2842 - Unveiling the Intricate Interplay of ROS, Autophagy and p53 in Radioresistance of Hepatocellular Carcinoma

Y. W. Lin¹, M. L. Kung², and M. H. Tai³; ¹Department of Radiation Oncology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ²Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ³Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan

Purpose/Objective(s): This study aimed to investigate the distinct responses exhibited by hepatocellular carcinoma (HCC) cell lines to irradiation, focusing on the dynamic impacts of gene expression, reactive oxygen species (ROS), autophagy, and mitochondrial functionality. Additionally, the study sought to explore the influence of p53 status on the cellular responses to radiation in different HCC cell lines. The primary objective was to identify transcriptional differences underlying the contrasting phenotypes of radioresistant and radionaïve HCC cells and to understand the interplay between cell survival and cell death post-radiation. Materials/

Methods: RNA sequencing was conducted on Huh-7 and Huh-7 irradiated resistant (Huh-7 IR) HCC cells to identify transcriptional differences. The study also involved the analysis of ROS levels, mitochondrial superoxide production, alterations in mitochondrial membrane potential, and gene expression in ROS, autophagy, cell cycle, cell senescence, and apoptosis pathways in HCC cells post-irradiation. Additionally, the responses of SK-Hep-1 (p53 wild type), Hep 3B (p53 deletion), Huh 7 (p53 mutation), and Huh-7 IR (p53 mutation) cells to radiation were evaluated in the context of their p53 status.
Results: The heat map of RNA sequencing results illustrates differentially expressed genes in Huh-7 and Huh-7 IR cells, with upregulated antioxidant genes and mTOR (a key regulator of autophagy and cell growth) in Huh-7 IR cells. Simultaneous downregulation of p53 and MDM2 in Huh-7 IR cells is noted, suggesting a disruption in this regulatory axis. Additionally, the upregulation of ATM and ATR in Huh-7 IR cells may indicate a more robust DNA damage response and repair capacity, contributing to resistance against radiation-induced cell death. Furthermore, the responses of SK-Hep-1, Hep 3B, and Huh 7 cells to radiation were influenced by their p53 status, with distinct patterns observed in ROS levels, mitochondrial superoxide production, and alterations in mitochondrial membrane potential post-irradiation. The radioresistant cell line, Huh 7-IR, exhibited enhanced capabilities in removing or neutralizing radiation-induced ROS.
Conclusion: The study provides comprehensive insights into the dynamic cellular responses following irradiation in HCC cell lines, considering the influence of p53 status. The findings underscore the potential interplay between cell survival and cell death, especially post-radiation, and highlight the importance of further investigations and time-course studies to deepen our understanding of these dynamic cellular responses, particularly in the context of p53 status. These results have significant implications for understanding the mechanisms of radioresistance and may contribute to the development of targeted therapeutic strategies for HCC patients undergoing radiation therapy.