To investigate the impact of serine/threonine-protein kinase 1(AKT1)-mediated autophagy in hepatocellular carcinoma (HCC) cells on their sensitivity to ¹²⁵I seed irradiation.

① iProX database and STRING12.0 website were utilized to analyze the proteomic data of HCC before and after ¹²⁵I seed irradiation to explore the differentially expressed proteins and associated functional connections. Meanwhile, The Cancer Genomics Atlas (TCGA) database was employed to analyze the relationship between AKT1 expression level and the survival of HCC patients. ② Human HCC cell lines HUH7 and Hep3B were exposed to continuous irradiation from ¹²⁵I radioactive seeds with an initial apparent activity of 0.8 mCi per seed for approximately 120 h, accumulating a total dose of 8 Gy, while the control cells were cultured under normal condition for 120 h. ③ Autophagy inhibitor, chloroquine (CQ) and inducer, rapamycin (RAPA) were used to treat the HCC cells respectively to establish the CQ group and the RAPA group. The lentiviral transfection technique was employed to construct the HCC cells with overexpressed AKT1, namely the AKT1 group. The HCC cells treated in the same way were continuously irradiated with ¹²⁵I seeds for 120 h to construct the CQ+¹²⁵I group, the RAPA+¹²⁵I group, and the AKT1+¹²⁵I group. ④The changes in microtubule-associated protein light chain 3 (LC3), p62, AKT1 and p-AKT1 were detected by Western blotting. Cell immunofluorescence assay was employed to observe the expression of autophagy related proteins, such as LC3. The colony forming ability and apoptotic rate were detected with plate cloning assay and flow cytometry.

① Continuous irradiation with ¹²⁵I seeds resulted in decreased expression of p62 and increased ratio of LC3Ⅱ/LC3Ⅰ (P<0.05) when compared with the negative control (NC) group. Immunofluorescence assay revealed more green fluorescence spots of LC3. When compared with the ¹²⁵I group, the CQ + ¹²⁵I group had significantly increased expression of p62 (P<0.01), decreased ratio of LC3Ⅱ/LC3Ⅰ(P<0.01), lower apoptotic rate (P<0.01), and more colony formations (P<0.01). In contrast, the results in the RAPA + ¹²⁵I group were opposite to those of the CQ + ¹²⁵I group. ② Analysis on the iProX database showed that the expression of AKT1 was decreased in the irradiated group, and analysis on the TCGA database indicated that high expression of AKT1 predicted a poor prognosis for HCC patients (P<0.01). ③After irradiation with ¹²⁵I seeds, the expression of AKT1 at both the RNA and protein levels was decreased in the ¹²⁵I group (P<0.01). After overexpression of AKT1, the level of autophagy was decreased (P<0.05). Irradiation of HCC cells with overexpressed AKT1 using ¹²⁵I seeds could partially restore the level of autophagy. In the AKT1 + ¹²⁵I group, the expression of AKT1, pAKT1 and p62 were all decreased, and the ratio of LC3Ⅱ/LC3Ⅰ was increased than the AKT1 group (P<0.05). ④ The apoptotic rate of the AKT1 + ¹²⁵I group was lower than that of the ¹²⁵I group (P<0.05) and higher than that in the AKT1 group (P<0.05). In HUH7 cells, the clonogenic ability of the AKT1 + ¹²⁵I group was higher than that of the ¹²⁵I group (P<0.05). In Hep3B cells, the clonogenic ability of the AKT1 + ¹²⁵I group was higher than that of the ¹²⁵I group, and the clonogenic ability of the AKT1 group was higher than that of the NC group (P<0.05).

¹²⁵I seed irradiation induce lethal autophagy in HCC cells by reducing the expression of AKT1, providing a new theoretical basis for the implantation of ¹²⁵I radioactive seeds in the treatment of HCC.