摘要
目的探讨同时抑制mTORCl激酶和GSK-3B激酶活性对黑素瘤细胞4EBPl的磷酸化、帽子依赖翻译、细胞存活和凋亡的影响。方法分别采用二甲基亚砜(DMSO组)、5nmol/L依维莫司(依维莫司组)、10gtm01/LAR—A014418(AR—A014418组)、5nmol/L依维莫司联合10μmol/LAR—A014418(联合处理组)处理A375细胞,Western印迹法检测4EBPl的磷酸化和生存素蛋白的表达,mTGTPpull—down实验检测elF4E和eIF4G的相互作用,CCK8法和流式细胞仪分别检测A375细胞的增殖和凋亡。结果依维莫司和AR—A014418对4EBPl磷酸化和生存素蛋白表达均有一定的抑制作用,两组p4EBPl—65分别为0.74±0.05和0.62±0.06,生存素蛋白分别为0.71±0.06和0.58±0.07,与DMSO组(分别为1.00±0.07和1.00±0.06)相比,均P〈0.001,且联合处理组对4EBPl磷酸化(0.14±0.04)和生存素蛋白表达(O.09±0.05)的抑制更为显著。m7GTPpull-down实验显示,依维莫司组(0.72±0.04)、AR—A014418组(0.67±0.05)、联合处理组(0.12±0.05)eIF4G相对值均低于DMSO组(1.00±0.06),而4EBPl相对值均高于DMSO组(分别为1.98±0.16、2.32±0.17、7.58±0.25、1.00±0.08),且联合处理组eIF4G降低和4EBPl增加最为显著。培养24h时,与DMSO组相比,依维莫司、AR-A014418、依维莫司联合AR—A014418均对A375细胞增殖有抑制作用(后3组抑制率分别为18.5%±1.3%、19.8%±1.8%、61.2%±2.1%),且联合处理组的抑制作用最为显著;培养48h时,依维莫司和AR—A014418对A375细胞增殖的抑制显示相同的趋势,且其抑制作用随时间的延长而增强。流式细胞仪检测显示,依维莫司和AR—A014418单独处理A375细胞24h对其凋亡有一定的促进作用(凋亡率分别为14.28%±2.18%、14.57%±2.35%),联合处理时细胞凋亡更为显著,凋亡率为55.18%±6.27%。结论联合使用依维莫司和AR—A014418显著抑制A375细胞中4EBPl的磷酸化,进而抑制elF4F蛋白复合物的形成,从而抑制帽子依赖的翻译,促进黑素瘤细胞的凋亡。
Objective To evaluate effects of simultaneous inhibition of mammalian target of rapamycin complex 1 (mTORC 1 ) kinase and glycogen synthase kinase-3β(GSK-3β) on phosphorylation of 4E-binding protein-1 (4EBP1), cap-dependent translation, as well as survival and apoptosis of melanoma cells. Methods Cultured A375 cells were classified into several groups to be treated with dimethyl sulfoxide (DMSO group), the mTORC1 kinase inhibitor everolimus at a concentration of 5 nmol/L (everolimus group), the GSK-3βkinase inhibitor AR-A014418 at a concentration of 10 μmol/L (AR-A014418 group), or 5 nmol/L everolimus and 10 μmol/L AR-A014418 (combined treatment group). After additional culture, Western-blot analysis was performed to measure protein expressions of phosphorylated 4EBP1 (p4EBP1) and survivin in A375 cells, m7GTP pull down assay to estimate interaction between eukaryotic initiation factor-4E (eIF4E) and eIF4G, cell counting kit 8 (CCK8) assay to evaluate cell proliferation, and flow cytometry to detect cell apoptosis. Results Both everolimus and AR-A014418 had inhibitory effects on 4EBP1 phosphorylation and survivin expression. The expressions of p4EBP1-65 and survivin were both significantly decreased in the everolimus group (0.74 ± 0.05 and 0.71± 0.06 respectively), AR-A014418 group (0.62 ± 0.06 and 0.58 ± 0.07 respectively) and combined treatment group (0.14±0.04 and 0.09 ± 0.05 respectively) compared with the DMSO group ( 1.00 ± 0.07 and 1.00 ± 0.06, respectively, all P 〈 0.001 ), with the most significant decrease observed in the combined
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2016年第4期271-275,共5页
Chinese Journal of Dermatology
基金
贵州省中药现代化专项项目