Mechanism Of Flubendazole On Proliferation Inhibition And Pro-apoptosis Of Cholangiocarcinoma Cells

Objective1.To observe the effect of Flubendazole(FLU)on cell activity,clone proliferation ability,migration ability,invasion ability,cell cycle and apoptosis promotion of(QBC939 and RBE)cells;2.To explore whether the effect of FLU on cell cycle(QBC939 and RBE)was related to the inhibition of Cyclin B1 and the promotion of p53 protein expression.3.To explore whether the effect of FLU on promoting apoptosis(QBC939 and RBE)is related to the Bcl-2 /caspase signaling pathway.Methods1.Human bile duct cancer cells(QBC939 and RBE)were routinely subcultured,and the relative inhibitory rate(IR)and half inhibitory dose(IC50)of FLU at different concentrations were detected by CCK8 assay.The blank control group and the drug experimental group(low concentration 0.25 M,high concentration 0.5 M)were determined.2.Cell proliferation were detected by cloning proliferation test,cell migration was detected by scratch test,and cell invasion was detected by Transwell test.3.Percentage of G1,S and G2 phase cells in QBC939 and RBE cells were detected by BD FACSCalibur.Annexin V-FITC/PI staining was used to detect the apoptosis rates of the cells by flow cytometry.4.Protein expressions of Cyclin B1 and p53 were detected by western blotting.The Bcl-2 /caspase signaling pathway was also detected.Results1.The results of CCK8 showed that Flubendazole(FLU)can significantly reduce the cell viability of QBC939 and RBE cell lines.2.The colony counts of colony formation experiments showed that the number of colonies of QBC939 and RBE cell lines in FLU treatment group(0.25μm and 0.5μm)were significantly lower than those in the blank control group,and the difference was statistically significant(P<0.01).The higher the concentration,the fewer the number of colonies.3.The results of scratch test showed that the mobility of QBC939 and RBE cell lines in FLU treatment group(0.25μm and 0.5μm)was significantly lower than that in the control group(P<0.01),and the higher the concentration,the slower the migration rate.4.Transwell results showed that the FBC treatment group(0.25μm and0.5μm)QBC939 and RBE cell lines were significantly different from the control group(P<0.01),and the concentration increased,the number of transmembrane cells decreased.5.The cell cycle results of flow cytometry showed that QBC939 and RBE cells in the FLU-treated group(0.25μm and 0.5μm)were concentrated in the G2/M phase,and the percentage of cells in G2/M phase increased from 12.71±0.72 and 12.00±0.08 to 82.75±3.53 and79.66±0.29.the statistical difference was significant(P<0.01),and the higher the concentration,the higher the G2/M percentage.6.The apoptosis rate of QBC939 and RBE cells in the FLU-treated group(0.25 μm and 0.5 μm)was significantly higher than that in the vehicle control group by flow cytometry after staining with the Annexin V-FITC/PI apoptosis assay kit(P< 0.01),the percentage of apoptosis increased from 1.34±0.12 and 1.21±0.05 to 7.33±0.33 and 3.44±0.23,respectively,and the higher the concentration,the higher the apoptotic rate.7.Western blotting revealed that FLU significantly down-regulated the Cyclin B1 protein of QBC939 and RBE cells;FLU significantly up-regulated P53 protein expression.8.Western blotting showed that the expression of Bcl-2 was down-regulated,and the expression of caspase-3 and caspase-9 in downstream genes was further enhanced.ConclusionsFlubendazole(FLU)inhibits the activity,proliferation,invasion and migration of bile duct cancer cells and promotes apoptosis.It is related to the inhibition of Cyclin B1 and the simultaneous enhancement of p53 expression,as well as the Bcl-2/caspase signaling pathway,which promotes apoptosis.