GNE-477对胶质瘤耐药细胞增殖和凋亡的影响

Effects of GNE-477 on proliferation and apoptosis of glioma drug-resistant cells

目的探讨磷脂酰肌醇3激酶(PI3K)/雷帕霉素靶蛋白(mTOR)新型抑制剂GNE-477对胶质瘤耐药细胞的增殖和凋亡的影响及其分子机制。方法通过浓度梯度递增法建立胶质瘤替莫唑胺(TMZ)耐药的模型细胞株,并命名为U87/TMZ。用不同浓度的GNE-477(0、4、8、16、32、64、128μmol/L)处理耐药细胞株之后,通过细胞计数试剂盒(CCK-8)法检测U87/TMZ细胞增殖活性;接着将细胞分为U87/TMZ+二甲基亚砜(DMSO)(对照组)和U87/TMZ+GNE-477(实验组),应用膜联蛋白V-PE/7-ADD染色和流式细胞仪来检测细胞凋亡,明确GNE-477对胶质胞瘤耐药细胞凋亡的影响。应用蛋白质印迹法(Western blot)检测两组之间凋亡相关基因[B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)]以及PI3K/mTOR信号通路关键靶点Akt和p-Akt的蛋白表达。结果CCK-8结果显示,U87+DMSO组吸光度(A)值(0.96±0.07)低于U87/TMZ+TMZ组A值(0.974±0.03),两组之间差异无统计学意义(t=0.32,P>0.05);U87+TMZ组A值0.46±0.09低于U87+DMSO组A值(0.96±0.07),差异有统计学意义(t=7.60,P<0.05)。GNE-477作用24 h后U87/TMZ细胞株增殖抑制浓度(IC50)为7.981μmol/L。流式细胞术结果显示,GNE-477处理U87/TMZ细胞株24 h后,GNE-477组细胞凋亡率为(34.75±2.02)%,高于对照组的总细胞凋亡率[(13.01±2.53)%],差异有统计学意义(t=11.63,P<0.05)。Western blot结果显示,GNE-477组的bax蛋白表达量(0.78±0.05)高于DMSO组(0.21±0.02),差异有统计学意义(t=18.83,P<0.05),GNE-477组bcl-2和p-Akt的蛋白表达量(0.21±0.02)和(0.10±0.01)低于DMSO组(1.13±0.09)和(0.3±0.03),差异有统计学意义(t=17.28、10.95,P<0.05),但是两组的Akt表达量(0.76±0.06)和(0.78±0.08)差异无统计学意义(t=0.38,P>0.05)。结论GNE-477可以克服多形性胶质母细胞瘤(GBM)细胞的化疗耐药性,抑制GBM耐药细胞的增殖,同时促进它的凋亡。

Objective To investigate the effect and molecular mechanism of the novel phosphatidylinositol 3 kinase(PI3K)/mammalian target of rapamycin(mTOR)inhibitor GNE-477 on proliferation and apoptosis of glioma resistant cells.Methods A model cell line of glioma temozolomide(TMZ)resistance was established by the concentration gradient increasing method named U87/TMZ.After treatment with GNE-477(0,4,8,16,32,64,128μmol/L),the proliferation activity of U87/TMZ cells was detected by cell counting kit-8(CCK-8)assay.Then the cells were divided into U87/TMZ+dimethylsurfoxide(DMSO)(control group)and U87/TMZ+GNE-477(treatment group).The apoptosis was detected by annexin v-PE/7-AAD staining and flow cytometry to clarify the effect of GNE-477 on apoptosis in glioma resistant cells.Western blotting was used to detect the protein expression of apoptosis-related genes(bcl-2,bax)and key targets of PI3K/mTOR signaling pathway Akt and p-Akt.Results CCK-8 assay showed that the absorbance(A)value of U87+DMSO group(0.96±0.07)was lower than that of U87/TMZ+TMZ group(0.974±0.03),but there was no statistically significant difference between the two groups(t=0.32,P>0.05);A value of U87+TMZ group(0.46±0.09)was lower than that in U87+DMSO group(0.96±0.07),and the difference was statistically significant(t=7.60,P<0.05).After treatment with GNE-477 for 24 h,the inhibitory concentration(IC50)of U87/TMZ cell line was 7.981μmol/L.Flow cytometry results showed that the apoptosis rate of U87/TMZ cell line treated by GNE-477 for 24 h was(34.75±2.02)%,higher than the control group[(13.01±2.53)%]with the difference being statistically significant(t=11.63,P<0.05).Western blotting results showed that Bax protein expression in GNE-477 group(0.78±0.05)was higher than that in DMSO group(0.21±0.02)with the difference being statistically significant(t=18.83,P<0.05).The protein expression levels of bcl-2 and p-Akt in GNE-477 group(0.21±0.02 and 0.10±0.01)were lower than those in DMSO group(1.13±0.09 and 0.30±0.03)with the difference being statistically significant(t=17.28,t=10.95,P<0.05),but there was no significant difference in the AKT expression between the two groups(0.76±0.06 and 0.78±0.08,t=0.38,P>0.05).Conclusion GNE-477 can overcome the chemoresistance of glioblastoma multiforme(GBM)cells and inhibit the proliferation of GBM resistant cells while promoting their apoptosis.