Epigallocatechin Gallate (EGCG) Inhibits the Sulfation of 1-Naphthol in a Human Colon Carcinoma Cell Line, Caco-2.

Abstract

Tea is a universally popular beverage, consumed by over two thirds of the world's population. Green tea is consumed mostly in Japan and China. The anti-mutagenic and anti-carcinogenic activity of green tea has been extensively examined. 1) Bioactivation of carcinogenic arylamine and heterocyclic amine metabolites by mammalian liver phenol sulfotransferases (P-STs) has been postulated as a key mechanism leading to aromatic carcinogenesis in humans. [2][3][4][5] To elucidate whether the anti-carcinogenic activity of green tea is related to ST activity, we previously investigated the effect of green tea on P-ST activity in the mouse intestine and in a human colon carcinoma cell line, Caco-2. We found that catechins, especially epigallocatechin gallate (EGCG), strongly inhibit in vitro P-ST activity in both types of cells. 6) In the present study, we examined the ability of catechins to inhibit P-ST activity in intact cells. We found that catechins, especially EGCG, inhibit the sulfation of 1-naphthol within intact cells as strongly as they do in vitro. MATERIALS AND METHODSMaterials 1-Naphthol and sulfate were obtained from Sigma (St. Louis, MO, U.S.A.). The catechins and reagents for HPLC were purchased from Wako Chemicals (Tokyo, Japan). Caco-2 cells were obtained at passage 40 from RIKEN Cell Bank, Japan.Cell Culture Caco-2 cells were grown in a 6-well plate (Iwaki, Japan) in 3 ml MEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin, 10 U/ml streptomycin and additional non-essential amino acids. The cells were kept at 37°C in a humidified atmosphere, containing 5% CO 2 . Cells were seeded in 6-well plates at a concentration of 5ϫ10 5 cells/ml and grown until confluence (5-6 d). Cells were cultivated for up to 3 weeks and their media was changed every 4-5 d.Analysis of Sulfation of 1-Naphthol in Intact Caco-2 Cells In order to examine the sulfation of 1-naphthol in intact Caco-2 cells, 1-naphthol was added to the medium of each well at a concentration of 200 mM. This value was chosen in light of the transport efficiency of 1-naphthol across the cell membrane and the K m value of P-ST activity (50 mM) in vitro. Following this, cells were incubated at 37°C. An aliquot (100 ml) was removed at various intervals and mixed with 5 ml of 200 mM p-nitrophenyl sulfate to provide an internal standard, then 10 ml of the mixture was injected onto the HPLC. The analysis was performed on an ODS column (CAPCELL PAK C 18 UG80, 250ϫ4.5 mm, Shiseido, Japan) at room temperature. The mobile phase was composed of 2 mM tetrabutylammonium hydrogen sulfate in water and acetonitrile (65 : 35). The flow rate was 1.3 ml/min with detection at 285 nm. The retention times of 1-naphthol and 1-naphthyl sulfate were 13.5 and 11.8 min, respectively. Linearity of the standard curve for 1-naphthyl sulfate was observed up to 100 mM. The effect of catechins on the sulfation of 1-naphthol was measured by the addition of EGCG, EGC, or EC at various concentrations into the culture medium. The IC 50 value for the concentration-...

Effect of Herbal Teas on Conjugation Reactions in a Human Colon Carcinoma Cell Line, Caco-2

“…This is supported by our finding in previous studies that epigallocatechin gallate (EGCG) is strong inhibitor of sulfoconjugation in Caco-2 cells. 4,5) Mild induction (20-30%) of sulfoconjugation was observed in Caco-2 cells following exposure to 5% hibiscus or lemongrass tea. Interestingly, low concentrations (0.1-1.0%) of hibiscus and lemongrass teas had the greatest inductive effect (Fig.…”

“…4) In this study, we extended our analysis to investigate glucuronidation of 1-naphthol in Caco-2 cells. Both conjugation reactions were detected in Caco-2 cells in a substrate dose-and culture time-dependent manner prior to 24 hr (data not shown).…”

“…Analyses of 1-Naphthyl Sulfate and Glucuronide ---Quantitation of 1-napthol conjugates was performed as described previously. 4) In brief, 1-Naphthol (200 µM) was added to the medium, after which the cells were incubated at 37°C. Aliquots (50 µl) were removed at various times, after which 30 µl of the mixture was filtered and injected into the HPLC apparatus, equipped with an ODS column (CAPCELL PAK C 18 UG80, 250 × 4.5 mm, Shiseido, Japan).…”

“…Previously, we have studied sulfoconjugation reactions in Caco-2 cells from a human colon carcinoma cell line and have observed inhibitory effects of catechins (green tea components) on sulfoconjugation. 4) To further identify potential herb-drug interactions, particularly those which might affect intestinal metabolism, we examined the effect of herbal teas on conjugation reactions (sulfoconjugation and glucuronidation) in Caco-2 cells.…”

Radical scavenging effects of 1-naphthol, 2-naphthol, and their sulfate-conjugates

“…1 for chemical structures). Notably, 1-Nap and 2-Nap may further be metabolized, particularly through sulfate conjugation in vitro and in vivo (Isozaki and Tamura, 2001;Ayala et al, 2015). Incidentally, in drug metabolism research, 1-Nap and 2-Nap are often used as prototype substrates for certain human SULTs, e.g., SUL-T1A1, SULT1A3, and SULT1E1 (Allali-Hassani et al, 2007).…”