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.