Inhibition of Tpa-Induced Protein Kinase C and Transcription Activator Protein –1 Binding Activities by Theaflavin-3,3’-Digallate from Black Tea in Nih 3t3 Cells

Tea is one of the most popular beverages in the world. Several reports have shown that both green tea and black tea were able to inhibit tumor cell proliferation in animal models. In this study, we investigated the inhibitory effects of black tea polyphenols including theaflavin (TF-1) , the mixture (TF-2) of theaflavin-3-gallate (TF-2a), and theaflavin-3'- gallate (TF-2b), theaflavin-3,3'-digallate (TF -3), thearubigin (TR), and a major green tea polyphenol (-)- epigallocatechin-3-gallate (EGCG) on 12- O- tetradecanoylphorbol 13-acetate (TPA)-induced protein kinase C (PKC) and transcription activator protein-1 (AP-1) binding activities in NIH3T3 cells. On analysis of PKC activity with partial purified preparation, TPA (100 ng/mL) treatment was able to elevate membrane-associated PKC activity ~3-fold, and treatment with TF-3 (20 M) and EGCG ( 20 M) showed 94.5% and 9.4% suppression on TPA-induced PKC activity, respectively. Translocation of PKC protein from cytosol to membrane was detected in TPA-treated NIH3T 3 cells , and TF-3 was able to block its translocation. By in vitro kinase assay using myelin basic protein (MBP) as a PKC- specific substrate, we found that TPA treatment was able to increase PKC kinase activity by detection of phosphorylated MBP protein and TF-3 showed strongest inhibitory effect on its phosphorylation while EGCG was shown to be less effective. We also analyzed the AP-1 binding activity by electrophoretic mobility shift assay and c-Jun gene expression by northern blot and western blot, the results showed that TF-3 is the most potent inhibitor on TPA-induced AP-1 binding activity and c-Jun gene expression among these five tea polyphenols. Our results might provide new molecular basis for understanding the inhibitory effects of tea polyphenols on TPA-mediated tumor promotion