The Inhibitory Effect And Molecular Mechanism Of Paris Polyphylla Monomer PP-26 On HepG2 Cells

Objective:To explore the molecular mechanism of PP-26 in regards to the proliferatory inhibition effects on Hepatocellular carcinoma cells in vitro and provide some experimental supports for the dedevelopment and utilization of Paris polyphylla monomer PP-26. Methods:1. The MTT assay was used to analyze the proliferative inhibition effect of PP-26 on L02, Hep G2, SW 1990 and LNCa P cells. We also used MTT assay to detect the growth inhibition effect of IC20 concentration of PP-26 combined with 5-FU on Hep G2 cells.2. Hoechst 33258 staining assay was used to detect the cytomorphological changes of Hep G2 cells induced by PP-26. Apoptosis was also assessed through a fluorescein Annexin V-FITC/PI double staining assay on Hep G2 cells.3. Flow cytometry with a PI staining assay was used to analyze the effects of PP-26 on the cell cycle distribution on Hep G2 cells.4. Western blotting analysis was used to detect the expression of cell cycle related proteins and apoptosis associated proteins including Akt signal pathway related proteins induced by PP-26 on Hep G2 cells. Result:1. MTT assay showed that the proliferation of Hep G2 cells, SW1990 cells, LNCa P cells was significantly inhibited with increasing PP-26 concentrations which showed little inhibitory effect on L02 cells. These results indicate that the numbers of Hep G2 cells, SW1990 cells, LNCa P cells were significantly reduced with PP-26 treatment in a doseand time-dependent manner. The half-maximal inhibitory concentration(IC50) value of PP-26 on Hep G2 cells, SW1990 cells and LNCa P cells for 24 h were 6.94±0.72,2.53±0.14,4.20±0.37 μmol/L, and on L02 cells, Hep G2 cells, SW1990 cells and LNCa P cells for 48 h were 6.98±0.99,1.91±0.45,1.90±0.07,2.45±0.11 μmol/L, and on L02 cells, Hep G2 cells, SW1990 cells and LNCa P cells for 72 h were 4.22±0.23,1.54±0.06,1.57±0.09,2.01±0.07 μmol/L, which indicated less cytotoxicity on L02 cells than those cancer cells. Our results also showed that the chemo-therapeutic effects of 5-FU on Hep G2 cells were enhanced when combined with IC20 concentration of PP-26, and the drug sensitivity was increased.2. The Hoechst 33258 staining showed that Hep G2 cells began to exhibit apoptotic characteristics after treatment with 3.2 μmol/L and 6.4 μmol/L PP-26 for 24 h, such as cell shrinkage, nuclear condensation and fragmentation and apoptotic bodies. The changes of cell morphology demonstrated that cell apoptosis occurred. Annexin V-FITC/PI staining showed that the percentage of apoptotic cells were increased with increasing concentrations of PP-26 on Hep G2 cells.3. Flow cytometry with a PI staining assay showed that the population of cells in the G2 phase increased with increasing concentrations of PP-26. These results indicate that cell cycle distribution was significantly arrested in the G2 phases upon PP-26 treatment. We also assessed the effect of PP-26 on cell cycle regulatory proteins by western blot analysis. The expression of cyclin D1, cycclin B1 and CDK4 was significantly reduced after treatment with increasing concentrations of PP-26 on Hep G2 cells for 24 h. However, the expression levels of Myt1、p21、p-cdc2(Tyr15)were increased. These results indicate that PP-26 induces G2 cell cycle arrest in Hep G2 cells.4. Western blotting assay showed that apoptosis-related proteins such as caspase 9、caspase 3、PARP increased after treatment with increasing concentrations of PP-26 on Hep G2 cells for 24 h. PP-26 suppressed the expression of anti-apoptotic Bcl-2, Mcl-1 and Bcl-x L and increased the expression of pro-apoptotic Bax. We examined the Akt signaling pathway, and our results showed that increasing PP-26 concentrations downregulated Phospho-Akt in a dose-dependent manner without affecting the total amount of Akt. The phosphorylation of downstream protein of Akt, p-GSK-3β and p-Foxo3 were also downregulated. The results demonstrated that Akt signal pathway was inhibited by PP-26 in Hep G2 cells.Conclusion:1. PP-26 inhibited the proliferation of Hep G2, SW1990 and LNCa P cells significantly while had little inhibitory effect on L02 cells. The chemo-therapeutic effects of 5-FU on Hep G2 cells were enhanced when combined with IC20 concentration of PP-26.2. PP-26 induces a G2 phase arrest in Hep G2 cells.3. PP-26 induces mitochondrial pathway-mediated cellular apoptosis in Hep G2 cells via inhibition of the Akt signaling pathway.