Time and dose effects of AKT inhibitor GSK2141795 on apoptosis of human hepatocellular carcinoma cell line Huh7 / 第二军医大学学报

ABSTRACT

Objective To explore the time and dose effects of AKT (a kind of protein serine/threonine kinase) inhibitor GSK2141795 on the apoptosis of human hepatocellular cell line Huh7. Methods Huh7 cells were treated with GSK2141795 at the concentrations of 0, 0.3, 1, 3, 10 and 30 µmol/L for 24 h. A concentration of 10 µmol/L GSK2141795 was selected to treat Huh7 cells for 0, 2, 6, 12, 24 and 48 h. The protein expression levels of AKT and phosphorylated AKTS473 (p-AKTS473) were determined by Western blotting and cell apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins (Bad, Bcl-2 and Caspase-9) were measured by qPCR and Western blotting. Results With the increase of GSK2141795 concentration, AKT protein level in Huh7 cells was gradually decreased and the p-AKTS473 protein level was gradually increased within the range of 0-10 µmol/L. With the prolongation of GSK2141795 treatment time, the AKT protein level was gradually decreased and the p-AKTS473 protein level was gradually increased within the range of 0-24 h. At 48 h of treatment, the AKT protein and p-AKTS473 protein expression levels were increased compared with 0 h. With the increase of GSK2141795 concentration and treatment time, the proportion of apoptotic cells was gradually increased, the expression levels of apoptotic molecules Bad and Caspase-9 were gradually increased, and the expression level of apoptotic antagonist Bcl-2 was gradually decreased. Conclusion AKT inhibitor GSK2141795 can effectively inhibit AKT protein expression, and induce apoptosis of Huh7 cells through Bad-Bcl-2 pathway in a dose-and time-dependent manner. In addition, the expression level of AKT protein in Huh7 cells can increase again after long-term stimulation by GSK2141795, suggesting the existence of a negative feedback signal loop.