Ester Bond-containing Tea Polyphenols Potently Inhibit Proteasome Activity in Vitro and in Vivo

It has been discovered that proteasome inhibitors are able to induce tumor growth arrest or cell death and that tea consumption is correlated with cancer prevention. Here, we show that ester bond-containing tea polyphenols, such as (−)−epigallocatechin-3-gallate (EGCG), potently and specifically inhibit the chymotrypsin-like activity of the proteasome in vitro (IC50 = 86–194 nm) and in vivo (1–10 μm) at the concentrations found in the serum of green tea drinkers. Atomic orbital energy analyses and high performance liquid chromatography suggest that the carbon of the polyphenol ester bond is essential for targeting, thereby inhibiting the proteasome in cancer cells. This inhibition of the proteasome by EGCG in several tumor and transformed cell lines results in the accumulation of two natural proteasome substrates, p27Kip1and IκB-α, an inhibitor of transcription factor NF-κB, followed by growth arrest in the G1 phase of the cell cycle. Furthermore, compared with their simian virus-transformed counterpart, the parental normal human fibroblasts were much more resistant to EGCG-induced p27Kip1 protein accumulation and G1 arrest. Our study suggests that the proteasome is a cancer-related molecular target of tea polyphenols and that inhibition of the proteasome activity by ester bond-containing polyphenols may contribute to the cancer-preventative effect of tea. It has been discovered that proteasome inhibitors are able to induce tumor growth arrest or cell death and that tea consumption is correlated with cancer prevention. Here, we show that ester bond-containing tea polyphenols, such as (−)−epigallocatechin-3-gallate (EGCG), potently and specifically inhibit the chymotrypsin-like activity of the proteasome in vitro (IC50 = 86–194 nm) and in vivo (1–10 μm) at the concentrations found in the serum of green tea drinkers. Atomic orbital energy analyses and high performance liquid chromatography suggest that the carbon of the polyphenol ester bond is essential for targeting, thereby inhibiting the proteasome in cancer cells. This inhibition of the proteasome by EGCG in several tumor and transformed cell lines results in the accumulation of two natural proteasome substrates, p27Kip1and IκB-α, an inhibitor of transcription factor NF-κB, followed by growth arrest in the G1 phase of the cell cycle. Furthermore, compared with their simian virus-transformed counterpart, the parental normal human fibroblasts were much more resistant to EGCG-induced p27Kip1 protein accumulation and G1 arrest. Our study suggests that the proteasome is a cancer-related molecular target of tea polyphenols and that inhibition of the proteasome activity by ester bond-containing polyphenols may contribute to the cancer-preventative effect of tea. Previous epidemiological studies have suggested that tea consumption may have a protective effect against human cancer (1Fujiki H. J. Cancer Res. Clin. Oncol... 1999; 125: 589-597Google Scholar, 2Kuroda Y. Hara Y. Mutat. Res... 1999; 436: 69-97Google Scholar, 3Yang C.S. Nutrition.. 1999; 15: 946-949Google Scholar, 4Ahmad N. Mukhtar H. Nutr. Rev... 1999; 57: 78-83Google Scholar). Recent animal studies have also demonstrated that green tea polyphenols could suppress the formation and growth of human cancers, including skin (5Katiyar S.K. Challa A. McCormick T.S. Cooper K.D. Mukhtar H. Carcinogenesis.. 1999; 20: 2117-2124Google Scholar, 6Wang Z.Y. Huang M.T. Ferraro T. Wong C.Q. Lou Y.R. Reuhl K. Iatropoulos M. Yang C.S. Conney A.H. Cancer Res... 1992; 52: 1162-1170Google Scholar), lung (7Xu Y. Ho C.T. Amin S.G. Han C. Chung F.L. Cancer Res... 1992; 52: 3875-3879Google Scholar), liver (8Nishida H. Omori M. Fukutomi Y. Ninomiya M. Nishiwaki S. Suganuma M. Moriwaki H. Muto Y. Jpn. J. Cancer Res... 1994; 85: 221-225Google Scholar), esophagus (9Wang Z.Y. Wang L.D. Lee M.J. Ho C.T. Huang M.T. Conney A.H. Yang C.S. Carcinogenesis.. 1995; 16: 2143-2148Google Scholar), and stomach (10Yamane T. Takahashi T. Kuwata K. Oya K. Inagake M. Kitao Y. Suganuma M. Fujiki H. Cancer Res... 1995; 55: 2081-2084Google Scholar). The major components of green and black tea include epigallocatechin-3-gallate (EGCG)1, epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EC), and their epimers (see Fig. 1 A). EGCG among those polyphenols has been most extensively examined because of its relative abundance and strong cancer-preventive properties (1Fujiki H. J. Cancer Res. Clin. Oncol... 1999; 125: 589-597Google Scholar, 11Balentine D.A. Wiseman S.A. Bouwens L.C. Crit. Rev. Food Sci. Nutr... 1997; 37: 693-704Google Scholar). EGCG has been shown to inhibit several cancer-related proteins, including urokinase (12Jankun J. Selman S.H. Swiercz R. Skrzypczak-Jankun E. Nature.. 1997; 387: 561Google Scholar), nitric-oxide synthase (13Lin Y.L. Lin J.K. Mol. Pharmacol... 1997; 52: 465-472Google Scholar), teromerase (14Naasani I. Seimiya H. Tsuruo T. Biochem. Biophys. Res. Commun... 1998; 249: 391-396Google Scholar), and tumor necrosis factor-α (15Okabe S. Ochiai Y. Aida M. Park K. Kim S.J. Nomura T. Suganuma M. Fujiki H. Jpn J. Cancer Res... 1999; 90: 733-739Google Scholar). However, nonphysiological concentrations of EGCG (i.e., concentrations higher than those found in human serum after tea consumption) were used in some earlier studies. Whether one or more of these proteins are the real molecular targets of EGCG and other tea polyphenols under physiological conditions needs further investigations.The 20S proteasome, a multicatalytic complex (700 kDa), constitutes the catalytic key component of the ubiquitous proteolytic machinery 26S proteasome (16Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature.. 1997; 386: 463-471Google Scholar, 17Maupin-Furlow J.A. Ferry J.G. J. Biol. Chem... 1995; 270: 28617-28622Google Scholar, 18Goldberg A.L. Science.. 1995; 268: 522-523Google Scholar, 19Baumeister W. Walz J. Zuhl F. Seemuller E. Cell.. 1998; 92: 367-380Google Scholar, 20Heinemeyer W. Fischer M. Krimmer T. Stachon U. Wolf D.H. J. Biol. Chem... 1997; 272: 25200-25209Google Scholar). There are three major proteasomal activities: chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities (16Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature.. 1997; 386: 463-471Google Scholar, 21Loidl G. Groll M. Musiol H.J. Huber R. Moroder L. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 5418-5422Google Scholar). The ubiquitin-proteasome system plays a critical role in the specific degradation of cellular proteins (22Hochstrasser M. Curr. Opin. Cell Biol... 1995; 7: 215-223Google Scholar), and two of the proteasome functions are to allow tumor cell cycle progression and to protect tumor cells against apoptosis (23Dou Q.P. Li B. Drug Resistance Updates.. 1999; 2: 215-223Google Scholar). The chymotrypsin-like but not trypsin-like activity of the proteasome is associated with tumor cell survival (24An B. Goldfarb R.H. Siman R. Dou Q.P. Cell Death Differ... 1998; 5: 1062-1075Google Scholar, 25Lopes U.G. Erhardt P. Yao R. Cooper G.M. J. Biol. Chem... 1997; 272: 12893-12896Google Scholar). Many cell cycle and cell death regulators have been identified as targets of the ubiquitin-proteasome-mediated degradation pathway. These proteins include p53 (26Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res... 1996; 56: 2649-2654Google Scholar), pRB (27Boyer S.N. Wazer D.E. Band V. Cancer Res... 1996; 56: 4620-4624Google Scholar), p21 (28Blagosklonny M.V. Wu G.S. Omura S. el-Deiry W.S. Biochem. Biophys. Res. Commun... 1996; 227: 564-569Google Scholar), p27Kip1 (29Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science.. 1995; 269: 682-685Google Scholar), IκB-α (30Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev... 1995; 9: 2723-2735Google Scholar), and Bax (31Li B. Dou Q.P. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 3850-3855Google Scholar).Here, we report for the first time that ester bond-containing tea polyphenols potently and selectively inhibit the proteasomal chymotrypsin-like but not trypsin-like activity in vitro andin vivo. Among the tea polyphenols examined, EGCG showed the strongest inhibitory activity against purified 20S proteasome, 26S proteasome of tumor cell extracts, and 26S proteasome in intact tumor cells. Furthermore, the inhibition of the proteasome in vivowas able to accumulate the natural proteasome substrates p27Kip1 and IκB-α as well as induce the arrest of tumor cells in the G1 phase. Finally, normal human WI-38 fibroblasts were more resistant to the EGCG treatment than their SV40-transformed counterpart. Previous epidemiological studies have suggested that tea consumption may have a protective effect against human cancer (1Fujiki H. J. Cancer Res. Clin. Oncol... 1999; 125: 589-597Google Scholar, 2Kuroda Y. Hara Y. Mutat. Res... 1999; 436: 69-97Google Scholar, 3Yang C.S. Nutrition.. 1999; 15: 946-949Google Scholar, 4Ahmad N. Mukhtar H. Nutr. Rev... 1999; 57: 78-83Google Scholar). Recent animal studies have also demonstrated that green tea polyphenols could suppress the formation and growth of human cancers, including skin (5Katiyar S.K. Challa A. McCormick T.S. Cooper K.D. Mukhtar H. Carcinogenesis.. 1999; 20: 2117-2124Google Scholar, 6Wang Z.Y. Huang M.T. Ferraro T. Wong C.Q. Lou Y.R. Reuhl K. Iatropoulos M. Yang C.S. Conney A.H. Cancer Res... 1992; 52: 1162-1170Google Scholar), lung (7Xu Y. Ho C.T. Amin S.G. Han C. Chung F.L. Cancer Res... 1992; 52: 3875-3879Google Scholar), liver (8Nishida H. Omori M. Fukutomi Y. Ninomiya M. Nishiwaki S. Suganuma M. Moriwaki H. Muto Y. Jpn. J. Cancer Res... 1994; 85: 221-225Google Scholar), esophagus (9Wang Z.Y. Wang L.D. Lee M.J. Ho C.T. Huang M.T. Conney A.H. Yang C.S. Carcinogenesis.. 1995; 16: 2143-2148Google Scholar), and stomach (10Yamane T. Takahashi T. Kuwata K. Oya K. Inagake M. Kitao Y. Suganuma M. Fujiki H. Cancer Res... 1995; 55: 2081-2084Google Scholar). The major components of green and black tea include epigallocatechin-3-gallate (EGCG)1, epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EC), and their epimers (see Fig. 1 A). EGCG among those polyphenols has been most extensively examined because of its relative abundance and strong cancer-preventive properties (1Fujiki H. J. Cancer Res. Clin. Oncol... 1999; 125: 589-597Google Scholar, 11Balentine D.A. Wiseman S.A. Bouwens L.C. Crit. Rev. Food Sci. Nutr... 1997; 37: 693-704Google Scholar). EGCG has been shown to inhibit several cancer-related proteins, including urokinase (12Jankun J. Selman S.H. Swiercz R. Skrzypczak-Jankun E. Nature.. 1997; 387: 561Google Scholar), nitric-oxide synthase (13Lin Y.L. Lin J.K. Mol. Pharmacol... 1997; 52: 465-472Google Scholar), teromerase (14Naasani I. Seimiya H. Tsuruo T. Biochem. Biophys. Res. Commun... 1998; 249: 391-396Google Scholar), and tumor necrosis factor-α (15Okabe S. Ochiai Y. Aida M. Park K. Kim S.J. Nomura T. Suganuma M. Fujiki H. Jpn J. Cancer Res... 1999; 90: 733-739Google Scholar). However, nonphysiological concentrations of EGCG (i.e., concentrations higher than those found in human serum after tea consumption) were used in some earlier studies. Whether one or more of these proteins are the real molecular targets of EGCG and other tea polyphenols under physiological conditions needs further investigations. The 20S proteasome, a multicatalytic complex (700 kDa), constitutes the catalytic key component of the ubiquitous proteolytic machinery 26S proteasome (16Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature.. 1997; 386: 463-471Google Scholar, 17Maupin-Furlow J.A. Ferry J.G. J. Biol. Chem... 1995; 270: 28617-28622Google Scholar, 18Goldberg A.L. Science.. 1995; 268: 522-523Google Scholar, 19Baumeister W. Walz J. Zuhl F. Seemuller E. Cell.. 1998; 92: 367-380Google Scholar, 20Heinemeyer W. Fischer M. Krimmer T. Stachon U. Wolf D.H. J. Biol. Chem... 1997; 272: 25200-25209Google Scholar). There are three major proteasomal activities: chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities (16Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature.. 1997; 386: 463-471Google Scholar, 21Loidl G. Groll M. Musiol H.J. Huber R. Moroder L. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 5418-5422Google Scholar). The ubiquitin-proteasome system plays a critical role in the specific degradation of cellular proteins (22Hochstrasser M. Curr. Opin. Cell Biol... 1995; 7: 215-223Google Scholar), and two of the proteasome functions are to allow tumor cell cycle progression and to protect tumor cells against apoptosis (23Dou Q.P. Li B. Drug Resistance Updates.. 1999; 2: 215-223Google Scholar). The chymotrypsin-like but not trypsin-like activity of the proteasome is associated with tumor cell survival (24An B. Goldfarb R.H. Siman R. Dou Q.P. Cell Death Differ... 1998; 5: 1062-1075Google Scholar, 25Lopes U.G. Erhardt P. Yao R. Cooper G.M. J. Biol. Chem... 1997; 272: 12893-12896Google Scholar). Many cell cycle and cell death regulators have been identified as targets of the ubiquitin-proteasome-mediated degradation pathway. These proteins include p53 (26Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res... 1996; 56: 2649-2654Google Scholar), pRB (27Boyer S.N. Wazer D.E. Band V. Cancer Res... 1996; 56: 4620-4624Google Scholar), p21 (28Blagosklonny M.V. Wu G.S. Omura S. el-Deiry W.S. Biochem. Biophys. Res. Commun... 1996; 227: 564-569Google Scholar), p27Kip1 (29Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science.. 1995; 269: 682-685Google Scholar), IκB-α (30Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev... 1995; 9: 2723-2735Google Scholar), and Bax (31Li B. Dou Q.P. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 3850-3855Google Scholar). Here, we report for the first time that ester bond-containing tea polyphenols potently and selectively inhibit the proteasomal chymotrypsin-like but not trypsin-like activity in vitro andin vivo. Among the tea polyphenols examined, EGCG showed the strongest inhibitory activity against purified 20S proteasome, 26S proteasome of tumor cell extracts, and 26S proteasome in intact tumor cells. Furthermore, the inhibition of the proteasome in vivowas able to accumulate the natural proteasome substrates p27Kip1 and IκB-α as well as induce the arrest of tumor cells in the G1 phase. Finally, normal human WI-38 fibroblasts were more resistant to the EGCG treatment than their SV40-transformed counterpart. We thank Drs. A. B. Pardee and R. H. Goldfarb for critical reading of this manuscript, Drs. D. C. Eichler and L. P. Solomonson for permission to use the HPLC and for valuable discussion about HPLC data, Dr. R. Lush III for initial HPLC analysis, and the Lipton Co. for providing the tea extracts. (−)−epigallocatechin-3-gallate (−)−epigallocatechin (−)−epicatechin-3-gallate (−)−epicatechin (−)−gallocatechin-3-gallate (−)−gallocatechin (−)−catechin-3-gallate (−)−catechin 7-amido-4-methyl-coumarin peptidyl-glutamyl peptide-hydrolyzing high performance liquid chromatography inhibitor of transciption factor NF-κB 70 kDa 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid benzyloxycarbonyl