Effects Of IFN-α2b And 5-FU On Cell Proliferation, Apoptosis And TRAIL Expression Of JEG-3 Cells

Objective:To observe the effects of IFN-α2b,5-FU and the combination of IFN-α2b plus 5-FU on cell proliferation,apoptosis and TRAIL expression of JEG-3 cells.To explore the feasibility and machanism of using IFN-α2b and IFN-α2b combined with 5-FU as therapeutic drugs for choriocarcinoma.Methods:(1)Inverted microscpe was used to observe the morphology changes of JEG-3 cells:JEG-3 cells were divided into 4 groups:the IFN-α2b group,which was treated by 5×10~4 IU/ml IFN-α2b;the 5-Fu group,which was treated by 0.5mg/ml 5-FU;the third group was treated with the combination of 5×10~4IU/ml IFN-α2b and 0.5mg/ml 5-FU;while the last group was not treated with either medicine but the complete medium.Each group was added to some DMSO with the same final concentration.48 hours later,we used inverted microscope to observe the morphology changes of cells and take photos.(2)CCK-8 assay was used to detect the growth inhibition ratio of JEG-3 cells:the concentrations of IFN-α2b in growth inhibition assays for IFN-α2b alone were 1×10~4IU/ml,5×10~4IU/ml,1×10~5IU/ml and 1×10~5IU/ml,and those of 5-FU were 0.1mg/ml,0.25 mg/ml,0.5mg/ml, and 1mg/ml.Both of the groups were treated for 24,48 and 72 hours respectively.5×10~4IU/ml IFN-α2b combined with 0.1mg/ml,0.25 mg/ml,0.5mg/ml,1mg/ml 5-FU respectively for 24 and 48 hours was the third group.The control group was added to complete medium.Each group contained DMSO with the same final concentration.Use Probit regression analysis to calculate IC50 of each drug at certain time.Figure out the value of "D" to see whether the combination of the two agents have cooperative effects.(3)Flow cytometry was used to investigate apoptotic rate and cell cycles of JEG-3 cells:the IFN-α2b group was treated with 5×10~4 IU/ml IFN-α2b,the 5-FU group was treated by 0.5mg/ml 5-FU,the combination group contained 5×10~4IU/ml IFN-α2b and 0.5mg/ml 5-FU,cells added to complete medium was set as the control group.Each group contained DMSO with the same final concentration.24 and 48 hours later,PI staining and flow cytometric techniques were used to detect apoptotic rate and cell cycles of JEG-3 cells respectively.(4)The expression of TRAIL in JEG-3 cells was detected by immunocytochemical SP method(Streptavidine-peroxidase conjugated method):the first group was treated with 5×10~4IU/ml IFN-α2b only,the second group was treated with 0.5mg/ml 5-FU only,the third group was treated by 5×10~4IU/ml IFN-α2b combined with 0.5mg/ml 5-FU,the control group was cultured by complete medium.The groups above were made to have the same final concentration of DMSO.We used hepatocellular carcinoma cells hepG-2 as positive control,while the negtive control was complete medium cultured JEG-3 cells.Immunocytochemical SP method was then applied to detect the expression of TRAIL 24 hour later.Results:(1)cell shrinkage,enlarged intercellular sapce and clear cell boudary were easy to be seen in medicine treated groups,especially in the drug combination group,and large quantity of dead suspended cells could be observed through inverted microscpe in the combination group.(2)The differences of the proliferative inhibition rates among various concentration groups of IFN-α2b were statistically significant.And the 5-FU groups were the same.Both IFN-α2b and 5-FU reduced cells growth in time-dependent and concentration-dependent manner.The combination of the two agents had cooperative effects on JEG-3 cell growth inhibition.(3)The cells in medicine treated groups had higher apoptitic rate than control group.IFN-α2b combined with 5-FU induced apoptosis of JEG-3 cells more effectively than single agent treated groups.The percentage of G0/G1 phase cells in medicine treated groups were increased,while which of cells in G2/M phase were decreased.The combined medicine treated group had higher percentage of cells in G0/G1 phase than single agent group.(4)The HSCORE of medicine treated groups were much higher than control group,and the group treated with the combination of the two medicines had stronger expression of TRAIL than any other group.Conclusions:1.Both IFN-α2b and 5-FU could inhibit cell proliferation of JEG-3cells,and their effects had time-dependent and concentration-dependent manner.The combination of IFN-α2b and 5-FU had synergistic effect on inhibition of JEG-3 cell proliferation.2.Both IFN-α2b and 5-FU could induce apoptosis of JEG-3 cells. IFN-α2b combined with 5-FU could increase the apoptotic rate of JEG-3 cells.3.Both IFN-α2b and 5-FU could induce TRAIL expression in JEG-3 cells.The combination of the two agents could promote the expression of TRAIL.4.We concluded that both IFN-α2b and 5-FU may inhibit cell growth through TRAIL induced apoptosis.The combination of the two drugs may have synergistic effect through this mechanism.