| Objective:To investigate the inhibitory effect of Temozolomide combined with valproic acid on the proliferation and apoptosis of pituitary adenoma cells AtT-20 and GT1-1 in mice,and to explore its possible molecular mechanism.Methods:1.Cell culture:Mouse pituitary adenoma cells AtT-20 and GT1-1 were cultured in DMEN/High Glucose medium containing 10%fetal bovine serum(FBS).The cells were cultured in cell incubator at 37? and 5%CO2.The growth status of the cells was observed.The eells were changed once every 1-2 days and half times,and passaged once every 3 days or so.Cells in logarithmic phase were taken for experiment.2.The effects of TMZ and VPA on the proliferation of AtT-20 and GT1-1 cells.AtT-20 and GT1-1 cells in logarithmic growth phase were divided into(1)TMZ group of AtT-20 cells:TMZ group was added with different concentrations(0.1,0.2,0.4,0.8,1.6,3.2 mM)to cells and cultured for 24,48 and 72 hours respectively;(2)VPA group of AtT-20 cells:VPA with different concentrations(2,4,8,16,32 and 64 mM)was added to cells and cultured for 24,48 and 72 hours;(3)GT1-1 cells were fine.Cell TMZ group:adding different concentrations of TMZ(same concentration)to cells and culturing for 24,48 and 72 hours respectively;(4)GT1-1 cell VPA group:adding different concentrations of VPA(same concentration)to cells,and culturing for 24,48 and 72 hours respectively.The effects of TMZ and VPA on the proliferation of AtT-20 and GT1-1 cells were detected by CCK-8 kit.3.The effect of TMZ combined with VP A on the activity of mouse pituitary adenoma cells AtT-20 and GT1-1.AtT-20 and GT1-1 cells in logarithmic growth phase were divided into(1)control group(complete medium);(2)TMZ group(1.6 mM TMZ);(3)VPA group(16 mM VP A);(4)TMZ+VPA group(1.6 mM TMZ and 16 mM VP A).After 72 hours of treatment,the effects of TMZ combined with VPA on the activity of AtT-20 and GT1-1 cells were detected by LDH kit.4.The effect of TMZ combined with VPA on apoptosis of pituitary adenoma cells AtT-20 and GT1-1 in mice.AtT-20 and GT1-1 cells in logarithmic growth phase were treated with complete culture medium,TMZ and VPA respectively.After 72 hours of treatment,the apoptotic effects of TMZ combined with VPA on AtT-20 and CT1-1 cells were detected by TUNEL staining and flow cytometry.Hoechst 33258 staining was used to detect the morphological effects of TMZ combined with VPA on AtT-20 and GT1-1 cells.5.Molecular mechanism of the effect of TMZ combined with VPA on apoptosis of mouse pituitary adenoma cells AtT·20 and GT1-1.AtT-20 and GT1-1 cells in logarithmic growth phase were treated with complete culture medium,TMZ and VPA respectively.After 72 hours of treatment,the effects of TMZ combined with VPA on cleaved Caspase-3,cleaved Caspase-9,Bax,Bcl-2,AIF,cleaved PARP,p53 and other apoptotic-related proteins in AtT-20 and GT1-1 cells were detected by Western blot..Results:1.The effects of TMZ and VPA on the proliferation of AtT-20 and GT1-1 cells.CCK-8 assay showed that the proliferation activity of ATT-20 and GT1-1 cells treated with TMZ and VPA was significantly lower than that of the control group(p<0.05).TMZ and VPA had significant inhibitory effect on the proliferation activity of the cells,and showed a significant dose-dependent and time-dependent manner.2.The effect of TMZ combined with VPA on the activity of mouse pituitary adenoma cells AtT-20 and GT1-1.The results of LDH assay showed that the cell viability of AtT-20 and GT1-1 cells treated with TMZ and VPA was significantly lower than that of the control group(p<0.05).The cell viability of AtT-20 and GT1-1 cells treated with TMZ and VPA was further decreased(p<0.05).3.The effect of TMZ combined with VPA on apoptosis of pituitary adenoma cells AtT-20 and GT1-1 in mice.TUNEL staining and flow cytometry showed that the apoptotic level of ATT-20 and GT1-1 cells treated with TMZ and VPA was significantly higher than that of control group(p<0.05).The apoptotic level of ATT-20 and GT1-1 cells treated with TMZ and VPA was further higher than that of control group(p<0.05).Hoechst 33258 staining results showed that after TMZ and VPA respectively acted on AtT-20 and GT1-1 cells,the number of uniformly dispersed stained nuclei(living nucleus)decreased,the number of densely stained granular fluorescence(apoptotic nucleus)increased,the apoptotic level increased significantly compared with the control group,and the apoptotic level further increased after TMZ and VPA combined application.4.Molecular mechanism of the effect of TMZ combined with VPA on apoptosis of pituitary adenoma cells AtT-20 and GT1-1 in mice.Western blot results showed that after TMZ and VPA respectively acted on AtT-20 and GT1-1 cells,the expression of Bax,AIF and p53 increased,the phosphorylation of caspase-3,caspase-9,PARP and other apoptotic-related proteins increased,while the expression of anti-apoptotic-related protein Bcl-2 decreased.The results were statistically significant(p<0.05).The expression levels of Bax,AIF and p53 apoptosis-related proteins and phosphorylation levels of caspase-3,caspase-9,PARP and other apoptosis-related proteins were significantly increased,and the expression level of Bcl-2 was further reduced.The results were statistically significant(p<0.05).Conclusions:1.TMZ and VPA can significantly inhibit the proliferation of pituitary adenoma cells AtT-20 and GT1-1 in a time-and dose-dependent.2.TMZ and VPA can significantly inhibit the activity of pituitary adenoma cells AtT-20 and GT1-1 and promote cell apoptosis.3.When TMZ and VPA were combined,they showed synergistic effect,which could further promote the apoptosis of pituitary adenoma cells and inhibit the activity of pituitary adenoma cells.4.The effects of TMZ and VPA on mouse pituitary adenoma cells AtT-20 and GT1-1 can promote the expression of Bax,AIF and p53 apoptosis-related proteins,promote the phosphorylation of caspase-3,easpase-9,PARP and other apoptosis-related proteins,and reduce the expression level of anti-apoptosis-related proteins Bel-2,so as to realize the promoting effect of TMZ and VPA on the apoptosis of AtT-20 and GT1-1 cells.When TMZ and VPA are combined,they can play a synergistic role and improve the effect,thus further promoting the apoptosis of pituitary adenoma cells.This indicates that the combined application of TMZ and VPA can promote the apoptosis of tumor cells through the regulation of apoptosis.. |
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