Knockdown of Opsin 3 (OPN3) Enhances G6PD Autophagic Degradation and Aggravates Oxidative Stress Damage to Radiosensitize Cervical Cancer Cells

Abstract

Background: Cervical cancer (CC) is the fourth most prevalent cancer among women worldwide, imposing a significant burden. While radiotherapy is a cornerstone in CC treatment, radioresistance remains a challenge, necessitating the exploration of mechanisms and novel targets for overcoming this challenge. Opsin 3 (OPN3) has been implicated in cancer treatment resistance, and the glucose-6-phosphate dehydrogenase (G6PD)-mediated glucose metabolism, along with oxidative stress, has been associated with radioresistance. This study aimed to explore the impact of OPN3 knockdown on the radioresistance of CC cells and the involvement of G6PD and oxidative stress in the underlying mechanisms. Methods: The HeLa cell line, a representative of CC, was utilized in this study. OPN3 knockdown was established through cell transfection. The subsequent effects on HeLa cell viability and apoptosis under ionizing radiation (IR) were assessed using cell counting kit-8 (CCK-8) assay, colony forming assay, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and western blot analysis to detect apoptosis-related proteins. Additionally, proteins associated with oxidative stress, DNA damage, and G6PD protein levels and activity were measured using enzyme-linked immunosorbent assay (ELISA) and western blot. Acquired radioresistant HeLa cells (RR-HeLa) were generated using IR. OPN3 knockdown was performed in RR-HeLa cells, and subsequent effects were assessed as described for HeLa cells. Reactive oxygen species (ROS) scavenger and G6PD inhibitor were employed to investigate the role of OPN3 in regulating oxidative stress and G6PD during radioresistance. Furthermore, in the G6PD degradation, autophagy inhibitor and proteasome pathway inhibitor were used to confirm the regulatory role of OPN3 on G6PD through autophagy. Results: OPN3 was upregulated in RR-HeLa cells (p < 0.001). OPN3 knockdown decreased the proliferation (p < 0.001), DNA damage (p < 0.001), and oxidative stress (p < 0.001) while reducing cell apoptosis (p < 0.001), glucose metabolism (p < 0.001), G6PD protein levels (p < 0.001), and activity (p < 0.001) in HeLa and RR-HeLa cells following IR treatment. OPN3 overexpression in HeLa cells increased proliferation post-IR treatment. The post-IR cell proliferation reduction induced by OPN3 knockdown was reversed by ROS inhibitor and G6PD overexpression (p < 0.001), while further decreased by G6PD inhibitor (p < 0.001). The OPN3 knockdown-induced decrease in G6PD was unchanged by the proteasome pathway inhibitor (p > 0.05) but was reversed by the autophagy inhibitor (p < 0.001), thereby reversing the post-IR cell proliferation (p < 0.001). Conclusion: OPN3 knockdown renders HeLa cells more radiosensitive and mitigates radioresistance by promoting the autophagic degradation of G6PD and exacerbating oxidative stress within the cells.