Elevated polyamines lead to selective induction of apoptosis and inhibition of tumorigenesis by ()-epigallocatechin-3-gallate (EGCG) in ODC/Ras transgenic mice
Barry Paul,
Candace S. Hayes,
Arianna Kim1,
Mohammad Athar1 and
Susan K. Gilmour2
Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA and 1 Department of Dermatology, Columbia University, 630W 168th Street, New York, NY 10032, USA
2 To whom correspondence should be addressed Email: gilmours{at}mlhs.org
 |
Abstract
|
---|
Tea polyphenolic constituents induce apoptosis in cancer cells but not in normal cells. To study the mechanism of this selective effect, we used the ornithine decarboxylase (ODC)/Ras double transgenic mouse model that develops spontaneous skin tumors due to over-expression of ODC and a v-Ha-ras transgene. Administration of the green tea polyphenol ()-epigallocatechin-3-gallate (EGCG) in the drinking water significantly decreased both tumor number and total tumor burden compared with untreated ODC/Ras mice without decreasing the elevated polyamine levels present in the ODC/Ras mice. EGCG selectively decreased both proliferation and survival of primary cultures of ODC over-expressing transgenic keratinocytes but not keratinocytes from normal littermates nor ras-infected keratinocytes. This decreased survival was due to EGCG-induced apoptosis and not terminal differentiation. Moreover, in skin from EGCG-treated ODC transgenic mice, caspase 3 (active form) was detected only in epidermal cells that possess very high levels of ODC protein. Since most transformed cells and tumor tissue possess higher levels of polyamines compared with normal cells or tissue, our data suggest that the elevated levels of polyamines in tumor cells sensitize them to EGCG-induced apoptosis. These results suggest that EGCG may be an effective chemopreventive agent in individuals with early, pre-neoplastic stages of cancer having higher levels of polyamines.
Abbreviations: BrdU, bromodeoxyuridine; EGCG, ()-epigallocatechin-3-gallate; ODC, ornithine decarboxylase
 |
Introduction
|
---|
Tea (Camellia sinensis) is one of the most popular beverages consumed in the world. Studies using both cell lines and a variety of animal models have shown that green tea and black tea inhibit tumorigenesis (14). Epidemiological studies have detected an association between consumption of tea and a lower risk of cancer in several types of tissue, including stomach, esophagus, prostate and lung (2,3,5). Green tea, which is widely consumed in many Asian countries, is chemically characterized by the presence of large amounts of polyphenolic compounds, of which ()-epigallocatechin-3-gallate (EGCG) is the most abundant. Administration of tea and tea components to animals has been shown to prevent the formation of chemically induced and UVB-induced skin tumors as well as both prostate and intestinal tumorigenesis in transgenic mice (69). A growing number of studies have shown that EGCG stimulates apoptosis in tumor cells compared with normal cells (911).
The majority of skin carcinogenesis studies with tea and tea polyphenols involve the use of chemical carcinogens, tumor promoters or UV irradiation to induce tumors. We have developed a simplified transgenic mouse model that does not require any prior treatment with a carcinogen or tumor promoting agent for spontaneous skin tumor development (12). Specifically, the expression of ornithine decarboxylase (ODC) and a mutated ras gene have been targeted to the skin of ODC/Ras transgenic mice to mimic the altered polyamine and Ras signaling that characterize many pre-neoplastic lesions (13). ODC is a key regulatory enzyme in the biosynthesis of polyamines that are essential for normal cell growth and differentiation. The polyamines putrescine, spermidine and spermine are some of the major cations present in cells. Whereas cellular mechanisms tightly control the expression of ODC in normal cells, alterations in the regulation of ODC is an early event in tumor development, leading to high basal levels of ODC expression and subsequent increased levels of polyamines. In addition to high levels of polyamines, many epithelial tumors exhibit aberrant Ras signaling either due to ras mutations or mutations in other genes that can also lead to chronic up-regulation of the Ras pathway (13).
Our previous studies have shown that crossing K6/ODC transgenic mice with TG.AC transgenic mice that express a v-Ha-ras transgene yield ODC/Ras double transgenic mice that develop spontaneous skin tumors by 2 months of age without any prior treatment with carcinogens or tumor promoters (12). It is significant that transgenic mice that only possess either the v-Ha-ras transgene or the ODC transgene will not develop tumors without a promotional stimulus such as TPA or wounding or a genetic initiating event such as treatment with the carcinogen dimethylbenz[a]pyrene, respectively. We have used this simplified animal model of carcinogenesis to test the potential inhibitory effect of oral administration of the green tea constituent EGCG on the spontaneous development of skin tumors resulting from aberrant expression of ODC and an activated Ha-ras gene. We have also evaluated the effect of EGCG on proliferation and apoptosis in primary cultures of normal keratinocytes in which polyamine levels are elevated.
 |
Materials and methods
|
---|
Animals and treatment schedules
K6/ODC transgenic mice on a C57Bl/6 background were generated as described by Megosh (14). ODC/Ras double transgenic mice were created by crossing K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice (12). At 3 weeks of age, administration of EGCG (Mitsui Norin, Tokyo, Japan) in the drinking water was initiated in ODC/Ras transgenic mice. EGCG was increased gradually over the first 2 weeks of treatment until the full dose of 0.045% (w/v) EGCG was obtained to slowly adjust the animals to EGCG in their drinking water. The treated ODC/Ras transgenic mice were maintained on water containing EGCG (prepared every other day) until death at 12 weeks of age, while control mice were fed tap water. Some K6/ODC transgenic mice and their normal littermates were administered EGCG in their drinking water for a total of 3 weeks with a step-wise increase of EGCG over the course of 14 days until a dose of 0.045% (w/v) EGCG was obtained for the final week. Two hours before death, all mice were injected i.p. with bromodeoxyuridine (Sigma Chemical, St Louis, MO) at a dose of 100 µg/g body wt.
At the time of death all tumors were counted and tumors >1 mm in diameter were removed, measured with calipers and weighed. The total weight of all tumors on each mouse was defined as total tumor burden. Tumors and non-tumor bearing skin were excised, and some tissue fixed in 4% p-formaldehyde for paraffin embedding. The remaining tissue was frozen in liquid nitrogen, ground with a mortar and pestle, and stored at 80°C for subsequent analysis.
ODC enzyme activity assay and polyamine analyses
To assay ODC enzymatic activity, frozen ground skin and tumor tissues were lysed in 25 mM TrisHCl (pH 7.5), 2.5 mM DTT, 0.1 mM EDTA and protease inhibitors including 1 µg/ml each of aprotinin, leupeptin, pepstatin, 1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 mM Pefabloc. Homogenates were assayed for ODC enzyme activity by quantifying the production of 14CO2 from L-[14C]ornithine (15). To assay whether EGCG has a direct effect on ODC activity, equal amounts of K6/ODC dermal homogenate were treated with either 0, 30, 60 or 100 µM EGCG on ice for 20 min and then assayed for ODC enzyme activity. A portion of ground tissue was also lysed in 0.2 N perchloric acid for measurement of polyamine levels. Putrescine, spermidine and spermine levels were determined by dansylation and separation on a reversed phase C18 high-performance liquid chromatography column (16). Polyamines were normalized to the DNA content of the tissue sample.
Primary cultures of epidermal cells
Primary cultures of epidermal cells were isolated from 3 to 4-day-old K6/ODC newborn pups and their normal littermates by a trypsin flotation procedure (17). K6/ODC transgenic pups were distinguished from their normal littermates by PCR genotyping for the K6/ODC transgene (14). Cells were plated at 3.0 x 106 cells/60-mm-dish or onto glass coverslips in low calcium keratinocyte media [EMEM w/o calcium chloride (BioWhittaker Walkersville, MD) supplemented with 8% chelex-treated fetal bovine serum and 0.05 mM calcium chloride] and grown at 35°C with 5% CO2. Some keratinocytes were transduced with a defective retrovirus containing the v-Ha-ras oncogene (18). The keratinocytes were cultured for 3 days before a 24-h treatment with varying concentrations of EGCG. In order to determine non-cytotoxic doses of EGCG, keratinocytes were cultured for 2 days, treated with 0, 1, 10, 20, 50 or 100 µM EGCG, and harvested by trypsinization 6, 12 or 24 h later. The number of trypan blue dye-excluding cells was determined using a hemocytometer. Cell viability was expressed as the percentage of non-stained viable cells among the total (dead and viable) population of cells.
To assay for [3H]thymidine incorporation, primary keratinocytes isolated from K6/ODC transgenic mice and their normal littermates were re-fed 24 h after plating in low calcium keratinocyte medium containing varying concentrations of EGCG. After 23 h, cells were pulsed for 1 h with 1.5 µCi [3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ). The cells were washed with cold PBS containing 0.1% unlabeled thymidine and scraped in 1 N sodium hydroxide. This lysate was acidified by adding an equal volume of 1 N perchloric acid, centrifuged at 10 000 g for 10 min, and washed three times with chilled 0.2 N perchloric acid. Pellets were heated in 0.5 N perchloric acid at 90°C for 20 min. After cooling, the lysate was centrifuged at 10 000 g for 15 min and the supernatant was saved. One-tenth of the supernatant was added to a 4-ml scintillation cocktail (ICN, Costa Mesa, CA) and counted. DNA quantification was performed using the method referenced above for polyamine analysis (16). Values were expressed as the percent incorporation of [3H]thymidine with respect to normal and K6/ODC cells that were not treated with EGCG.
Immunochemistry
Primary keratinocytes grown on glass coverslips were treated with the indicated dose of EGCG 3 days after plating and harvested 24 h later. The coverslips were washed with cold PBS and fixed for 20 min with 4% p-formaldehye in PBS. Goat serum was used to suppress non-specific binding. Coverslips were incubated for 1 h at room temperature in a humidity chamber with an affinity-purified rabbit polyclonal antibody that reacts with the mouse p20 subunit of caspase 3 but does not react with the precursor form (R&D Systems, Minneapolis, MN). The coverslips were then incubated with a biotinylated anti-rabbit secondary antibody followed by incubation with an avidinhorseradishperoxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Immunoreactive cells were localized by incubating with a diaminobenzidine chromagen solution and then counterstained with hematoxylin. At least 1000 cells were counted for each coverslip with three coverslips per treatment group, and the percent of stained cells was reported.
Tissues were fixed in 4% p-formaldehyde in PBS overnight and then embedded in paraffin. Sections were deparaffinized, hydrated and then treated with 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven at half power for 10 min. Sections were incubated with a caspase 3 primary antibody (1:1500), followed by biotinylated secondary antibody, and then an avidinhorseradishperoxidase complex (Vectastain Elite ABC kit). Immunoreactive cells were localized by incubating the sections with a chromagen solution containing diaminobenzidine and peroxide and then counterstaining with hematoxylin. Bromodeoxyuridine (BrdU) incorporation in cells undergoing DNA synthesis was detected in skin sections using a rat monoclonal anti-BrdU antibody (Zymed Laboratories, San Francisco, CA). The proliferative index was determined by multiplying the number of BrdU-positive cells/1000 epithelial cells by 100.
Immunoblot analyses
For immunoblots, tissue or keratinocytes were homogenized in RIPA buffer [50 mM TrisHCl pH 7.4, 150 mM NaCl, 0.25% (w/v) deoxycholic acid, 1% (w/v) NP-40, 1 mM EDTA, containing 1 µg/ml each of aprotinin, leupeptin, pepstatin, 1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 mM Pefabloc], and passed through a syringe needle multiple times after a 45-min incubation on ice. Lysates were clarified by centrifugation at 13 000 r.p.m. for 10 min, and the protein content was determined using the Bio-Rad D/C protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated by 15% SDSPAGE and transferred to PVDF membranes (Millipore, Bedford, MA). Immunoblots were incubated for 1 h at room temperature in blocking solution (PBS with 10% non-fat dry milk and 0.05% Tween 20) followed by incubation with the primary antibody diluted in PBS with 0.1% milk and 0.05% Tween 20 for 2 h at room temperature. Blots were probed with a rabbit polyclonal anti-keratin-1, anti-loricrin and anti-involucrin antibody (Covance, Richmond, CA), a rabbit polyclonal anti-Bcl-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or a rabbit polyclonal anti-Bax antibody and a mouse monoclonal anti-Ras antibody (Upstate Biotechnology, Lake Placid, NY). A monoclonal anti-ß-actin antibody (Sigma, St Louis, MO) was used as a protein loading control. The immunoblots were developed with a horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) followed by detection using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
Statistical analysis
The tumor data were organized into frequency distributions for each study group and the non-parametric Wilcoxon Rank Sum test was used to determine exact P values. All other analyses used the unpaired t-test or ANOVA. All statistical calculations were done using the Statview software package (SAS, Cary, NC).
 |
Results
|
---|
Inhibitory effect of EGCG on tumor burden and tumor number in ODC/Ras double transgenic mice
To test whether treatment with EGCG prevents the formation of spontaneous skin tumors in the ODC/Ras transgenic mouse model, mice were given EGCG in their drinking water for 9 weeks. To acclimate the mice, EGCG was gradually increased to a final concentration of 0.045% in their drinking water over the first 2 weeks of treatment. We chose to administer EGCG through the drinking water because topical administration would localize treatment to only a defined area of the dorsal skin of the mouse during a time when spontaneous tumors develop randomly over their entire skin surface. Treatment was initiated at 3 weeks of age, before the occurrence of spontaneous tumors that develop between 6 and 10 weeks of age. All mice were killed at 12 weeks of age, and the skin and skin tumors examined.
Compared with water-fed ODC/Ras double transgenic mice, EGCG-treated ODC/Ras mice exhibited a significant reduction in both the median number of spontaneous tumors (Figure 1a) and the median total tumor burden (Figure 1b). After 9 weeks of treatment, the water-fed control group had more tumors per mouse (median 5.5 tumors/mouse; range 08) than the EGCG-treated animals (median 2 tumors/mouse; range 03), showing a 64% inhibition (P < 0.001). Treatment of mice with EGCG also significantly reduced the total tumor burden per mouse by 91% (P < 0.001). The reduction in tumor burden was not solely due to the fewer number of tumors as evidenced by a significant reduction (P < 0.01) in the median average mass of each tumor (0.045 g/tumor in the EGCG-treated group and 0.28 g/tumor in the water-fed control group). Some ODC/Ras transgenic mice were treated for an additional month with EGCG resulting in no regression of tumors (data not shown). However, these tumors remained small and cornified and not well vascularized. Analyses showed that the inhibitory effect of EGCG treatment on tumor development in ODC/Ras transgenic mice was not sex-dependent.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1. EGCG decreases the number of tumors and tumor burden in ODC/Ras transgenic mice. ODC/Ras double transgenic mice were administered EGCG (gray bars) in their drinking water (n = 14) or water alone (black bars) (n = 19) beginning at 3 weeks of age. At 12 weeks of age, tumors were removed, counted, and weighed. (a) Total number of spontaneous tumors at time of death. (b) Total tumor burden at time of death.
|
|
Histological characterization of the tumors revealed that EGCG oral treatment of the ODC/Ras transgenic mice had no effect on the types of tumor, the majority of which were keratoacanthomas. However, the tumors were more keratinized and developed more slowly in the EGCG-treated mice compared with the control water-fed mice. Whereas 25% of the ODC/Ras tumors converted to malignant squamous cell carcinomas within the short 6-week period from when the tumors first appeared, the conversion of tumors to malignant squamous cell carcinomas was completely blocked in the EGCG-treated mice.
EGCG has no effect on polyamine levels in skin
Several reports have demonstrated that green tea and its polyphenolic constituents can inhibit ODC enzyme activity and subsequent polyamine levels (19,20). The polyamines putrescine, spermidine and spermine are some of the major cations present in cells. Indeed it has been suggested that polyamine depletion by green tea could be a mechanism for its antitumorigenic activities (21). Although administration of EGCG in the drinking water resulted in a 50% reduction in ODC activity in the non-tumor bearing skin of ODC/Ras transgenic mice, there was no significant change in the polyamine levels in EGCG-treated ODC/Ras mice compared with water-fed animals (Figure 2a and b). The EGCG-induced reduction in ODC activity was not a direct effect on the ODC enzyme as EGCG had no inhibitory effect on ODC activity when added to a lysate of ODC/Ras transgenic skin (data not shown). Although ODC activity was lower in ODC/Ras transgenic mice following EGCG treatment, ODC activity remained significantly elevated in EGCG-treated ODC/Ras transgenic mouse skin as compared with skin from transgenic littermate mice possessing the v-Ha-ras transgene but no K6/ODC transgene (Figure 2b). Thus, the elevated epidermal ODC activity in EGCG-treated animals was sufficient to maintain the high levels of polyamines in ODC/Ras transgenic skin. Moreover, there was no significant difference in ODC or polyamine levels in skin tumors from EGCG-treated ODC/Ras transgenic mice compared with non-treated ODC/Ras mice (Figure 2c and d). Thus, the data indicate that the EGCG antitumor effect was not due to a decrease in overall polyamine levels in the skin of ODC/Ras transgenic mice.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2. EGCG effect on ODC enzyme activity and polyamine levels in skin and skin tumors in ODC/Ras transgenic mice. ODC/Ras transgenic mice and their normal littermates with no K6/ODC transgene were treated orally with either EGCG (n = 4) or water (n = 4) as the sole source of drinking water for 9 weeks and then killed at 12 weeks of age. Non-tumor-bearing skin lysates (a and b) and skin tumor lysates (c and d) were assayed for polyamine levels (a and c), including putrescine (black bar), spermidine (gray bar) and spermine (white bar) and for ODC enzyme activity (b and d). Values are means ± SD, and are representative of three separate experiments.
|
|
Inhibitory effect of EGCG on proliferation rate in primary keratinocytes with increased polyamine levels
In order to determine whether EGCG affects the growth of epidermal cells with elevated levels of ODC and/or an activated v-Ha-ras, primary keratinocytes were isolated from the skin of K6/ODC transgenic mice and their normal littermates. Primary K6/ODC transgenic keratinocytes were both age and genetically identical to normal keratinocytes, but transgenic keratinocytes possessed higher levels of ODC/polyamines (22). Using trypan blue dye-exclusion to test for cell viability, we found that high doses (50 and 100 µM) of EGCG significantly increased cell death and lower doses (120 µM) resulted in no cytotoxicity in either normal or ODC over-expressing cells (data not shown). We tested the effect of 24 h of non-cytotoxic doses of EGCG on the incorporation of [3H]thymidine in primary cultures of keratinocytes (Figure 3). Whereas EGCG had no inhibitory effect on [3H]thymidine incorporation in normal keratinocytes, EGCG significantly inhibited [3H]thymidine incorporation in K6/ODC transgenic keratinocytes up to 60% at 20 µM EGCG (P<0.001). In contrast, 20 µM EGCG stimulated DNA synthesis in normal keratinocytes (P < 0.01) as has been reported previously (23).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3. [3H]Thymidine incorporation in primary keratinocytes treated with EGCG. Primary keratinocytes isolated from K6/ODC transgenic mice (gray bars) and their normal littermates (black bars) were treated with 0, 1, 10 or 20 µM EGCG for 24 h. Cell proliferation was assessed by the amount of [3H]thymidine incorporation (c.p.m.) normalized to µg DNA. Values are means ± SD of at least three samples. *P<0.01.
|
|
We examined whether EGCG selectively inhibited the growth of K6/ODC transgenic keratinocytes by blocking the expression of the ODC transgene. K6/ODC transgenic keratinocytes were plated in the presence of the specific inhibitor,
-difluoromethylornithine (DFMO), to suppress ODC activity. DFMO was removed after 24 h, permitting a rise in ODC activity. However, EGCG failed to inhibit the subsequent rise in ODC enzyme activity in the K6/ODC transgenic keratinocytes following washout of the DFMO (data not shown).
EGCG selectively increases apoptosis in keratinocytes with high levels of polyamines
Primary keratinocytes that are no longer cycling are committed to cell death either by terminal differentiation or apoptosis. A 24-h exposure to 20 µM EGCG had no effect on the survival of normal keratinocytes, but it decreased the total number of adherent K6/ODC transgenic keratinocytes by 49% (Figure 4). EGCG treatment did not induce terminal differentiation markers, such as keratin 1, involucrin or loricrin, in ODC over-expressing keratinocytes (Figure 5). Immunocytochemical studies revealed that a 24-h exposure to EGCG increased the percent of caspase 3 (active form)-positive cells, by 73% in K6/ODC transgenic keratinocytes (P < 0.001; Figure 4). This effect was selective for ODC over-expressing keratinocytes because treatment of normal keratinocytes with EGCG had little or no effect on caspase 3-positive cells (P > 0.05; Figure 4b). Immunoblot analyses showing increased levels of pro-apoptotic Bax protein and decreased levels of anti-apoptotic Bcl-2 confirmed the apoptotic effect of EGCG (10 or 20 µM) on keratinocytes with elevated levels of ODC but not in normal keratinocytes (Figure 5). Bax levels were increased in v-Ha-ras-infected keratinocytes in comparison with non-infected cells (Figure 5). However, retroviral infection of a v-Ha-ras transgene did not further increase the percent cells positive for either caspase 3 or Bax in EGCG-treated K6/ODC or normal keratinocytes compared with keratinocytes that were not ras-infected.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4. EGCG induces apoptosis in keratinocytes and skin of K6/ODC transgenic mice. Primary keratinocytes were isolated from K6/ODC transgenic mice and their normal littermates and half the cells were infected with a v-Ha-ras retrovirus. Three days later, the cells were treated with 20 µM EGCG and harvested 24 h later. Some keratinocytes were plated on coverslips for immunocytochemistry using an anti-caspase 3 (active form) antibody. The relative cell density is expressed as the relative number of cells following ras infection and/or EGCG treatment divided by the number of keratinocytes not ras infected and not EGCG treated. The values for the percent caspase 3-stained are the percent of caspase 3-stained cells on three separate coverslips/treatment group ± SD, and are representative of two experiments. At least 6000 total cells were counted per group. *P< 0.01.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5. EGCG selectively increases the ratio of Bax to Bcl-2 in ODC over-expressing keratinocytes. Primary keratinocytes were isolated from K6/ODC transgenic mice and their normal littermates and half the cells were infected with a v-Ha-ras retrovirus. Three days later, the cells were treated with 10 or 20 µM EGCG and harvested 24 h later. Equal amounts of cell lysates were resolved by SDSPAGE and transferred to PVDF membranes. Bax, Bcl-2, keratin-1, involucrin, loricrin, Ras and ß-actin protein were detected by immunoblotting.
|
|
To determine whether EGCG treatment induces apoptosis in the skin of mice with elevated levels of polyamines but no activated Ras protein, K6/ODC transgenic mice and their normal littermates were treated with EGCG in their drinking water for 3 weeks. Immunohistochemical studies revealed that EGCG treatment selectively induced the appearance of caspase 3-positive cells in epithelial cells (0.44%) in the transgenic mouse skin but not in normal littermate skin (0%) (P < 0.001; Table I). Moreover, the proliferation index, as measured by percent BrdU-labeled nuclei (Table I), was depressed by EGCG treatment in transgenic skin (26.7 vs 15.3%; P < 0.001) but not in normal skin (0.71 vs 0.51%; P > 0.05). Taken as a whole, the data suggest that non-transformed keratinocytes expressing high levels of ODC/polyamines are more sensitive to the apoptotic effect of EGCG, and that their growth is preferentially inhibited by EGCG.
 |
Discussion
|
---|
This study demonstrates that EGCG efficiently suppresses the formation of skin tumors in mice that are genetically initiated with a v-Ha-ras transgene and genetically promoted with elevated levels of ODC and polyamines. Studies using cultured cells have suggested ODC inhibition, resulting in decreased cellular polyamine levels, as a possible mechanism of tea-mediated cell growth inhibition (1921). Our studies using an animal model over-expressing epidermal ODC have demonstrated that EGCG can effectively inhibit skin tumor development without altering polyamine levels.
A novel finding in this study is that elevated polyamines, that typify transformed cells, are sufficient to sensitize otherwise normal keratinocytes to undergo growth arrest and apoptosis following EGCG treatment. EGCG treatment decreased [3H]thymidine incorporation in ODC over-expressing keratinocytes and BrdU incorporation in the skin of orally treated K6/ODC transgenic mice. In addition, topical administration of EGCG decreased BrdU incorporation in K6/ODC transgenic skin as well (data not shown). This growth inhibitory effect of EGCG was selective for keratinocytes with elevated polyamine levels both in vivo and in vitro. In contrast, a growth stimulatory effect was seen in keratinocytes that did not possess higher polyamine levels as EGCG stimulated DNA synthesis in normal, non-transgenic keratinocytes.
Green tea polyphenols such as EGCG have been shown to induce apoptosis via multiple pathways including inhibition of NF-
B activation, binding to Fas, activation of tumor necrosis factor
-mediated signaling pathway, cell cycle arrest at G0/G1, and binding to and suppressing anti-apoptotic Bcl-2 family proteins (10,11,24). Low concentrations of EGCG have been shown to preferentially inhibit the growth of many types of transformed cells but not normal cells, primarily via induction of apoptosis (911). In agreement with previous reports, we found that non-toxic doses of EGCG did not decrease cell survival of primary cultures of normal keratinocytes. However, identical concentrations of EGCG depressed both proliferation and survival of primary cultures of keratinocytes with elevated levels of polyamines. This decreased survival was due to EGCG-induced apoptosis and not terminal differentiation in ODC over-expressing keratinocytes. Moreover, in skin from EGCG-treated K6/ODC transgenic mice, caspase 3 (active form) protein was detected only in the epithelial cells lining follicular cysts that develop following hair follicle degeneration and hair loss. Since the dermal follicular cysts in K6/ODC skin possess very high levels of ODC protein (25), elevated levels of polyamines appear to sensitize otherwise normal keratinocytes to EGCG-induced apoptosis. Although the percent caspase 3 (active form)-positive cells was low in the skin of EGCG-treated K6/ODC mice, a similarly low percent of caspase 3-stained cells has been reported in UVB-induced tumors in mice topically treated with EGCG (9).
Previously, we have shown that expression of p53 and its transcriptional target, Bax, are increased in ODC over-expressing K6/ODC transgenic skin compared with skin from normal littermates (25). Similarly, we detected higher levels of Bax protein in the skin of ODC/Ras transgenic mice (data not shown). It is possible that the polyamine-induction of p53 and pro-apoptotic proteins such as Bax contributes to the sensitization of keratinocytes to EGCG-induced apoptosis. Moreover, it has been reported that EGCG can antagonize the anti-apoptotic effects of Bcl-2 family proteins by binding and inhibiting Bcl-2 and Bcl-xL (24). Accordingly, the Bax/Bcl-2 ratio increases in ODC over-expressing keratinocytes that are induced to undergo apoptosis following EGCG treatment. Very few tumors developed in EGCG-treated ODC/Ras transgenic mice, and it is likely that the decreased survival and growth inhibition of ODC over-expressing tumor cells in EGCG-treated mice correlated with an increased Bax:Bcl-2 ratio.
Our data suggest that EGCG-induced apoptosis is selective for cells with elevated polyamines and is not due to transformation. Primary cultures of keratinocytes that over-express ODC are not transformed and are not tumorigenic in nude mice (16). Retroviral v-Ha-ras infection of ODC over-expressing keratinocytes did not further increase the number of caspase 3-positive cells or the expression of the pro-apoptotic gene Bax following EGCG treatment. As most transformed cells and tumor tissue possess higher levels of polyamines compared with normal cells or tissue, our data suggest that a possible mechanism for the EGCG inhibitory effect on tumorigenesis in ODC/Ras transgenic mice is that elevated levels of polyamines in tumor cells sensitize them to EGCG-induced apoptosis. These observations imply that EGCG may be an excellent chemopreventive agent in individuals at high risk for cancer and in early, pre-neoplastic stages of cancer, such as people diagnosed with actinic keratoses or intestinal adenomas that possess elevated levels of polyamines. Moreover, chemopreventive strategies that lower polyamine levels may diminish the selective apoptotic effect of EGCG. Future studies are needed to investigate this possibility, and whether other epithelial tissue types are sensitized by high levels of polyamines to undergo apoptosis following exposure to EGCG.
 |
Acknowledgments
|
---|
We thank Tom O'Brien, Cheryl Hobbs, Allan Conney and Chris Sell for helpful discussions. We thank Loretta Rossino for manuscript preparation. Supported by grants CA070739 and CA97249 from the National Cancer Institute.
 |
References
|
---|
- Wang,Z.Y., Huang,M.T., Ferraro,T., Wong,C.Q., Lou,Y.R., Reuhl,K., Iatropoulos,M., Yang,C.S. and Conney,A.H. (1992) Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice. Cancer Res., 52, 11621170.[Abstract]
- Lambert,J.D. and Yang,C.S. (2003) Mechanisms of cancer prevention by tea constituents. J. Nutr., 133, 3262S3267S.[Abstract/Free Full Text]
- Conney,A.H. (2003) Enzyme induction and dietary chemicals as approaches to cancer chemoprevention: the Seventh DeWitt S. Goodman Lecture. Cancer Res., 63, 70057031.[Abstract/Free Full Text]
- Bickers,D.R. and Athar,M. (2000) Novel approaches to chemoprevention of skin cancer. J. Dermatol., 27, 691695.[Medline]
- Bushman,J.L. (1998) Green tea and cancer in humans: a review of the literature. Nutr. Cancer, 31, 151159.[Medline]
- Gupta,S., Hastak,K., Ahmad,N., Lewin,J.S. and Mukhtar,H. (2001) Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc. Natl Acad. Sci. USA, 98, 1035010355.[Abstract/Free Full Text]
- Orner,G.A., Dashwood,W.M., Blum,C.A., Diaz,G.D., Li,Q. and Dashwood,R.H. (2003) Suppression of tumorigenesis in the Apc (min) mouse: down-regulation of beta-catenin signaling by a combination of tea plus sulindac. Carcinogenesis, 24, 263267.[Abstract/Free Full Text]
- Lu,Y.P., Lou,Y.R., Xie,J.G., Yen,P., Huang,M.T. and Conney,A.H. (1997) Inhibitory effect of black tea on the growth of established skin tumors in mice: effects on tumor size, apoptosis, mitosis and bromodeoxyuridine incorporation into DNA. Carcinogenesis, 18, 21632169.[Abstract]
- Lu,Y.P., Lou,Y.R., Xie,J.G., Peng,Q.Y., Liao,J., Yang,C.S., Huang,M.T. and Conney,A.H. (2002) Topical applications of caffeine or ()-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice. Proc. Natl Acad. Sci. USA, 99, 1245512460.[Abstract/Free Full Text]
- Ahmad,N., Gupta,S. and Mukhtar,H. (2000) Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells. Arch. Biochem. Biophys., 376, 338346.[CrossRef][ISI][Medline]
- Yang,G.Y., Liao,J., Li,C., Chung,J., Yurkow,E.J., Ho,C.T. and Yang,C.S. (2000) Effect of black and green tea polyphenols on c-jun phosphorylation and peroxide production in transformed and non-transformed human bronchial cell lines: possible mechanisms of cell growth inhibition and apoptosis induction. Carcinogenesis, 21, 20352039.[Abstract/Free Full Text]
- Smith,M.K., Trempus,C.S. and Gilmour,S.K. (1998) Cooperation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin. Carcinogenesis, 19, 14091415.[Abstract]
- Vojtek,A.B. and Der,C.J. (1998) Increasing complexity of the Ras signaling pathway. J. Biol. Chem., 273, 1992519928.[Free Full Text]
- Megosh,L., Gilmour,S.K., Rosson,D., Soler,A.P., Blessing,M., Sawicki,J.A. and O'Brien,T.G. (1995) Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase. Cancer Res., 55, 42054209.[Abstract]
- O'Brien,T.G., Simsiman,R.C. and Boutwell,R.K. (1975) Induction of the polyamine biosynthetic enzymes in mouse epidermis by tumor promoting agents. Cancer Res., 35, 16621670.[Abstract]
- Clifford,A., Morgan,D., Yuspa,S.H., Soler,A.P. and Gilmour,S. (1995) Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res., 55, 16801686.[Abstract]
- Yuspa,S.H. and Harris,C.C. (1974), Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp. Cell. Res., 86, 95105.[ISI][Medline]
- Roop,D.R., Lowy,D.R., Tambourin,P.E., Strickland,J., Harper,J.R., Balaschak,M., Spangler,E.F. and Yuspa,S.H. (1986) An activated Harvey ras oncogene produces benign tumours on mouse epidermal tissue. Nature, 323, 822824.[ISI][Medline]
- Agarwal,R., Katiyar,S.K., Zaidi,S.I. and Mukhtar,H. (1992) Inhibition of skin tumor promoter-caused induction of epidermal ornithine decarboxylase in SENCAR mice by polyphenolic fraction isolated from green tea and its individual epicatechin derivatives. Cancer Res., 52, 35823588.[Abstract]
- Steele,V.E., Kelloff,G.J., Balentine,D., Boone,C.W., Mehta,R., Bagheri,D., Sigman,C.C., Zhu,S. and Sharma,S. (2000) Comparative chemopreventive mechanisms of green tea, black tea and selected polyphenol extracts measured by in vitro bioassays. Carcinogenesis, 21, 6367.[Abstract/Free Full Text]
- Bachrach,U. and Wang,Y.C. (2002) Cancer therapy and prevention by green tea: role of ornithine decarboxylase. Amino Acids, 22, 113.[CrossRef][ISI][Medline]
- Shore,L.J., Peralta Soler,A. and Gilmour,S.K. (1997) Ornithine decarboxylase expression leads to translocation and activation of protein kinase CK2 in vivo. J. Biol. Chem., 272, 1253612543.[Abstract/Free Full Text]
- Chung,J.H., Han,J.H., Hwang,E.J., Seo,J.Y., Cho,K.H., Kim,K.H., Youn,J.I. and Eun,H.C. (2003) Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes. FASEB J., 17, 19131915.[Abstract/Free Full Text]
- Leone,M., Zhai,D., Sareth,S., Kitada,S., Reed,J.C. and Pellecchia,M. (2003) Cancer prevention by tea polyphenols is linked to their direct inhibition of antiapoptotic Bcl-2-family proteins. Cancer Res., 63, 81188121.[Abstract/Free Full Text]
- Gilmour,S.K., Birchler,M., Smith,M.K., Rayca,K. and Mostochuk,J. (1999) Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin. Cell Growth Differ., 10, 739748.[Abstract/Free Full Text]
Received June 3, 2004;
revised August 5, 2004;
accepted September 5, 2004.