Ataxia telangiectasia and Rad3-related protein (ATR) plays a crucial role in cancer and has become a promising target for cancer therapy. Daphnegiravone D (DGD), which could induce apoptosis and oxidative stress in hepatocellular carcinoma (HCC) cells, but the detailed target protein was still unclear. The study provided that the possible target of DGD against HCC cells was determined by isobaric labels for relative and absolute quantification (iTRAQ) assay. In all changed proteins the fold change of ATR was particularly significant. The results from GO, KEGG and PPI analysis showed that DNA damage, cell cycle, apoptosis, DNA repair related pathways changed and ATR was exactly related to them. Moreover, the mRNA and protein of ATR were both decreased in a concentration-dependent manner, and the results of molecular docking also verified the binding. Additionally, cellular thermal shift assay (CETSA) suggested that DGD could directly target at ATR protein. Furthermore, the knockdown of ATR could increase apoptosis and reactive oxygen species (ROS) which induced by DGD. Since ATR inhibitors were generally used in combination with chemotherapy drugs (especially DNA damage drugs) in clinical trials, we investigated the combined application of DGD and oxaliplatin. The results showed that DGD combined with OXA also increased the apoptosis and ROS production of Hep3B cells over either drug alone. Taken together, this study revealed that DGD targeting ATR could be a promising therapeutic strategy for the treatment of liver cancer.
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