Theaflavin-3,3′-digallate (TF3), a flavan-3-ol derivative found in black tea, exhibits anti-tumor activity, but its mechanism of action in hepatocellular carcinoma (HCC) remains to be elucidated. Here we systematically delineate how TF3 targets Pin1 to suppress HCC through an integrated approach combining computational simulations, enzyme assay and cell-based assays. TF3 spontaneously occupies the active site of Pin1 with a docking score of −8.9 kcal/mol, inhibiting its PPIase activity (IC50 = 60.33 μmol/L) and yielding a binding constant (Ka) of 3.1 × 105 mol/L. Drug affinity responsive target stability (DARTS) assays further corroborated that TF3 directly engages Pin1 within HCC cells. Functionally, TF3 potently suppressed the viability of HepG2, SK-Hep-1 and Huh-7 cells in both dose- and time-dependent manners (IC50 = 61.22, 14.09 and 69.85 μmol/L at 24 h, respectively), and exhibited a modest selectivity window against the viability of L02 and THLE-2 cells (IC50 = 133.43 and 90.29 μmol/L at 24 h, respectively). In addition, TF3 triggers mitochondrial-mediated apoptosis, evidenced by ROS accumulation, loss of mitochondrial membrane potential, an elevated Bax/Bcl-2 ratio, cytochrome c release and enhanced PARP cleavage, and induces G2/M phase arrest. It also robustly inhibits HCC cell proliferation, invasion and migration, coinciding with downregulation of proteins governing cell cycle progression and invasive behavior. Transcriptome profiling coupled with enrichment analysis discovered that TF3 treatment differentially regulated 5009 genes, which were prominently enriched in pathways linked to apoptosis, cell cycle control, MAPK and PI3K/AKT signaling pathways. Western blotting analysis revealed that TF3 selectively suppresses phosphorylation of p38 and the PI3K/AKT cascade, activating JNK phosphorylation. In summary, our findings indicate that TF3 suppresses HCC proliferation by targeting Pin1, with attendant modulation of the MAPK and PI3K/AKT pathways, thereby presenting a potential candidate for targeted HCC therapy.
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