Carcinogenesis vol.19 no.12 pp.2201–2204, 1998
SHORT COMMUNICATION
(–)-Epigallocatechin-3-gallate inhibition of ultraviolet B-induced AP-1 activity
Margaret Barthelman, Warner B.Bair III, Kim Kramer Stickland, Weixing Chen, Barbara N.Timmermann1, Susanne Valcic1, Zigang Dong2 and G.Tim Bowden3 Department of Radiation Oncology, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, AZ 85724, 1Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, 2Hormel Institute, University of Minnesota, Austin, MN, USA 3To
whom correspondence should be addressed Email:
[email protected]
Green tea polyphenols have been shown to inhibit cancer in a variety of tumor models, including ultraviolet B (UVB)induced non-melanoma skin cancer. In green tea extracts, the major dry mass constituent is the family of catechins, of which (–)-epigallocatechin-(3)-gallate (EGCG) is considered to be important for the chemopreventive activity. EGCG has been shown to have antioxidant properties, but there has been little progress toward identifying the specific targets and mechanisms of its action. Using cultured human keratinocytes, we show that UVB-induced AP-1 activity is inhibited by EGCG in a dose range of 5.45 nM to 54.5 µM. EGCG is effective at inhibiting AP-1 activity when applied before, after or both before and after UVB irradiation. EGCG also inhibits AP-1 activity in the epidermis of a transgenic mouse model. This work begins to define a mechanism by which EGCG could be acting to inhibit UVB-induced tumor formation.
Many epidemiological studies have suggested a possible causal relationship between green tea consumption, primarily in Asian countries, and a reduced risk for some cancers (1,2). Animal and tissue culture models have taken this hypothesis further, showing chemopreventive effects of green tea polyphenols (GTP) against tumor initiation, promotion, and progression (1–6). GTP and its major constituent, (–)-epigallocatechin-(3)gallate (EGCG), have been shown to inhibit mutagenesis and tumorigenesis induced by chemicals and ultraviolet B (UVB) irradiation (1,3,5,6) and metastasis of B16 melanoma cells to mouse lung (7). EGCG is the most abundant of the green tea compounds tested, both in terms of mass and chemopreventive activity (1–3,5). The fact that EGCG is effective with low toxicity and low cost but can also inhibit existing tumors (6,8– 10) makes it a promising chemopreventive and chemotherapeutic agent. GTP and EGCG have antioxidant properties. This may explain their inhibition of UV- and 12-O-tetradecanoylphorbol13-acetate (TPA)-induced skin damage, including edema, hypAbbreviations: AP-1, activator protein-1; DMSO, dimethyl sulfoxide; EGCG, (–)-epigallocatechin-3-gallate; EGF, epidermal growth factor; GTP, green tea polyphenols; OA, okadaic acid; PBS, phosphate buffered saline; TNFα, tumor necrosis factor alpha; TPA, 12-O-tetradecanoyl phorbol-13-acetate; TRE, TPA response element; UVB, ultraviolet B. © Oxford University Press
erplasia, H2O2 production and inflammation (3,11,12). In JB6 cells, EGCG has recently been shown to inhibit epidermal growth factor (EGF) and TPA-induced AP-1 activity, a marker for skin tumor promotion (13). Through a sealing effect, EGCG blocks binding of tumor promoters to their receptors (14,15). In BALB/3T3 cells, EGCG is hypothesized to inhibit okadaic acid (OA) induced TNFα expression and release by inhibiting AP-1 DNA binding and interaction between OA and its cellular receptor (15). GTP and EGCG inhibit chemical initiation by inducing detoxifying enzymes and inhibiting metabolic activation by cytochrome P450, thereby inhibiting DNA adduct formation (1,16). Such processes would not explain the inhibitory effect of EGCG against a physical carcinogen such as UV or ionizing radiation. In non-melanoma skin cancer models, EGCG applied topically to mouse skin inhibits UVB-induced complete photocarcinogenesis and immunosuppression (6,11). Oral GTP has been shown to block both UVB-induced initiation and promotion (4,5). EGCG has also been shown to block cell transformation induced by TPA, EGF and ionizing radiation (13,17). UVB radiation is a causative factor in human non-melanoma skin cancer. We have previously shown that UVB irradiation induces AP-1 activity, both in vitro and in vivo (18,19). The AP-1 transcription factor complex is made of Jun homodimers and Jun–Fos heterodimers which bind to and transactivate from a cis element called the TPA-response element (TRE). AP-1 regulates transcription of many genes involved in carcinogenesis, and is itself upregulated during tumor promotion and progression (20–25). Agents that inhibit AP-1 activity have been shown to be efficacious against carcinogenesis (18–25). We used a human keratinocyte cell line, HCL14, that contains signature UVB mutations in both p53 alleles. These cells represent skin cells that have been damaged, perhaps initiated, by UVB exposure. HCL14 cells were previously derived from HaCaT cells that were transfected with a reporter luciferase construct driven by the human collagenase promoter, which contains a single TRE (18). We have used this model to study agents that may inhibit UVB-induced AP-1 activity. Cells were serum-starved for 14 h, then EGCG was added at a concentration of 5.45 µM (in 0.1% DMSO added to serum-free DMEM) and pre-incubated for 10 h. Plates were aspirated, rinsed twice with PBS, and irradiated to 250 J/m2 (without medium) using a pair of Westinghouse FS20-T12 bulbs. Fresh medium containing 5.45 µM EGCG was then immediately returned to the cells for an additional 20 h until extracts were harvested for the luciferase assay, as previously described (18). Under these conditions, UVB-irradiation alone induces a 60–100-fold increase in AP-1 transactivation as measured by luciferase activity (18). Addition of 5.45 µM EGCG inhibited this induction by 75% (Figure 1). To show that our system is sensitive to EGCG across a range of doses, we treated HCL14 cells, as above, with varying concentrations of EGCG over five orders of magnitude. As seen in Figure 2, we observed a dose-dependent inhibition of 2201
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Fig. 1. EGCG inhibition of UVB-induced AP-1 transactivation. After a starvation period, HCL14 cells were pre-treated with EGCG (or 0.1% DMSO control) for 10 h, UVB irradiated (250 J/m2), then post-treated for an additional 20 h, all under serum-free conditions. Cell extracts were harvested for luciferase assays. Bars represent the means of seven separate experiments of 3–4 plates per group. Error bars are SEMs.
Fig. 2. EGCG dose-dependent inhibition of UVB-induced AP-1 transactivation. After a starvation period, HCL14 cells were pre-treated with EGCG (or 0.1% DMSO control) for 10 h, UVB irradiated (250 J/m2), then post-treated for an additional 20 h, all under serum-free conditions. Cells were treated with the various doses of EGCG indicated (5.45 nM to 54.5 µM or 0.1% DMSO control) in serum-free media. Cell extracts were harvested for luciferase assays. Bars represent the means of three separate plates, and the results in this figure are representative of six separate experiments. Error bars are SEMs. A two way analysis of variance model was used to test the difference in luciferase activities. In the experiment at each dose level of EGCG in UVB irradiated cells there was a statistically signficant (P , 0.05) reduction in luciferase activity compared with UVB irradiated cells not treated with EGCG.
UVB-induced AP-1 activation. The data shown in Figure 2 are representative of six independent experiments. Statistically significant inhibition (P , 0.05) of UVB-induced AP-1 activation was seen in this experiment at all dose levels of EGCG. In four of six experiments a statistically significant (P , 0.05) reduction in luciferase activity was observed at all dose levels of EGCG. The range of doses used in previous in vitro inhibition studies (10,13,15) was 1–100 µM. At the highest dose of EGCG used (54.5 µM), we do see some cell morphology changes and a dark red precipitant on the plate, but no decrease in cell viability (data not shown). At all doses used, EGCG does not cause any toxicity in our cells (data not shown), regardless of UVB treatment. 2202
Fig. 3. EGCG inhibition of UVB-induced AP-1 activity in mouse skin. Transgenic B6D2 mice were generated to express luciferase driven by a pair of AP-1 response elements. After three pre-treatments of 5 mg topically applied EGCG or acetone control, mice were dorsally irradiated (0 or 10 kJ/m2), then post-treated with a single 5 mg application (or acetone). One day later, skin epidermis was harvested for luciferase assays.
To begin to study the mechanism of EGCG inhibition of UVB-induced mouse skin carcinogenesis, EGCG inhibition of AP-1 activity was assayed in transgenic mice. Transgenic B6D2 mice were generated to contain a luciferase reporter driven by two TRE elements (19,24). Mice were treated topically three times in a week with 5 mg of EGCG in acetone. The area of the skin treated was ~12 cm2. The mice were then irradiated with 10 kJ/m2 of UVB, followed 30 min later by a final application of 5 mg EGCG. One day later, skin epidermis was harvested for luciferase activity. In mouse skin epidermis, UVB induced a nearly 40-fold increase in luciferase activity, as compared with acetone treated controls (Figure 3). Treatment with topical EGCG reduced this UVB-induction of AP-1 transactivation activity by 60%. By inhibiting AP-1 activity in UVB-irradiated mouse skin, EGCG may be preventing nonmelanoma skin cancer at the level of tumor promotion. There have been reports that EGCG may exert some of its anti-cancer effects through a simple sunscreening of UVB radiation (6,16). We investigated whether EGCG was effective subsequent to irradiation, to avoid sunscreening effects. We treated HCL14 cells with medium containing 5.45 mM EGCG prior to (10 h) or following (20 h) UVB irradiation (Figure 4). EGCG effectively inhibited UVB-induced AP-1 activity at all times it was applied. The inhibition appeared to be greater when applied after irradiation, as compared with pre-incubation. By treating cells with EGCG before or after irradiation, we further defined when EGCG activity is required to block AP-1 activity. In doing so, we see that the mechanism of inhibition extends beyond any sunscreening or UVB-absorbing effects of EGCG. We have shown that EGCG inhibits AP-1 transactivation, a marker of tumor promotion, that is induced by a physical carcinogen, UVB. Previously, GTP was shown to inhibit UVBinduced ornithine decarboxylase and cyclooxygenase, and restore UVB-inhibited catalase, GSH and glutathione peroxidase in epidermal cells (16). Such effects are likely linked to its antioxidant abilities, which would counteract UVB-induced free radicals, lipid peroxidation, changes in endogenous antioxidants, membrane damage and hyperproliferation (16,26–28). Interestingly, EGCG has been shown to interact directly with the membrane in liposome studies, where it has been shown to inhibit PKC activity (14). UVB signals, however, are transduced through other cascades (29–31), including atypical
Green tea polyphenols and inhibition of cancer
Fig. 4. Selective times of EGCG treatment and their effects on UVBinduced AP-1 transactivation. HCL14 cells were starved and then pretreated with 5.45 µM EGCG (1 Pre-EGCG) or 0.1% DMSO control (– Pre-EGCG). Cells were irradiated (250 J/m2) or mock-irradiated followed by a post-treatment of 5.45 µM EGCG (1 Post-EGCG) or 0.1% DMSO control (– Post-EGCG). Cells were harvested at 20 h post-irradiation for luciferase assays. Bars represent the means of six separate experiments of 3–4 plates per group. Error bars are SEMs.
PKC, which are also EGCG sensitive. EGCG has also been shown to inhibit the release of TNFα, a cytokine, known to be released by keratinocytes subsequent to UV irradiation (15,32). EGCG, perhaps through its antioxidant activity, could be acting at numerous points in the UVB signaling cascade, including Ras/Raf and MAPK/JNK/p38 activity which mediate UVB-induced AP-1 activity (29–31). This has been seen in JB6 cells, where EGCG inhibits EGF- and TPA-induced c-Jun phosphorylation (13). However, Yu et al. found a dosedependent increase in JNK1 and ERK2 activity in unstimulated HepG2 cells following addition of EGCG at doses ~10-fold higher than those used here (10). EGCG has also been found to induce junD, c-fos, and fosB mRNA only after okadaic acid stimulation in BALB/3T3 cells (15), but stimulates c-fos and c-jun expression in unstimulated HepG2 cells (10). Oral green tea has been found to inhibit NNK-induced c-myc, cHa-ras, and c-raf expression in mouse lung (33), but had no effect on c-myc in cultured HepG2 cells (10). Taken together, this may indicate that EGCG activity is dependent on the state and the nature of the cellular model. Based on our findings, we will be examining the regulation of AP-1 activity and levels of AP-1 family members, in particular c-fos, which is hypothesized to play a key role in regulation of AP-1 activity following UVB irradiation and oxidative stress (10,34,35; W.Chen et al., submitted). We will go on to investigate the UVB signal transduction cascade to further define upstream targets of EGCG inhibition. This work begins to give a mechanistic explanation to a natural product with clinical potential as a chemoprevention agent. Acknowledgements We are grateful to Cindy Calley for statistical assistance and Anne Cione for secretarial support. This work was supported by US Public Health Service grant CA 27502, Cancer Center Core grant CA 23074, NIEHS Center grant ES 06694 and the Schick Memorial Fund.
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