Effect Of Mannan Peptide Combined With 5-Fu On The Proliferation And Apoptosis Of HepG2 Cells

Abstract:To investigate the influence of Mannan Peptide combined with 5-Fu on the proliferation and apoptosis of HepG2 cells. Moreover, to study the expression of Bcl-2 and Caspase-3 protein on HepG2 cells, to further explore the related mechanism.Method:Devided the HepG2 cells into four groups:control group,5-Fu group, Mannan Peptide group and drug group. The morphology of every group cells were observed under inverted phase contrast microscope. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to examine the cell cycle, proliferation index (PI) and apoptosis ratio of every group cells. The expressions of Bcl-2 and Caspase-3 protein in every group cells were analyzed by the immunocytochemical method.Result:(1) Microscope showed that cells alter round,contract and adherence rarefaction after exposed Mannan Peptide. After exposure that combined with 5-Fu, microscope showed cells presented different changes, including cell shrinkage, plasma membrane blebbing, nuclear condensation and formation of apoptotic bodies. (2) The inhibition effect of 5-Fu plus Mannan Peptide was significantly stronger than Mannan Peptide alone, and in a time and dose-dependent manner. (3) The flow cytometry showed that every group cells were treated with drug for up to 48 hour, compared with control group,5-Fu group and Mannan Peptide group, the cell percent of combination group was significantly increased in G0/G1 stage and decreased in S stage, cell proliferation index decreased, cell proliferation was arrested in G1/S stage. The flow cytometry used AV/PI method showed that the apoptosis ratio of combination group was 33.92±2.26% after exposure to drug 48 hours, compare with control group,5-Fu group and Mannan Peptide group, the apoptosis ratio of combination group was significantly increased (P<0.01).(4)Immunocytochemical analysis showed that after exposure to drug 48 hours, compare with control group, the expression of Bcl-2 protein in 5-Fu group cells decrease and Caspase-3 protein increase(P<0.01).The expression of Bcl-2 protein in Mannan Peptide group cells also decrease, but the expression of Caspase-3 protein have no obvious changes. Compare with control group and 5-Fu group, the combination of Mannan Peptide and 5-Fu could significantly increase Caspase-3 protein level and decrease Bcl-2 protein level (P<0.01).Conclusions:(1) 5-Fu can inhibit the increase of HepG2 cell and the inhibition was enhanced by combined with Mannan Peptide of different concentration. (2) The combination of Mannan Peptide and 5-Fu can synergetically inhibit the proliferation and induce apoptosis in human hepatoma cell lines HepG2, and the cells were markedly arrested in G1/S stage. (3) Bcl-2 and Caspase-3 protein may play an very important role in the process of Hep2 cell growth and apoptosis induction by the combination of Mannan Peptide and 5-Fu.