Ultrastructural Analysis of Apoptosis Induced By Zinc Oxide Nanosphere in Human Hepatoma Huh7 Through Mitochondria Dysfunction
Abstract The efficiently of hepatocellular carcinoma therapy predominantly depends on advancement of nanomedicine. Zinc oxide nanostructure is promising material in nanomdicine field due to their unique properties. Zinc oxide Nanosphere (ZnO Ns) was fabricated with a size 50 nm diameter by a developed Sol-gel approach, using non-toxic biotemplate yeast extract. The physicochemical properties of zinc oxide nanosphere were estimated by Fourier-transform infrared spectrum (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscope (TEM). Liberated zinc ions released from the zinc oxide nanosphere suspended medium was determined by ICP-AS. The viability was tested by using HuH7 and Vero cells.ZnO nanosphere was more effectively on cell line than released Zn ions. The cell cycle arrested at G1/S. Also, the apoptosis assay by using Annexin-V/PI showed that apoptosis of HuH7 by ZnO nanosphere is concentration and time-dependent. The mechanism of apoptosis was evaluated via using techniques such as RT-PCR and flow cytometry. The results revealed significance up-regulated of Bax, P53, and Cytochrome C, while a Bcl2 results displayed significance down-regulated.Caspase 3 assay results showed that the apoptosis mechanism was intrinsic and extrinsic pathways. Also, ZnO nanosphere and free Zn+2 ions induced oxidative stress via increasing reactive oxygen species (ROS) and lipid peroxidation. The ultrastructural analysis of HuH7 cell was occurred by TEM.Transmission electron microscope analysis of HuH7 after treated with ZnO nanosphere at different times revealed a chromatin condensation of the nuclear periphery fragmentation appearance. Interestingly, the apoptosis of HuH7 cells induced by Zinc oxide nanosphere, showed the canonical ultrastructure features of apoptotic nuclei, and fragmented by budding. Furthermore, There were many vacuoles that filled in the cytoplasm, majority lipid droplets, which resembling those observed in foamy cells, and vesicles with intact membranes, which were recognized as swollen mitochondria.