β-Sitosterol-assisted silver nanoparticles activates Nrf2 and triggers mitochondrial apoptosis via oxidative stress in human hepatocellular cancer cell line.

Cancer nanomedicine is an emerging field of cancer therapeutics. Incidence of hepatocellular carcinoma (HCC) is increasing worldwide, and currently, it is the second leading cause of cancer-related deaths. This study investigates the cytotoxic potential of β-sitosterol-assisted silver nanoparticles (BSS-SNPs) in HepG2 cells. The silver nanoparticles were synthesized using β-sitosterol as a reducing and stabilizing agent. The characterization of BSS-SNPs was done by UV-visible spectrophotometry and transmission electron microscope (TEM) analysis. HepG2 cells were treated with different concentrations of BSS-SNPs for 24 hr, and cytotoxicity was evaluated by MTT assay. Intracellular ROS was investigated by 2',7'-dichlorofluorescin diacetate staining. The nuclear factor erythroid 2-related factor 2 (Nrf-2) protein expression was investigated by immunofluorescence staining. Morphology-related to apoptotic changes were analyzed by annexin V staining. Intrinsic apoptosis pathwayrelated molecular markers were investigated by western blotting and PCR analysis. Spectrophotometry analysis confirmed a strong absorption peak at 420 nm, which showed the successful synthesis of BSS-SNPs. The TEM analysis indicated the spherical-, rod-, and hexagonal BSS-SNPs with the size ranges from 5 to 55 nm. BSS-SNPs significantly inhibited the proliferation and induced ROS and Nrf-2 expression in HepG2 cells. BSS-SNPs treatment caused apoptosis-related morphological changes and upregulated the pro-apoptotic markers such as bax, p53, cytochrome c, and caspases-9, -3 and downregulated bcl-2 expressions. Our findings suggest that BSS-SNPs might serve as potential drug candidates for HCC.