Abstract:

Objective To discuss the effect of Fuzheng Ruanjian Anticancer Formula on the malignant biological behaviors of the hepatocellular carcinoma HepG2 cells by requlating protein kinase B（Akt）/murine double minute 2（MDM2）/P53 signaling pathway. Methods The HepG2 cells were treated with 0， 0.05， 0.10， 0.20， 0.40， 0.80， 1.60， 3.20， and 6.40 g·mL^-1 Fuzheng Ruanjian Anticancer Formula for 48 h. CCK-8 method was used to detect the survival rates of the HepG2 cells in various groups， and the concentrations of Fuzheng Ruanjian Anticancer Formula for the subsequent experiments were screened. The HepG2 cells were divided into control group， low dose of Fuzheng Ruanjian Anticancer Formula group （0.2 g·mL^-1）， medium dose of Fuzheng Ruanjian Anticancer Formula group （0.4 g·mL^-1）， high dose of Fuzheng Ruanjian Anticancer Formula group （0.8 g·mL^-1）， SC79 group （8 mg·L^-1 SC79）， and high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group （0.8 g·mL^-1 Fuzheng Ruijian Anticancer Formula+8 mg·L^-1 SC79）. CCK-8 method was used to detect the proliferation activities of the HepG2 cells in various groups； clone formation assay was used to detect the clone formation rates of the HepG2 cells in various groups； flow cytometry was used to detect the apoptotic rates of the HepG2 cells in various groups； Transwell chamber assay was used to detect the numbers of migration and invasion HepG2 cells in various groups； Western blotting method was used to detect the expression levels of proliferating cell nuclear antigen （PCNA）， cysteine aspartate specific proteinase （Caspase-3）， matrix metalloproteinase （MMP）-2， MMP-9， phosphorylated Akt （p-Akt）， phosphorylated MDM2 （p-MDM2）， and P53 proteins in the HepG2 cells in various groups. Results As the increasing of concentrations of Fuzheng Ruanjian Anticancer Formula （0， 0.05， 0.10， 0.20， 0.40， 0.80， 1.60， 3.20， and 6.40 g·mL^-1）， the surival rates of the HepG2 cells were gradually decreased （P<0.05）， and 0.2， 0.4， and 0.8 g·mL^-1 Fuzheng Ruanjian Anticancer Formula were selected for the subsequent experiments. The CCK-8 assay results showed that compared with control group， the proliferation activities of the HepG2 cells in low， medium， and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased （P<0.05）， in a dose-dependent manner， while the proliferation activity of the cells in SC79 group was significantly increased （P<0.05）. Compared with high dose of Fuzheng Ruanjian Anticancer Formula group， the proliferation activity of the HepG2 cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group was significantly increased （P<0.05）. The clone formation assay results showed that compared with control group， the clone formation rates of the HepG2 cells in low， medium， and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased （P<0.05） in a dose-dependent manner， while the clone formation rate of the cells in SC79 group was significantly increased （P<0.05）； compared with high dose of Fuzheng Ruanjian Anticancer Formula group， the clone formation rate of the cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group was significantly increased （P<0.05）. The flow cytometry results showed that compared with control group， the apoptotic rates of the HepG2 cells in low， medium， and high doses of Fuzheng Ruijian Anticancer Formula groups were significantly increased （P<0.05） in a dose-dependent manner， while the apoptotic rate of the cells in SC79 group was significantly decreased （P<0.05）； compared with high dose of Fuzheng Ruanjian Anticancer Formula group， the apoptotic rate of the cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group was significantly decreased（P<0.05）. The Transwell chamber assay results showed that compared with control group， the numbers of migration and invasion HepG2 cells in low， medium， and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased （P<0.05） in a dose-dependent manner， while the numbers of migration and invasion cells in SC79 group were significantly increased （P<0.05）； compared with high dose of Fuzheng Ruanjian Anticancer Formula group， the numbers of migration and invasion HepG2 cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group were significantly increased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of PCNA， MMP-2， MMP-9， p-Akt， and p-MDM2 proteins in the cells in low， medium， and high doses of Fuzheng Ruanjian Anticancer Formula groups were significantly decreased （P<0.05） in a dose-dependent manner， while the expression levels of Caspase-3 and P53 proteins were significantly increased （P<0.05） in a dose-dependent manner， while the expression levels of PCNA， MMP-2， MMP-9， p-Akt， and p-MDM2 proteins in the cells in SC79 group were significantly increased （P<0.05）， and the expression levels of Caspase-3 and P53 proteins were significantly decreased （P<0.05）； compared with high dose of Fuzheng Ruanjian Anticancer Formula group， the expression levels of PCNA， MMP-2， MMP-9， p-Akt， and p-MDM2 proteins in the cells in high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group were significantly increased （P<0.05）， while the expression levels of Caspase-3 and P53 proteins were significantly decreased （P<0.05）. Conclusion Fuzheng Ruanjian Anticancer Formula may inhibit the proliferation， migration， and invasion of the HepG2 cells and promote the apoptosis， and its mechanism may be related to suppressing the Akt/MDM2 signaling pathway and upregulating the P53 proteim expression.

Key words: Fuzheng Ruanjian Anticancer Formula, Protein kinase B, Mouse double minute 2, P53, Liver neoplasm, Cell proliferation, Apoptosis