3095 - Caffeine-Enhanced Radiation-Mediated G2/M Phase Arrest through ATM/?H2AX/Chk2 Signaling in Human Hepatoma Cells

Purpose/Objective(s): Radiotherapy is a cornerstone in the treatment of hepatocellular carcinoma (HCC), but its efficacy is often limited by intrinsic radioresistance. Caffeine, a known modulator of DNA damage response pathways, has been suggested to enhance radiation sensitivity in various cancers. This study aims to investigate whether caffeine can potentiate radiation-induced DNA damage, promote G2/M phase arrest, and increase apoptosis in human hepatoma cells through the ATM/?H2AX/Chk2 pathway.

Materials/Methods: Human hepatoma Huh7 cells were treated with caffeine at concentrations of 2.5, 5, and 10 mM and exposed to 8 Gy of ionizing radiation. Cells were harvested at 24 and 48 hours post-treatment. Cell viability was measured using MTT and colony formation assays. Flow cytometry was performed to analyze cell cycle distribution, while apoptosis was assessed using TUNEL assay and caspase-3 activation analysis. DNA damage levels were quantified by comet assay and ?H2AX immunofluorescence staining. Western blot analysis was conducted to evaluate the activation of key DNA damage response proteins, including ATM, ?H2AX, and Chk2.

Results: Caffeine significantly enhanced the radiosensitivity of human hepatoma cells in a dose- and time-dependent manner. MTT and colony formation assays showed a pronounced decrease in cell viability following caffeine pre-treatment in irradiated cells, with the most significant reduction observed in the 10 mM caffeine + IR group. Flow cytometry analysis revealed a marked accumulation of cells in the G2/M phase, indicating enhanced checkpoint activation and prolonged cell cycle arrest. TUNEL staining and caspase-3 activation analysis demonstrated a substantial increase in apoptosis in caffeine-pretreated cells post-irradiation. DNA damage assessments via comet assay and ?H2AX immunofluorescence staining indicated increased levels of DNA fragmentation and persistent ?H2AX foci formation in the caffeine + IR group, suggesting impaired DNA repair. Western blot analysis confirmed the activation of the ATM/?H2AX/Chk2 pathway, with increased phosphorylation of ATM, ?H2AX, and Chk2 in caffeine-pretreated irradiated cells. These findings suggest that caffeine enhances radiation-induced cytotoxicity by reinforcing DNA damage signaling, delaying DNA repair, and driving apoptosis through G2/M checkpoint activation.

Conclusion: This study provides compelling evidence that caffeine acts as a potent radiosensitizer in human hepatoma cells by augmenting radiation-induced DNA damage and apoptosis through ATM/?H2AX/Chk2 pathway activation. The results highlight the potential of caffeine as an adjunct to radiotherapy for hepatocellular carcinoma. Given these promising findings, further in vivo studies and clinical evaluations are warranted to explore its translational potential.