| Abstract: | 茶紅素（Theaflavins）與(-)-表沒食子酸兒茶素-3-沒食子酸酯（(-)-Epigallocatechin3-gallate(EGCG)）分別為紅茶與綠茶中含量最豐且生物活性最強之兒茶素(catechins)。近來研究發現EGCG會藉由抑制chymotrypsin-like(ChT-L)蛋白脢體(proteasome)的活性，來抑制腫瘤細胞的生長。其中的作用機制，可能為EGCG化學結構中的gallic-ester部份，與ChT-L蛋白脢體的活性中心形成共價結合，進而抑制了ChT-L蛋白脢體的活性。基於這個假設，我們推測EGCG的結構衍生物茶紅素，亦能透過類似的機制來抑制ChT-L蛋白脢體的活性。為了證明我們的推論，theaflavin(TF1), theaflavin-3-gallate(TF2a),theaflavin-3'-gallate(TF2b)和theaflavin-3,3'-gallate(TF3)等茶紅素對蛋白脢體活性的抑制作用首先被檢查。初步的實驗數據說明在invitro的實驗中，這些茶紅素都能對20S或26S蛋白脢體的ChT-L活性產生不同程度的抑制作用，其中以TF3的抑制效果最強；分析腫瘤細胞株如JurkatT,U937,HL60和MCF-7之細胞萃取液中之ChT-L蛋白脢體活性，亦發現TF3對活性抑制的影響程度最大。此外，實驗中發現茶多酚亦影響peptidylglutamylpeptide hydrolase(PGPH)蛋白脢體的活性。值得注意的是，這些茶多酚化學結構中的gallic-ester組成愈多，其抑制ChT-L蛋白脢體活性的程度也就愈加明顯，由此推測gallic-ester結構確實參與蛋白脢體活性抑制之作用。雖然invitro的實驗證明茶紅素能夠抑制ChT-L蛋白脢體的酵素活性，且這種抑制作用能夠減緩腫瘤細胞株的生長速率，但是仍然沒有足夠的證據可以證明這些茶多酚與ChT-L蛋白脢體活性中心之間有直接的交互作用。因此，為了找出茶紅素與蛋白脢體活性中心直接結合的證據，我們先將各種茶多酚分別耦合在CNBr-activatedSepharose4Bbeads上，再以親合性色層分析法，由腫瘤細胞萃取液分離出能與茶多酚結合的蛋白，續以二維電泳與質譜儀鑑定之，我們希望能夠藉此化學蛋白質體學(chemicalproteomics)的方法，來找出是否有任何蛋白脢體之subunits與茶多酚結合的證據，並推衍出茶多酚在癌症預防上的分子作用機制。
Theaflavins and (-)-epigallocatechin-3-gallate (EGCG) are major active polyphenols in black tea and green tea, respectively. Recent studies have shown that EGCG exerts its anti-tumor activity by the inhibition of chymotrypsin-like (ChT-L) proteasome activity. This inhibition might result from the interaction of gallic-ester moiety of EGCG with the catalytic residue of proteasome which forms the covalent adduct. Based on this hypothesis, we suggest that the gallic-ester containing theaflavins might inhibit the ChT-L proteasome activity similar to EGCG. To this end, the inhibitory effects of the theaflavins including theaflavin (TF1), theaflavin-3-gallate (TF2a), theaflavin-3’-gallate (TF2b) and theaflavin-3,3’-gallate (TF3) on the proteasome activity were firstly examined. In this study, we display that TF3 strongly inhibited ChT-L activities of purified 20S and 26S proteasomes in vitro, and the 26S proteasomes in Jurkat T, U937, HL60 and MCF-7 cancer cell extracts compared with other tea polyphenols. Additionally, all tea polyphenols, especially TF3, also inhibited peptidyl glutamyl peptide hydrolase (PGPH) activities of 26S proteasome as well as ChT-L activities in MCF-7 cells. Interestingly, the more inhibitory effect on ChT-L activity in cancer cell extracts by tea polyphenols; the more suppressing influence on cancer cell viability, suggesting that tea polyphenols might lead to anti-proliferating effect on cancer cells by proteasome inhibition. Compared with well-characterized polyphenols on proteasome inhibition, the ester bond-bearing compounds exhibited more potently inhibitory effects on ChT-L activity than those without ester bond(s), indicating that ester-bond moiety of tea polyphenols is essential for proteasome inhibition. Furthermore, the gallic acid and n-propyl gallate also slightly inhibited proteasome activities suggesting that the galloyl moiety of tea polyphenols also contributes to the proteasome inhibition. Although in vitro proteasome assay indicates that theaflavins and EGCG directly inhibit 20S ChT-L proteasome activity, no evidence can directly verify that the binding of these tea polyphenols to the catalytic core of proteasome exists. Next, we will find out the facts that can demonstrate the docking of the tea polyphenol to the proteasome. We employ a chemical proteomics strategy that is grounded on the affinity capture of cellular targets in combination with protein identification by mass spectrometry. Tea polyphenols, coupled to the CNBr-activated Sepharose 4B beads, was used as ligands for the affinity purification of cellular targeted proteins. We hope that the subunits of the 26S proteasome can be identified by this method if the direct binding of tea polyphenols to the proteasome subsists. On the other hand, the selectivity of these tea polyphenol proteasome inhibitors will be established to deduce the molecular actions of these tea polyphenols on cancer prevention. |
