Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3'-digallate.
Our previous study showed that tea polyphenols inhibited MAP kinase and AP-1 activities in mouse epidermal JB6 cells and the corresponding H-ras-transformed cell line 30.7b Ras 12. The present study investigated the mechanisms of this inhibition. The cells were incubated with (-)-epigallocatechin-3-gallate (EGCG) or theaflavin-3,3'-digallate (TFdiG) (20 mM) for different times, and the cell lysate was analyzed by immunoblotting. EGCG treatment decreased the levels of phospho-Erk1/2 and -MEK1/2 time-dependently (by 60% at 60 min). TFdiG lowered their levels by 38%-50% at 15 min. TFdiG effectively decreased total Raf-1 protein levels, most likely through lysosomal degradation. EGCG did not affect protein levels or the activity of Raf-1 significantly but decreased its association with MEK1 as determined by co-immunoprecipitation. In addition, EGCG and TFdiG (10 mM) inhibited the phosphorylation of Elk-1 by isolated phospho-Erk1/2 in vitro. This inhibition of Erk1/2 activity is Elk-1 concentration-dependent and ATP concentration-independent, which suggests that EGCG and TFdiG interfere with the binding of the protein substrate to the kinase. The presently demonstrated specific mechanisms of inhibition of MAP kinases by EGCG and TFdiG may help us to understand the effects of tea consumption on cancer, inflammatory diseases, and cardiovascular diseases.
Chung
J Y
JY
Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Park
J O
JO
Phyu
H
H
Dong
Z
Z
Yang
C S
CS
D000255:Adenosine Triphosphate / Q000378:metabolism; D044946:Biflavonoids; D002384:Catalysis; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology*; D002460:Cell Line; D004268:DNA-Binding Proteins; D005419:Flavonoids; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D013662:Tea; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1
D044946:Biflavonoids; D004268:DNA-Binding Proteins; D005419:Flavonoids; D010636:Phenols; D011108:Polymers; D011518:Proto-Oncogene Proteins; D013662:Tea; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; C077197:theaflavin digallate; D005707:Gallic Acid; D000255:Adenosine Triphosphate; D002392:Catechin; C045651:epigallocatechin gallate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras)