Zhao Lijing, Teng Bo, Wen Lianji, Feng Qingjie, Wang Hebin, Li Na, Wang Yafang, Liang Zuowen
Department of Otolaryngology Head and Neck Surgery, The Second Hospital, Jilin University Changchun, 130041, China ; Department of Pathophysiology, Norman Bethune Medical School, Jilin University Changchun, 130021, China.
Department of Otolaryngology Head and Neck Surgery, The Second Hospital, Jilin University Changchun, 130041, China.
Int J Clin Exp Med. 2014 Feb 15;7(2):337-47. eCollection 2014.
The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, against all class I phosphatidylinositol3-kinase (PI3K) and other members of the PI3K-like kinase family. The study was to determine the effect of AZD8055 on proliferation and apoptosis on Hep-2, a human laryngeal cancer cell line and to investigate the underlying mechanism(s) of action. Hep-2 cells were treated with AZD8055 for 24, 48 or 72 h. MTT was used to determine cell proliferation. Rhodamine 123 and TUNEL staining were used to determine mitochondrial membrane potential and cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Protein expressions were examined by western blotting. Treatment with AZD8055 inhibited proliferation and induced apoptosis in Hep-2 cells in a dose- and time-dependent manner. During the prolonged treatment with AZD8055, AZD8055 inhibits the mammalian target of rapamycin mTOR. Further experiments showed which signaling cascade p-4EBP1 and substrate EIF4E as well as downstream proteins were down regulated. Furthermore, our study showed that the expression profiles of various BH3-only proteins including Bid, Bad, and Bim, apoptosis regulatory protein cleaved caspase3 was up regulated in a time-dependent manner in Hep-2 cells treated with AZD8055. Thus, in vitro, AZD8055 potently inhibits proliferation and induces apoptosis in head and neck squamous cell carcinoma.
雷帕霉素哺乳动物靶点（mTOR）激酶形成两种多蛋白复合物，即mTORC1和mTORC2，它们调节细胞生长、存活和自噬。mTORC1的变构抑制剂，如雷帕霉素，已被广泛用于研究肿瘤细胞的生长、增殖和自噬，但临床应用有限。在此，我们描述了AZD8055，一种新型的mTOR激酶活性ATP竞争性抑制剂，它能抑制所有I类磷脂酰肌醇3激酶（PI3K）和PI3K样激酶家族的其他成员。本研究旨在确定AZD8055对人喉癌细胞系Hep-2增殖和凋亡的影响，并探讨其潜在作用机制。用AZD8055处理Hep-2细胞24、48或72小时。采用MTT法测定细胞增殖。用罗丹明123和TUNEL染色法测定线粒体膜电位，并通过荧光激活细胞分选（FACS）分析细胞凋亡。通过蛋白质印迹法检测蛋白质表达。AZD8055处理以剂量和时间依赖性方式抑制Hep-2细胞的增殖并诱导其凋亡。在长时间用AZD8055处理期间，AZD8055抑制雷帕霉素哺乳动物靶点mTOR。进一步实验表明p-4EBP1和底物EIF4E以及下游蛋白的哪些信号级联被下调。此外，我们的研究表明，在用AZD8055处理的Hep-2细胞中，包括Bid、Bad和Bim在内的各种仅含BH3结构域蛋白的表达谱以及凋亡调节蛋白裂解的caspase3以时间依赖性方式上调。因此，在体外，AZD8055能有效抑制头颈部鳞状细胞癌的增殖并诱导其凋亡。
