in response to many activators because phorbol esters, potent activators of PKC, are NF-
B inducers and purified PKC releases NF-
B from I
B
when incubated with cytosolic extracts (4042). EGCG or theaflavins are known to inactivate PKC (43,44). Therefore, inhibition of TPA-induced I
B
phosphorylation by these polyphenols may result from inactivation of PKC. Moreover, these polyphenols also inhibited NF-
B sequence-specific DNA-binding activity. These effects of the polyphenols were observed in the same concentration range as for inhibition of cell transformation (6). Because NF-
B is closely involved in oncogenesis (4547), the results of the present study suggest that, in addition to inhibition of AP-1 activation, inhibition of NF-
B activation is also important in the anti-promotion effects of these polyphenols.
Li et al. (15) reported that a small molecular inhibitor of NF-
B, which prevented I
B release from NF-
B, also inhibited TPA-induced AP-1 transactivation and cell transformation in JB6 cells. Moreover, they suggested that NF-
B may act as a mediator of AP-1 transactivation in TPA-induced transformation because NF-
B transactivation by TPA responds earlier than AP-1 transactivation (15). In addition, dominant negative Jun expression in mouse (16) or human (48) keratinocytes blocked not only AP-1 but also NF-
B activation when tumor progression was also blocked. These findings suggest that both AP-1 and NF-
B may be involved in signaling tumor promoter-induced cell transformation. Interestingly, interaction of transcription factors, including AP-1 and NF-
B, has been described (36,49). Components of AP-1, c-Jun or c-Fos, but not JunB or JunD, as well as components of NF-
B (p65 but not p50 or cRel), interact and synergize in both AP-1- and NF-
B-dependent gene transactivation (49). The interaction involves the bZip domain of c-Jun or c-Fos and the rel homology domain of p65. p65/c-Jun or p65/c-Fos interaction-induced transactivation appears specific for the AP-1/TPA response element (49). EGCG and theaflavins inhibit TPA-induced phosphorylation of I
B
, thus blocking nuclear translocation and activation of NF-
B. Because AP-1 and NF-
B activation is important for tumor-induced neoplastic transformation (1012,4547,50), inhibition of transcription factor interaction-induced transactivation through blocking nuclear translocation of NF-
B by EGCG and theaflavins may also play a key role in their anti-tumor promotion effects. Thus, the anti-tumor promotion effects of these polyphenols were suggested to result from inhibition of NF-
B, AP-1 and transcription factor interaction pathways, which are closely involved in tumor promotion.
In summary, this study has provided suggestions for the mechanisms of the anti-tumor promotion action of EGCG and theaflavins. Our experiments indicate that inhibition of NF-
B transactivation may be involved in inhibition of tumor promoter-induced neoplastic transformation in JB6 cells in addition to suppression of AP-1 transactivation. Inhibition of NF-
B activation by EGCG and theaflavins is mediated through inhibition of I
B phosphorylation. These results provide insight into the biological actions of tea and the molecular basis for the development of new chemoprotective reagents for cancer.
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Notes
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1 To whom correspondence should be addressed Email: zgdong{at}smig.net 
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Acknowledgments
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This work was supported by grant CA81064 from the National Cancer Institute and the Hormel Foundation.
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Received February 25, 2000;
revised June 19, 2000;
accepted June 30, 2000.