Ultrastructural analysis of zinc oxide nanospheres enhances anti-tumor efficacy against Hepatoma

Amr Hassan; Fawziah A Al-Salmi; Tamer M M Abuamara; Emadeldin R Matar; Mohamed E Amer; Ebrahim M M Fayed; Mohamed G A Hablas; Tahseen S Mohammed; Haytham E Ali; Fayez M Abd El-Fattah; Wagih M Abd Elhay; Mohammad A Zoair; Aly F Mohamed; Eman M Sharaf; Eldessoky S Dessoky; Fahad Alharthi; Hussam Awwadh E Althagafi; Ahmed I Abd El Maksoud

doi:10.3389/fonc.2022.933750

Ultrastructural analysis of zinc oxide nanospheres enhances anti-tumor efficacy against Hepatoma

Front Oncol. 2022 Oct 27:12:933750. doi: 10.3389/fonc.2022.933750. eCollection 2022.

Authors

Amr Hassan¹, Fawziah A Al-Salmi², Tamer M M Abuamara³, Emadeldin R Matar⁴, Mohamed E Amer³, Ebrahim M M Fayed³, Mohamed G A Hablas³, Tahseen S Mohammed⁵, Haytham E Ali³, Fayez M Abd El-Fattah⁶, Wagih M Abd Elhay³, Mohammad A Zoair⁷, Aly F Mohamed⁸, Eman M Sharaf⁹, Eldessoky S Dessoky², Fahad Alharthi², Hussam Awwadh E Althagafi¹⁰, Ahmed I Abd El Maksoud¹¹

Affiliations

¹ Department of Bioinformatics, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat, Egypt.
² Biology Department, College of Sciences, Taif University, Taif, Saudi Arabia.
³ Department of Histology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
⁴ Departments of Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
⁵ Department of Public Health and Community Medicine, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
⁶ Department of Anatomy and Embryology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
⁷ Department of Physiology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
⁸ Research and development department, Egyptian Organization for Biological Products and Vaccines [Holding Company for Vaccine and Sera Production (VACSERA)], Giza, Egypt.
⁹ Department of Bacteriology, Immunology, and Mycology, Animal Health Research Institute (AHRI), Shebin El Kom, Egypt.
¹⁰ Biology Department, Faculty of Science and Arts, Al-Baha University, Al-Mikhwah, Saudi Arabia.
¹¹ Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat, Egypt.

Abstract

Zinc oxide nanomaterial is a potential material in the field of cancer therapy. In this study, zinc oxide nanospheres (ZnO-NS) were synthesized by Sol-gel method using yeast extract as a non-toxic bio-template and investigated their physicochemical properties through various techniques such as FTIR, XR, DLS, and TEM. Furthermore, free zinc ions released from the zinc oxide nanosphere suspended medium were evaluated by using the ICP-AS technique. Therefore, the cytotoxicity of ZnO nanospheres and released Zn ions on both HuH7 and Vero cells was studied using the MTT assay. The data demonstrated that the effectiveness of ZnO nanospheres on HuH7 was better than free Zn ions. Similarly, ZnO-Ns were significantly more toxic to HuH7 cell lines than Vero cells in a concentration-dependent manner. The cell cycle of ZnO-Ns against Huh7 and Vero cell lines was arrested at G₂/M. Also, the apoptosis assay using Annexin-V/PI showed that apoptosis of HuH7 and Vero cell lines by ZnO nanospheres was concentration and time-dependent. Caspase 3 assay results showed that the apoptosis mechanism may be intrinsic and extrinsic pathways. The mechanism of apoptosis was determined by applying the RT-PCR technique. The results revealed significantly up-regulated Bax, P53, and Cytochrome C, while the Bcl₂ results displayed significant down-regulation and the western blot data confirmed the RT-PCR data. There is oxidative stress of the ZnO nanospheres and free Zn⁺² ions. Results indicated that the ZnO nanospheres and free Zn⁺² ions induced oxidative stress through increasing reactive oxygen species (ROS) and lipid peroxidation. The morphology of the HuH7 cell line after exposure to ZnO nanospheres at different time intervals revealed the presence of the chromatin condensation of the nuclear periphery fragmentation. Interestingly, the appearance of canonical ultrastructure features of apoptotic morphology of Huh7, Furthermore, many vacuoles existed in the cytoplasm, the majority of which were lipid droplets, which were like foamy cells. Also, there are vesicles intact with membranes that are recognized as swollen mitochondria.

Keywords: Bax; G2/M transition; P53; Zn+2 ion; apoptotic morphology; reactive oxygen species; zinc oxide nanosphere.