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Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes¹

Department of Dermatology, Seoul National University College of Medicine, and Laboratory of Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea

²Correspondence: Department of Dermatology, Seoul National University Hospital, 28 Yongon-dong, Chongno-Gu, Seoul 110-744, Korea. E-mail: hceun{at}snu.ac.kr

SPECIFIC AIMS

Recently, epigallocatechin-3-gallate (EGCG), a major constituent polyphenol in green tea, was found to have a pronounced growth inhibitory effect on cancer cells, but not on normal cells. In this study, we investigated the effects of EGCG on the proliferation and UV-induced apoptosis of normal human epidermal keratinocytes and the signaling pathways through which EGCG acts in vitro and in vivo.

PRINCIPAL FINDINGS

1. EGCG induced keratinocyte proliferation in human skin in vivo and in vitro
The topical application of 10% EGCG to aged human skin three times a week for 6 wk increased the epidermal thickness. The number of Ki-67 positive keratinocytes in the basal cell layer also increased. These results indicate that the topical application of EGCG stimulates keratinocyte proliferation, which increases the epidermal thickness of human skin in vivo (Fig. 1 A). Treatment of cultured keratinocytes with EGCG for 5 days significantly increased their proliferation in a dose-dependent manner up to 1.0 µM of EGCG (Fig. 1B ).

View larger version (55K): [in this window] [in a new window]   Figure 1. EGCG increased the epidermal thickness of aged human skin in vivo and stimulated the proliferation of human epidermal keratinocytes. A) 10% EGCG or its vehicle (70% propylene glycol, 30% ethanol) was applied to the buttock skin of 5 elderly men, 3 times a week for 6 wk, under occlusion. EGCG- or vehicle-treated skin samples were obtained by punch biopsy. The skin sections were stained with H&E and immunostained with anti-Ki-67 antibody. The average epidermal thickness was measured using an image analyzer and the number of Ki-67 positive keratinocytes per mm of the basal cell layer was calculated. The pictures are representatives of the 5 individuals; values shown are the means ± SE of the 5 individuals. B) Cultured human epidermal keratinocytes were treated with various concentrations of EGCG (0–100 µM) every 2 days for 5 days and an MTT assay performed. Values are means ± SE from 6 wells and the graph is a representative result from triplicate experiments. *P < 0.05, **P < 0.01 compared with vehicle-treated group.

2. EGCG stimulated keratinocyte proliferation through the phosphorylation of Bad by activating Erk- and Akt-dependent pathways
Treatment with EGCG increased the phosphorylation of Erk and Akt 1 h post-treatment in cultured normal keratinocytes, while the level of total Erk and Akt was unchanged. EGCG increased phosphorylation at Ser112 and Ser136 of Bad protein, while the total Bad protein was unchanged (Fig. 2 ).

View larger version (38K): [in this window] [in a new window]   Figure 2. EGCG phosphorylated Ser112 and Ser136 of Bad protein through Erk and Akt pathways, respectively. Cultured keratinocytes were treated with EGCG (0–0.5 µM) for 1 h. Western blot analysis was performed. All bands shown are representative of triplicate experiments. A) The MEK 1 inhibitor PD98059 (20 µM) inhibited EGCG-induced phosphorylation at Ser112 of Bad. Cultures were incubated with MEK 1 inhibitor 1 h before treatment with EGCG until harvesting. B) The PI-3 kinase inhibitor LY294002 (20 µM) inhibited EGCG-induced phosphorylation at Ser136 of Bad. The cultures were incubated with LY294002 1 h before treatment with EGCG until harvesting. *P < 0.05 compared with vehicle-treated group, P <0.05 compared with each EGCG-treated group, respectively.

The MEK inhibitor PD98059 (20 µM) inhibited the EGCG-induced phosphorylations of Erk and Ser112 of Bad, but the phosphorylation of Ser136 of Bad was unaffected (Fig. 2A ). The PI-3 kinase inhibitor LY294002 (20 µM) inhibited the EGCG-induced phosphorylation of Akt and of Ser136 but did not affect the phosphorylation of the Ser112 (Fig. 2B ). When cultured keratinocytes were treated with PD98059 or LY294002, EGCG-induced proliferation was inhibited. Therefore, our data suggest that EGCG activates both Erk- and Akt-dependent pathways and that this results in phosphorylation of Ser112 and Ser136 of Bad protein, respectively. Moreover, this increased phosphorylation of Bad protein plays roles in the stimulation of keratinocyte proliferation by EGCG. We confirmed these effects of EGCG on Bad phosphorylation in human skin in vivo.

3. EGCG increased Bcl-2 expression and decreased Bax expression
EGCG significantly increased Bcl-2 protein expression in a dose-dependent manner 24 h post-treatment in cultured keratinocytes. Conversely, treatment of cultured keratinocytes with EGCG inhibited Bax protein expression in a dose-dependent manner. These results indicate that EGCG increased the ratio of Bcl-2 to Bax, resulting in the increased proliferation of epidermal keratinocytes. We confirmed that EGCG had the same effect on Bcl-2 and Bax expression in human skin in vivo.

4. ECCG inhibited UV-induced apoptosis by inducing Bad phosphorylation and increasing the ratio of Bcl-2 to Bax
EGCG prevented UV-induced apoptosis in epidermal keratinocytes both in vitro and in vivo. UV irradiation increased Erk and Akt phosphorylation levels in cul-tured keratinocytes and induced the phosphorylation of Bad at both Ser112 and Ser136. Pretreatment with EGCG prior to UV irradiation augmented the UV-induced phosphorylation of Erk and Akt and the UV-induced phosphorylation of both Ser112 and Ser136 of Bad. The MEK 1 inhibitor PD98059 (20 µM) inhibited the augmentation of the Ser112 phosphorylation, but not of the Ser136, of Bad protein by EGCG. The PI-3 kinase inhibitor LY294002 (20 µM) inhibited the augmentation of the Ser136 phosphorylation, but not that of Ser112 by EGCG. These results indicate that EGCG prevents UV-induced apoptosis by augmenting Bad phosphorylation through Erk and Akt activation. On the other hand, UV irradiation significantly decreases the ratio of Bcl-2 to Bax in cultured keratinocytes; EGCG can reverse this UV-induced decrease, which resulted in the prevention of UV-induced apoptosis. We demonstrated that these effects are present in human skin in vivo.

5. Differential effects of EGCG on normal keratinocyte and squamous carcinoma cell proliferation
In normal keratinocytes, EGCG enhanced proliferation at 0.5 µM but did not affect it significantly at 50 µM.However, in squamous carcinoma cells, EGCG decreased proliferation at both concentrations dose-dependently. Treatment with EGCG also modulated the activities of Erk and Akt and expression of Bcl-2 and Bax differently in normal keratinocytes and squamous carcinoma. The phosphorylations of Erk and Akt were increased by EGCG at 0.5 µM, but not at 50 µM, in normal keratinocytes, which led to increased phosphorylation of Bad at Ser112 and Ser136, respectively. On the other hand, phosphorylation of Erk was increased, but the phosphorylation of Akt and Bad at Ser112 and at Ser136 was decreased by EGCG in a dose-dependent manner in squamous carcinoma cells. EGCG increased the ratio of Bcl-2 to Bax in normal keratinocytes, but decreased it in squamous carcinoma cells. Taken together, these results suggest that the observed differences between normal keratinocytes and squamous cancer cells, in terms of the proliferation induced by treatment of EGCG, may be due to the different signaling events induced by EGCG.

CONCLUSIONS AND SIGNIFICANCE

We found, for the first time, that topical EGCG application to aged human skin induces epidermal keratinocytes proliferation and results in increased epidermal thickness. We also found that EGCG inhibits the UV-induced apoptosis of human epidermal keratinocytes in vivo and in vitro and that EGCG affects the proliferation of normal keratinocytes and squamous carcinoma cells differently.

EGCG promotes the survival of keratinocytes and inhibits UV-induced apoptosis by a dual mechanism (Fig. 3 ): 1) increased phosphorylation at Ser112 and Ser136 of Bad protein through Erk-dependent and Akt-dependent pathways, respectively, and 2) by increasing the ratio of Bcl-2 to Bax. The suppression of Bad-mediated cell death caused by EGCG occurs rela-tively early, 1 h post-EGCG treatment, whereas the contribution made by the Bcl-2/Bax ratio is detected significantly later, 24 h after treatment. Therefore, these two proposed molecular mechanisms associated with EGCG-induced cell proliferation promote the survival of keratinocytes with different kinetics. ECGC acts differently on cancer cells, and may protect against cancers by inhibiting proliferation and inducing apoptosis even at physiological concentrations. Our data suggest that EGCG can be used topically to induce an anti-aging effect and an anti-cancer effect in human skin.

View larger version (20K): [in this window] [in a new window]   Figure 3. Proposed molecular mechanisms of EGCG-induced cell proliferation and survival in human epidermal keratinocytes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0914fje; doi: 10.1096/fj.02-0914fje

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