AKT2-knockdown suppressed viability with enhanced apoptosis, and attenuated chemoresistance to temozolomide of human glioblastoma cells in vitro and in vivo
AKT2-knockdown suppressed viability with enhanced apoptosis, and attenuated chemoresistance to temozolomide of human glioblastoma cells in vitro and in vivo
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DOI：
10.2147/ott.s83795
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发表时间：
2015-01-01
影响因子：
4
通讯作者：
Lu, Yicheng
中科院分区：
文献类型：
--
作者：
Cui, Yong;Lin, Jing;Lu, Yicheng
AKT 2激酶（蛋白激酶B β）在高级别胶质瘤中过表达。AKT 2基因的上调先前已在遭受化疗失败和肿瘤进展的胶质母细胞瘤患者中观察到。在这项研究中，我们的目的是评估AKT 2对人胶质母细胞瘤细胞系U251的生存力和化疗耐药性的影响。用靶向AKT 2的短发夹RNA（shRNA）稳定转染U251细胞系。然后用替莫唑胺（TMZ）处理检查低表达AKT 2的U251细胞的活力，并在体外和肿瘤植入小鼠中测试细胞凋亡。接下来，通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质印迹分析测量几种耐药相关分子的表达。结果显示，AKT 2-shRNA转染的细胞的半数抑制浓度（IC 50）显著低于Lenti-GFP转染和未转染的对照组，AKT 2-shRNA和TMZ联合处理的小鼠肿瘤生长无论在质量还是体积上都受到明显抑制。与此同时，TMZ处理的肿瘤细胞的凋亡显着增强AKT 2敲低后，通过流式细胞术和原位末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）分析测量。此外，TMZ处理的胶质母细胞瘤U251细胞中的AKT 2抑制上调了凋亡效应器caspase-3，而它下调了抗凋亡蛋白Bcl-2，DNA修复蛋白MGMT和药物外排泵蛋白MRP 1。我们的研究确定AKT 2是胶质母细胞瘤中呈现化疗耐药的重要基因，并且是增强胶质瘤化疗临床效果的潜在靶点。
The AKT2 kinase (protein kinase B beta) is overexpressed in high-grade gliomas. Upregulation of the AKT2 gene has been previously observed in glioblastoma patients suffering from chemotherapy failure and tumor progress. In this study, we aimed to evaluate the effect of AKT2 on viability and chemoresistance in the human glioblastoma cell line U251. The U251 cell line was stably transfected with short hairpin RNA (shRNA) targeting AKT2. U251 cells underexpressing AKT2 were then examined for viability with temozolomide (TMZ) treatment, and tested for cell apoptosis both in vitro and in tumor-implanted mice. Next, expressions of several chemoresistance-related molecules were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis. The results showed that the 50% inhibitory concentration (IC50) of AKT2 shRNA-transfected cells was significantly lower compared with Lenti-GFP-transfected and nontransfected controls and that the tumor growth of the AKT2-shRNA and TMZ combined-treated mice was obviously suppressed in either mass or volume. Concomitantly, the apoptosis of TMZ-treated tumor cells was significantly enhanced after knockdown of AKT2, as measured by flow cytometry and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Furthermore, AKT2-inhibition in TMZ-treated glioblastoma U251 cells upregulated apoptotic effector caspase-3, whereas it downregulated antiapoptotic protein Bcl-2, DNA repairing protein MGMT, and drug efflux pump protein MRP1. Our study identified AKT2 as an important gene in presenting chemoresistance in glioblastoma, and a potential target to potentiate the clinical effect of chemotherapy in glioma treatment.