Effect of genistein on the bioavailability and intestinal cancer chemopreventive activity of (-)-epigallocatechin-3-gallate

Carcinogenesis vol.29 no.10 pp.2019–2024, 2008 doi:10.1093/carcin/bgn182 Advance Access publication August 5, 2008 Effect of genistein on the bioavailability and intestinal cancer chemopreventive activity of (-)-epigallocatechin-3-gallate Joshua D.Lambert1,3,
, Seok-Joo Kwon1, Jihyeung Ju1, Mousumi Bose1, Mao-Jung Lee1, Jungil Hong2, Xingpei Hao1 and Chung S.Yang1 1 Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA and 2 Division of Food Science, College of Natural Science, Seoul Women’s University, Seoul 139-774, Korea 3 Present address: Department of Food Science, The Pennsylvania State University, 332 Food Science Building, University Park, PA 16802, USA
To whom correspondence should be addressed. Tel: þ1 814 865 5223; Fax: þ1 814 863 6132; Email: The green tea (Camellia sinensis) catechin, (-)-epigallocatechin3-gallate (EGCG), has shown cancer-preventive activity in animal models. Previously, we have reported the bioavailability of EGCG in rats and mice. Here, we report that cotreatment of HT-29 human colon cancer cells with genistein (from soy) increased cytosolic EGCG by 2- to 5-fold compared with treatment with EGCG only. Inclusion of genistein, at non-cytotoxic concentrations, increased the growth inhibitory effects of EGCG against HT-29 cells (up to 2-fold at 20 mM genistein). Intragastric coadministration of EGCG (75 mg/kg) and genistein (200 mg/kg) to CF-1 mice resulted in an increase in plasma half-life (t1/2 148.7 ± 16.4 versus 111.5 ± 23.4 min) and exposure (AUC0/N 183.9 ± 20.2 versus 125.8 ± 26.4 mg/ml 3 min) of EGCG. Cotreatment with genistein also increased the maximal concentration (Cmax), 6 h exposure (AUC0/360 min), and t1/2 of EGCG in the small intestine by 2.0-, 4.7- and 1.4-fold, respectively, compared with mice treated with EGCG only. Contrary to our expectations, the combination of 0.01% EGCG in the drinking fluid and 0.2% genistein in the diet enhanced intestinal tumorigenesis in male adenomatous polyposis coli (APC)min/1 mice. This combination increased the multiplicity of tumors in the medial and distal small intestine: the largest increase was in tumors >2 mm in diameter (4.3-fold compared with controls). This study demonstrates that although genistein can enhance EGCG bioavailability and in vitro growth inhibitory activity, this combination enhances tumorigenesis in the APCmin/1 mouse. These results further show the need for careful cancer prevention studies in animal models and for caution when interpreting data from in vitro studies. Introduction Previously, we have reported that the absolute bioavailability of EGCG in CF-1 mice and Sprague–Dawley rats is 26.5 and 1.6%, respectively (3,4). EGCG undergoes methylation, glucuronidation and sulfation in vivo and is largely present as the glucuronide in the plasma of treated mice (1,5,6). Studies in our laboratory and others have suggested that EGCG might also be a substrate for multidrug resistance-related protein (MRP) 1 and 2 (7–9). Cotreatment of Madin–Darby canine kidney cells overexpressing human MRP1 and MRP2 with EGCG and selective inhibitors of MRP1 and MRP2 resulted in a 10-fold increase in the cytosolic concentration of EGCG in both cases (8). Zhang et al. (9) have reported that cotreatment of Caco-2 cells with green tea catechins, including EGCG, and MK571, an inhibitor of MRP, resulted in 5.7-fold decrease in basolateral-to-apical flux of EGCG compared with cells treated only with EGCG. Modulation of the factors affecting EGCG bioavailability might increase plasma and tissue levels of this compound and increase its cancer-preventive activity. Genistein (Figure 1), an isoflavone from soybeans (Glycine max, Fabaceae), is common in the diet and has been studied for a number of potential health effects including cancer prevention and antiinflammatory activity (10–12). Genistein has also been shown to modulate MRP-mediated efflux (13,14). We have previously reported that cotreatment of MRP-overexpressing cells with [3H]-EGCG and genistein or related isoflavones results in increased cell-associated radioactivity compared with treatment with EGCG alone (8). This measurement includes both membrane-bound and intracellular EGCG and its metabolites: the effect of genistein on the intracellular levels of EGCG, strictly defined, has not been previously reported. The interaction between EGCG and genistein is also of interest due to the likelihood of co-occurrence in the diet. Whereas laboratory studies have shown that EGCG and green tea prevent cancer, epidemiological studies have been less conclusive (2,15–17). One possible confounding variable is differences in diet among individuals and between populations. Other dietary factors could influence the bioavailability and biological activity of EGCG. Careful studies are needed to understand these potential interactions. The effects of genistein on EGCG cancer chemopreventive effects and bioavailability have not been previously reported. In the present study, we hypothesized that genistein could increase the cytosolic concentration and growth inhibitory activity of EGCG in vitro and increase the plasma and tissue levels, as well as the cancer-preventive activity, of EGCG in vivo. Herein, we report the results of our studies. Materials and methods Tea (Camellia sinensis) is second only to water in terms of worldwide popularity as a beverage. (-)-Epigallocatechin-3-gallate (EGCG, Figure 1) is the major catechin component of green tea and is believed to be a major active constituent. Studies with animal models have shown that green tea and EGCG have preventive activity against cancer of the oral cavity, esophagus, stomach, intestine, colon, liver, lung, prostate, skin and other sites (1). Studies with human cancer cell lines have shown that EGCG possesses a number of activities related to cancer prevention such as inhibition of mitogen-activated protein kinase cascades, DNA methyltransferase, epidermal growth factor receptor signaling and others. It is not known, however, whether these potential mechanisms of action occur in animals or in humans because of the limited oral bioavailability of EGCG (1,2). Chemicals EGCG (100% pure) was provided by Mitsui Norin Co., Ltd (Fujieda City, Japan). b-D-glucuronidase (G-7896, EC 3.2.1.31, from Escherichia coli with 9  106 U/g solid) and sulfatase (S-9754, EC 3.1.6.1, from Abalone entrails with 2.3  10 5 U/g solid) purchased from Sigma Chemical Co. (St Louis, MO). Genistein (.95% pure) was purchased from AB Chem Technologies (Franklin Park, NJ). All other reagents were of the highest grade commercially available. Dosing solutions of EGCG and genistein were prepared in 0.9% NaCl. For analytical purposes, a standard stock solution of EGCG, epigallocatechin, epicatechin and epicatechin-3-gallate (10 lg/ml each) was prepared in 11.4 mM ascorbic acid-0.13 mM ethylenediaminetetraacetic acid (pH 3.8) and stored at 80°C (...truncated)