Zirconia Nanoparticles Induce HeLa Cell Death Through Mitochondrial Apoptosis and Autophagy Pathways Mediated by ROS.

Hang Dong,Qinghai Wang,Xinmin Nie,Rong Gui,Xueyuan Huang,Qiangqiang Zhao,Yinghui Shang,Haiting Liu,Jian Li,Wansong Chen

doi:10.3389/fchem.2021.522708

Abstract

Zirconia nanoparticles (ZrO2 NPs) are commonly used in the field of biomedical materials, but their antitumor activity and mechanism is unclear. Herein, we evaluated the anti-tumor activity of ZrO2 NPs and explored the anti-tumor mechanism. The results of in vitro and in vivo experiments showed that the level of intracellular reactive oxygen species (ROS) in HeLa cells was elevated after ZrO2 NPs treatment. Transmission electron microscopy (TEM) showed that after treatment with ZrO2 NPs, the mitochondria of HeLa cells were swollen, accompanied with the induction of autophagic vacuoles. In addition, flow cytometry analysis showed that the apoptotic rate of HeLa cells increased significantly by Annexin staining after treatment with ZrO2 NPs, and the mitochondrial membrane potential (MMP) was reduced significantly. The proliferation of HeLa cells decreased as indicated by reduced Ki-67 labeling. In contrast, TUNEL-positive cells in tumor tissues increased after treatment with ZrO2 NPs, which is accompanied by increased expression of mitochondrial apoptotic proteins including Bax, Caspase-3, Caspase-9, and Cytochrome C (Cyt C) and increased expression of autophagy-related proteins including Atg5, Atg12, Beclin-1, and LC3-II. Treating HeLa cells with N-acetyl-L-cysteine (NAC) significantly reduced ROS, rate of apoptosis, MMP, and in vivo anti-tumor activity. In addition, apoptosis- and autophagy-related protein expressions were also suppressed. Based on these observations, we conclude that ZrO2 NPs induce HeLa cell death through ROS mediated mitochondrial apoptosis and autophagy.

Highlights

ZrO2 NPs are the main raw material for synthetic casting sand, refractory and porcelain
Observed under Transmission electron microscopy (TEM), ZrO2 NPs were monodispersed with diameters averaging about 25 nm (Figure 1A), which were larger than the previous reported green synthesized Nanozirconia using leaf extract of Eucalyptus globulus (E. globulus) (Balaji, et al, 2017)
Based on dynamic light scattering (DLS) data (Figure 1B), ZrO2 NPs averaged 25 nm in size, which was consistent with the data of TEM

Summary

Introduction

ZrO2 NPs are the main raw material for synthetic casting sand, refractory and porcelain. They are common biomedical materials used for biosensors, implants, joints and oral prosthesis replacement (Lohbauer, et al, 2010; Qeblawi, et al, 2010). Reduced graphene oxide modified by ZrO2 NPs are used for highly sensitive electrochemical sensors for anticancer drugs (Venu, et al, 2018). Iron-manganese-doped sulfated zirconia nanoparticles have been shown to induce HepG2 and MDA-MB-231 cell death, while they are less effective at killing normal Chang cells and HUVECs (Al-Fahdawi, et al, 2015).

Methods

Results

Conclusion