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

Borgia Sherin Rebecca, Flavian; Sharmila, Ramanathan; Pon Nivedha, Rajamanickam

doi:10.31557/APJCP.2026.27.7.2589

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

Document Type : Research Articles

Authors

¹ PG and Research Department of Biotechnology, Bishop Heber College (Autonomous), Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.

² PG and Research Department of Biotechnology and Bioinformatics, Holy Cross College (Autonomous), Tamil Nadu, India.

10.31557/APJCP.2026.27.7.2589

Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Keywords