Effects Of Berberine On Growth Inhibition And Apoptosis Induction Of Hepatocellular Carcinoma Cells: An Experimental Study

Objective: To study the expression of p53,CyclinDl and Caspase-3 protein on HepG2 cells. Moreover, to investigate the growth inhibition and apoptosis induction of berberine on HepG2 cells and to further explore its possible antitumor mechanism.Methods: (l)The cell culture method was used to prepare the coverslips of HepG2 cells. The expression and location of p53 protein, CyclinDl and Caspase-3 was observed by immunocellularchemical staining.(2)There are three groups in this experimental study , i.e. experimental group, positive-control group and negative-control group. In experimental group, HepG2 cells were treated by berberine of various concentrations from 0 to 72h;in positive-control group, HepG2 cells were treated by 5-Fu of various concentrations for the same time;in negative-control group, HepG2 cells were only treated by PBS. The inhibitive rate of HepG2 cells were tested by MTT analysis. The immunocellularchemical staining methods were used to observe the expression of p53 protein, CyclinD1 and Caspase-3 on HepG2 cells. The HepG2 cells apoptosis were determined with flow cytometry and agarose gel electrophoresis. Meanwhile, the same indexes were studied on human Chang liver cells with the methods mentioned above.Results: (1) Both of p53 and CyclinDl protein were prominently expressed in cellular nucleus. The Caspase-3 was hardly expressed in HepG2 cells. (2) MTT analysis showed that, compared with control group, berberine could inhibit the growth of HepG2 cells dependent upon the berberine concentration ranged from 1.0μmol/L to 100μmol/L and treated time from 12 to 72 hours. When HepG2 cells was treated by 100μmol/L berberine for 72 hours, the inhibiting rate(39.2%) was the highest. (3) According to the results of immunocellularchemical staining, theexpressions of p53 and CyclinDl decreased significantly following treatment by berberine and 5-Fu, in contrast to the increased expression of Caspase-3. There was no significant difference between the groups treated by berberine and 5-Fu in these expressions on HepG2 cells. (4) Flow cytometry showed that berberine could prevent the cell-cycle of HepG2 cell from progressing with a notable arrest of G0/G1-phase-cells along with a decreased number of S-phase-cells. Compared with negative control group (6.3%), the apoptotic rate(34.6%) of HepG2 cells in experimental group was significantly increased. Agarose gel electrophoresis testing displayed a typical apoptosis ladder strip in the experimental group.Conclusions: (1) P53 and CyclinDl protein were highly expressed in the human HepG2 cells and their location were on the cell's nucleus. However, the expression of Caspase-3 protein was very weak in the cells. (2) Berberine can significantly inhibit the growth of HepG2 cells in a limited range of concentrations and induce HepG2 cells to getting apoptosis. Also, it can be found that ,by the flow cytometry analysis, the HepG2 cells were markedly arrested in G0/G1 phase.(3)Caspase-3 and CyclinDl play a very important role in the process of HepG2 cell growth inhibition and apoptosis induction by berberine treatment.