Isorhamnetin-3-O-Neohesperidoside Exerts Anti-Hepatocellular Carcinoma Activity by Targeting Multiple Oncogenic...

The objective of this study was to investigate the inhibitory effects and underlying molecular mechanisms of Isorhamnetin-3-O-neohesperidoside (IHN), a naturally occurring O-methylated flavonol, against human hepatocellular carcinoma (HepG2) cells, with a particular focus on its impact on cell proliferation, apoptosis, metabolic regulation, and oxidative stress.

Human liver cancer HepG2 cells were treated with varying concentrations of IHN for 24 and 48 hours. The cytotoxic and anti-proliferative effects were assessed using standard cytotoxicity assays. Flow cytometry was performed to analyze cell cycle distribution and apoptosis induction. Metabolic assays evaluated glucose uptake and lactate dehydrogenase A (LDH-A) activity to assess the Warburg effect. Oxidative stress markers were analyzed by measuring reactive oxygen species (ROS), lipid peroxidation, and the glutathione (GSH/GSSG) ratio. Quantitative real-time PCR (qRT-PCR) was used to determine the expression of key regulatory genes.

IHN exhibited potent, dose- and time-dependent cytotoxicity against HepG2 cells, with Half-maximal inhibitory concentration (IC₅₀) values decreasing from 161.22 μM (24 h) to 95.87 μM (48 h). IHN- induced G0/G1 cell cycle arrest accompanied by the upregulation of Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) and promoted apoptosis through the intrinsic pathway, as shown by increased expression of BCL2-associated X Protein (BAX), Caspase-3 (CASP-3), and Caspase-9 (CASP-9). Metabolically, IHN suppressed the Warburg effect, leading to reduced glucose uptake and decreased LDH-A activity. This was associated with a marked increase in ROS levels and lipid peroxidation, along with depletion of GSH/GSSG and downregulation of antioxidant genes, indicating severe oxidative stress.

IHN exerts significant anti-hepatocellular carcinoma activity by targeting multiple oncogenic pathways. Its mechanism involves cell cycle arrest, intrinsic apoptosis induction, metabolic suppression, and redox imbalance. These findings suggest that IHN is a promising multi-target natural compound that exploits the metabolic and oxidative vulnerabilities of HepG2 cells, highlighting its potential as a novel therapeutic candidate for hepatocellular carcinoma.