Mao-Jung Lee

Mechanism of Action of (−)-Epigallocatechin-3-Gallate: Auto-oxidation–Dependent Inactivation of Epidermal Growth Factor Receptor and Direct Effects on Growth Inhibition in Human Esophageal Cancer KYSE 150 Cells

(-)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the phosphorylation of epidermal growth factor receptor (EGFR). We observed similar effects of EGCG in esophageal squamous cell carcinoma KYSE 150 cells and epidermoid squamous cell carcinoma A431 cells. Pretreatment of KYSE 150 cells with EGCG (20 micromol/L) for 0.5 to 24 hours in HAMs F12 and RPMI 1640 mixed medium at 37 degrees C, before the addition of EGF, resulted in a decreased level of phosphorylated EGFR (by 32-85%). Prolonged treatment with EGCG (8 or 24 hours) also decreased EGFR protein level (both by 80%). EGCG treatment for 24 hours also caused decreased signals of HER-2/neu in esophageal adenocarcinoma OE19 cells. These effects of EGCG were prevented or diminished by the addition of superoxide dismutase (SOD, 5 units/mL), or SOD plus catalase (30 units/mL), to the cell culture medium. A similar phenomenon on inactivation of EGFR was observed in A431 cells as well. Under culture conditions for KYSE 150 cells, EGCG was unstable, with a half-life of approximately 30 minutes; EGCG dimers and other oxidative products were formed. The presence of SOD in the culture medium stabilized EGCG and increased its half-life to longer than 24 hours and some EGCG epimerized to (+)-gallocatechin-3-gallate. A mechanism of superoxide radical-mediated dimerization of EGCG and H2O2 formation is proposed. The stabilization of EGCG by SOD in the culture medium potentiated the activity of EGCG in inhibiting KYSE 150 cell growth. The results suggest that in cell culture conditions, the auto-oxidation of EGCG leads to EGFR inactivation, but the inhibition of cell growth is due to other mechanisms. It remains to be determined whether the presently observed auto-oxidation of EGCG occurs in vivo. In future studies of EGCG and other polyphenolic compounds in cell culture, SOD may be added to stabilize EGCG and to avoid possible artifacts.

Bioavailability issues in studying the health effects of plant polyphenolic compounds

Polyphenolic compounds are common in the diet and have been suggested to have a number of beneficial health effects including prevention of cancer, cardiovascular disease, diabetes, and others. For some dietary polyphenols, certain benficial effects are suggested by epidemiological studies, some are supported by studies in animal models, and still others are extrapolated from studies in vitro. Because of the relatively poor bioavailability of many of these compounds, the molecular basis of these beneficial effects is not clear. In the present review, we discuss the potential health benefits of dietary polyphenols from the point of view of bioavailability. Tea catechins, curcumin, and proanthocyanidins are used as examples to illustrate some of the problems that need to be resolved. Further research on both the biological activity and bioavailability of dietary polyphenols is needed to properly assess their usefulness for the prevention and treatment of disease.

Delivery of Tea Polyphenols to the Oral Cavity by Green Tea Leaves and Black Tea Extract

Catechins and theaflavins, polyphenolic compounds derived from tea (Camellia sinensis, fam. Theaceae), have been reported to have a wide range of biological activities including prevention of tooth decay and oral cancer. The present study was undertaken to determine the usefulness of green tea leaves and black tea extract for the delivery of catechins and theaflavins to the oral cavity. After holding either green tea leaves (2 g) or brewed black tea (2 g of black tea leaves in 100 ml) in the mouth for 2–5 min and thoroughly rinsing the mouth, high concentrations of catechins (Cmax = 131.0–2.2 μm) and theaflavins (Cmax = 1.8–0.6 μm) were observed in saliva in the 1st hour. Whereas there was significant interindividual variation in the peak levels of catechins and theaflavins, the overall kinetic profile was similar, with t1/2 = 25–44 min and 49–76 min for catechins and theaflavins, respectively (average coefficient of variation in t1/2 was 23.4%). In addition to the parent catechin and theaflavin peaks, five unidentified peaks were also observed in saliva after black tea treatment. Hydrolysis of theaflavin gallates, apparently by salivary esterases, was observed in vitro and in vivo. These results indicate that tea leaves can be used as a convenient, slow-release source of catechins and theaflavins and provide information for the possible use of tea in the prevention of oral cancer and dental caries.

DOSE-DEPENDENT LEVELS OF EPIGALLOCATECHIN-3-GALLATE IN HUMAN COLON CANCER CELLS AND MOUSE PLASMA AND TISSUES

Epigallocatechin-3-gallate (EGCG; molecular formula: C22H18011)is the most abundant catechin in green tea (Camellia sinensis Theaceae). Both EGCG and green tea have been shown to have cancer-preventive activity in a number of animal models, and numerous mechanisms have been proposed based on studies with human cell lines. EGCG has been shown to undergo extensive biotransformation to yield methylated and glucuronidated metabolites in mice, rats, and humans. In the present study, we determined the concentration-dependent uptake of EGCG by HT-29 human colon cancer cells (20–600 μM) and the dose dependence of EGCG plasma and tissue levels after a single dose of EGCG (50–2000 mg/kg i.g.) to male CF-1 mice. The cytosolic levels of EGCG were linear with respect to extracellular concentration of EGCG after treatment of HT-29 cells for 2 h (915.3–6851.6 μg/g). In vivo, EGCG exhibited a linear dose relationship in the plasma (0.03–4.17 μg/ml), prostate (0.01–0.91 μg/g), and liver (0.09-18.3 μg/g). In the small intestine and colon, however, the levels of EGCG plateaued between 500 and 2000 mg/kg i.g. These results suggest that absorption of EGCG from the small intestine is largely via passive diffusion; however, at high concentrations, the small intestinal and colonic tissues become saturated. The levels of 4″-O-methyl-EGCG and 4′,4″-di-O-methyl-EGCG parallel those of EGCG with respect to dose. The present study provides information with respect to what concentrations of EGCG are achievable in mice and may guide dose selection for future cancer chemoprevention studies with EGCG.

Peracetylation as a Means of Enhancing in Vitro Bioactivity and Bioavailability of Epigallocatechin-3-Gallate

(–)-Epigallocatechin-3-gallate (EGCG) is the widely studied catechin in green tea (Camellia sinensis). Previously, we have reported the low bioavailability of EGCG in rats and mice. As a means of improving the bioavailability of EGCG, we have prepared a peracetylated EGCG derivative (AcEGCG) and herein report its growth inhibitory activity and cellular uptake in vitro, as well as bioavailability in mice. AcEGCG exhibited enhanced growth inhibitory activity relative to EGCG in both KYSE150 human esophageal (IC50 = 10 versus 20 μM) and HCT116 human colon cancer cells (IC50 = 32 versus 45 μM). AcEGCG was rapidly converted to EGCG by HCT116 cells, and treatment of cells with AcEGCG resulted in a 2.8- to 30-fold greater intracellular concentration of EGCG as compared with treatment with EGCG. AcEGCG was also more potent than EGCG at inhibiting nitric oxide production (4.4-fold) and arachidonic acid release (2.0-fold) from lipopolysaccharide-stimulated RAW264.7 murine macrophages. Intragastric administration of AcEGCG to CF-1 mice resulted in higher bioavailability compared with administration of equimolar doses of EGCG. The plasma area under the curve from 0 to infinity (AUC0→∞) of total EGCG was 465.0 and 194.6 [(μg/ml) · min] from the administration of AcEGCG and EGCG, respectively. The t1/2 of EGCG was also increased following administration of AcEGCG compared with EGCG (441.0 versus 200.3 min). The AUC0→∞ and t1/2 were also increased in small intestinal (2.8- and 4.3-fold, respectively) and colonic tissues (2.4- and 6.0-fold, respectively). These data suggest that acetylation represents a means of increasing the biological potency in vitro, increasing the bioavailability of EGCG in vivo, and may improve cancer-preventive activity.

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice.

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.

Mixed tocopherols prevent mammary tumorigenesis by inhibiting estrogen action and activating PPAR-γ

Purpose: Tocopherols are lipophilic antioxidants present in vegetable oils. Although the antioxidant and anticancer activities of α-tocopherol (vitamin E) have been studied for decades, recent intervention studies with α-tocopherol have been negative for protection from cancer in humans. The tocopherols consist of four isoforms, which are the α, β, γ, and δ variants, and recent attention is being given to other isoforms. In the present study, we investigated the inhibitory effect of a tocopherol mixture rich in γ- and δ-tocopherols against mammary tumorigenesis. Experimental Design: Female Sprague Dawley rats were treated with N-methyl-N-nitrosourea (NMU), and then fed diets containing 0.1%, 0.3%, or 0.5% mixed tocopherols rich in γ- and δ-tocopherols for 9 weeks. Tumor burden and multiplicity were determined, and the levels of markers of inflammation, proliferation, and apoptosis were evaluated in the serum and in mammary tumors. The regulation of nuclear receptor signaling by tocopherols was studied in mammary tumors and in breast cancer cells. Results: Dietary administration of 0.1%, 0.3%, or 0.5% mixed tocopherols suppressed mammary tumor growth by 38%, 50%, or 80%, respectively. Tumor multiplicity was also significantly reduced in all three mixed tocopherol groups. Mixed tocopherols increased the expression of p21, p27, caspase-3, and peroxisome proliferator activated receptor-γ, and inhibited AKT and estrogen signaling in mammary tumors. Our mechanistic study found that γ- and δ-tocopherols, but not α-tocopherol, activated peroxisome proliferator activated receptor-γ and antagonized estrogen action in breast cancer. Conclusion: The results suggest that γ- and δ-tocopherols may be effective agents for the prevention of breast cancer.

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.

A γ-Tocopherol–Rich Mixture of Tocopherols Inhibits Colon Inflammation and Carcinogenesis in Azoxymethane and Dextran Sulfate Sodium–Treated Mice

We investigated the effects of a γ-tocopherol–rich mixture of tocopherols (γ-TmT, containing 57% γ-T, 24% δ-T, and 13% α-T) on colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)–treated mice. In experiment 1, 6-week-old male CF-1 mice were given a dose of AOM (10 mg/kg body weight, i.p.), and 1 week later, 1.5% DSS in drinking water for 1 week. The mice were maintained on either a γ-TmT (0.3%)–enriched or a standard AIN93M diet, starting 1 week before the AOM injection, until the termination of experiment. In the AOM/DSS–treated mice, dietary γ-TmT treatment resulted in a significantly lower colon inflammation index (52% of the control) on day 7 and number of colon adenomas (9% of the control) on week 7. γ-TmT treatment also resulted in higher apoptotic index in adenomas, lower prostaglandin E2, leukotriene B4, and nitrotyrosine levels in the colon, and lower prostaglandin E2, leukotriene B4, and 8-isoprostane levels in the plasma on week 7. Some of the decreases were observed even on day 7. In experiment 2 with AOM/DSS– treated mice sacrificed on week 21, dietary 0.17% or 0.3% γ-TmT treatment, starting 1 week before the AOM injection, significantly inhibited adenocarcinoma and adenoma formation in the colon (to 17-33% of the control). Dietary 0.3% γ-TmT that was initiated after DSS treatment also exhibited a similar inhibitory activity. The present study showed that γ-TmT effectively inhibited colon carcinogenesis in AOM/DSS–treated mice, and the inhibition may be due to the apoptosis-inducing, anti-inflammatory, antioxidative, and reactive nitrogen species–trapping activities of tocopherols.

δ-Tocopherol Is More Active than α- or γ-Tocopherol in Inhibiting Lung Tumorigenesis In Vivo

In contrast to strong epidemiologic, preclinical, and secondary clinical evidence for vitamin E (tocopherols) in reducing cancer risk, large-scale clinical cancer-prevention trials of α-tocopherol have been negative. This vexing contrast helped spur substantial preclinical efforts to better understand and improve the antineoplastic activity of tocopherol through, for example, the study of different tocopherol forms. We previously showed that the γ-tocopherol–rich mixture (γ-TmT) effectively inhibited colon and lung carcinogenesis and the growth of transplanted lung-cancer cells in mice. We designed this study to determine the relative activities of different forms of tocopherol in a xenograft model, comparing the anticancer activities of δ-tocopherol with those of α- and γ-tocopherols. We subcutaneously injected human lung cancer H1299 cells into NCr nu/nu mice, which then received α-, γ-, or δ-tocopherol or γ-TmT in the diet (each at 0.17% and 0.3%) for 49 days. δ-Tocopherol inhibited tumor growth most strongly. γ-Tocopherol and γ-TmT (at 0.3%) also inhibited growth significantly, but α-tocopherol did not. δ-Tocopherol also effectively decreased oxidative DNA damage and nitrotyrosine formation and enhanced apoptosis in tumor cells; again, γ-tocopherol also was active in these regards but less so, and α-tocopherol was not. Each supplemented diet increased serum levels of its tocopherol – up to 45 μmol/L for α-tocopherol, 9.7 μmol/L for γ-tocopherol, and 1.2 μmol/L for δ-tocopherol; dietary γ- or δ-tocopherol, however, decreased serum α-tocopherol levels, and dietary α-tocopherol decreased serum levels of γ-tocopherol. Each dietary tocopherol also increased its corresponding side-chain–degradation metabolites, with concentrations of δ-tocopherol metabolites greater than γ-tocopherol and far greater than α-tocopherol metabolites in serum and tumors. This study is the first in vivo assessment of δ-tocopherol in tumorigenesis and shows that δ-tocopherol is more active than α- or γ-tocopherol in inhibiting tumor growth, possibly through trapping reactive oxygen and nitrogen species and inducing apoptosis; δ-tocopherol metabolites could contribute significantly to these results. Cancer Prev Res; 4(3); 404–13. ©2011 AACR.