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. MATERIALS AND 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.
Subject(s)
Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Muscle Proteins , Phenols/pharmacology , Proto-Oncogene Proteins c-bcl-2 , Tea/chemistry , Adenocarcinoma/pathology , Apoptosis/drug effects , Calpain/analysis , Carcinoma/pathology , Carrier Proteins/metabolism , Caspases/analysis , Cell Line , Cysteine Endopeptidases/drug effects , G1 Phase/drug effects , Guanosine Triphosphate/chemical synthesis , Guanosine Triphosphate/isolation & purification , Humans , Intracellular Signaling Peptides and Proteins , Jurkat Cells , Male , Microfilament Proteins/metabolism , Multienzyme Complexes/drug effects , Phenols/chemical synthesis , Phenols/isolation & purification , Polymers/pharmacology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins/metabolism , Stereoisomerism , Transcriptional Elongation Factors , bcl-2-Associated X ProteinABSTRACT
[reaction: see text] A brief stereospecific synthesis of cryptophycin 1 is described in which (R)-mandelic acid serves as the sole source of asymmetry for unit A. The key step is a hetero-Diels-Alder cycloaddition.
