3,3’,5,5’-Tetramethoxybiphenyl-4,4’-diol exerts a cytotoxic effect on hepatocellular carcinoma cell lines by inducing morphological and ultrastructural alterations, G2/M cell cycle arrest and death by apoptosis via CDK1 interaction

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with frequent recurrence and chemoresistance, underscoring the need for new treatment strategies. 3,3’,5,5’-Tetramethoxybiphenyl-4,4’-diol (TMBP) showed cytotoxicity against lung cancer cell lines without harming normal cells. Thus, we investigated the antitumoral effect of TMBP on HCC cell lines, HuH7.5 (p53-mutant) and HepG2/C3A (p53-wild type). Cells were treated with TMBP (12.5–150 µM) for 24 and 48 h, and metabolic cellular activity (MTT) were used to determine the 50 % inhibitory concentration (IC 50 ) values. TMBP cytotoxicity were assessed by Trypan blue assay, scanning and transmission electron microscopy. Cell migration (wound healing), total ROS (H 2 DCFDA), mitochondrial dysfunction (TMRE), lipid droplets (Nile Red), and autophagic vacuoles (MDC) were assessed. Flow cytometry characterized cell cycle distribution and cell death. Caspase 3/7 activity and CASP3 expression confirmed apoptosis. Molecular docking and gene expression analysis validated TMBP-CDK1 interaction. TMBP reduced cell viability, with IC 50 values of 68 and 55 µM (HuH7.5) and 50 and 42 µM (HepG2/C3A) at 24 and 48 h. TMBP induced severe morphological alterations, impaired migration, increased ROS, mitochondrial dysfunction, increased lipid droplets and autophagic vacuoles. TMBP also led to G2/M arrest and apoptosis, likely via CDK1 inhibition through hydrogen bonding at Tyr15. These findings highlight TMBP as a promising therapeutic candidate targeting CDK1 in HCC. • TMBP promotes severe morphological alterations and impaired migration in HCC cells. • TMBP-treated HCC cells present increased ROS/NOx levels along with loss of ∆Ψm. • TMBP-induced stress led to increased lipid droplets and autophagic vacuoles. • TMBP induces G2/M cell cycle arrest and apoptosis in HCC-treated cells. • TMBP inhibits CDK1 protein activation via hydrogen bond with Tyr15 residue.