Abstract:

Objective To discuss the inhibitory effect of the bradykinin B1 receptor （B1R） antagonist ELN441958 on the growth of hepatocellular carcinoma HepG2 cells and its regulatory mechanism on the protein kinase B （Akt）/forkhead box O3a （FoxO3a） signaling pathway， and to clarify the anti-tumor effect of the B1R antagonist ELN441958 in liver cancer. Methods After the HepG2 cells were treated with different doses （0， 2.5， 5.0， 10.0， and 20.0 μmol·L^-1） of ELN441958 for 24， 48， and 72 h， 0 μmol·L^-1 ELN441958 was regared as control group. CCK-8 method was used to detect the inhibitory rate of proliferation of the cells in various groups. The HepG2 cells were divided into control group， 5.0 μmol·L^-1 ELN441958 group， 10.0 μmol·L^-1 ELN441958 group， and 15.0 μmol·L^-1 ELN441958 group； 5-ethynyl-2'-deoxyuridine （EdU） staining was used to detect the EdU positive cell rates of the HepG2 cells in various groups； flow cytometry was used to detect the apoptotic rates of the cells in various groups and the proportions of the cells at different cell cycles； immunofluorescence method was used to detect the expression level of B1R protein in the HepG2 cells in various groups； Western blotting method was used to detect the expression levels of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cyclin D1 （Cyclin D1）， cyclin-dependent kinase 4 （CDK4）， B1R， Akt， phosphorylated Akt （p-Akt）， FoxO3a， and phosphorylated FoxO3a （p-FoxO3a） proteins in the HepG2 cells in various groups. The HepG2 cells were divided into control group， B1R agonist des-Arg9-BK （1.0 μmol·L^-1 des-Arg9-BK） group， and BK+ELN441958 group； Western blotting method was used to detect the expression levels of Bcl-2， Bax， Cyclin D1， CDK4， Akt， p-Akt， FoxO3a， and p-FoxO3a proteins in the HepG2 cells in various groups. The HepG2 cells were divided into control group， Akt agonist SC79 group， and SC79+ELN441958 group； Western blotting method was used to detect the expression levels of Bcl-2， Bax， Cyclin D1， CDK4， Akt， p-Akt， FoxO3a， and p-FoxO3a proteins in the HepG2 cells in various groups. Results The CCK-8 assay results showed that compared with control group， when the action time was the same， the inhibitory rate of proliferation of the HepG2 cells was significantly increased with the increase of ELN441958 dose （P<0.05 or P<0.01）； when the dose of ELN441958 was the same， the inhibitory rate of proliferation of the HepG2 cells was significantly increased with the extension of action time （P<0.05 or P<0.01）. The half maximal inhibitory concentration （IC₅₀） values of ELN441958 at 24， 48， and 72 h were （21.4±1.1）， （10.5±0.3）， and （3.2±0.3） μmol·L?1， respectively. Compared with control group， the EdU positive cell rates of the HepG2 cells in 5.0， 10.0， and 15.0 μmol·L?1 ELN441958 groups were significantly decreased （P<0.05 or P<0.01）. The flow cytometry results showed that compared with control group， the apoptotic rates of the HepG2 cells in different doses of ELN441958 groups were significantly increased （P<0.05 or P<0.01）， and the percentages of the cells at G0/G1 phase were significantly increased （P<0.05 or P<0.01）. The Western blotting results showed that compared with control group， the； the expression levels of Bcl-2， CDK4， and Cyclin D1 proteins in the HepG2 cells in 5.0， 10.0 and 15.0 μmol·L^-1 ELN441958 groups were significantly decreased （P<0.01）， and the expression level of Bax protein was significantly increased （P<0.05 or P<0.01）. The immunofluorescence results showed that compared with control group， the fluorescence intensity of B1R in the HepG2 cells in 15.0 μmol·L^-1 ELN441958 group was significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression level of B1R， p-Akt， and p-FoxO3a proteins in the HepG2 cells in 10.0 and 15.0 μmol·L^-1 ELN441958 groups were significantly decreased （P<0.01）； the expressions p-Akt， and p-FoxO3a proteins were decreased （P<0.05 or P<0.01） compared with B1R agonist des-Arg9-BK group， the expression levels of Bcl-2， CDK4， Cyclin D1， B1R， p-Akt， and p-FoxO3a proteins in the HepG2 cells in BK+ELN441958 group were significantly decreased （P<0.01）， and the expression level of Bax protein was significantly increased （P<0.01）； compared with SC79 group， the expression levels of Bcl-2， CDK4， Cyclin D1， B1R， p-Akt， and p-FoxO3a proteins in the HepG2 cells in SC79+ELN441958 group were significantly decreased （P<0.01）， and the expression level of Bax protein was significantly increased （P<0.01）. Conclusion The B1R antagonist ELN441958 can inhibit the proliferation of HepG2 cells， and induce the apoptosis and G₀/G₁ phase cell cycle arrest of HepG2 cells， and its mechanism may be related to the regulation of Akt/FoxO3a signaling axis.

Key words: ELN441958, Liver neoplasms, Protein kinase B, Forkhead box O3a, Apoptosis, Cell cycle