Catechins are the major constituents in green tea, accounting for 30-42% of the dry weight, and more than 50% of the total catechins are EGCG (2
). Abundant studies have suggested that tea consumption has protective effects against cancer and many other diseases in animal models (1
). Epidemiological studies, though inconclusive, have also suggested an association between tea consumption and a lower risk of cancer in humans. One study suggested a significantly reduced risk of breast cancer among green tea drinkers (40
). Phase II clinical trials have indicated effectiveness of green tea extract against UV-induced skin injuries, oxidative DNA damage, and prostate cancer (41
). Most studies of EGCG in isolated cells have demonstrated effective concentrations ranging from 10 to 100μM for inhibition of tumor cell growth (3
). In contrast, EGCG concentrations found in human plasma through tea consumption are only in the high nanomolar range (42
). Thus, it may be difficult to achieve, through tea consumption, effective concentrations of EGCG that have anti-cancer activity; this may contribute to the inconclusive results of some epidemiological investigations. However, better understanding of the possible mechanisms might allow design of more bioavailable EGCG derivatives which could be developed for chemotherapeutic purposes.
All of the data we presented here and elsewhere (7
) are consistent with the notion that EGCG is an inhibitor of hsp90 function through its interaction with the C-terminal end of this protein, in particular at or near an ATP binding site. ATP plays an important role in hsp90 chaperone function in terms of stabilizing the hsp90 structure as well as completion of the chaperone cycle (29
). There are two ATP binding sites, one on the N-terminus and the other on the C-terminus (27
). The binding of ATP to the N-terminal site induces the association of the N-terminal domains forming a closed conformation and resulting in stabilization of client protein binding (27
). Binding of GA to the N-terminal ATP site prevents this closed conformation, and permits destabilization of hsp90-client protein complexes. The precise function of the C-terminal ATP binding site is less understood. Previous studies have indicated that N-terminal ATP binding is required for the C-terminal site to become available for nucleotide binding (27
). Our finding that GA enhances the ability of AC88 to immunoprecipitate hsp90 is consistent with the N-terminal ATP binding site being involved in the unmasking of the C-terminal site. ATP was found to stabilize a C-terminal hsp90 fragment in a trypsinolytic footprinting (22
), suggesting that ATP binding modifies the conformation of the C-terminal region. Novobiocin, which binds to the C-terminus of hsp90, is believed to bind to this ATP binding site residing within residues 663-676 (28
), and this binding has been shown also to lead to the destabilization of hsp90-client protein interactions. Thus, it has been suggested that hsp90 function is regulated by the cyclic binding, hydrolysis and/or exchange of ATP at these two sites resulting in hsp90 conformations available for and stabilizing the interactions with client proteins and cochaperones.
In the present study, EGCG was found to protect a C-terminal hsp90 fragment from lysis by trypsin, in similar fashion as both ATP and novobiocin (22
). EGCG also impaired the ability of AC88, whose epitope was mapped to residues 661-676 (25
), to immunoprecipitate hsp90, and competed with hsp90 or its C-terminal fragment to bind to ATP-agarose. These data indicate that EGCG binds at or near the C-terminal ATP binding site on hsp90. Consistent with this, we further demonstrated that EGCG effectively blocks hsp90 dimerization as well as chaperone function determined by the ability to mediate refolding of a denatured client, luciferase. Together, these data indicate that the mechanism of EGCG acting as an hsp90 inhibitor is through its ability to bind at or near to the C-terminal ATP binding site of hsp90, disrupting ATP binding, inducing an inappropriate conformational change, and inhibiting hsp90 chaperone function. A recent study found that EGCG decreases the K+
-ATP channel’s sensitivity to ATP, and suggested that the 5’OH group in the B-ring in EGCG is critical for binding to the ATP binding pocket (44
). EGCG was also found to compete with ATP for binding to a glucose-regulated protein 78 (GRP78), an hsp70 family member (5
). In addition, EGCG was found to bind to an ATP binding site on insulin-like growth factor-1 receptor (IGF-1R) (6
). It remains to be determined whether the ability of EGCG to affect ATP binding sites in a variety of proteins may be a general feature of EGCG and other EGCG-like compounds.
To show more effectively the ability of EGCG to modify chaperone activity, we examined in more detail the mechanism whereby EGCG inhibits the transcriptional activity of the AhR, an hsp90 client protein. Based on current knowledge, our previous data (7
), and the data we present here, we propose a model in which the AhR exists in multiple forms within the cytoplasm coupled with hsp90, but also determined by the relative absence or presence of XAP2 and/or p23 (). TCDD binding initiates transformation of the cytosolic AhR to a nuclear AhR-Arnt heterodimer which then binds to AhRE. The binding of EGCG to hsp90 stabilizes an AhR complex that includes at least hsp90 and XAP2, and possibly p23. However, the interaction of EGCG with the hsp90-AhR complex also apparently modifies AhR conformation to expose the NLS sequence resulting in translocation of the AhR complex into the nucleus (7
). The stabilization of the AhR-hsp90-XAP2 complex prevents TCDD-mediated AhR-Arnt association and blocks AhR-mediated transcriptional activity.
Fig 7 A proposed model for the AhR complex response to TCDD and its modification by EGCG. In the cytoplasm, the AhR forms a complex with hsp90, XAP2, and p23. TCDD binding initiates the translocation of AhR to the nucleus, its dissociation from XAP2, p23, and (more ...)
GA, which binds to the N-terminal ATP binding site of hsp90, has also been found to block TCDD-induced transcription by affecting the AhR complex. In contrast to EGCG, GA destabilizes the hsp90-AhR interaction, induces AhR nuclear translocation and release of p23, does not prevent the TCDD-induced AhR-Arnt heterodimerization, but leads to increased proteosomal degradation of the AhR (25
). Molybdate, which binds the C-terminus of hsp90, enhances the association of hsp90-AhR complex, prevents AhR nuclear translocation, and prevents TCDD-induced Arnt hetero-dimerization and AhR degradation (45
). Our studies, presented here and elsewhere (7
), indicate that EGCG stabilizes the AhR complex, induces AhR nuclear translocation, but prevents the TCDD-induced Arnt heterodimerization and degradation of the AhR. Notably, EGCG was also shown to increase the nuclear accumulation of Nrf-2 (NF-E2-related factor). However, contrary to that of AhR, the DNA binding and transcriptional activity of Nrf-2 were enhanced by EGCG treatment in human breast epithelial (MCF10A) cells (46
). Therefore, EGCG appears to act uniquely among the hsp90 inhibitors. The uniqueness of EGCG as an hsp90 inhibitor is also examplified by its modification on the hsp90-cochaperone complex. In these investigations, we illustrated that EGCG alters the interaction of hsp90 with the cochaperones. Differently from any other hsp90 inhibitors, EGCG stabilizes the association of hsp70, Cyp40 and XAP2 to hsp90. Notably, another C-terminal hsp90 inhibitor, novobiocin significantly reduces the interaction of hsc70, FKBP52 and p23 with hsp90 (22
). Generally, it is thought that hsp70 binds to hsp90 in an “intermediate” complex through HOP, and dissociates with HOP from the late complex that contains TPR co-chaperones, such as CyP40 (26
). Our observations suggest that EGCG could stabilize a novel hsp90-co-chaperone complex which may contribute to the different effects of EGCG compared with other hsp90 inhibitors.
Our data further indicate that EGCG binds not only to AhR-bound hsp90 but also purified hsp90. This suggests that EGCG is likely to affect other hsp90 client proteins through binding to hsp90 and inhibition of hsp90 functions such as dimerization and chaperone activity. Shown in the current investigation, as an example of hsp90 client proteins, AhR activity is affected by EGCG but not the level of AhR protein. However, the effects of EGCG on other hsp90 client proteins may not be the same as for the AhR, as shown in and elsewhere (1
). In our investigations, we demonstrated that EGCG decreases the level of the hsp90 client proteins ErbB2, Raf-1 and pAKT in SKOV3 cells. Other data have shown that EGCG alters levels and/or activity of many hsp90 client proteins including ErbB2, AKT, CDK4, and Raf-1 both in vivo
and in vitro
in several different cell types (1
). Much evidence derived from the use of the GA-derived hsp90 inhibitors and novobiocin has shown these client proteins to be effective biomarkers of hsp90 inhibition (48
). Clearly, further research is needed to define effects of EGCG on different client proteins and the mechanisms for these effects. We recognize that the ability of EGCG to affect hsp90 function may not be the only mechanism whereby EGCG exerts its anti-cancer effect, as many mechanisms have been postulated (3
). However, the ability to affect hsp90 may be a common mechanism for several of them that have been proposed. Interestingly, several proteins reported to bind to EGCG are either hsp90 client proteins (vimentin, IGF-1R, Grp78) (4
) or closely related (laminin receptor) (49
) to hsp90. Furthermore, inhibition of hsp90 disrupts their signaling pathways or leads to their degradation respectively (50
). Additional studies are necessary to determine whether hsp90 plays a role in their binding to EGCG.
It is known that different clients have different binding sites on hsp90, and also likely require different interactions with cochaperone proteins other than or in addition to XAP2, hsp70 and Cyp40 (26
). Moreover, different types of tumor cells are likely to depend on different, select and/or multiple hsp90 client protein-dependent signaling pathways for their growth and survival. As such, the ultimate client protein-dependent signaling pathway affected by EGCG that leads to inhibition of tumor growth may be tumor cell specific. Critical experiments providing conclusive evidence that inhibition of hsp90 is responsible for the anti-tumor effects of EGCG are likely to be of many types, cell specific, and reasonably complex.
That EGCG acts as an hsp90 inhibitor is a novel finding. Since hsp90 inhibitors such as 17-AAG, have been intensively studied for their anti-cancer effects, developing derivatives or other hsp90 inhibitors that are selective and more bioavailable becomes advantageous for identifying agents with increased therapeutic efficacy. Our identification here of the specific region on hsp90 with which EGCG interacts provides some momentum for research that may ultimately lead to the use of EGCG and/or of its derivatives for chemotherapeutic purpose.