Deficiency of Grx1 leads to high sensitivity of HeLaS3 cells to oxidative stress via excessive accumulation of intracellular oxidants including ROS

Abstract

Oxidative stress is often initiated by excess reactive oxygen species (ROS) production, resulting in macromolecular damage, which is implicated in many disease states. Glutaredoxin 1 (Grx1) is an antioxidant enzyme that plays an important role in redox signaling and redox homeostasis. In the present study, we generated HeLaS3 cell lines deficient in Grx1 by the CRISPR/CAS9 system to clarify how Grx1 affects the physiological activities of HeLaS3 cells to respond to oxidative stress. First, the survival assay revealed that Grx1-deficient HeLaS3 cells were more sensitive to γ-ray irradiation, heat shock and H₂O₂ exposure than HeLaS3 wild-type cells. Next, the intracellular redox state was investigated using a fluorescent probe (2′-7′dichlorofluorescin diacetate), and the oxidized state of total proteins and a peroxidase Prx2 were measured by Western blot analysis. Exposure to γ-ray irradiation, heat shock and H₂O₂ significantly induced more accumulation of intracellular oxidants including ROS and higher levels of oxidized proteins in Grx1-deficient HeLaS3 cells. Furthermore, MitoSox Red staining demonstrated that Grx1 deficiency causes a higher level of oxidants production in mitochondria. Moreover, Grx1-deficient HeLaS3 cells had a higher cytochrome c level and higher apoptosis rate (Annexin-V/FITC and EthD-III staining assay) upon oxidative stress. These results suggested that Grx1 deficiency lead to mitochondrial redox homeostasis disruption and apoptotic cell death upon oxidative stress. In addition, the results of proliferation assay and MitoTracker staining assay (multinuclear cell formation rate) suggested that oxidative stress exposure inhibits cell proliferation maybe by affecting cytoplasmic division in Grx1-deficient HeLaS3 cells.

Keywords:

• Glutaredoxin 1
• reactive oxygen species
• γ-ray irradiation
• redox homeostasis
• oxidative stress

Author contributions

Tingyi Zhao and Qiu-Mei Zhang-Akiyama designed the study. Tingyi Zhao performed the experiments. Tingyi Zhao drafted the manuscript. Tingyi Zhao and Qiu-Mei Zhang-Akiyama refined the manuscript.

Disclosure statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Data availability statement

All data used to support the findings of this study are included within the article.

Additional information

Funding

This work was financially supported in part by a Grants-in-Aid for Scientific Research [16K00545 and 19K12320] from the Ministry of Education, Grants for Excellent Graduate Schools, MEXT, Culture, Sports, Science and Technology of Japan (to Qiu-Mei Zhang-Akiyama).