Bufalin Induces Apoptosis Of Human Glioma Stem Cells And Improves The Sensitivity Of Temozolomide

Purpose: To clarify the inhibition of cell growth and proliferation of bufalin on glioma cells,the suppression of human glioma stem cells,inducing apoptosis and mechanism of human glioma stem cells,and to explore the effect of bufalin on the sensitivity of temozolomide.Material and method:Different concentrations of bufalin were added to human glioma U87 MG,LN-229 cells and hepatocellular LO2,the cell survival rate of bufalin on after 24,48 and 72 hours was examined by MTT.Colony formation observed the effect of different concentration of bufalin on glioma cells.Proliferation.The morphology of the glioma stem-like cells were observed by an inverted optical microscope after the glioma cells cultured in low adhesion cell culture plates and serum-free medium.The morphological changes,the volume and quantity of stem cells were observed after the treatment of bufalin.PI fluorescence staining was used to observe the death of glioma stem-like cells after the treatment of bufalin.Flow cytometry was used to detect the apoptosis of glioma stem-like cells.Western blot was used to detect the expression level of apoptosis related proteins in glioma stem-like cells.The proliferation and apoptosis of glioma stem-like cells were detected by MTT,flow cytometry and western blot after bufalin and temozolomide alone and in combination.Results:1.MTT assay showed that bufalin treatment led the cell viability of U87 MG and LN-229 cells a significantly decreased with a dose and time dependent manner.The half-maximal inhibitory concentration(IC50)values of U87 MG cells were 85.3,34.5 and 16.7 n M for 24,48 and 72 h.Similarly,the IC50 values of LN-229 cells were 184.2,96.1 and 32.7 n M for 24,48 and 72 h.The inhibitory effect of bufalin on hepatocellular LO2 was not obvious.The number of clones of U87 MG and LN-229 cells was lower than that in the control group,and 20 n M bufalin was significantly less than 10 n M and the control group.And in the 20 n M group of U87 MG cells,cell clones were almost invisible.2.U87 MG and LN-229 cells can be formed into spheroids and the expression of stem cell marker protein is significantly higher than that of normal cells.The form of glioma stem-like cells spheroids after the treatment of bufalin were significantly smaller than those in the control group,and the number of spheroids was significantly reduced.After the treatment of bufalin,the glioma stem-like cell spheroids became significantly looser and even had cell debris around them.PI staining showed that glioma stem-like cells died more with the concentration of bufalin increased.The flow cytometry was used to detect the glioma stem-like cells treated by bufalin,and found that the apoptosis ratio was significantly higher than that in the control group,and there is a dose-time dependence.Western blot showed thet the expression of apoptotic protein was increased with the increase of bufalin's concentrate.3.MTT assay showed that the survival rate of glioma stem-like cells after the treatment of combined bufalin and temozolomide was significantly lower than that of the single drug group.The cell apoptosis rate of the combined drug group was also higher than that of the single drug group.Western blot showed that the expression of apoptotic protein p-bad and Cleaved caspase-9 was increased,and the expression of anti-apoptosis protein bcl-2 and bcl-xl was significantly decreased.Conclusion:1.Bufalin inhibits the growth and proliferation of glioma cells.Bufalin has almost no toxic side effects on hepatocellular LO2.2.Bufalin inhibits glioma stem-like cells' activity and kills glioma stem-like cells.Bufalin induces apoptosis of glioma stem-like cells.3.Bufalin increased the sensitivity of glioma stem cells to temozolomide,and the mechanism may be to up-regulate the expression of pro-apoptotic protein and down-regulate the expression of anti-apoptotic protein.