Effect of curcomol on proliferation and apoptosis of glioma by regulating the phosphatidylinositol 3 kinase/protein kinase B signaling pathway
Objective To explore the mechanism of curcumol on proliferation and apoptosis of gli-oma through phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)signaling pathway.Methods Different concentrations of curcumol(0,25,50,100,200 µmol/L)were used to intervene human astro-blastoma cells(U87MG)and the cell growth was observed by researcher;In addition,the cell viability was tested by cell counting kit(CCK-8).Logarithmic U87MG cells were inoculated into caudate nucleus of nude mice which is contribute to establish glioma in situ model.In terms of the model nude mice,which were randomly divided into model group(equal volume of saline),low dose of curcumol group(20 mg/kg),middle dose of curcumol group(40 mg/kg)and high dose of curcumol group(80 mg/kg),10 mice in each group were treated by continuous gavage for 3 weeks.And then the researcher had an observation of the changes of the mice's behavior.Calculate relative tumor volume and relative tumor inhibition rate;His-topathology of tumor was observed by hematoxylin-eosin(HE)staining;Immunohistochemical(IHC)analy-sis of proliferating cell nuclear antigen(PCNA)expression and tumor cell proliferation index;The apoptosis level of tumor cells was stained by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining.Comparison of the expression of PI3K/Akt pathway related proteins and downstream cy-clin D1,B cell lymphoma/lewkmia-2(bcl-2)and bcl-2 related X protein(bax)protein by Western blot-ting.The independent samples ttest was used for comparison of differences between the two groups,and one-way ANOVA analysis was used for comparison of differences between multiple groups.Results After applying the method of different concentrations of curcumol to intervene human astroblastoma cells(U87MG),and with the increasing of the the dosing concentration,floating fragments appeared in cells,besides,the number of apoptotic cells increasd at the same time.CCK-8 results show that,the cell survival rate were control group(1.00±0.07)％,25 μmol/L group(1.10±0.04)％,50 μmol/L group(0.67± 0.04)％,100 µmol/L group(0.58±0.06)％,200 μmol/L group(0.24±0.01)％,respectively.The cell survival rate of each research group was lower than that of the control group,the difference was statisti-cally significant(F=323.402,P＜0.05).The results of calculating tumor volume and tumor inhibition rate showed that,the tumor volume of model group was(42.50±27.78)mm3,the tumor volume and tumor inhibition rate of each treatment group were low dose of curcumol group[(31.85±20.89)mm3,25.20％],middle dosegroup[(21.49±13.42)mm3,49.54％],highdosegroup[(13.73±12.05)mm3,67.75％],respectively.The tumor volume of each group was smaller than that of the model group,the difference was statistically significant(F=2.147,P＜0.05).The tumor volume of high dose of cur-cumolgroup was significantly smaller than that of model group[(13.73±12.05)mm3 vs.(42.50±27.78)mm3,t=2.130,P＜0.05].IHC results showed that the tumor cell proliferation index of control group,model group,low dose of curcumol group,middle dose of curcumolgroup and high dose of curcumolgroup were(16.67±1.52)％,(40.00±1.73)％,(33.67±2.52)％,(25.67±4.17)％,(18.00±2.64)％,respectively.The tumor cell proliferation index of each treatment group was lower than that of model group,the difference was statistically significant(F=32.607,P＜0.05).The proliferation index of tumor cells in the high dose group was significantly lower than that in the model group[(18.00±2.64)％vs.(40.00± 1.73)％,t=12.050,P＜0.05].TUNEL staining showed that the apoptosis rates of tumor cells in each group were(65.80±4.37)％,(13.47±1.87)％,(23.13±4.17)％,(38.57±5.85)％and(53.00± 5.70)％,respectively.The apoptosis rate of tumor cells in each treatment group was higher than that in the model group,and the difference was statistically significant(F=60.625,P＜0.05),what's more,the apoptosis rates of tumor cells in the high dose group were significantly higher than those in the model group[(53.00±5.70)％vs.(13.47±1.87)％,t=-11.398,P＜0.05].The results of Western Blot showed that the relative expression of PI3K,Akt protein in the control group,the model group,the low dose group,the middle dose group and the high dose group were(0.10±0.01,0.72±0.06),(0.49±0.02,1.94±0.02),(0.39±0.01,1.69±0.17),(0.32±0.05,1.41±0.28),(0.14±0.03,1.11± 0.28),respectively.The relative expression of PI3K,Akt was lower than that of the model group,which indicates the difference was statistically significant(F=3.150,17.944,P＜0.05).The expression of PI3K,Akt in the high dose group was lower than that of the model group[(0.14±0.03,1.11±0.28)vs.(0.49±0.02,1.94±0.69),t=2.823,4.973,P＜0.05].The relative expression of Cyclin D1 in control group,model group,low dose group,middle dose group and high dose group were(0.33±0.07),(1.12±0.08),(0.89±0.14),(0.71±0.10),(0.50±0.10),respectively.The relative expressionof Cyclin D1 in each treatment group was lower than that in the model group,the difference was statistically significant(F=27.851,P＜0.05).The expression of Cyclin D1 in high dose group was significantly low-er than that in model group[(0.50±0.10)vs.(1.12±0.08),t=8.010,P＜0.05].Apoptosis-related protein Bcl-2,Bax was expressed(0.42±0.02,0.95±0.08),(1.18±0.15,0.42±0.11),(0.96± 0.16,0.51±0.13),(0.72±0.08,0.62±0.09),(0.57±0.08,0.73±0.10)in the control group,model group,low dose group,middle dose group and high dose group,respectively.The expression of bcl-2 was lower than that in the control group,and the expression of bax was higher than that in the control group,which also indicate that the difference was statistically significant(F=23.907,11.864,P＜0.05).The expression of bcl-2 in high dose group was significantly lower than that in model group[(0.57±0.08)vs.(1.18±0.15),t=6.391,P＜0.05].The expression of bax in high dose group was significantly higher than that in model group[(0.73±0.10)vs.(0.42±0.11),t=3.663,P＜0.05].Conclusion Curcumol,the research target,inhibits the proliferation of glioma cells effevtively and pro-mote their apoptosissignificantly;And the mechanism of curcumol may be play an important in anti-tumor by regulating the PI3K/Akt signaling pathway.
GliomaCurcumolPhosphatidylinositol 3 kinase/protein kinase B signaling path-wayProliferationApoptosis
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