Green tea acts as an anticancer agent, especially in colon cancer. Although the molecular mechanism of this anticancer effect is still not entirely understood, most green tea effects are believed to result from EGCG.23
In addition to its chemopreventive activity, EGCG is known to possess antiangiogenic properties through inhibition of proangiogenic factors including VEGF and bFGF.24,25
Our results show that EGCG suppresses the protein levels of bFGF and VEGF in colorectal cancer cells, which could account for reduced angiogenesis and hence hamper tumor growth and metastasis. It has been previously reported that EGCG down-regulates bFGF expression,25
but the exact mechanism had not been determined. In this study, we investigated the molecular mechanism involved in the suppression of bFGF by EGCG and found that this occurred specifically through posttranslational modification. We also examined the transcriptional regulation of bFGF by EGCG, but we could not identify any changes in mRNA level () or mRNA stability in the presence of EGCG (data not shown) in LoVo cells. Thus, posttranslational regulation of bFGF by EGCG may fully account for the EGCG-induced bFGF suppression.
EGCG possesses a strong antioxidant activity26
as well as prooxidant activity in cell culture systems, producing H2
in the media.27
Therefore, we investigated whether the prooxidative activity may cause bFGF suppression in our cell culture. GSH is one of the major intracellular antioxidants. When GSH and EGCG are added to LoVo cells, bFGF protein is only partially recovered, which indicates that oxidation, presumably H2
generation by EGCG, contributes to EGCG-induced bFGF suppression but not to a major extent (). To elucidate further the molecular mechanism of EGCG in signaling pathways, we tested for involvement of different pathways using kinase inhibitors. We found that lactacystin, an inhibitor of proteasomal degradation, inhibited EGCG-induced bFGF suppression (). These results were also confirmed using the recombinant bFGF proteins (). LoVo cells were transfected with the expression vectors containing either bFGF
and then treated with EGCG for 1 hour, followed by cycloheximide treatment for 0, 1, 6, and 24 hours. There was no significant change in vehicle-treated LacZ transfected samples, whereas the recombinant bFGF proteins were rapidly degraded in the presence of vehicle and EGCG. These results highlight the involvement of the ubiquitin-proteasome pathway as a novel mechanism facilitated by EGCG. There was, however, no change in β
-catenin levels, even though it is known to be degraded by the ubiquitin-proteasome pathway (, left panel
). One consideration is that LoVo cells contain a mutant adenomatous polyposis coli tumor suppressor protein that would not bind to β
-catenin in mediating ubiquitination. However, after 24 hours, β
-catenin seems to be degraded, indicating that other pathways may be involved in β
-catenin degradation in the presence of EGCG.
The ubiquitin-enriched fractions were also obtained from EGCG-treated samples, and we found that EGCG increased the ubiquitin activity in a dose-dependent manner (). This result was confirmed by immunoprecipitation of bFGF and LacZ with ubiquitin and V5 antibodies (). Ubiquitin is one of the most conserved eukaryotic proteins, and it conjugates other proteins through a well-defined enzymatic pathway.28
The ubiquitin-proteasome pathway is now being recognized as an important regulatory system in cancer pathways and, in fact, in many cellular processes.29
Proteins are conjugated with ubiquitin and subsequently transferred to the 26S proteasome, which is a multicatalyticsubunit complex consisting of a 20S barrel-shaped proteolytic core and a 19S cap-like regulatory complex.30
Our data support this mechanism of degradation, showing that EGCG facilitates ubiquitination of bFGF and specifically triggers trypsin-like activity of the 20S proteasome, leading to degradation of bFGF. In contrast, chymotrypsin-like activity of the proteasome is associated with tumor cell survival, and EGCG has been known to decrease proteasome activity via chymotrypsin-like activity of the 20S proteasome.31
It is therefore very surprising that EGCG increases only trypsin-like activity of the 20S proteasome. However, our data strongly support the hypothesis that EGCG increases trypsin-like activity of the 20S proteasome. First of all, proteasome inhibitors (Epoxomicin and AW9155) other than lactacystin did not show any effect on EGCG-induced bFGF suppression (). When these inhibitors were compared in their activity, only lactacystin possesses the inhibitory effect on trypsin-like activity. Another explanation of lactacystin specificity in EGCG-mediated bFGF degradation is that, unlike other proteasome inhibitors, lactacystin has reactive hydroxyl groups that might compete with EGCG for activation of 20S proteasomal activities. To our knowledge, this is the first report that EGCG specifically enhances trypsin-like activity of the 20S proteasome. In addition, our data provide a rationale to develop anticancer drugs that increase ubiquitination of angiogenic protein and also increase trypsin-like activity of the 20S proteasome.
An in vivo study using APCMin/+ mice treated with EGCG or ECG indicated that intestinal tumor load and polyp numbers were significantly reduced only in the EGCG-treated mice; there were no significant changes in the mice provided ECG. Although it is possible that the concentration and solubility of ECG reduced its effectiveness, EGCG was clearly shown to suppress bFGF to a greater extent (). EGCG-mediated reductions in bFGF levels in APCMin/+ mouse tumors, by ELISA assay, correlated with apparently fewer Factor VIII-labeled blood vessels in the tumors, suggesting an inhibitory effect on angiogenesis in the EGCG-treated mice. Thus, EGCG indeed suppressed bFGF expression associated with a reduction in tumorigenesis in APCMin/+ mice.
The concentration of green tea catechins in human plasma has been reported to reach no higher than 1 μ
mol/L even with consumption of large amounts of the beverage.32
However, higher levels are expected to be present in lumen of the gastrointestinal tract. Although the 50 μ
mol/L EGCG dose in cell culture reflects a higher range of plasma concentration, it is possible that this concentration can be reached in the intestinal tract. The exact effective concentration remains to be determined; however, the bioavailability and degradation as well as metabolic effects of catechins in the cell culture should be considered.
Our study raised many intriguing and important questions that require clarification in the future. In particular, the molecular feature of EGCG that serves as a signal trigger for ubiquitination and the sites of ubiquitination of bFGF in the presence of EGCG should be examined. In addition, the specific enhancing activity of EGCG on trypsin-like activity of the 20S proteasome remains to be elucidated. Overexpression of bFGF is very common in human colorectal cancer cells and in adenomas derived from mouse models, and these findings provide a molecular mechanism that details regulation of bFGF by EGCG.