Decoding the Chemico‐Biological Pharmacology of Wogonin Against Hepatocellular Carcinoma: An Integrative Computational Chemistry, Molecular Dynamics Simulation, and Experimental Investigation of ROS‐Orchestrated Apoptotic and Antimetastatic Mechanism
Guihu Wang, Mengchen Zhu, Aziz Kubaev, An JiangBackground
Hepatocellular carcinoma (HCC) is a highly fatal malignancy with frequent recurrence, necessitating effective multitarget therapeutic strategies. We herein investigated the anti‐HCC potency of wogonin through integrated computational and experimental approaches.
Methods
Gaussian 16 was employed to evaluate molecular stability, reactivity, HOMO–LUMO gap, molecular descriptors, and molecular electrostatic potential (MEP) through density functional theory (DFT) calculations. Drug‐likeness, pharmacokinetics, and toxicity were predicted using pkCSM, SwissADME, ADMET AI, and ProTox‐II. Wogonin targets were identified using SwissTargetPrediction, SEA, SuperPred, and CTD, while HCC‐associated genes were retrieved from GeneCards. Hub genes were identified using CytoHubba, followed by enrichment analysis and construction of a compound–target–pathway network in Cytoscape. Molecular docking with TP53, CTNNB1, and STAT3 was performed using CB‐Dock2, and interactions were analyzed in Discovery Studio. The stability of the wogonin–TP53 complex was evaluated by a 100 ns molecular dynamics simulation using Desmond through RMSD, RMSF, radius of gyration (Rg), hydrogen bonds, MM/GBSA, principal component analysis (PCA), and free energy landscape (FEL) analyses. Experimental validation was performed in HepG2 and THLE‐2 cells treated with wogonin (0–160 μM). Cell viability, colony formation, morphology, apoptosis, ROS generation, cell cycle progression, cell migration, cell invasion, and TP53, CTNNB1, and STAT3 protein expression were assessed using standard biochemical and cell‐based assays.
Results
DFT analysis indicated favorable electronic stability and chemical reactivity of wogonin, while ADMET prediction suggested acceptable pharmacokinetic and safety profiles. Network pharmacology identified TP53, CTNNB1, and STAT3 as key hub genes enriched in apoptosis, oxidative stress, inflammation, PI3K/AKT, NF‐κB, STAT3, and cancer‐related pathways. Molecular docking demonstrated strong binding to all three targets, and molecular dynamics simulations confirmed stable ligand–protein interactions throughout the 100 ns trajectory. In vitro, wogonin dose‐dependently reduced HepG2 cell viability and clonogenicity with lower cytotoxicity toward THLE‐2 cells. It also induced morphological alterations, cellular apoptosis, intracellular ROS accumulation, G2/M cell cycle arrest, suppressed cell migration and cell invasion, and modulated TP53, CTNNB1, and STAT3 protein expressions.
Conclusion
Wogonin exerts potent multitarget anti‐HCC activity by inducing apoptosis, ROS‐mediated oxidative stress, and G2/M cell cycle arrest and inhibiting cell migration and invasion through modulation of TP53/CTNNB1/STAT3‐associated oncogenic signaling, highlighting its potential as a multitarget therapeutic candidate against HCC.