Investigation of the Anticancer Effects and Senescence Induction of Hesperetin Combined with Cisplatin in Hepatocellular Carcinoma and Embryonic Fibroblast Cell Lines.

[OBJECTIVE] Hepatocellular carcinoma (HCC), the most common form of liver cancer, often develops in individuals with chronic liver diseases, especially cirrhosis. Cisplatin (Cisp), a chemotherapy agent commonly used in HCC treatment, is effective but is known to damage normal cells, including fibroblasts. Hesperetin (HST), a citrus flavanone found abundantly in citrus fruits, has demonstrated antioxidant, anti-inflammatory, and anticancer properties. This study aimed to investigate the synergistic cytotoxic effects and selective induction of senescence by HST in combination with Cisp in HepG2 cancer cells and NIH-3T3 fibroblast cells.

[METHODS] The cytotoxic effects of HST were assessed using the MTT assay to determine cell viability. The antiproliferative properties were evaluated using colony formation assays. Senescence was assessed using SA-β-gal staining, while flow cytometry was used to analyze cell cycle distribution and apoptosis. Protein expression related to proliferation and apoptosis was determined via Western blot analysis.

[RESULTS] MTT assay results indicated that both HST and Cisp reduced HepG2 cell viability in a dose-dependent manner, with IC values of 258 ± 2.47 µM and 5 ± 1.83 µM, respectively. Their combination (HST: 33-130 µM; Cisp: 0.6-2.5 µM) showed synergistic effects (combination index, CI < 1) co-treatment with HST (65 and 130 µM) significantly enhanced senescence in HepG2 cells. Clonogenic assays showed inhibition of colony formation, supported by reduced expression of p-ERK1/2 and Cyclin D1. Flow cytometry revealed increased apoptosis and G2/M phase arrest, with upregulation of Bax and caspase-3, and downregulation of Bcl-xL. In NIH-3T3 cells, HST showed minimal cytotoxicity (IC50 > 500 µM), and co-treatment with Cisp reduced senescence markers.

[CONCLUSION] These results suggest that HST and Cisp co-treatment synergistically reduces cancer cell viability while protecting normal fibroblasts from senescence, supporting its potential as a co-chemotherapeutic agent in HCC treatment, while also serving as a protective agent against senescence in healthy tissues.