Effect And Mechanism Of Isoliquiritigenin On Cell Proliferation And Differentiation Of SHG44 Human Brain Glioma Stem Cells

Objective: To investigate the effect and menchanism of Isoliquiritigenin on cell proliferation and differentiation of SHG44 human glioma stem cells, in order to provide some experimental basis for the application of Isoliquiritigenin on the treatment of the human brain glioma.Methods:(1) SHG44 human brain glioma stem cells were cultured in serum-free medium supplemented with EGF、FGF and B27, and in the medium of Culture dish, culture bottle and suspension cell culture plate. CD133﹑Nestin and Bcl-2 were detected using immunofluorescence analysis. The morphology, size and number of the stem cell balls were compared under the inverted microscope, and the morphology of stem cells was observed by scanning electron microscope.(2) After comparative study, SHG44 human brain glioma stem cells were cultured in serum-free medium supplemented with EGF、FGF and B27, used the suspension cell culture plate. The experiment included control group(including equivalent amount of serum-free DMEM / F12 medium with DMSO), different concentration of isoliquiritin group(5~160 μmol/L). The Inhibition of proliferation of the cell for 12, 24, 48 and 72 hours cells were detected by CCK-8 method. And the formation of glioma stem cell spheres with the seventh day under the drug action.(3) After screening by concentration and time, The experiment included control group(including equivalent amount of serum-free DMEM / F12 medium with DMSO), different concentration of isoliquiritin group(10~160 μmol/L),blocking agent group(DAPT 2μmol/L),isoliquiritin + blocking agent group(10~160 μmol/L + 2.0 μmol/L DAPT). Glial fibrillary acidic protein(GFAP),beta tubulin III and nestin were used for observing the expression situation of differentiation marker matter after 24~72 hours of the effect of different isoliquiritin by immunofluorescence staining. Nestin, GFAP and beta-Tubulin III were detected in 72 hours after the effect of different isoliquiritin by Western blot.The cell cycles of 72 hours cells treatmented with different isoliquiritin were detected by flow cytometry. Notch1, RBP-JK and Hes1 of 72 hours cells treatmented with different isoliquiritin and blocking agent were detected by Real-time PCR.Results:(1) In the serum-free medium, a minority of SHG44 human glioma stem cells showed the capacity of self-renew and proliferation, these cells generated free-floating neurosphere-like, the espression of CD133 and nestin and Bcl-2 were detected in human glioma stem cells. By statistical analysis, the glioma stem cell balls, which were purified by the suspension culture plate culture, were larger and more(P<0.05) than those which were purified by Culture dish and culture bottle. For the suspension culture plate, the glioma stem cell balls were larger and more(P<0.05), and were more regular, with the increase of the number of purification. Under the inverted microscope, the glioma stem cells were round shape, and the scanning electron microscope showed spherical shape, which showed longer spindle shape and more fine projections, and Cell and cell through the antenna communication.(2) For 12-48 hours, along with the isoliquiritigenin concentration increase, The stronger the cell activity(P<0.05).For 72 hours, with concentration increases, the inhibitory effect is stronger(P<0.05). Every other day dosing until seventh day: By statistical analysis, the number of glioma stem cells decreased and the diameter decreased(compared with the control group), and P <0.05; When the concentration of isoliquiritin was less than 40 μmol/L, compared with 160 μmol/L, the number and diameter of stem cells were statistically significant(P<0.05); When the concentration of isoliquiritin was greater than 40 μmol/L, compared with 160 μmol/L,the number of stem cells was not statistically significant(P > 0.05).(3) In control group, there was no obvious differentiation of glioma stem cells. In the 48~24 hours, the more differentiated cells were increased with the concentration, and the stem cells decreased. After 72 hours, with the increase of the concentration, differentiated cells and stem cells were decreased. The results of Western blot: after 72 hours when the effect of isoliquiritin: Compared with the control group, with isoliquiritin concentration increased the expression of Nestin protein decreased gradually(P<0.05); Compared with the control group, 10,40,160 μmol/L group, the expression level of GFAP protein was significantly up-regulated(P<0.05),and the 40 μmol/L group, the expression of GFAP protein concentration were higher than other groups(P < 0.05); Compared with the control group, 10,40,160 μmol/L group beta-Tubulin Ⅲ protein expression levels were up-regulated(P < 0.05),and 10 μmol/L group III beta-Tubulin protein expression group was higher than other concentration(P<0.05). Flow cytometry results: after 72 hours when the effect of isoliquiritin, Compared with the control group, with isoliquiritin concentration increased the proportion of cells in G0/G1 phase increased significantly(P<0.05). The results of PCR Real-time: after Notch1 pathway blocker effect, Compared with the control group, different isoliquiritin group and blocker group, the expression of Notch1,RBP-JK and Hes1 gens decreased significantly(P<0.05); Compared with isoliquiritigenin group, the expression of Notch1, RBP-JK and Hes1 mRNA in isoliquiritigenin plus DAPT group and blocker group significantly decreased(P<0.05);Compared with blocker group, the expression of Notch1, RBP-JK and Hes1 gens were decreased significantly in isoliquiritigenin plus DAPT group(P<0.05).Conclusions:(1) Cyagen 6 hole suspension cell culture plate is an ideal carrier of culture glioma stem cells.(2) Isoliquiritigenin can induce SHG44 glioma stem cells into astrocytes and neuron cell differentiation, and can inhibit the proliferation of glioma stem cells.(3) The down-regulation of Notch1, RBP-JK and Hes1 in Notch1 signaling pathway may related to the process of Isoliquiritigenin induced SHG44 glioma stem cells differentiate into astrocytes and neurons.