| Objective:To reveal the synergistic anti-tumor effects of proliferation and growth and mechanisms of recombinant human endostatin (Endostar, YH-16)combined with cytotoxic drug 5-fluorouracil(5-FU).Methods:After culture of high-invasive choricarcinoma cells (JEG-3), we observed the anti-tumor proliferation and apoptosis effects of YH-16 alone and combined with 5-Fu In JEG-3 cell, using the methods including MTT and Flow cytometry respectively;The expression of vascular endothelial growth factor-B (VEGF-B) was analyzed by Reverse-tanscriptase-polymerase chainreaction (RT-PCR).Results:(1)We observed the cell growth inhibition rate of JEG-3 cells by using MTT:the concentrations of YH-16 in growth inhibition assays for YH-16 alone were 50ug/ml,100ug/ml,200ug/ml and 400ug/ml, and those of 5-FU were O.lmg/ml,0.25 mg/ml,0.5mg/ml and 1 mg/ ml. All of the groups were treated for 24,48 and 72 hours respectively. In the same concentration, with the time of various drugs action, the inhibition of growth rates were increased, the differences between the various concentration groups were statistically significant, P<0.05. At the same time, as the drug concentration increased, YH-16 inhibit growth of JEG-3 cells on time-dependent in a certain range, the growth inhibition rate of 200ug/ml YH-16 group is stronger than 50ug /ml,100ug/ml groups, but weaker than 400ug/ml groups, the difference between them was statistically significant, P<0.05.5-FU groups reduced cells growth in time-dependent and concentration-dependent manner. At the same time, as the drug concentration increased, the growth inhibition rate of JEG-3 cells were stronger, the difference between them was statistically significant, P <0.05. Cell growth inhibition rate had a relationship with concentration and reaction time of 5-Fu groups,P<0.05. The cell growth inhibition rate of YH-16 combined with 5-FU interaction of JEG-3 cells was much higher than that of corresponding concentration of YH-16 and 5-FU group, after 24,48 and 72 hours. The differences are statistically significance. P<0.05. With the 5-FU concentration, the cell growth inhibition rate increased. The combination of the two agents had synergistic effects on JEG-3 cell growth inhibition.(2)By using Flow Cytometry, we observed the apoptosis and cell-cycle of YH-16,5-FU alone and combined on the JEG-3 cells:the role of the apoptosis of drug intervention group was significantly higher than the control group after 48 hours, P<0.05. The apoptosis rate of the combination group were significantly higher than 5-FU and YH-16 single-drug group, there was significant difference between them, P<0.05. After 48 hours, the percentage of G0/G1 phase cells of different drugs in the intervention group was more than the control group, the percentage of G2/M phase cell was less than the control group, P<0.05. the percentage of G0/G1 phase cells of YH-16 combined with 5-FU group were more than the single drug group, P<0.05.(3) To detect the expression level of VEGF-B of JEG-3 cells of YH-16,5-FU single group and combined intervention group by RT-PCR: comparison of the expression of VEGF-B mRNA within JEG-3 cells, the various drug intervention group were less than the control group, combined treatment group was lower than the other groups, there was significant difference between them, P<0.05.Conclusions:1. YH-16 can directly inhibit the growth of JEG-3 cells, arreste JEG-3 cells in the G0/G1 phase, inhibit JEG-3 cell proliferation, induce apoptosis, the mechanism may be related to reduced the expression of VEGF-B of JEG-3 cells.2. YH-16 combined with 5-Fu had synergistic effects on JEG-3 cell growth inhibition and induce apoptosis, it may enhance the curative effect of 5-Fu treatment chemotherapy. |
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