1076
Notes
Biol. Pharm. Bull. 24(9) 1076—1078 (2001)
Vol. 24, No. 9
Epigallocatechin Gallate (EGCG) Inhibits the Sulfation of 1-Naphthol in a Human Colon Carcinoma Cell Line, Caco-2 Takayo ISOZAKI and Hiro-omi TAMURA* Kyoritsu College of Pharmacy, Minato-ku, Tokyo 105–8512, Japan. Received April 9, 2001; accepted June 5, 2001 We have previously reported that epigallocatechin gallate (EGCG) strongly inhibits the in vitro phenol sulfotransferase (P-ST) activity of a human colon carcinoma cell line, Caco-2. In the present study, we examined the ability of EGCG to inhibit the sulfation of 1-naphthol in intact Caco-2 cells. Sulfation of 1-naphthol was detected in Caco-2 cells after 2 h of incubation, and was observed to continue for 24 h, resulting in an accumulation of sulfated 1-naphthol. Sulfation was strongly inhibited by the addition of EGCG to the culture medium. The IC50 of EGCG was calculated to be 20 m M; this value is similar to that obtained from in vitro assays (14 m M) [Ref. Tamura et al., Biol. Pharm. Bull., 23, 695, (2000)]. These results indicate that catechins are capable of inhibiting P-ST activity in intact cells as well as in vitro. We believe that the inhibitory activity of catechins might be the mechanism by which catechins (and green tea) exert anti-carcinogenic activity against procarcinogenic compounds that require P-ST activation in vivo. Key words sulfotransferase; inhibition; EGCG; Caco-2; green tea
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.2–5) 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.
in vitro. Following this, cells were incubated at 37 °C. An aliquot (100 m l) was removed at various intervals and mixed with 5 m l of 200 m M p-nitrophenyl sulfate to provide an internal standard, then 10 m l of the mixture was injected onto the HPLC. The analysis was performed on an ODS column (CAPCELL PAK C18UG80, 25034.5 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 1naphthyl sulfate were 13.5 and 11.8 min, respectively. Linearity of the standard curve for 1-naphthyl sulfate was observed up to 100 m M. The effect of catechins on the sulfation of 1naphthol was measured by the addition of EGCG, EGC, or EC at various concentrations into the culture medium. The IC50 value for the concentration–activity curve was calculated using a curve-fit program for Macintosh. RESULTS AND DISCUSSION
MATERIALS AND METHODS Materials 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% CO2. Cells were seeded in 6-well plates at a concentration of 53105 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 m M. This value was chosen in light of the transport efficiency of 1-naphthol across the cell membrane and the Km value of P-ST activity (50 m M) ∗ To whom correspondence should be addressed.
Sulfation of 1-Naphthol in Caco-2 Cells Previously, we reported a high level of P-ST activity in Caco-2 cells assayed in vitro.6) In this report, we measured the P-ST activity of intact cells. As is shown in Fig. 1A, 1-naphthyl sulfate was detected 2 h after 200 m M of 1-naphthol was added to the media of cells cultured for 3 weeks. The level of 1-naphthyl sulfate increased linearly over the first 24 h. Fig. 1B illustrates that cells cultured for longer periods produced more 1-naphthyl sulfate, even though the number of cells within each culture remained constant throughout each culture period (data not shown). One study reports that the in vitro P-ST activity of Caco-2 cells increases with cell age.7) From these results, it is assumed that the amount of accumulated 1-naphthyl sulfate within the media of Caco-2 cells reflects the cells’ P-ST activity. Effect of Catechins on the Sulfation of 1-Naphthol in Caco-2 Cells Previously, we demonstrated that catechins and green tea strongly inhibit P-ST activity within the mouse intestine and Caco-2 cells in vitro. We attributed the anti-cancer activity of green tea to the inhibitory action of catechins. It is possible that such catechin-induced inhibition of P-ST
e-mail:
[email protected]
© 2001 Pharmaceutical Society of Japan
September 2001
Fig. 1.
1077
Sulfation of 1-Naphthol in Intact Caco-2 Cells
(A) HPLC analysis. Samples were mixed with an internal standard (200 m M 2-nitrophenyl sulfate), and then subjected to reverse phase HPLC analysis. The retention times for 1naphthol and 1-naphthyl sulfate were 13.5 (peak 3) and 11.8 (peak 2) min, respectively. Peak 1 represents the internal standard, 2-nitrophenyl sulfate. (B) Accumulation of 1-naphthyl sulfate in culture medium. At various intervals after the addition of 1-naphthol, the concentration of 1-naphthyl sulfate was determined. Cells were cultured for various periods (j, 1 week; m, 2 weeks; d, 3 weeks). All points represent the average of results from three culture wells. Values obtained at each point fell within 5% of each other.
Fig. 2.
Inhibition of 1-Naphthol Sulfation in Caco-2 Cells by EGCG
Effect of EGCG on sulfation was determined by adding EGCG to the culture medium with 200 m M 1-naphthol in 3 week cultured cells. (A) Accumulation of the conjugate within the culture medium with (s), or without (d), 500 m M EGCG. Each point represents the average of results from 3 culture wells. Values fell within 5% of each other at each point. (B) Dose-dependence of EGCG inhibition. An IC50 of 20 m M was calculated.
Fig. 3.
Effects of EGC and EC on Sulfation in Caco-2 Cells
The concentration of 1-naphthyl sulfate was measured after the addition of either EGC or EC (500 m M) to the culture medium with 200 m M 1-naphthol of 3-week cultured cells. Values represent the average of 3 samples and all values fell within 5% of each other. p,0.05 (∗) represents a significant difference from the EGCG-treated sample at 24 h, based on Student’s t-test.
activity might reduce the bioactivation of carcinogenic arylamine and heterocyclic amine metabolites, thereby reducing the rate of aromatic carcinogenesis in vivo. To evaluate this hypothesis, the effect of catechins on 1-naphthol sulfation in Caco-2 cells was investigated. Since the concentration of EGCG in a standard green tea preparation is over 1 mM EGCG,6) we chose 500 m M EGCG as the maximal dose for this study. As shown in Fig. 2, the sulfation of 1-naphthol was inhibited by EGCG in a dose-dependent manner. The IC50 of EGCG was calculated to be 20 m M, which is similar to the value calculated from in vitro assays (14 m M) in a previous study.6) This suggests that the inhibition of P-ST activity by EGCG occurs in a similar manner in both intact cells and in vitro. Figure 3 shows that EGC and EC were poorer inhibitors than EGCG. Previously, we noted a similar trend in vitro.6) This further suggests that inhibition of P-ST activity by catechins in intact Caco-2 cells occurs through the same mechanism as it does in vitro (via competitive inhibition). Recent studies indicate that a large array of benzyl and allylic alcohols are activated to mutagenic metabolites by
1078
STs.3) Considering this, along with our own data, we propose that the inhibition of P-ST activity by catechins (and green tea) can reduce the bioactivation of procarcinogenic phenolic compounds within cells, thereby reducing the rate of carcinogenesis. Several reports have shown that the concentration of EGCG in the blood after two or three cups of green tea can reach about 1 m M.8,9) Furthermore, a high concentration of EGCG has been detected in rat small intestine and colon mucosa following oral administration.10) These findings support our hypothesis; however, further investigation must be carried out to confirm it. Recently, Otake et al. reported that the dietary polyphenols, quercetin and resveratrol, caused a potent reduction in the estrogenic ST activity of intact normal human mammary epithelial cells.11) The physiological importance of interactions between STs and dietary compounds in vivo needs to be clarified in the future. Acknowledgements This work was supported, in part, by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a
Vol. 24, No. 9
grant from the Tokyo Biochemical Research Foundation. REFERENCES 1) Kuroda Y., Hara Y., Mutation Res., 436, 69—97 (1999). 2) Yamazoe Y., Abu-Zeid M., Gong D., Ataiano N., Kato R., Carcinogenesis, 10, 1675—1679 (1989). 3) Glatt H., FASEB J., 11, 314—321 (1997). 4) Surh Y.-J., Chem.-Biol. Interact., 109, 221—235 (1998). 5) Glatt H., Engelke C. E. H., Pabel U., Teuber W., Jones A. L., Coughtrie M. W. H., Andrae U., Falany C., Meinl W., Toxicol. Lett., 112, 113, 341—348 (2000). 6) Tamura H., Matsui M., Biol. Pharm. Bull., 23, 695—699 (2000). 7) Baranczyk-Kuzma A., Garren J. A., Hidalgo I. J., Borchart R. T., Life Sci., 49, 1197—1206 (1991). 8) Yang C. S., Nature (London), 389, 134—135 (1997). 9) Nakagawa K., Okuda S., Miyazawa T., Biosci. Biotech. Biochem., 61, 1981—1985 (1997). 10) Nakagawa K., Miyazawa T., J. Nutr. Sci. Vitaminol., 43, 679—684 (1997). 11) Otake Y., Nolan A. L., Walle U. K., Walle T., J. Steroid Biochem. Mol. Biol., 73, 256—270 (2000).
