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Prednisolone is an anti-inflammatory drug used to treat a number of conditions, including liver disease and cancer. Numerous studies have demonstrated that glucocorticoids such as prednisolone modified by ionizing radiation can promote anticancer activity in cancer cells. To the best of our knowledge, however, the effect of ionizing radiation on prednisolone structure and cancer cells has not yet been identified. The present study created a novel prednisolone derivative using γ-irradiation, and its anticancer properties were investigated in liver cancer cells. The present study confirmed the structure of the new prednisolone derivative using liquid chromatogram-mass spectrometry. MTT assays determined the cytotoxic effects of γ-irradiated (IR)-prednisolone in liver cancer cells. Flow cytometry analysis evaluated apoptosis, mitochondrial membrane potential and cell cycle distribution. Western blotting was used to analyze the proteins associated with apoptosis. The chromatogram profile revealed that IR-prednisolone produced a number of peaks compared with the single peak of the original prednisolone. In contrast to prednisolone, the MTT results showed that IR-prednisolone significantly prevented the growth of liver cancer cells. IR-prednisolone promoted apoptosis and arrested the cell cycle at the G0/G1 stage in Huh7 cells. IR-prednisolone also altered the mitochondrial membrane potential and activated caspase-associated proteins, which activated the intrinsic apoptotic signaling pathway. In conclusion, IR-prednisolone promoted anticancer effects in liver cancer cells via apoptosis activation. The present study demonstrated that IR-prednisolone may be a potential anticancer agent against liver cancer, although specific molecules have yet to be identified. [ABSTRACT FROM AUTHOR]