title: Synthetic analogs of green tea polyphenols as proteasome inhibitors.
abstract: BACKGROUND
  Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study.
  
  
  METHODS
  GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay.
  
  
  RESULTS
  (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells.
  
  
  CONCLUSIONS
  Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.
authors.affiliation: Drug Discovery Program, H Lee Moffitt Cancer Center & Research Institute, Departments of Interdisciplinary Oncology and Biochemistry & Molecular Biology, College of Medicine,University of South Florida, Tampa, FL 33612-9497, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Smith
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wang
authors.forename: Aslamuzzaman
authors.initials: A
authors.lastname: Kazi
authors.forename: Lian-Hai
authors.initials: LH
authors.lastname: Li
authors.forename: Tak-Hang
authors.initials: TH
authors.lastname: Chan
authors.forename: Q Ping
authors.initials: QP
authors.lastname: Dou
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D017209:Apoptosis / Q000187:drug effects; D002154:Calpain / Q000032:analysis; D002277:Carcinoma / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism; D020169:Caspases / Q000032:analysis; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000187:drug effects; D009124:Muscle Proteins; D010636:Phenols / Q000138:chemical synthesis / Q000302:isolation & purification / Q000494:pharmacology*; D011108:Polymers / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D013237:Stereoisomerism; D013662:Tea / Q000737:chemistry*; D035602:Transcriptional Elongation Factors; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D002352:Carrier Proteins; C114720:Elp1 protein, human; C491968:Ikbkap protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D009097:Multienzyme Complexes; D009124:Muscle Proteins; D010636:Phenols; D011108:Polymers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C058992:Tagln protein, mouse; D013662:Tea; D035602:Transcriptional Elongation Factors; D051028:bcl-2-Associated X Protein; D006160:Guanosine Triphosphate; D002154:Calpain; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex