Prolonged radiation exposure alters cellular proteome network in human hepatocellular carcinoma cells

Background: Hepatocellular carcinoma (HCC), leading cause of cancer-related mortality globally, presents a major challenge as majority of patients are diagnosed at later stages, rendering them ineligible for effective interventions such as liver resection and transplantation. Radiation therapy (RT) is growing as a well-tolerated non-invasive local ablative treatment option; however, its effectiveness is restricted by therapy-induced radioresistance, resulting in tumor recurrence, enhanced invasion, and alterations in the extracellular matrix. This study aims to examine the impact of prolonged radiation exposure on the molecular dynamics and cellular behavior of HCC cells, with particular emphasis on the function of invadopodia in acquired resistance and increased invasiveness, to pave the way for development of novel therapeutic strategies for improved patient outcomes. Methodology: Radioresistant HCC cell lines were created by exposing them to a cumulative dose of 20 Gy of radiation (2 Gy daily for ten-days) followed by functional assays to measure colony formation, invasion, and migration. LC-MS was used to identify and quantify proteins in both the radiation-treated and control groups. Western blotting and immunofluorescence analysis were carried out to verify the differentially expressed proteins. Results: Functional assays showed a significant increase in colony formation which indicates radioresistant HCC cells had enhanced proliferation capabilities as compared to normal cancer cells. In addition, these cells demonstrated a significant increase in their invasive and migratory abilities, suggesting that radioresistant cells not only exhibit improved survival but also have a higher potential for metastatic spread. Proteomic profiling analysis showed a differential expression of proteins between the radiation-treated and control groups. When compared to the control group, which included 5,201 proteins, the radiation-treated cells contained a total of 5,342 proteins. Additionally, 5,007 proteins were found to be present in both groups. Significant differences were observed, especially in proteins related to cell proliferation, cellular organization, metabolism, stress response, signal transduction, and other biological processes. Drebrin, plectin, and filamin-A were selected for further analysis due to their considerable upregulation among the differentially expressed proteins. Microscopic investigation revealed the existence of invadopodia, actin-rich protrusions, in radioresistant cells compared to control HCC cells. F-actin staining demonstrated more prominent invadopodia structures in radioresistant specimens compared to the control group. Conclusion: This study suggests that prolonged radiation exposure influences multiple cellular and proteome level changes to promote aggressive tumorigenic and metastatic behavior of HCC cells.