Gallic acid enhances radiosensitivity in pancreatic ductal adenocarcinoma models in association with CRL4-DCAF1-related CDK1 destabilization and ROS-associated DNA damage amplification.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains poorly responsive to radiotherapy (RT). This study examined whether gallic acid (GA) enhances PDAC radiosensitivity in association with CDK1 destabilization and ROS-associated DNA damage amplification. METHODS: GEO transcriptomic datasets and TCGA-PAAD clinical data were integrated to prioritize CDK1, followed by tissue and cell-line validation. Low-dose clonogenic experiments assessed radiosensitization at 50 μM under limited short-term cytotoxicity, whereas mechanistic interrogation was performed predominantly at 150 μM and should therefore be interpreted as exposure-specific evidence. CDK1 stability and ubiquitination were evaluated using cellular assays and a purified cell-free CRL4-DCAF1 reconstitution. Functional relevance was tested using complete CDK1-overexpression and DCAF1 knockdown/rescue matrices with RT-only controls, antioxidant interventions and orthotopic PANC-1-luc xenografts. RESULTS: CDK1 was consistently upregulated in three GEO PDAC datasets. At 50 μM, GA shifted radiation dose-survival curves in PANC-1 and MIA PaCa-2 cells, with SER10 values of 1.35 (95% CI, 1.23-1.60) and 1.39 (95% CI, 1.31-1.52). In predominantly 150 μM cellular mechanistic assays, GA+RT increased oxidative stress and DNA damage-associated responses while reducing Rad51-associated repair foci. In the purified system, component-dependent CDK1 ubiquitination was observed; 50 μM GA increased total and K48(Ub) signals, whereas 150 μM increased only the K48(Ub) signal after correction. ROS elevation preceded significant CDK1 loss, and NAC or MitoTEMPO attenuated ROS, CDK1 loss, γ-H2AX and K48(Ub). Complete genetic matrices showed genotype-by-GA interactions without material RT-only survival effects. In vivo, GA+RT suppressed orthotopic tumor progression, prolonged survival and altered CDK1-related pharmacodynamic markers without overt short-term toxicity under the tested schedule. CONCLUSIONS: Across the tested PDAC models, GA was associated with enhanced radiation response and ROS-linked, CRL4-DCAF1-related CDK1 ubiquitination and destabilization. Purified reconstitution established CDK1 substrate competence in the defined ligase system without assigning direct molecular-glue activity to GA.