NPS-2143对胶质瘤增殖与迁移能力的影响及机制

Effects of NPS-2143 on proliferation and migration ability of glioma cells and its mechanism

目的 探究NPS-2143对胶质瘤U87、U251和Hs683细胞增殖与迁移能力的影响及机制。方法 将胶质瘤细胞分为3组,不加药物的对照组(Control组)、6μmol/L和8μmol/L NPS-2143处理组,通过细胞增殖实验CCK-8和细胞平板克隆形成实验检测不同浓度NPS-2143对U87和U251细胞活力与增殖的影响,通过流式细胞术实验检测不同浓度NPS-2143对细胞周期和凋亡的影响,Western blot检测细胞凋亡相关蛋白(Bax、Caspase 3)和细胞周期相关蛋白(P21、Cyclin D1)的表达水平;通过细胞划痕实验和细胞迁移实验检测NPS-2143对U87和Hs683细胞迁移的影响,Western blot法检测上皮间质转化(EMT)相关蛋白(N-cadherin、MMP2、MMP9)的表达水平;通过透射电镜观察NPS-2143对U87细胞中自噬囊泡数量的影响,Western blot检测细胞自噬相关蛋白(LC3、P62)和AKT-mTOR信号通路相关蛋白(p-AKT、p-mTOR)的表达水平。结果 与Control组比较,NPS-2143处理组胶质瘤细胞活力与增殖能力降低,且呈时间、浓度依赖性(P<0.01);与Control组比较,NPS-2143处理组胶质瘤细胞G1期细胞比例和凋亡细胞比例升高(P<0.05);与Control组比较,NPS-2143处理组胶质瘤细胞中P21蛋白表达水平增加,Cyclin D1蛋白表达水平下降,细胞凋亡蛋白Bax和Caspase 3表达水平增加(P<0.01);与Control组比较,NPS-2143抑制胶质瘤细胞的迁移能力(P<0.01),胶质瘤细胞中N-cadherin、MMP2、MMP9的表达水平降低(P<0.01),LC3、P62、p-AKT、p-mTOR蛋白表达水平升高(P<0.01);与Control组比较,NPS-2143使胶质瘤细胞内自噬囊泡增加(P<0.01)。结论 NPS-2143能抑制胶质瘤的增殖与迁移,其机制可能与激活AKT-mTOR通路有关。

Objective To explore the effect and mechanism of NPS-2143 on proliferation and migration of glioma U87 and U251,and Hs683 cells.Methods The glioma cells were divided into 3 groups:the Control group(without medical treatment),the 6μmol/L and the 8μmol/L NPS-2143 treatment group.The effects of NPS-2143 on the viability and proliferation of U87 and U251 cells were investigated by using cell counting kit-8(CCK-8)and colony formation experiments after the treatment with different concentrations of NPS-2143.Flow cytometer(FCM)was used to detect the effects of different concentrations of NPS-2143 on cycle and apoptosis of U87 and U251 cells.The expression levels of apoptosis-related proteins(Bax,Caspase 3)and cell cycle related proteins(P21,Cyclin D1)in U87 and U251 were detected by Western blot;The migration capability of U87 and Hs683 cells treated with different concentrations of NPS-2143 was detected by wound healing and Transwell experiments.The expression levels of epithelial-mesenchymal transition(EMT)related proteins(N-cadherin,MMP2,MMP9)in U87 and Hs683 cells were detected by Western blot;the expression levels of autophagy proteins(LC3,P62)and AKT-mTOR pathway related proteins(p-AKT,p-mTOR)in U87 and U251 cells were detected by Western blot.The changes of the number of autophagy vesicles in glioma U87 cells treated with NTP-2143 were observed by transmission electron microscopy.Results The viability and proliferation ability of glioma cells were reduced,with dependence of time and concentration(P<0.01),the proportaion of G1 phase and apoptotic glioma cells increased(P<0.05)and the expression level of P21,Bax,and Caspase 3 increased(P<0.01)while the level of Cyclin D1 decreased(P<0.01)in the NPS-2143 treatment group,compared with the Control group.NPS-2143 inhibited the migration ability of glioma cells(P<0.01)and decreased the expression levels of N-cadherin,MMP2,and MMP9(P<0.01),and also increased the expression levels of autophagy proteins LC3,P62,p-AKT,and p-mTOR(P<0.01),compared with the Control group.The number of autophagy vesicles obviously increased in glioma U87 cells treated with NPS-2143,compared with the Control group(P<0.01).Conclusion NPS-2143 may inhibit the proliferation and migration of glioma cells by blocking autophagy through mediating the AKT-mTOR pathway.